The sugar beet gene encoding the sodium proton exchanger 1 (BvNHX1) is regulated by a MYB
A NAC gene regulating senescence improves grain protein, zinc, and iron content in wheat
multilayer endosperm.However,most of the iron in rice seed,for example,is associated with the embryo and the aleurone layer,not the endosperm, suggesting that VIT1-mediated iron storage in the embryo may play the same role in developing endospermic plants as that described here for Arabidopsis.Furthermore,unlike other Fe trans-porters characterized to date such as IRT1,which can transport Cd as well as Fe(21),VIT1does not appear to transport Cd.Cd levels in seeds from lines overexpressing VIT1were low(<0.1part per million),with no significant difference compared to wild-type seeds(P<0.05).Therefore,any potential biotechnological applications of VIT1 will not have to consider unwanted accumulation of this toxic heavy metal.Our study demonstrates the power of com-bining mutant analysis with a technique that can both image and determine the elemental com-position of living plant material.Although2D imaging with x-ray fluorescence has been used before to image the distribution of metals in plant tissues(22,23),including Arabidopsis seed(24), our ability to render3D images at high resolution allowed us to determine that Fe was associated with the provascular system throughout the seed and should prompt more studies on spatial distribution of metals in biological samples.Our study also highlights the role of the vacuole in seed iron storage and suggests that the vacuole offers another avenue for increasing the iron content of plant-based diets.References and Notes1.C.Curie,J.-F.Briat,Annu.Rev.Plant Biol.54,183(2003).2.F.Goto,T.Yoshihara,N.Shigemoto,S.Toki,F.Takaiwa,Nat.Biotechnol.17,282(1999).3.P.Lucca,R.Hurrell,I.Potrykus,J.Am.Coll.Nutr.21,184S(2002).4.L.-Q.Qu,T.Yoshihara,A.Ooyama,F.Goto,I.Takaiwa,Planta222,225(2005).5.A.Pich,R.Manteuffel,S.Hillmer,G.Scholz,W.Schmidt,Planta213,967(2001).6.F.Raguzzi,E.Lesuisse,R.R.Crichton,FEBS Lett.231,253(1988).7.H.-P.Bode,M.Dumschat,S.Garotti,G.F.Fuhrmann,Eur.J.Biochem.228,337(1995).8.L.Li,O.S.Chen,D.M.Ward,J.Kaplan,J.Biol.Chem.276,29515(2001).9.O.S.Chen,J.Kaplan,J.Biol.Chem.275,7626(2000).10.M.Schmid et al.,Nat.Genet.37,501(2005).11.J.L.Bowman,S.G.Mansfield,in Arabidopsis:An Atlas ofMorphology and Development,J.Bowman,Ed.(Springer-Verlag,New York,1994),pp.351–361.12.D.Eide,M.Broderius,J.Fett,M.L.Guerinot,Proc.Natl.Acad.Sci.U.S.A.93,5624(1996).13.N.J.Robinson,C.M.Proctor,E.L.Connolly,M.L.Guerinot,Nature397,694(1999).14.E.E.Rogers,M.L.Guerinot,Plant Cell14,1787(2002).15.E.P.Colangelo,M.L.Guerinot,Plant Cell16,3400(2004).16.J.N.Lott,M.M.West,Can.J.Bot.79,1292(2001).17.M.S.Otegui,R.Capp,L.A.Staehelin,Plant Cell14,1311(2002).18.S.R.Sutton et al.,in Applications of SynchrotronRadiation in Low-Temperature Geochemistry andEnvironmental Science,P.A.Fenter,M.L.Rivers,N.C.Sturchio,S.R.Sutton,Eds.(Mineralogical Society ofAmerica,Washington,DC,2002),vol.49,pp.429–483.19.W.H.Stuppy,J.A.Maisano,M.W.Colbert,P.J.Rudall,T.B.Rowe,Trends Plant Sci.8,2(2003).nquar et al.,EMBO J.24,4041(2005).21.G.Vert et al.,Plant Cell14,1223(2002).22.T.Punshon,nzirotti,S.Harper,P.M.Bertsch,J.Burger,J.Environ.Qual.34,1165(2005).23.D.H.McNear Jr.et al.,Environ.Sci.Technol.39,2210(2005).24.L.W.Young,N.D.Westcott,K.Attenkofer,M.J.T.Reaney,J.Synchrotron Radiat.13,304(2006).25.S.Kumar,K.Tamura,I.Jacobsen,M.Nei,MEGA2:Molecular Evolutionary Genetics Analysis Version2.0.(Pennsylvania and Arizona State Universities,UniversityPark,PA,and Tempe,AZ,2000).26.J.S.Busse,R.F.Evert,Int.J.Plant Sci.160,1(1999).27.We thank F.Ausubel,C.R.McClung,and D.Salt forcritically reading the manuscript;hner and D.Saltfor ICP-MS analysis;and vanway for help withmicroscopy.This work was supported by grants from theNSF(DBI0077378and IBN-0419695to M.L.G.;DBI0420126to J.R.E.)and NIH(DK30534to J.K.).T.P.wassupported by a training fellowship from the DartmouthNIH–National Institute of Environmental Health ScienceSuperfund Basic Research Program Project(P42ES07373)through the Center for Environmental Health Sciences atDartmouth.A portion of this work was performed atBeamline X26A,National Synchrotron Light Source,Brookhaven National Laboratory.X26A is supported by theU.S.Department of Energy(DOE)–Geosciences andDOE–Office of Biological and Environmental Research,Environmental Remediation Sciences Division.Supporting Online Material/cgi/content/full/1132563/DC1Materials and MethodsFigs.S1and S2Table S1ReferencesMovie S117July2006;accepted6October2006Published online2November2006;10.1126/science.1132563Include this information when citing this paper.A NAC Gene Regulating Senescence Improves Grain Protein,Zinc,and Iron Content in WheatCristobal Uauy,1*Assaf Distelfeld,2*Tzion Fahima,2Ann Blechl,3Jorge Dubcovsky1†Enhancing the nutritional value of food crops is a means of improving human nutrition and health. We report here the positional cloning of Gpc-B1,a wheat quantitative trait locus associated with increased grain protein,zinc,and iron content.The ancestral wild wheat allele encodes a NAC transcription factor(NAM-B1)that accelerates senescence and increases nutrient remobilization from leaves to developing grains,whereas modern wheat varieties carry a nonfunctional NAM-B1 allele.Reduction in RNA levels of the multiple NAM homologs by RNA interference delayed senescence by more than3weeks and reduced wheat grain protein,zinc,and iron content by more than30%.T he World Health Organization estimates that more than2billion people havedeficiencies in key micronutrients such as Zn and Fe and more than160million children under the age of5lack adequate protein(1), leading to an economic burden for society(2). The two major types of wheat,tetraploid wheats [diploid cell(2n)=28],used for pasta,and hexaploid wheats(2n=42),used primarily for bread,account for~20%of all calories con-sumed worldwide.Annual wheat production is estimated at620million tons of grain(3),translating into approximately62million tonsof protein.Increasing grain protein content(GPC)has been hindered by environmental ef-fects,complex genetic systems governing thistrait,and a negative correlation with yield(4).Less progress has been made in increasing Zn andFe content,the focal point of the HarvestPlusglobal initiatives(5).Wild emmer wheat[Triticum turgidum ssp.dicoccoides(DIC)]is the ancestor of cultivatedpasta wheat(T.turgidum ssp.durum)and apromising source of genetic variation in protein,Zn,and Fe content(6,7).A quantitative traitlocus(QTL)for GPC was mapped on chromo-some arm6BS in a population of recombinantinbred lines derived from the T.turgidum ssp.durum cultivar Langdon(LDN)and the chro-mosome substitution line LDN(DIC6B)(8).This locus was associated with GPC increases of~14g kg−1in both tetraploid and hexaploid lines(8–10).Olmos et al.(11)mapped this QTL asa simple Mendelian locus,Gpc-B1(Fig.1A),which was later located within a0.3-cM interval(12).Molecular markers Xuhw89and Xucw71within this region flank a245-kb physicalcontig,including Gpc-B1(13).Tetraploid and hexaploid wheat lines carry-ing this245-kb DIC segment show delayedsenescence and increased GPC and grain micro-nutrients(14,15).The complete sequencing ofthis region(DQ871219)revealed five genes1Department of Plant Sciences,University of California,One Shields Avenue,Davis,CA95616,USA.2Institute ofEvolution,University of Haifa,Mt.Carmel,Haifa31905,Israel.3United States Department of Agriculture,Agricul-tural Research Service,Western Regional Research Center,800Buchanan St.,Albany,CA94710,USA.*These authors contributed equally to this work.†To whom correspondence should be addressed.E-mail:jdubcovsky@Fig. 1.Map-based cloning of Gpc-B1.(A )QTL for grain protein on wheat chromosome arm 6BS (11).(B )Se-quenced B-genome physical contig.The position and orienta-tion of five genes are indicated by arrows.(C )Fine mapping of Gpc-B1.The x ’s indi-cate the positions of critical recombination events flanking Gpc-B1.Vertical lines represent polymorphism mapped in the critical lines.A single gene with three exons (green rectan-gles)was annotated within the 7.4-kb region flanked by the closest recombination events.The open arrowhead in-dicates the transcription initiation site.(D )Graphical genotypes of critical recombinant substitution lines used for fine-mapping of Gpc-B1.Blue bars represent LDN markers;red bars represent DIC markers.(E )Flag-leaf chlorophyll content of recombinant substitution lines segregating for Gpc-B1(14).Asterisks indicate significant differences (P <0.01).Phenotypes of critical recombinant substitution lines:(F )chlorophyll at 20days after anthesis(DAA),(G )grain protein,(H )Zn,and (I )Fe concentrations.Blue and red bars indicate the presence of the LDN and DIC alleles at TtNAM-B1,respectively.(J )First 18nucleotides of DIC and LDN TtNAM-B1alleles and their corresponding amino acid translation.The LDN allele carries a 1-bp insertion (red T)that disrupts the reading frame (indicated by red amino acid residues).Error bars represent standard error of themean.Fig.2.(A )Expression profile of the different TtNAM genes relative to ACTIN in tetraploid wheat recombinant substitution line 300carrying a func-tional TtNAM-B1gene.Units are values linear-ized with the 2(–DD CT)method,where CT is the threshold cycle.(B and C )Relative transcript level of endogenous TaNAM genes in T 2plants (L19-54)segre-gating for transgenic (n =12,white)and nontransgenic (n =11,black)TaNAM RNAi constructs at (B)4and (C)9days after anthe-sis.Asterisks indicate significant differences (P <0.05).(D )Flag-leaf chlorophyll content profile of transgenic (n =22T 1plants)and nontransgenic controls (n =10T 1plants).(E )Representative trans-genic (left)and non-transgenic (right)plants50DAA.(F )Main spike and peduncles of representative transgenic and nontransgenic plants 50DAA.Error bars represent standard error of themean.(Fig.1B)(16).A high-resolution genetic map, based on approximately9000gametes and new molecular markers(table S1),was used to determine the linkage between these genes and the Gpc-B1locus.Three recombinant substitu-tion lines with recombination events between markers Xuhw106and Xucw109delimited a 7.4-kb region(Fig.1,C and D)(16).The re-combinant lines carrying this DIC segment se-nesced on average4to5days earlier(P<0.01, Fig.1,E and F)and exhibited a10%to15% increase in GPC(Fig.1G),Zn(Fig.1H),and Fe (Fig.1I)concentrations in the grain(P<0.01). Complete linkage of the7.4-kb region with the different phenotypes suggests that Gpc-B1is a single gene with multiple pleiotropic effects.The annotation of this7.4-kb region(Fig. 1C)identified a single gene encoding a NAC domain protein,characteristic of the plant-specific family of NAC transcription factors (17).NAC genes play important roles in de-velopmental processes,auxin signaling,defense and abiotic stress responses,and leaf senescence (18,19).Phylogenetic analyses revealed that the closest plant proteins were the rice gene ONAC010(NP_911241)and a clade of three Arabidopsis proteins including No Apical Meri-stem(NAM)(figs.S1and S2).On the basis of these similarities,the gene was designated NAM-B1(DQ869673).To indicate the species source,we have added a two-letter prefix(e.g., Ta and Tt for T.aestivum and T.turgidum genes, respectively).Comparison of the parental TtNAM-B1se-quences revealed a1-bp substitution within the first intron and a thymine residue insertion at position11,generating a frame-shift mutation in the LDN allele(DQ869674,Fig.1J).This frame shift resulted in a predicted protein having no similarity to any GenBank sequence and lacking the NAC domain.The wild type TtNAM-B1allele was found in all42wild emmer accessions examined (T.turgidum ssp.dicoccoides)(table S2)and in 17of the19domesticated emmer accessions (T.turgidum ssp.dicoccum).However,57culti-vated durum lines(T.turgidum ssp.durum)(20) (table S3)lack the functional allele,which suggests that the1-bp frame-shift insertion was fixed during the domestication of durum wheat. The wild-type TaNAM-B1allele was also absent from a collection of34varieties of hexaploid wheat(T.aestivum ssp.aestivum),representingdifferent market classes and geographic loca-tions.Twenty-nine of these showed no poly-merase chain reaction(PCR)amplificationproducts of the TaNAM-B1gene,which suggeststhat it is deleted,whereas the remaining fivelines have the same1-bp insertion observed inthe durum lines(table S4).In addition to the mutant TtNAM-B1copy,thedurum wheat genome includes an orthologouscopy(TtNAM-A1)on chromosome arm6AS anda paralogous one(TtNAM-B2)91%identical atthe DNA level to TtNAM-B1on chromosomearm2BS(21)(fig.S3and table S5).These twocopies have no apparent parisonsat the protein level of the five domains char-acteristic of NAC transcription factors(17)revealed98%to100%protein identity(fig.S2)between barley,wheat,rice,and maize homologs.Quantitative PCR(16)showed transcriptsfrom the three TtNAM genes at low levels in flagleaves before anthesis,after which their levelsincreased significantly toward grain maturity(Fig.2A).Transcripts were also detected in green spikesand peduncles.The similar transcription profilesand near-identical sequences of TtNAM-A1,B1,and B2suggest that the4-to5-day delay insenescence and the10%to15%decrease in grainprotein,Zn,and Fe content observed in LDN arelikely the result of a reduction in the amount offunctional protein rather than the complete loss-of-function of a specific gene.To test this hypothesis,we reduced the tran-script levels of all NAM copies using RNA in-terference(RNAi).An RNAi construct(16)wastransformed into the hexaploid wheat varietyBobwhite,selected for its higher transformationefficiency relative to tetraploid wheat.The RNAiconstruct targeted the3′end of the four TaNAMgenes found in hexaploid wheat(TaNAM-A1,D1,B2,and D2),outside the NAC domain,toavoid interference with other NAC transcriptionfactors(fig.S4and table S6)(22).We identified two independent transgenicplants(L19-54and L23-119)with an expectedstay-green phenotype.Quantitative PCR analy-sis of transgenic L19-54plants showed a sig-nificant reduction in the endogenous RNAlevels of the different TaNAM copies(22)at4and9days after anthesis(P<0.05)(Fig.2,Band C)compared with control lines.Transgenicplants reached50%chlorophyll degradation inflag leaves24days later than their nontransgenicsibs(P<0.001)(Fig.2D),and their main spikepeduncles turned yellow more than30days laterthan the controls(Fig.2,E and F).The presence of the RNAi transgene also hadsignificant effects on grain protein,Zn and Feconcentrations.Transgenic plants showed areduction of more than30%in GPC(P<0.001),36%in Zn(P<0.01),and38%in Fe(P<0.01)concentration compared with the nontransgeniccontrols(Table1).No significant differences wereobserved in grain size(P=0.41),suggesting thatthe extra days of grain filling conferred by thereduced TaNAM transcript level did not translateinto larger grains in our greenhouse experiments(23).Similar results were obtained for the secondtransgenic event,L23-119(fig.S5and table S7).These results suggest that the reduced grainprotein,Zn,and Fe concentrations were the re-sult of reduced translocation from leaves,ratherthan a dilution effect caused by larger grains.This hypothesis was confirmed by analyzing theresidual nitrogen,Zn,and Fe content in the flagleaves.We analyzed both transgenic eventstogether(due to greater variability in flag leavescompared with the grains)and confirmed higherlevels of N(P=0.01),Zn(P<0.01),and Fe(P<0.01)in the flag leaves of transgenic plantscompared with the nontransgenic sister lines(table S8).This supports a more efficient N,Zn,and Fe remobilization in plants with higherlevels of functional TaNAM transcripts.These results confirm that a reduction inRNA levels of the TaNAM genes is associatedwith a delay in whole-plant senescence;a de-crease in grain protein,Zn,and Fe concen-trations;and an increase in residual N,Zn,andFe in the flag leaf.These multiple pleiotropiceffects suggest a central role for the NAM genesas transcriptional regulators of multiple pro-cesses during leaf senescence,including nutrientremobilization to the developing grain.The differences observed between the trans-genic and nontransgenic plants for these traitswere larger than those observed between theLDN and DIC alleles.The RNA interference onall functional TaNAM homologs may result in alarger reduction of functional transcripts than thesingle nonfunctional TtNAM-B1allele in tetra-ploid recombinant lines carrying the LDN allele.The cloning of Gpc-B1provides a direct linkbetween the regulation of senescence and nu-trient remobilization and an entry point to char-acterize the genes regulating these two processes.This may contribute to their more efficient mani-pulation in crops and translate into food withenhanced nutritional value.References and Notes1.World Health Organization,Meeting of Interested Parties:Nutrition(2001;http://www.who.int/mipfiles/2299/MIP_01_APR_SDE_3.en.pdf).2.R.M.Welch,R.D.Graham,J.Exp.Bot.55,353(2004).3.United Nations Food and Agriculture Organization,FoodOutlook(4,2005;/documents).4.N.Simmonds,J.Sci.Food Agric.67,309(1995).Table1.Characterization of grain and senescence-related traits of transgenic Bobwhite T1plants (event L19-54)segregating for the presence(transgenic,n=22plants)or absence(nontransgenic, n=10plants)of the TaNAM RNAi W,thousand kernel weight;DAA,days afteranthesis.GPC (%)Zn(ppm)Fe(ppm)TKW(g)Dry peduncle(DAA)Dry spike(DAA)Transgenic13.2752.4537.4030.2372.553.0 Nontransgenic19.0882.5060.8331.2738.437.2 Difference–5.81–30.09–23.42–1.04+34.1+15.8 P value<0.001<0.01<0.010.41<0.001<0.001/index.html.6.L.Avivi,“High protein content in wild tetraploid Triticumdicoccoides Korn,”S.Ramanujam,Ed.,5th Int.Wheat Genet.Symp.,New Delhi,Indian Soc.Genet.Plant Breed.(1978).7.I.Cakmak et al.,Soil Sci.Plant Nut.50,1047(2004).8.L.R.Joppa,C.Du,G.E.Hart,G.A.Hareland,Crop Sci.37,1586(1997).9.A.Mesfin,R.Frohberg,J.Anderson,Crop Sci.39,508(1999).10.P.W.Chee,E.M.Elias,J.Anderson,S.F.Kianian,Crop Sci.41,295(2001).11.S.Olmos et al.,Theor.Appl.Genet.107,1243(2003).12.A.Distelfeld et al.,Funct.Integr.Genomics4,59(2004).13.A.Distelfeld,C.Uauy,T.Fahima,J.Dubcovsky,NewPhytol.169,753(2006).14.C.Uauy,J.Brevis,J.Dubcovsky,J.Exp.Bot.57,2785(2006).15.A.Distelfeld et al.,Physiol.Plant.,in press.16.Materials and methods are available as supportingmaterial on Science Online.17.H.Ooka et al.,DNA Res.10,239(2003).18.A.N.Olsen,H.A.Ernst,L.L.Leggio,K.Skriver,TrendsPlant Sci.10,79(2005).19.Y.Guo,S.Gan,Plant J.46,601(2006).20.M.Maccaferri,M.C.Sanguineti,P.Donini,R.Tuberosa,Theor.Appl.Genet.107,783(2003).21.The paralogous TtNAM-A2copy was not detected in theLDN tetraploid BAC library or by PCR with2A genomespecific primers derived from T.urartu,where theTuNAM-A2gene is present.22.The Bobwhite TaNAM-B1gene is deleted as determinedby PCR with four sets of independent NAM-B1specificprimers(table S5).Therefore,no expression data areincluded for TaNAM-B1in the transgenic plants.23.Field experiments including Gpc-B1isogenic linesshowed a more variable effect of the DIC chromosomeregion(including TtNAM-B1)on grain size(14).24.We thank G.Hart and L.Joppa for the original mappingmaterials;L.Li,L.Valarikova,and L.Beloborodov forexpert technical assistance;R.Thilmony for criticalreading of the manuscript;and the National Small GrainCollection,M.Sanguineti,J.Dvorak,and E.Nevo forgermplasm used in this study.This research wassupported by the National Research Initiative of USDA’sCooperative State Research,Education,and ExtensionService,CAP Grant No.2006-55606-16629,by BARD,the United States–Israel Binational Agricultural Researchand Development Fund,Grant -3573-04C,and bya Vaadia-BARD fellowship.GenBank accession numbersare DQ871219and DQ869672to DQ869679.Supporting Online Material/cgi/content/full/314/5803/1298/DC1Materials and MethodsFigs.S1to S6Tables S1to S9References9August2006;accepted20October200610.1126/science.1133649Evolutionary History of Salmonella Typhi Philippe Roumagnac,1François-Xavier Weill,2Christiane Dolecek,3Stephen Baker,4Sylvain Brisse,2Nguyen Tran Chinh,5Thi Anh Hong Le,6Camilo J.Acosta,7*Jeremy Farrar,3 Gordon Dougan,4Mark Achtman1†For microbial pathogens,phylogeographic differentiation seems to be relatively common.However, the neutral population structure of Salmonella enterica serovar Typhi reflects the continued existence of ubiquitous haplotypes over millennia.In contrast,clinical use of fluoroquinolones has yielded at least15independent gyrA mutations within a decade and stimulated clonal expansion of haplotype H58in Asia and Africa.Yet,antibiotic-sensitive strains and haplotypes other than H58still persist despite selection for antibiotic resistance.Neutral evolution in Typhi appears to reflect the asymptomatic carrier state,and adaptive evolution depends on the rapid transmission of phenotypic changes through acute infections.M any bacterial taxa can be subdivided into multiple,discrete clonal groupings(clo-nal complexes,or ecotypes)that have diverged and differentiated as a result of clonal replacement,selective sweeps,periodic selection, and/or population bottlenecks(1).Geographic isolation and clonal replacement can also result in phylogeographic differences between bacterial pathogens from different parts of the world(2), even within young,genetically monomorphic pathogens(3)(supporting online material text)such as Mycobacterium tuberculosis(4)and Y ersinia pestis(5).Typhi is a genetically monomorphic(6), human-restricted bacterial pathogen that causes21 million cases of typhoid fever and200,000deaths per year,predominantly in southern Asia,Africa,and South America(7).Typhi also enters a carrierstate in rare individuals[such as Mortimer’sexample of“Mr.N the milker”(8)],who can shedhigh levels of these bacteria for decades in theabsence of clinical symptoms.Genome sequencesare available from strains CT18(9)and Ty2(10),but the global diversity,population genetic struc-ture,and evolutionary history of Typhi were poorlyunderstood.It has been speculated that Typhievolved in Indonesia,which is the exclusive sourceof isolates with the z66flagellar antigen(11).We investigated the evolutionary history andpopulation genetic structure of Typhi by mutationdiscovery(12)within200gene fragments(~500base pairs each)from a globally representativestrain collection of105strains.The200genesincluded121housekeeping genes;50genesencoding cell surface structures,regulation,andpathogenicity;and29pseudogenes.Size variationof a poly-T6-7homopolymeric stretch within onegene fragment was inconsistent with other phylo-genetic patterns(homoplasies)and this fragmentwas excluded from further analysis.The other1991Max-Planck-Institut für Infektionsbiologie,Department ofMolecular Biology,Charitéplatz1,10117Berlin,Germany. 2Institut Pasteur,UnitéBiodiversitédes Bactéries Patho-gènes Emergentes,28rue du Docteur Roux,75724Paris cedex15,France.3Oxford University Clinical Research Unit, Hospital for Tropical Diseases,190Ben Ham Tu,District5, Ho Chi Minh City,Vietnam.4The Wellcome Trust Sanger Institute,Wellcome Trust Genome Campus,Hinxton,Cam-bridge CB101SA,UK.5Hospital for Tropical Diseases,190 Ben Ham Tu,District5,Ho Chi Minh City,Vietnam.6Institut National d’Hygiène et d’Épidémiologie,Hanoi1000,Vietnam. 7International Vaccine Institute(IVI),Kwanak Post Office Box 14,Seoul151-600,Korea.*Present address:GlaxoSmithKline,King of Prussia,PA 19406–2772,USA.†To whom correspondence should be addressed.E-mail: achtman@mpiib-berlin.mpg.de Fig.1.Minimal span-ning tree of105globalisolates based on se-quence polymorphismsin199gene fragments(88,739base pairs).Thetree shows59haplo-types(nodes)based on88BiPs,the continentalsources of which areindicated by colors with-in pie charts.The num-bers along some edgesindicate the number ofBiPs that separate thenodes that they connect;unlabeled edges reflectsingle BiPs.The ge-nomes of the CT18andTy2strains have beensequenced(GenBank ac-cession codes AL513382and AE014613,respec-tively).z66refers to aflagellar variant that iscommon in Indonesia(11).Ty2CT1822222222233332451234 5 strainsAfricaAsiaSouth AmericaH45: ancestral haplotype2z662H45z66z66H58H1。
拟南芥alpha-Dioxygenase 2原核表达,纯化及亚细胞定位预测
,2002 a,b)。van der Biezen .(1996)首次利用转
座子标签法从在番茄中分离到一个新基因，该基因
突变导致生长缓慢、植株矮小柔弱、结实率低等，因
而命名为
。Sanz (1998)通过差异显示
技术从烟草中分离到参与细菌诱导的超敏反应过程
的新基因，并命名为 （pathogeninduced oxyge
spectively. A fusion protein about 98 kD was detected by SDSPAGE and Western blot in the induced recombinant BL21 (DE3)RIPL
codon+ strain, and it accounts for 38% of the total bacterial proteins. The target protein was purified by the GST chromatography col
第5 期
张美祥等: 拟南芥 Alphadioxygenase 2 原核表达、纯化及亚细胞定位预测
817
们蛋白序列的相似性为 71.4%。拟南芥 DOX1 在蛋
白序列、表达模式与最初的从烟草中分离到的
相似，其表达受病原及抗逆过程诱导(Koeduka
,2005)，拟南芥 DOX2 与最初从番茄中分离到
nase），到目前为止研究者已先后从番茄、烟草、拟南
芥、水稻、小麦等植 物中分离 到 18 个 同源基 因
（Hamberg
,2005）， 并 将 其 统 一 归 类 为 al
phadioxygenase。
拟南芥中脂肪酸 alphadioxygenase 存在两个拷
eutrema salsugineum 基因组
盐芥（Eutrema salsugineum）是自然生长于农田区盐渍化土壤的盐生植物，与拟南芥（Arabidopsis thaliana）近缘、同属十字花科，具有高耐盐性、生活周期短和基因组结构小等优点，能够在500 mmol/L NaCl的高盐环境下完成生活史，是研究植物耐盐性的理想模式植物。
盐芥基因组中有7个串联重复基因编码PHT1;3，且实验证明至少其中6个基因参与了磷吸收。
在盐胁迫下盐芥中多数PHT1基因表达上调，而拟南芥中多数PHT1基因的表达受到抑制，包括PHT1;9在内的多个基因在两个物种中呈现相反的表达模式。
进一步分析发现EsPHT1;9和AtPHT1;9均参与磷从根到茎的转运，且在低磷和盐胁迫下，地上部分的总磷与钾含量呈显著正相关，提示磷和钾存在协同转运机制。
EsPHT1;9及其启动子提高拟南芥对盐和低磷双重胁迫的抗性。
花花柴耐盐相关基因NHX的克隆与分析
关键词
Ｎ Ｘ； Ｒ Ａ； Ｈ 总 Ｎ 分子 克隆 ； 分析 文献标 识码 ： Ａ Ｄ Ｉ１ ．９９ ｊ ｓ．０ ９— ５ ８２ １ ．４ ０６ Ｏ ：０ ３６ ／ｉ ｎ １０ ０ ６ ．０ １０ ．０ ｓ
中图分类号 ：８ ２ Ｑ １
Ｃｌｎ ｎ ｎ ａｙ ｉ ｆＳ ｌ ｒｅ ａ ｔＲｅａｅ ｎ ｏ ｉ ｇ ａ ｄ Ａｎ ｌｓｓｏ ａｔＴｏｌｒ ｎ ｌｔｄ Ｇｅ ｅ ＮＨＸ ｎＫａ ｅｉ ｉ ａ ｐ ｃ ｉ ｒ ｌｎ ａ ｃ ｓ ｉａ
植 物花花柴为材料 ， 利用分子克隆技术 ， 从总 Ｒ Ａ中克隆编码 Ｎ Ｈ Ｎ ａ ／ 逆 向转运蛋 白基 因。依测序结 果判断 已从花花柴 ｃ － Ｄ
Ｎ 中克隆到 １５ ｐ的 Ｎ Ｘ基因片段 ， 对该 序列进行数据库搜索和对序列 比对分析 。结果显示一致性达 到 ６ ．７ ， Ａ ２ ３ｂ Ｈ 并 ８ ７ ％ 可以
摘要 植物 Ｎ Ｘ属于 Ｎ Ｈ Ｈ ａ ／ 逆向转运蛋 白 Ｎ Ｅ Ｎ Ｈ ／ ＨＸ亚家族 的成员 ， 为阳离子逆 向转运蛋 白家族 的一个亚类 ， 具有调节 细
胞内 ｐ Ｈ值和 Ｎ 的浓度及维持 细胞 内离 子稳态等多种功能 。为进一步开发新疆盐生植物耐盐相关基 因资源 ， ａ 本研究 以盐 生
（ ｅ ａｏａｏｙｏ ｉ ｏｉａ Ｒ ｓｕｃ ｒｔ ｔ ｎａ ｄＵｉｚｔ ｎｏ ａｉ ａｉ， ３Ｋ ｙＬ ｂ ｒｔ ｆ ｏ ｇ ｌ ｅｏ ｒｅＰｏｅ ｉ ｎ ｔｉ ｉ ｆ ｒ Ｂ ｓ ｒ Ｂ ｌ ｃ ｃｏ ｌａ ｏ Ｔ ｍ ｎ
Ｘ ｎｉｎ ｒｄ ｃ ｏ ｉｊ ｇＰｏ ｕｔ ｎ＆ Ｃ ｎｔ ｃｉ ｒｕ ， ｌ ， ｉ ｉ ｇ８ ３ ０ ） ａ ｉ ｏ ｓ ｕ ｔ ｎＧ ｏ ｐ Ａ ａ Ｘｎ ａ ４ ３ ０ ｒ ｏ ｒ ｊｎ
拟南芥β-1
（ 疆 大学 生命 科 学与 技 术 学 院 ． 疆 生 物 资 源与 基 因 工程 重 点 实验 室 ， 鲁木 齐 新 新 鸟 ８０４ ） ３０６
摘
要 ： 目的 】 究 拟 南 芥 Ｂ １３ 【 研 一 ， 一葡 聚糖 酶 基 因 （ Ｇ ） 不 同组 织 及 胁 迫 条 件 下 的 表 达 规 律 。 【 法 】 Ｂ １在 方 研
究 以拟 南 芥 为 材 料 ， ２ Ｓｒ Ｎ 以 ８ Ａ为 内 参 ， 用 半 定 量 Ｒ Ｒ 利 Ｔ—Ｐ Ｒ法 研 究 了低 温 （ ℃ ） 高 温 （ ７ ） Ｎ Ｃ 胁 迫 Ｃ ４ 、 ３℃ 、ａ１
（０ ｍ ｌＬ 和 与 之 等 渗 的 Ｐ Ｇ ２ ．％ ） ２ｏｍ ｏ ） ／ Ｅ （１５ 干旱 胁 迫 处 理 在 不 同组 织 （ 、 、 、 ） Ｂ １ 表 达 情 况 。 【 叶 薹 花 果 中 Ｇ 的 结 果 】 １正 常植 株 中 ， Ｇ 在 不 同组 织 的表 达 量 不 同 ： 、 ＞叶 ＞薹 。 （ ） 迫处 理 对 Ｂ Ｉ的 表 达 量 有 影 响 。 （） Ｂ １ 花 果 ２胁 Ｇ
胁 迫 后 表 达 量 下 降 ； 果 实 中 ， Ｇ 在 Ｐ Ｇ胁 迫 后 表 达 量 稍 有 增 加 ， ３ ℃ 和盐 胁 迫 表 达 稍 有 下 降 。【 论 】 在 Ｂ１ Ｅ 而 ７ 结
在正 常植 株 中 ， Ｇ 的 表达 具 有 差 异 性 ； Ｇ 对 各 种胁 迫 处 理 的 响应 不 同。 Ｂ １ Ｂ １
Ｅ ｐ ｅｓ ｎＡ ａ ｓ ｆ ｘ ｒｓｉ ｎ ｌ ｉ ｏ ｏ ｙ ｓ Ｐ一１３一ｇｕ ａ ａｅｇｎ ｊ ｎＤ ｆ ｒｎ ， ｌｃｎ ｓ ｅ ｅ（５ １ ｉ ｅｅ ｔ 『 ）ｉ Ｇ
Ｔｉｓ ｓ ｕ ｄ ｒ Ｖａ ｉ ｕ ｉ ｔｃ Ｓ ｒ ｓ ｅ ｎ Ａｒ ｂ ｄ ｐ ｉ ｈ ｌ ｎ ． ｓ ｕｅ ｎ ｅ ｒｏ ｓ Ａｂ ｏ ｉ ｔ ｅ ｓ ｓ ｉ ａ ｉｏ ｓｓ ｔ ａ ｉ ａ Ｌ ａ
过量表达大叶补血草LgSOS1基因对拟南芥耐盐性的影响
（ ｏ ｌ ｇ ｆＬ ｆ ｃｅ ｃ ， ｈ ｈ ｚ Ｕｎ ｖ ｒ ｉｙ Ｓ ｉ ｅ ｉ ８ ２ ０ ） Ｃ ｌ ｅ ｏ ｉｅ Ｓ ｉ ｎ ｅ Ｓ ｉ ｅ ｉ ｅ ｉ ｅ ｓｔ ， ｈ ｈ ｚ ， ３ ０ ３
ｓ ｐａ ａ ｅ ｒ ｅ ｒ ｔ ｄ ｆｏｍ ａｌｐｈｙ ｅ Ｌｉ ｎｉ ｈ ｏ ｔ ｍｏ ｕｍ ｇｍ ｅ ｉ ｉ （Ｗ ｉｄ１ Ｋ ｕ ｚ ｗ ｅ ｅ ｎｓ ｒｅ ｎ ｏ ｌ ｎｔ ｅ ｔ ｒ ＣＡ Ｍ ＢＩ １ ０１ ｌｎ ｉ ｌ ．） ｎｔபைடு நூலகம்ｅ， ｒ ｉ ｅ ｔ ｄ ｉ ｔ ｐ ａ ｖ ｃ ｏ ｐ Ａ ３ ．Ａ ｒ ｂ ｄｏ ｉ ａ ｉ ｐｓｓ
na盐胁迫下降低细胞内na含量并显著增强转基因植物的编码质膜na明确其在na蓝藻质膜na烟草f1本研究将大叶补血草sos1基因转入到拟南芥中进行过量表达后nacl胁迫下转基因植株的长势好于野生型并且转基因植株的鲜重干重均高于野生型说明盐胁迫下转基因植株有较强的耐盐表型保持了良好150200mmolnacl条件下转基因植株地上部分na含量显著低于野生型p005在盐胁迫下sos1将na转运出细胞与植物的耐盐性直接相关
Ａｂ ｔａｔ Ｔｈ ＤＮＡｓｏ Ｓ０Ｓ１（ ｎ ｎ ｃ ｅｓｏ ．Ｅ ８ ４ ８ ， ｌｓ ｍｂ ａ ｅＮａ Ｈ ａ ｔ ｏ ｔｒｇ ｎ Ｓ １ ｓｒ ｃ ： ｅｃ ｆＬｇ Ｇｅ Ｂａ ｋ ａ ｃ ｓｉｎＮｏ Ｕ７ ０ ５ ） ａｐａ ｍａｍｅ ｒ ｎ ／ ｎ ｉ ｒｅ ｅ ｅＬｇ ＯＳ ｐ
ｃ ｎ ｒ ｌ ｆＣａ Ｖ ５ ｒ ｍｏ ｅ ， ｎ ｙ ｒ ｍｙ ｉ－ ｅ ｉｔ ｎ ｌ ｎ ｓｗｅ ｅ ｏ ｔ ｉｅ ． Ｒ ｎ ｌ ｓｓ ｉ ｄｃ ｔ ｄ ｔ ａ Ｓ ｏ ｔ ｏ Ｍ ｏ ３ Ｓ ｐ ｏ ｔ ｒ ａ ｄ ｈ ｇ ｏ ｃｎ ｒ ｓｓ ａ ｔｐ ａ ｔ ｒ ｂ ａ ｎ ｄ ＰＣ ａ ａ ｙ ｉ ｎ ｉａ ｅ ｈ ｔＬｇ ＯＳ ｅ ｅ ｗａ １ｇｎ ｓ ｉ ｔ ｇ ａ ｅ ｎ ｏ ｔ ｅ ｇ ｎ ｍｅ ｏ ａ ｉ ｏ ｓ ｓ ＲＴ－ ＣＲ ｎ ｌ ｓｓ ｒ ｖ ａ ｅ ｈ ｔｔ ａ ｓ ｅ ｉ ｌ ｎ ｓ ｏ ｅ — ｘ ｒ ｓ ｅ ｈ Ｓ ｎ ｅ ｒ ｔｄ ｉ ｔ ｈ ｅ ｏ ｆＡｒ ｂ ｄ ｐ ｉ． Ｐ ａ ａ ｙ ｉ ｅ ｅ ｌ ｄ ｔ ａ ｒ ｎ ｇ ｎ ｃｐ ａ ｔ ｖ ｒｅ ｐ ｅ ｓ ｄ ｔ ｅ Ｌｇ ＯＳ１ ｉ ｈ ｎ ｔｅ
黄芪多糖调控TXNIP
生命科学仪器 2023年第21卷/第6期研究报告65项目来源:河北省中医药管理局中医药类科研指令性计划课题(2021444)黄芪多糖调控T X N I P /N L R P 3炎症小体对高糖诱导的人视网膜内皮细胞凋亡的作用机制陶雯璇 李君卿 张元坤 张京红(张家口市第四医院,河北张家口075000)摘要 目的探究黄芪多糖调控T X N I P /N L R P 3炎症小体对高糖诱导的人视网膜内皮细胞凋亡的作用机制㊂方法将h R E C s 细胞随机分为C o n t r o l 组㊁H G 组㊁A P S 组和A P S +p c D N A 3.1-T X N I P 组㊂采用C C K-8和流式细胞仪检测细胞存活率和凋亡率;D C F H-D A 荧光探针检测活性氧(R O S)水平;检测细胞中氧化应激和炎症因子水平;蛋白质印迹法检测细胞中T X N I P ㊁N L R P 3㊁I L -1β和ca s p a s e -1蛋白表达㊂结果和C o n t r o l 组相比,H G 组细胞存活率㊁S O D 活性和G S H-P x 含量明显降低,细胞凋亡率㊁R O S 相对荧光强度㊁M D A 含量㊁I L -1β和IL -18含量及细胞中T X -N I P ㊁N L R P 3㊁I L -1β㊁c a s p a s e -1蛋白表达明显增加(P <0.05);和H G 组相比,A P S 组细胞存活率㊁S O D 活性和G S H -P x 含量明显增加,细胞凋亡率㊁R O S 相对荧光强度㊁M D A 含量㊁I L -1β和IL -18含量及细胞中T X N I P ㊁N L R P 3㊁I L -1β㊁c a s p a s e -1蛋白表达明显降低(P <0.05);和A P S 组相比,A P S +p c D N A 3.1-T X N I P 组细胞存活率㊁S O D 活性和G S H-P x 含量明显降低,细胞凋亡率㊁R O S 相对荧光强度㊁M D A 含量㊁I L -1β和IL -18含量及细胞中T X N I P ㊁N L R P 3㊁I L -1β㊁c a s p a s e -1蛋白表达明显增加(P <0.05)㊂结论黄芪多糖可抑制高糖诱导的视网膜内皮细胞凋亡㊁炎症反应和氧化应激损伤,其作用机制可能和抑制T X N I P /N L R P 3通路激活有关㊂关键词 黄芪多糖;高糖诱导的视网膜内皮细胞;凋亡;T X N I P /N L R P 3通路M e c h a n i s m o f a s t r a g a l u s p o l y s a c c h a r i d e m o d u l a t i o n o f T X N I P /N L R P 3i n f l a m m a t o r y ve s i c l e s o n h i g h g l u c o s e -i n d u c e d a p o pt o s i s i n h u m a n r e t i n a l e n d o t h e l i a l c e l l s T a o W e n x u a n L i J u n q i n g Z h a n g Y u a n k u n Z h a n g J i n g h o n g (Z h a n g j i a k o u F o u r t h H o s p i t a l ,Z h a n g ji a k o u 075000,C h i n a )ʌA b s t r a c t ɔO b je c t i v e :T o i n v e s t i g a t e t h e m e c h a n i s m of A s t r ag a l u s p o l y s a c ch a ri d e m o d u l a t i n g T X N I P /N L R P 3i n f l a m m a t o r y v e s i c l e s o n h i g h g l u c o s e -i n d u c e d a p o p t o s i s i n h u m a n r e t i n a l e n d o t h e l i a l c e l l s .M e t h o d s :T h e h R E C s c e l l s w e r e r a n d o m l yd i v i de d i n t o C o n t r o l g r o u p ,H G g r o u p ,A P S g r o u p ,a n d A P S +p c D N A 3.1-T X N I P g r o u p s .C e l l s u r v i v a l a n d a p o pt o s i s w e r e d e t e c t e d b y C C K -8a n d f l o w c y t o m e t r y ,r e s p e c t i v e l y ;r e a c t i v e o x y g e n s p e c i e s (R O S )l e v e l s w e r e d e t e c t e d b y DC F H -D A f l u o r e s c e n t p r o b e ;o x i d a t i v e s t r e s s a n d i n f l a m m a t o r yf a c t o r l e v e l s w e r e d e t e c t e d i n t h e c e l l s ;a n d T X N I P ,N L R P 3,I L -1β,a n d c a s p a s e -1p r o t e i n e x p r e s s i o n w e r e d e t e c t e d i n t h e c e l l s b y p r o t e i n b l o t t i n g .R e s u l t s :C o m p a r e d w i t h t h e C o n -t r o l g r o u p ,t h e c e l l s u r v i v a l r a t e ,S O D a c t i v i t y a n d G S H-P x c o n t e n t w e r e s i g n i f i c a n t l y l o w e r i n t h e H G g r o u p,a n d t h e a p o p t o s i s r a t e ,r e l a t i v e f l u o r e s c e n c e i n t e n s i t y o f R O S ,M D A c o n t e n t ,I L -1βa n d I L -18c o n t e n t ,a n d t h e e x p r e s s i o n o f T X N I P ,N L R P 3,I L -1β,a n d c a s p a s e -1p r o t e i n s i n t h e c e l l s w e r e s i g n i f i c a n t l y i n c r e a s e d (P <0.05);C o m p a r e d w i t h t h e H G g r o u p ,t h e c e l l s u r v i v a l r a t e ,S O D a c t i v i t y a n d G S H-P x c o n t e n t i n t h e A P S g r o u p w e r e s i g n i f i c a n t l yi n c r e a s e d ,a n d t h e a p o p t o s i s r a t e ,r e l a t i v e f l u o r e s c e n c e i n t e n s i t y o f R O S ,M D A c o n t e n t ,I L -1βa n d I L -18c o n t e n t ,a n d c e l l u l a r e x p r e s -s i o n o f T X N I P ,N L R P 3,I L -1β,a n d c a s p a s e -1p r o t e i n s w e r e s i g n i f i c a n t l y d e c r e a s e d (P <0.05);C o m p a r e d w i t h t h e A P S g r o u p ,c e l l s u r v i v a l ,S O D a c t i v i t y a n d G S H-P x c o n t e n t w e r e s i g n i f i c a n t l y l o w e r i n t h e A P S +p c D N A 3.1-T X N I P g r o u p ,a n d a p o p t o s i s r a t e ,r e l a t i v e f l u o r e s c e n c e i n t e n s i t y o f R O S ,M D A c o n t e n t ,I L -1βa n d I L -18c o n t e n t ,a n d c e l l u l a r e x -p r e s s i o n o f T X N I P ,N L R P 3,I L -1β,a n d c a s p a s e -1p r o t e i n s w e r e s i g n i f i c a n t l y i n c r e a s e d (P <0.05).C o n c l u s i o n :A s t r a g a -l u s p o l y s a c c h a r i d e i n h i b i t s h i g h g l u c o s e -i n d u c e d a p o p t o s i s ,i n f l a m m a t o r y r e s p o n s e a n d o x i d a t i v e s t r e s s d a m a ge i n r e t i n a l e n -d o t h e l i a l c e l l s ,a n d i t s m e c h a n i s m of a c t i o n m a y b e r e l a t e d t o t h e i n h i b i t i o n o f T X N I P /N L R P 3p a t h w a y ac t i v a t i o n .ʌK e y wo r d s ɔA s t r a g a l u s p o l y s a c c h a r i d e ;h i g h g l u c o s e -i n d u c e d r e t i n a l e n d o t h e l i a l c e l l s ;a p o p t o s i s ;T X N I P /N L R P 3p a t h -w a y中图分类号:R 45 文献标识码:A D O I :10.11967/2023211213糖尿病视网膜病变(d i a b e t i c r e t i n o p a t h y,D R )是糖尿病常见的一种微血管病变,主要病变为人视网膜内皮细胞(h u m a n r e t i n a l e n d o t h e l i a l c e l l s ,h R E C s)功能障碍,其也是视力丧失的主要原因,影响患者的生活质量[1]㊂D R 发病机制复杂,近年来研究发现[2],慢性视网膜炎症是导致D R 发病的主要因素,当白细胞浸润到视网膜释放炎性递质,通过破坏血-视网膜屏障(b l o o d r e t i n a l b a r r i e r ,B R B )使血管渗漏和新血管形成㊂核苷酸结合寡聚化结构域样受体蛋白3(n u c l e o t i d e -b i n d i n g o l i go m e r i z a t i o n d o -m a i n -l i k e r e c e pt o r p r o t e i n 3,N L R P 3)炎症小体会破坏B R B 参与D R 进程[3];N L R P 3还能通过结合研究报告生命科学仪器 2023年第21卷/第6期66其介质硫氧还蛋白相互作用蛋白(t h i o r e d o x i n -i n -t e r a c t i n g pr o t e i n ,T X N I P )激活N L R P 3炎症小体[4]㊂由此可见,抑制T X N I P /N L R P 3通路激活可能是治疗D R 的新靶点㊂黄芪多糖(a s t r a g a l u s p o l y s a c h a r -i n ,A P S)是黄芪中一种活性最强的有效成分,现代研究证实[5],A P S 具有多种生物学功能,如抗氧化㊁抗炎㊁抗凋亡㊁改善微循环等,但其对D R 的作用及机制报道较少㊂因此,本研究通过D R 体外实验模型,旨探究A P S 能否调控T X N I P /N L R P 3炎症小体影响D R 发生发展㊂1 材料与方法1.1 试剂与仪器 人视网膜内皮细胞(h R E C s)购自中国科学院上海细胞库㊂黄芪多糖(国药准字Z 20040085,天津赛诺制药有限公司,规格250m g/瓶);T X N I P ㊁N L R P 3㊁I L -1β和ca s p a s e -1抗体(英国A n c a m 公司);T X N I P 过表达载体(pc D N A 3.1-T X N I P )(广州瑞博生物公司);细胞活力/毒性(C E L L C O U N T I N G K I T-8,C C K-8)试剂盒㊁氧化应激指标检测试剂盒及炎症因子E L I S A 检测试剂盒(南京健成生物研究所);流式细胞仪检测试剂盒(美国B D 公司);低温高速离心机(型号:M i -c r o 17R ,德国T h e r m o 公司);酶标仪(型号C M a x -P l u s ,美国M o l e c u l a r D e v i c e s 公司);显微镜(型号A E 2000型,美国M o t i c 公司)㊂1.2 细胞培养及分组 将h R E C s 培养于R P M I -1640培养基中,含10%胎牛血清㊁100U /m L 青霉素和链霉素,将培养基置于37ħ㊁5%C O 2的培养箱24h ,收集处于对数期的细胞㊂将h R E C s 细胞随机分为C o n t r o l 组(h R E C s 细胞培养于葡萄糖含量为5m m o l /L 的正常培养基)㊁H G 组(h R E C s 细胞培养于含25m m o l /L 葡糖糖培养基)㊁A P S 组(h R E C s 细胞培养于含25m m o l /L 葡糖糖和300g/L 黄芪多糖培养基[8])和A P S+p c D N A 3.1-T X N I P 组(将h R E C s 细胞转染4μg p c D N A 3.1-T X N I P 质粒,后培养于含25m m o l /L 葡糖糖和300g/L 黄芪多糖培养基),各组细胞均培养48h 后进行后续研究㊂1.3 C C K -8检测细胞增殖能力 将h R E C s 细胞接种于96孔板中,每孔细胞密度为1.5ˑ104个/m L ,培养细胞于37ħ环境及质量分数为5%C O 2的培养箱,分别在培养24h ㊁48h ㊁72h 时将C C K -8溶液(100μL )加入到孔板中,后培养细胞于37ħ环境及质量分数为5%C O 2的培养箱,3.5h 后于酶标仪450n m 处测定酶标仪D O 值㊂该实验重复3次取均值㊂1.4 流式细胞仪检测细胞凋亡 将h R E C s 细胞接种于96孔板中,每孔细胞密度为1.5ˑ104个/m L ,培养细胞于37ħ环境及质量分数为5%C O 2的培养箱24h ,分别将1ˑb i n d i n g bu f f e r ㊁10μL a n n e x i n V 和20μL P I 加入到孔板中培养,后将原有的培养基弃掉,将提前配制好的A n n e x i n V /P I 染液加入到孔板中,常规孵育,将胰蛋白酶裂解液分别加入到孔板的每孔中,裂解细胞呈悬液于流式细胞仪上检测㊂该实验重复3次取均值㊂1.5 2,7-二氯荧光素二乙酸酯(2',7'-D I C H L O -R O F L U O R E S C I N D I A C E T A T E ,D C F H-D A )荧光探针检测活性氧(R E A C T I V E O X Y G E N S P E C I E S,R O S)水平将h R E C s 细胞接种于6孔板中孵育1.5h ,P B S 洗涤细胞后,加入提前用P B S 稀释为10μm o l/L 的D C F H-D A 荧光探针,孵育于37ħ避光的环境中,30m i n 后用P B S 再次洗涤细胞,P B S 重悬收集细胞,通过荧光显微镜观察细胞中R O S 水平㊂该实验重复3次取均值㊂1.6 检测细胞中氧化应激和炎症因子水平 将各组h R E C s 中加入R I P A 裂解细胞,根据试剂盒说明书,分别检测细胞裂解液中S O D ㊁M D A ㊁G S H-P x㊁I L -1β㊁I L -18水平㊂1.7 蛋白质印迹法检测细胞中T X N I P ㊁N L R P 3㊁I L-1β和ca s p a s e -1蛋白表达 用B C A 法测定细胞中总蛋白浓度㊂热水煮沸蛋白样品(终浓度为2μg㊃m L -1)10m i n ,保存于-20ħ环境㊂取蛋白上样50μg ,加入将上样缓冲液后煮沸,将变性的蛋白样品用S D S -P A G E 分离,后转移蛋白样品于P V D F 膜上,加入质量分数为5%的脱脂牛奶(5%)封闭,1h 后用T B S T 溶液清洗P V D F 膜,后加入一抗(1:1000),在4ħ避光环境下孵育一抗过夜;后将H R P标记的二抗(1:5000)加入细胞中,常温孵育2h㊂将E C L 发光液加入细胞显影,曝光后对各组细胞蛋白条带用I m a g e L a b 软件分析㊂1.8 统计学分析 实验数据采用G r a p h p a d p r i a m 8.0软件进行统计学分析㊂数据以均值ʃ标准差(M e a n ʃS D )表示㊂不同组间数据采用重复测量方差和单因素方差(o n e -w a y A N O V A )分析,各组间两两比较采用L S D-t 检验,实验结果以P <0.05表示差异有统计学意义㊂2 结果2.1 各组细胞增殖和凋亡率比较 和C o n t r o l 组相比,H G 组细胞存活率降低,细胞凋亡率增加(t 存活率=10.80,t 凋亡率=13.04,P<0.0001);和H G 组相比,A P S 组细胞存活率增加,细胞凋亡率降低(t 存活率=9.408,t 凋亡率=8.846,P <0.0001);和A P S 组相比,A P S +p c D N A 3.1-T X N I P 组细胞存活率降低,细胞凋亡率增加(t 存活率=8.318,t 凋亡率=8.126,P <0.0001)(图1)㊂2.2 各组细胞R O S 水平比较 H G 组细胞中R O S相对荧光强度高于C o n t r o l 组(t=16.91,P<0.0001);A P S 组细胞中R O S 荧光相对强度低于H G 组(t =12.93,P <0.0001);A P S +p c D N A 3.1-T X N I P 组细胞中R O S 相对荧光强度高于A P S 组(t生命科学仪器 2023年第21卷/第6期研究报告67=12.50,P <0.0001)(图2)㊂A :各组细胞存活率比较;B:流式细胞仪检测细胞凋亡;C :各组细胞凋亡率比较㊂v s C o n t r o l 组*P <0.05;v s H G 组#P <0.05;v s A P S 组әP <0.05㊂图1 各组细胞增殖和凋亡率比较n =6A :D C F H-D A 荧光探针检测细胞中R O S 水平;B :各组细胞中R O S 水平比较㊂和C o n t r o l 组相比*P <0.05;和H G 组相比#P <0.05;和A P S 组相比әP <0.05㊂图2 各组细胞R O S 水平比较n =62.3 各组细胞中氧化应激指标比较 和C o n t r o l 组相比,H G 组细胞中S O D 活性和G S H-P x 含量降低,M D A 含量增加(t S O D =10.78,t G S H-P x=10.87,t M D A =12.38,P <0.0001);和H G 组相比,A P S 组细胞中S O D 活性和G S H-P x 含量增加,M D A 含量降低(t S O D =10.63,t G S H-P x =7.801,t M D A =7.757,P<0.0001);和H G 组相比,A P S+p c D -N A 3.1-T X N I P 组细胞中S O D 活性和G S H-P x含量降低,M D A 含量增加(t S O D =9.362,t G S H-P x =7.549,t M D A =8.571,P <0.0001)(图3)㊂A :细胞中S O D 活性;B :细胞中M D A 含量比较;C :细胞中G S H-P x 含量比较㊂和C o n t r o l 组相比*P <0.05;和H G 组相比#P <0.05;和A P S 组相比әP <0.05㊂图3 各组细胞S O D 活性和M D A ㊁G S H-P x 含量比较n =62.4 各组细胞中炎症因子含量比较 和C o n t r o l 组相比,H G 组细胞中I L -1β和IL -18含量增加(t I L -1β=11.78,t I L -18=13.49,P<0.0001);和H G 组相比A P S 组细胞中I L -1β和IL -18含量均降低(t I L -1β=8.791,t I L -18=9.094,P<0.0001);和A P S 组相比,A P S +p c D N A 3.1-T X N I P 组细胞中I L -1β和IL -18含量增加(t I L -1β=7.832,t I L -18=8.585,P <0.0001)(图4)㊂A :细胞中I L -1β含量比较;B :细胞中I L -18含量比较㊂和C o n t r o l 组相比*P <0.05;和H G 组相比#P <0.05;和A P S 组相比әP <0.05㊂图4 各组细胞中I L -1β和IL -18含量比较n =62.5 各组细胞中T X N I P ㊁N L R P 3㊁I L-1β㊁c a s p a s e -1蛋白表达比较 和C o n t r o l 组相比,H G 组细胞中T X N I P ㊁N L R P 3㊁I L -1β㊁c a s p a s e -1蛋白表达均增加(t T X N I P =11.56,t N L R P 3=9.311,t I L -1β=9.640,t c a s p a s e -1=12.93,P<0.0001);和H G 组相比,A P S 组细胞中TX N I P ㊁N L R P 3㊁I L -1β㊁c a s p a s e -1蛋白均降低(t T X N I P =8.993,t N L R P 3=6.114,t I L -1β=6.020,t c a s p a s e -1=10.47,P<0.0001);和A P S 组相比,A P S +p c D N A 3.1-T X N I P 组细胞中T X N I P ㊁N L R P 3㊁IL -1β㊁c a s p a s e -1蛋白均增加(t T X N I P =9.242,t N L R P 3=6.123,t I L -1β=6.015,t c a s p a s e -1=10.65,P <0.0001)(图5)㊂A :细胞中T X N I P ㊁N L R P 3㊁I L -1β㊁c a s p a s e -1蛋白条带图;B :细胞中T X N I P 蛋白表达比较;C :细胞中N L R P 3蛋白表达比较;D :细胞中I L-1β蛋白表达比较;E :细胞中c a s pa s e -1蛋白表达比较㊂和C o n t r o l 组相比*P <0.05;和H G 组相比#P <0.05;和A P S 组相比әP <0.05㊂图5 各组细胞中T X N I P ㊁N L R P 3㊁I L -1β㊁c a s pa s e -1蛋白表达比较n =63 讨论目前,临床目前常采用激光凝术㊁玻璃体手术等方法治疗D R ,虽能改善患者症状,但由于会损伤视力及使产生不适使患者不易接受㊂因此,探究有效治疗D R 药物对改善患者生活质量尤为重要㊂S u n 等[6]学者研究发现,A P S 治疗糖尿病大鼠,可有效改善心肌组织损伤㊂彭涛等[7]研究证实,A P S 对急性高眼压大鼠视网膜可发挥保护作用㊂赖莉研究报告生命科学仪器 2023年第21卷/第6期68等[8]研究证实,A P S 可通过介导T r a f 6/T A K 1信号通路保护视网膜㊂还有研究发现[9],A P S 可通过抑制多种因素诱导的血管内皮细胞凋亡㊂本研究结果显示,H G 组h R E C s 存活率明显降低,凋亡率明显增加,A P S 干预后,h R E C s 存活率明显增加,凋亡率明显降低,提示A P S 可保护视网膜内皮细胞损伤,对D R 发挥治疗作用㊂闫丰华等[10]研究证实,黄芪多糖可减轻大鼠糖尿病视网膜损伤,从而对视网膜发挥保护作用㊂研究发现[11],高糖环境下视网膜内皮细胞中的氧化应激和D R 发生发展密切相关,当细胞内的氧化平衡状态被破坏,导致细胞过度凋亡,进而影响内皮细胞的功能㊂R O S ㊁S O D ㊁M D A 和G S H-P x指标是检测氧化应激反应的常用标志物㊂本研究结果显示,A P S 可抑制高糖诱导h R E C s 中M D A 含量和R O S 含量,增加S O D 活性和G S H-P x 含量,提示A P S 可提高高糖诱导h R E C s 抗氧化能力,减少氧化损伤㊂汪洋等[12]研究发现,达格列净对人视网膜血管内皮细胞氧化应激和凋亡发挥抑制作用,从而通过减轻细胞损伤保护D R ㊂炎性细胞因子可介导D R 进程,当视网膜炎症和白细胞黏附于D R 微血管时,可对血-视网膜屏障的破坏发挥促进作用㊂研究报道[13],I L-1β在D R 发展中发挥重要作用,而I L -1β的成熟及释放都受到N L R P 3炎症小体的调控㊂N L R P 3炎症小体异常激活可通过活化c a s p a s e -1和I L -1β等效应分子介导机体的炎症和免疫过程㊂Z h e n g 等[14]研究证实,阻断糖尿病视网膜中N L R P 3炎症小体的形成可抑制其损伤㊂由此,N L R P 3炎症小体可能是治疗D R 的重要靶点㊂T X N I P 是硫氧还蛋白的内源性抑制蛋白,当其与硫氧还原蛋白的活性部分结合时,会导致细胞内活性氧化物积累,进而导致机体损伤;T X N I P 还能通过激活N L R P 3炎症小体对炎症发挥促进作用㊂冯梅等[15]通过体内实验发现,富氢水可通过抑制T X N I P /N L R P 3通路的激活降低D R 大鼠视网膜血管通透性,减轻视网膜神经元损伤㊂本研究结果显示,A P S 可抑制高糖诱导h R E C s 中T X N I P ㊁N L R P 3㊁I L -1β㊁c a s p a s e -1蛋白表达,由此猜测,A P S 可能通过抑制T X N I P /N L -R P 3通路的激活减轻高糖诱导h R E C s 凋亡㊁炎症和氧化应激损伤,对视网膜发挥保护作用㊂为了验证这一猜想,本研究采用过表达T X N I P 和A P S 共同干预高糖诱导h R E C s ,结果显示,pc D N A 3.1-T X -N I P 可减弱A P S 对高糖诱导h R E C s 凋亡㊁炎症和氧化应激损伤的抑制作用㊂综上所述,黄芪多糖可抑制高糖诱导的视网膜内皮细胞凋亡㊁炎症反应和氧化应激损伤,其作用机制可能和抑制T X N I P /N L R P 3通路激活有关㊂由于时间和成本等因素,本研究未设置单独的过表达T X N I P 分组或抑制T X N I P 组,可能使本研究结果存在一定局限性,在今后的研究中会增加单独过表达T X N I P 组或敲减T X N I P 组进行对照,进一步证实黄芪多糖能通过调控T X N I P /N L R P 3通路对高糖诱导的视网膜内皮细胞发挥作用,进而为临床治疗糖尿病视网膜病变提供更多的实验依据㊂参考文献[1]Y a o X ,Z h a o Z ,Z h a n g W ,e t a l .S pe c i a l i z e d R e t i n a l E n d o t h e l i a l C e l l s M o d u l a t e B l o o d -R e t i n a B a r r i e r i n D i a b e t i c R e t i n o p a t h y[J ].D i a b e t e s ,2024,73(2):225-236.[2]D h a r m a r a ja n S ,C a r r i l l o C ,Q i Z ,e t a l .R e t i n a l i n f l a m m a t i o n i n m u r i n e m o d e l s o f t y p e 1a n d t y p e 2d i ab e t e s w i t h d i a b e t ic r e t i n o pa -t h y [J ].D i ab e t o l o g i a ,2023,66(11):2170-2185.[3]S i n g h L P ,Y u m n a mc h a T ,S w o r n a l a t a D e v i T .M i t o p h a gi c F l u x D e r e g u l a t i o n ,L ys o s o m a l D e s t a b i l i z a t i o n a n d N L R P 3I n f l a m m a -s o m e A c t i v a t i o n i n D i a b e t i c R e t i n o p a t h y:P o t e n t i a l s o f G e n e T h e r -a p y T a r g e t i n g T X N I P a n d T h e R e d o x S y s t e m [J ].O ph t h a l m o l R e s R e p,2018,3(1):O R R T -126.[4]L i a n L ,L e Z ,W a n g Z ,e t a l .S I R T 1I n h i b i t s H i gh G l u c o s e -I n -d u c e d T X N I P /N L R P 3I n f l a m m a s o m e A c t i v a t i o n a n d C a t a r a c tF o r m a t i o n [J ].I n v e s t O p h t h a l m o l V i s S c i ,2023,64(3):16.[5]J i a n g X ,L i Y ,F u D ,e t a l .C a v e o l i n -1a m e l i o r a t e s a c e t a m i n o -p h e n -a g g r a v a t e d i n f l a m m a t o r y d a m a g e a n d l i p i d d e po s i t i o n i n n o n -a l c o h o l i c f a t t yl i v e r d i s e a s e v i a t h e R O S /T X N I P /N L R P 3p a t h -w a y [J ].I n t I m m u n o ph a r m a c o l ,2023,114:109558.[6]S u n S ,Y a n g S ,Z h a n g N ,e t a l .A s t r a g a l u s p o l ys a c c h a r i d e s a l l e -v i a t e s c a r d i a c h y p e r t r o p h y i n d i a b e t i c c a r d i o m y o p a t h y v i a i n h i b i t i n gt h e B M P 10-m e d i a t e d s i g n a l i n g p a t h w a y [J ].P h yt o m e d i c i n e ,2023,109:154543.[7]彭涛,于丹丹,谢美娜,等.黄芪多糖对高眼压大鼠视网膜神经节细胞凋亡的影响[J ].中国临床药理学杂志,2020,36(10):1344-1346.[8]赖莉,覃晖.黄芪多糖通过T R A F 6/T A K 1信号通路调节视网膜神经节细胞的炎症反应[J ].中国中医眼科杂志,2019,29(6):434-437.[9]王忠庆,蔡帆,诸波,等.黄芪多糖对大鼠缺氧/复氧诱导的心肌细胞自噬及凋亡抑制作用的机制探讨[J ].中国循环杂志,2022,37(2):185-192.[10]闫丰华,焦禄安,郑加军,等.黄芪多糖对糖尿病模型大鼠视网膜病变及血清胱抑素C 的影响[J ].热带医学杂志,2019,19(7):813-816,804.[11]H a y d i n ge r C D ,O l i v e r G F ,A s h a n d e r L M ,e t a l .O x i d a t i v e S t r e s s a n d I t s R e g u l a t i o n i n D i a b e t i c R e t i n o p a t h y[J ].A n t i o x i d a -n t s (B a s e l ),2023,12(8):1649.[12]汪洋,王可,刘宝兰.达格列净对高糖诱导人视网膜血管内皮细胞凋亡及氧化应激的影响[J ].国际眼科杂志,2022,22(3):378-382.[13]R a m a n K S ,M a t s u b a r a J A.D y s r e gu l a t i o n o f t h e N L R P 3I n f l a m -m a s o m e i n D i a b e t i c R e t i n o p a t h y a n d P o t e n t i a l T h e r a p e u t i c T a r -ge t s [J ].O c u l I m m u n o l I nf l a m m ,2022,30(2):470-478.[14]Z h e ng X ,Wa n J ,T a n G .T h e m e c h a n i s m s o f N L R P 3i n f l a m m a -s o m e /p y r o p t o s i s a c t i v a t i o n a n d t h e i r r o l e i n d i ab e t ic r e t i n o p a t h y.F r o n t I m m u n o l .2023A pr 25;14:1151185.[15]冯梅,江霞,王艳丽,等.富氢水对糖尿病视网膜病变大鼠硫氧还蛋白相互作用蛋白/核苷酸结合寡聚化结构域样受体蛋白3通路及视网膜血管通透性的影响[J ].安徽医药,2022,26(7):1367-1373.。
嗜糖假单胞菌麦芽四糖酶基因在地衣芽孢杆菌中的异源表达
50㊀2021Vol.47No.1(Total 421)DOI:10.13995/ki.11-1802/ts.025483引用格式:楼志华,刘翔,张劲楠.嗜糖假单胞菌麦芽四糖酶基因在地衣芽孢杆菌中的异源表达[J].食品与发酵工业,2021,47(1):50-54.LOU Zhihua,LIU Xiang,ZHANG Jingnan.Heterologous expression of maltotetraose-forming amylase from Pseud-omonas saccharophila in Bacillus licheniformis [J].Food and Fermentation Industries,2021,47(1):50-54.嗜糖假单胞菌麦芽四糖酶基因在地衣芽孢杆菌中的异源表达楼志华1,2∗,刘翔1,张劲楠11(江苏省奥谷生物科技有限公司,江苏常州,213300)2(溧阳维信生物科技有限公司,江苏常州,213300)摘㊀要㊀为了考察重组地衣芽孢杆菌的异源表达特性,进一步提高麦芽四糖酶的发酵水平,以适用于工业化生产,研究构建了木糖诱导型的重组地衣芽孢表达系统,表达了来源于嗜糖假单胞菌的麦芽四糖酶基因,并进行了初步发酵优化㊂结果显示,构建的表达系统经过核酸电泳以及十二烷基硫酸钠-聚丙烯酰氨凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis ,SDS-PAGE )鉴定均正确,该系统在发酵过程中经过木糖诱导,重组酶能够成功地分泌至发酵液中,然后通过对诱导剂添加时间㊁诱导温度和发酵时间优化后,摇瓶水平的最大酶活力可达(168.2ʃ9.41)U /mL ㊂最佳的发酵条件为,在24h 添加诱导剂,诱导温度30ħ,发酵至稳定期结束停止发酵㊂该研究为重组地衣芽孢杆菌的异源表达研究以及麦芽四糖酶的工业化应用提供了参考㊂关键词㊀麦芽四糖酶;地衣芽孢杆菌;嗜糖假单胞菌;发酵优化;异源表达;木糖诱导第一作者:硕士,工程师(通讯作者,E-mail:muxinhua23@)收稿日期:2020-08-26,改回日期:2020-09-21㊀㊀麦芽四糖酶(glucan 1,4-α-maltotetraohydro-lase),又名麦芽四糖淀粉水解酶,可以连续地催化淀粉状多糖中非还原型末端麦芽四糖残基的水解,主要产物为麦芽四糖[1]㊂而麦芽四糖因其独特的理化特性,一度被誉为最有希望的麦芽低聚糖[2],在食品加工行业有着广泛㊁重要的应用㊂麦芽四糖酶基因主要来源于斯氏假单胞菌(Pseudomonas stutzeri )和嗜糖假单胞菌(Pseudomonas saccharophila ),二者来源的麦芽四糖酶均为α构型㊂相比而言,嗜糖假单胞菌来源的麦芽四糖酶的热稳定性和pH 稳定性均较好,在40ħ以下和pH 5.5~11均能长时间保持稳定;在最适温度(55ħ)㊁最适pH(6.7)以及在其他宿主内表达后酶活力也均较高[3]㊂这些因素表明嗜糖假单胞菌来源的麦芽四糖酶更适合于工业化应用,也因而备受学者的关注㊂国内,ZHOU㊁赵云等[4-5]在大肠杆菌中克隆表达了嗜糖假单胞菌来源的麦芽四糖酶,但获得的重组酶大部分是包涵体,重组酶活力并不高㊂2019年,张梓芊㊁杨亚楠等[6-7]在Bacillus subtilis 中构建了表达系统胞外表达了嗜糖假单胞菌来源的麦芽四糖酶,发现其比活力为980.49U /mg,通过发酵优化后,在摇瓶水平重组酶活力可高达147U /mL㊂这些报道中,重组酶酶学性质基本一致,在芽孢杆菌中表达可获得更高的活力㊂国外,美国杰能科公司上市的麦芽四糖酶SAS3则以地衣芽孢杆菌为宿主进行重组表达[8],并且在地衣芽孢杆菌发酵产相关淀粉酶方面形成了技术封锁,因此导致国内纯化的麦芽四糖价格极高㊂地衣芽孢杆菌是优良的食品安全级表达宿主,被美国FDA 认定为食品安全级菌株(Generally Recognized As Safe,GRAS)已有近40年的历史,而且该菌产酶能力高,胞外蛋白分泌能力大约是枯草芽孢杆菌的2倍[9]㊂然而,国内目前还未发现有关重组地衣芽孢杆菌表达麦芽四糖酶的研究㊂本研究拟构建地衣芽孢杆菌表达系统,对来源于嗜糖假单胞菌的麦芽四糖酶基因进行木糖诱导表达,通过果聚糖合酶信号肽使麦芽四糖酶分泌至胞外,并对构建的重组菌进行初步发酵条件优化,以期为地衣芽孢杆菌的异源表达以及麦芽四糖酶的工业化应用提供参考㊂1㊀材料与方法1.1㊀菌株和质粒本研究所用敲除了α-淀粉酶基因amyL 和碱性蛋白酶基因aprE 的地衣芽孢杆菌Bacillus licheniformis CICIM B1391(BLΔAE)㊁E.coli JM109以及大肠杆菌-芽孢杆菌穿梭质粒pHY300-PLK,均购于江南大学,由江苏省奥谷生物科技有限公司保藏㊂重组质粒pBL-SY2由江南大学石贵阳教授惠赠,该重组质粒携带了来源于B.licheniformis CICIM B1391自身的木糖异构酶启动子及其调控蛋白基因㊁枯草芽孢杆菌果聚糖合酶信号肽基因sacBss㊁B.licheniformis ATCC14580的麦芽糖淀粉酶基因以及木糖异构酶基因的终止子ter㊂嗜糖假单胞菌麦芽四糖酶基因序列由NCBI数据库查寻获得,序列号为:X16732.1,将该序列和终止子ter一并委托上海生物工程有限公司合成,合成后的片段插入至质粒pET28a中,即pET28a-G4-ter㊂重组质粒pBLxys㊁pBLG4,重组地衣芽孢杆菌BLG4由本研究构建㊂1.2㊀工具酶、引物和试剂含DNA聚合酶的Taq和Pfu预混液㊁T4DNA连接酶,Thermo Fisher公司;各种限制性内切酶㊁PCR产物纯化试剂盒㊁质粒提取试剂盒,TAKARA有限公司;引物由上海生物工程有限公司合成,引物序列见表1;酵母粉和蛋白胨,安琪酵母有限公司;其他试剂为国产分析纯或生化试剂㊂表1㊀本研究所使用的引物Table1㊀Primers used in this study引物名称引物序列(5 -3 )a酶切位点PxylF CC AAGCTTTTAAAATCTCTCATTCATAAACCGTTCC HindⅢPxylsR GG GGTACCATGATGATGATGATGATGCG KpnⅠPxylsF GG GGTACCATGAGCCACATCCTGC KpnⅠPterR GCTCTAGAGGGTAAAAAACCATTCACTC XbaⅠ㊀㊀注:a下划线序列为酶切位点1.3㊀DNA操作技术质粒提取㊁DNA片段纯化㊁回收等均参照TAKARA 试剂盒说明书㊂PCR扩增反应㊁DNA琼脂糖凝胶电泳㊁酶切㊁连接以及转化子筛选等均参照分子克隆实验指南[10]㊂1.4㊀木糖诱导分泌表达载体的构建首先以重组质粒pBLSY2为模板,以PxylF㊁Pxy-lsR为引物,扩增含木糖异构酶启动子及其调控蛋白基因和信号肽的基因片段Blxyl-sacBss,反应条件:95ħ10min,95ħ30s,54ħ30s,72ħ2min,30个循环,72ħ10min㊂然后分别同pHY300-PLK经Hind Ⅲ和KpnⅠ酶切后通过胶回收克隆至pHY300-PLK,获得木糖诱导分泌表达的重组质粒pBLxys后转化至E.coli JM109,即E.coli JM109/pBLxys㊂再以pET28a-G4-ter为模板,以引物PxylsF㊁PterR扩增麦芽四糖酶结构基因,反应条件与上述一致,再分别同pHY300-PLK 经KpnⅠ和XbaⅠ酶切后通过胶回收克隆至pBLxys,即为木糖诱导分泌表达麦芽四糖酶的重组质粒pBLG4,经过转化子筛选后,送去测序,测序正确后即获得携带pBLG4重组质粒的E.coli JM109/pBLG4㊂1.5㊀重组地衣芽孢杆菌的构建培养E.coli JM109/pBLxys㊁E.coli JM109/pBLG4,提取重组质粒pBLxys㊁pBLG4后,按文献[13]所述电转方法分别将其转入BLΔAE,从而获得重组地衣芽孢杆菌BLXYLS和BLG4㊂1.6㊀麦芽四糖酶酶活定义及测定麦芽四糖酶酶活力单位(U)定义为:在pH7.0和50ħ的反应条件下,每分钟分解可溶性淀粉生成相当于1μmoL葡萄糖所需的酶量㊂麦芽四糖酶酶活测定方法参照文献[5]㊂1.7㊀发酵优化实验LBG培养基(g/L):葡萄糖10.0,蛋白胨FP321 10.0,酵母粉FM4085.0,NaCl10.0㊂发酵培养基(g/L):麦芽糊精90,蛋白胨FP321 20,酵母粉FM40810,玉米浆干粉5,(NH4)2HPO4 10,K2HPO4㊃3H2O9.12,KH2PO41.36,CaCl20.50, MgSO4㊃7H2O0.50㊂在进行发酵优化实验时,均使用挡板摇瓶进行发酵,发酵前均加入终质量浓度为20mg/L的四环素㊂每组实验做3个平行㊂2㊀结果与分析2.1㊀重组表达载体的构建根据NCBI显示,嗜糖假单胞菌麦芽四糖酶基因序列(G4-ter)总长约1.7kbp,编码551个氨基酸,理论上蛋白大小为59.9kDa,而终止子序列总长为0.1 kbp,因此KpnⅠ和XbaⅠ酶切后应获得约1.8kbp 和6.3kbp大小的2条核酸条带㊂Blxyl-sacBss基因片段长度约为1.4kbp,经HindⅢ和KpnⅠ酶切后应获得约6.7kbp和1.4kbp大小的2条核酸条带㊂对获得的克隆宿主E.coli JM109/pBLG4提取重组质粒pBLG4,质粒大小约8.1kbp,构建重组表达载体图见图1㊂然后分别用HindⅢ㊁HindⅢ和KpnⅠ㊁KpnⅠ和XbaⅠ酶切,电泳鉴定结果如图2,结合测序结果,可以充分说明重组表达载体构建正确㊂2.2㊀重组麦芽四糖酶的表达将对照菌BLXYLS和重组菌BLG4分别进行摇瓶发酵,接种后8h均加入10g/L木糖进行诱导,然后继续培养30h,离心后收集发酵液上清,分别检测二者胞外麦芽四糖酶活力㊂结果显示,只有重组菌BLG4检测到活力,而对照菌BLXYLS未检测到活力㊂将二者发酵液上清进行十二烷基硫酸钠-聚丙烯酰氨凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)分析,结果见图3㊂可以2021年第47卷第1期(总第421期)51㊀52㊀2021Vol.47No.1(Total 421)Blxyl -木糖异构酶启动子及其调控蛋白基因;sacBss -枯草芽孢杆菌果聚糖合酶信号肽基因;G4-嗜糖假单胞菌麦芽四糖酶基因;ter -木糖异构酶基因的终止子图1㊀重组表达载体pBLG4Fig.1㊀Diagram of recombinant expression vector pBLG4M-Maker;1-pBLG4/Hind Ⅲ;2-pBLG4/Hind Ⅲ+Kpn Ⅰ;3-pBLG4/Kpn Ⅰ+Xba Ⅰ图2㊀重组表达载体pBLG4验证电泳图Fig.2㊀Electropherogram of recombinant expression vector pBLG4发现在约60kDa处出现一明显的表达条带,这与理论推测的蛋白大小基本一致,表明重组菌成功地将麦芽四糖酶分泌到了发酵液中㊂M-Maker;1-BLXYLS;2-BLG4图3㊀发酵液上清液SDS-PAGE 电泳图Fig.3㊀SDS-PAGE electropherogram of fermentationbroth supernatant2.3㊀发酵条件优化2.3.1㊀诱导剂添加时间对发酵的影响重组菌接种后,在37ħ㊁250r /min 下进行培养,分别在接种后的8㊁12㊁16㊁20㊁24㊁28㊁32h 取样检测OD 600,并添加10g /L 木糖进行诱导,发酵至24h 时补加30g /L 麦芽糊精,发酵至48h 时添加10g /L 的木糖,然后继续发酵至72h 结束㊂发酵结束后,取样检测OD 600和发酵液中的麦芽四糖酶活力,结果如表2㊁图4所示㊂结果显示,诱导剂添加时间对重组菌生长影响较小,但对发酵结束时胞外麦芽四糖酶酶活有显著影响㊂随着诱导剂添加时间的延迟(8~24h),发酵结束时检测到的胞外酶活呈递增趋势,当发酵24h 添加诱导剂时,发酵结束时胞外酶活力高达(138.2ʃ11.2)U /mL;而当28㊁32h 添加诱导剂时,发酵结束时酶活有所降低㊂事实上,这与木糖操纵子在地衣芽孢杆菌中的转录特性有关,据文献[12-13]报道,在对数生长期到稳定期的转换期间木糖操纵子转录活性显著增加,并且这种较高的转录活性会维持近12h㊂根据检测到的菌体量来看,发酵至24h 后,重组菌的生长速率明显降低,该时间点对应文献中对数生长期到稳定期的转换期间,因此,在发酵24h 时诱导能够检测到较高的麦芽四糖酶酶活㊂2.3.2㊀诱导温度对发酵的影响重组菌接种后,在37ħ㊁250r /min 下进行培养,当培养至24h 时,添加10g /L 木糖进行诱导,并添加30g /L 麦芽糊精,分别转移至25㊁30㊁37㊁42ħ,发酵至48h 时添加10g /L 的木糖和30g /L,然后继续发酵至72h 结束㊂发酵结束后,取样检测OD 600和发酵液中的麦芽四糖酶活力,结果如图5所示㊂可以发现,诱导温度对重组菌生长和产酶均有显著的影响㊂在25~37ħ,随着温度升高,发酵结束后的菌体量也越来越高,而42ħ时,菌体量又有所降低,这说明重组菌最适生长温度为37ħ㊂当诱导温度为30ħ时,胞外酶活力最高可达(161.4ʃ10.4)U /mL,37ħ次之,42ħ时胞外酶活力最低㊂重组菌在温度胁迫下的产酶特性是可以预见的㊂温度低时,菌体整体代谢速率较慢,酶的合成速率也会降低[14]㊂而温度较高时,尽管菌体代谢速率增加,但地衣芽孢杆菌分泌的其他蛋白酶也会增加[15],对重组酶的水解速率也会增加,另外重组酶自身的稳定性也可能有所下降㊂所以,诱导温度变化对重组菌生长和重组酶酶活力均有较大的影响㊂2021年第47卷第1期(总第421期)53㊀表2㊀不同时间添加诱导剂发酵结束时的菌体量Table 2㊀Biomass at the end of fermentation under different induction time诱导剂添加时间/h 8121620242832发酵结束时OD 600值50.2ʃ2.0849.6ʃ3.7050.6ʃ3.2150.8ʃ2.8851.8ʃ3.0453.3ʃ3.6352.1ʃ3.41图4㊀诱导剂不同添加时间对重组菌表达麦芽四糖酶的影响Fig.4㊀Effect of different adding time of inducer on the expression of maltotetraose in recombinant bacteria图5㊀诱导温度对重组菌表达麦芽四糖酶的影响Fig.5㊀Effect of induction temperature on the expression ofmaltotetraose in recombinant bacteria2.3.3㊀发酵时间对发酵的影响根据2.3.1和2.3.2的结果,在37ħ㊁250r /min下对重组菌进行培养,当培养至24h 时,添加2%木糖进行诱导,并转移至30ħ继续发酵㊂诱导后每隔12h 检测菌体量OD 600和麦芽四糖酶酶活㊂结果如图6所示㊂可以看到,发酵至84h 时,胞外检测到的最大酶活力为(168.2ʃ9.41)U /mL,之后酶活力明显开始下降㊂显然,重组地衣芽孢杆菌胞外酶活力开始下降的时间要略晚于稳定期,这与2.3.1小节所记录的特性相符,表明木糖诱导型重组地衣芽孢杆菌发酵至稳定期结束时停止发酵最为合适㊂在后续应用过程中,对重组酶的酶学性质进行了简单研究㊂以淀粉液为化液底物反应时,其最适pH 为7.0,最适反应温度为50ħ,并且在反应48h 后,相对酶活力仍能达50%以上,结合异淀粉酶进行转化,麦芽四糖转化率可达72.4%,其特性与文献报道[5-7]基本一致,表明重组酶的酶学性质未发生明显图6㊀重组地衣芽孢杆菌发酵过程曲线Fig.6㊀Growth and maltotetraose production curves ofrecombinant Bacillus licheniformis改变,有良好的应用前景㊂目前,国内对麦芽四糖酶的发酵还处于基础研究阶段,多见于在大肠杆菌㊁枯草芽孢杆菌中的表达,在地衣芽孢杆菌中的表达未见报道㊂杨亚楠等[7]在以枯草芽孢杆菌表达来源于嗜糖假单胞麦芽四糖酶的表达时,摇瓶水平酶活力可力达147U /mL,本研究摇瓶水平酶活力可达(168.2ʃ9.41)U /mL,较之有所提高,但提高有限㊂多项研究显示,地衣芽孢杆菌胞外蛋白分泌量可达枯草芽孢杆菌的2倍[9],这表明地衣芽孢的表达性能仍未被完全开发,其优势尚未完全发挥,可提升的空间仍然很大㊂据报道[1],美国杰能科公司也是通过重组地衣芽孢杆菌分批补料发酵,表达嗜糖假单胞菌的麦芽四糖酶,根据该酶的比酶活[7]及地衣芽孢杆菌的产酶特性,保守估计其发酵水平应在10000U /mL 左右㊂就本研究结果来看,尽管继续通过发酵优化能够继续提升发酵水平,但提升效果可能有限,还需要充分挖掘地衣芽孢杆菌的表达潜力,提升其表达能力,诸如在密码子偏好性[16],酶蛋白修饰[17],蛋白酶基因继续敲除[18],高效启动子开发[19-20]等方面均可继续进行深入研究㊂3㊀结论本研究利用地衣芽孢杆菌木糖诱导分泌表达载体,对来源于嗜糖假单胞菌麦芽四糖基因进行了表达,重组酶成功地分泌至胞外,能够在发酵液上清液中检测到酶活力,而且目标蛋白大小符合理论大小㊂对重组菌进行了发酵条件优化:在24h 添加诱导剂,诱导温度30ħ,发酵至稳定期结束停止发酵,摇瓶水54㊀2021Vol.47No.1(Total 421)平可高达(168.2ʃ9.41)U /mL㊂参考文献[1]㊀ZANGY,ANDERSEN J H,DINOVI M,et al.Maltotetraohydrolase from Pseudomonas stutzeri expressed in Bacillus licheniformis [M ].71th.Geneva:World Health Organization,2016.[2]㊀唐玉,孙俊良,梁新红,等.麦芽四糖研究新进展[J].河南科技学院学报(自然科学版),2013,41(4):14-16.TANG Y,SUN J L,LIANG H X,et al.New advances in maltote-traose[J ].Journal of Henan Institute of Science and Technology (Natural Sciences Edition),2013,41(4):14-16.[3]㊀赵云.麦芽四糖淀粉酶基因克隆㊁表达及活性研究[D].上海:华东师范大学,2013.ZHAO Y.Cloning,expression and activity study of a α-4-amylase enzyme [D].Shanghai:East China Normal University,2013.[4]㊀ZHOU J H,BABA T,TAKANO T,et al.Properties of the enzyme ex-pressed by the Pseudomonas saccharophila maltotetraohydrolase gene (mta )in Escherichia coli [J].Carbohydrate Research,1992,223:255-261.[5]㊀赵云,朱蓓霖,汪正华,等.麦芽四糖淀粉酶基因优化表达及酶学性质分析[J].中国生物工程杂志,2013,33(5):100-106.ZHAO Y,ZHU B L,WANG Z H,et al.Optimization of expression and the characterization of a α-4-amylase enzyme [J].China Bio-technology,2013,33(5):100-106.[6]㊀张梓芊.来源于Pseudomonas saccharophila 的麦芽四糖生成酶的分泌表达及其结构与性质研究[D].无锡:江南大学,2019.ZHANG Z Q.Secretion expression of maltotetraose-forming amylase from Pseudomonas saccharophila and analysis of its structure and en-zymatic properties[D].Wuxi:Jiangnan University,2019.[7]㊀杨亚楠.Pseudomonas saccharophila 麦芽四糖淀粉酶的重组表达㊁发酵优化及其应用研究[D].无锡:江南大学,2019.YANG Y N.Recombinant expression,fermentation optimization and application of maltotetraose amylase from Pseudomonas saccharophila [D].Wuxi:Jiangnan University,2019.[8]㊀BERG C T,DERKX P M F,FIORESI C,et al.Modified amylase from Pseudomonas saccharophila :Denmark,DK1907538[P].2008-04-09.[9]㊀PHENGNUAM T,SUNTORNSUK W.Detoxification and anti-nutrientsreduction of Jatropha curcas seed cake by Bacillus fermentation [J].Journal of Bioscience and Bioengineering,2013,115(2):168-172.[10]㊀SAMBROOKJ,RUSSELL D W.分子克隆实验指南[M].北京:科学出版社,2008.SAMBROOK J,RUSSELL D W.Molecular Cloning:A LaboratoryManual[M].Beijing:Science Press,2008.[11]㊀LI Y R,GU Z H,ZHANG L,et al.Inducible expression of trehalose synthase in Bacillus licheniformis [J].Protein Expression and Puri-fication,2017,130:115-122.[12]㊀LI Y R,JIN K,ZHANG L,et al.Development of an inducible se-cretory expression system in Bacillus licheniformis based on an engi-neered xylose operon[J].Journal of Agricultural and Food Chemis-try,2018,66(36):9456-9464.[13]㊀LI Y R,LIU X,ZHANG L,et al.Transcriptional changes in the xy-lose operon in Bacillus licheniformis and their use in fermentation optimization[J].International Journal of Molecular Sciences,2019,20(18):4615-4630.[14]㊀VOIGT B,SCHROETER R,JÜRGEN B,et al.The response of Ba-cillus licheniformis to heat and ethanol stress and the role of theSigB regulon[J].Proteomics,2013,13(14):2140-2161.[15]㊀李宗文,李由然,顾正华,等.地衣芽胞杆菌FLP /FRT 基因编辑系统的构建及验证[J].生物工程学报,2019,35(3):123-136.LI Z W,LI Y R,GU Z H,et al.Development and verification of anFLP /FRT system for gene editing in Bacillus licheniformis [J].Chi-nese Journal of Biotechnology,2019,35(3):123-136.[16]㊀ZHOU J W,LIU H,DU G C,et al.Production of α-cyclodextringlycosyltransferase in Bacillus megaterium MS941by systematic co-don usage optimization[J].Journal of Agricultural and Food Chem-istry,2012,60(41):10285.[17]㊀朱静,周健.产碱菌麦芽四糖淀粉酶的化学修饰[J].生物化学与生物物理学报,1995,27(3):299-303.ZHU J,ZHOU J.Chemical modification of maltotetrose-forming am-ylase from Alcaligenes sp.[J].Acta Biochimica Et Biophysica Sini-ca,1995,27(3):299-303.[18]㊀潘力,刘欣,王斌.一种枯草芽孢杆菌重组菌株及其制备方法与应用:中国,CN106085937A[P].2016-06-15.PAN L,LIU X,WANG B.Recombination of Bacillus subtilis and itspreparation method and application:China,CN106085937A [P ].2016-06-15.[19]㊀SONG W,NIE Y,MU X Q,et al.Enhancement of extracellular ex-pression of Bacillus naganoensis pullulanase from recombinant Ba-cillus subtilis :Effects of promoter and host[J].Protein Expressionand Purification,2016,124:23-31.[20]㊀XIAO F X,LI Y R,ZHANG Y P,et al.Construction of a novel sug-ar alcohol-inducible expression system in Bacillus licheniformis [J].Applied Microbiology and Biotechnology,2020,104(12):1-17.Heterologous expression of maltotetraose-forming amylase from Pseudomonassaccharophila in Bacillus licheniformisLOU Zhihua 1,2∗,LIU Xiang 1,ZHANG Jingnan 11(Jiangsu OGO Biotechnology Co.,Ltd.,Changzhou 213300,China)2(Liyang VisionBio Technology Co.,Ltd.,Changzhou 213300,China)ABSTRACT ㊀To investigate the heterologous expression in Bacillus licheniformis and improve the fermentation level of maltotetraose-forming amylase,a xylose-inducible expression system was constructed in B.licheniformis CICIM B1391to express the maltotetraose-form-ing amylase from Pseudomonas saccharophila ,and a preliminary fermentation optimization was conducted.It was observed that maltote-traose-forming amylase was successfully secreted into the fermentation broth induced by xylose.After optimized induction time,temperature and fermentation time,the maximum enzyme activity reached to (168.2ʃ9.41)U /mL in shake flask fermentation.The optimal fermenta-tion conditions were adding the inducer at 24h,the induction temperature of 30ħ,and ending fermentation at the end of stable period.Key words ㊀maltotetraose-forming amylase;Pseudomonas saccharophila ;recombinant expression;fermentation optimization。
桃NHX基因家族的全基因组鉴定与表达分析
山东农业科学 ２０２１，５３（１２）：８～１６ＳｈａｎｄｏｎｇＡｇｒｉｃｕｌｔｕｒａｌＳｃｉｅｎｃｅｓ ＤＯＩ：１０．１４０８３／ｊ．ｉｓｓｎ．１００１－４９４２．２０２１．１２．００２收稿日期：２０２１－０８－１６基金项目：山东省农业良种工程项目“优质高档桃突破性新品种选育”（２０２０ＬＺＧＣ００７）作者简介：李淼（１９８９—），男，山东泰安人，博士，助理研究员，主要从事桃分子育种研究。
Ｅ－ｍａｉｌ：ｌｉｍｉａｏ６５４３＠１６３．ｃｏｍ通信作者：张安宁（１９７４—），男，山东泰安人，硕士，研究员，主要从事桃栽培和育种研究。
Ｅ－ｍａｉｌ：ｚａｎ＿ｈｏｐｅ＠１６３．ｃｏｍ桃ＮＨＸ基因家族的全基因组鉴定与表达分析李淼１，李桂祥１，刘伟１，高晓兰１，王孝友２，张安宁１（１．山东省果树研究所，山东泰安 ２７１０００；２．蒙阴县果业发展服务中心，山东蒙阴 ２７６５００） 摘要：本研究利用生物信息学方法从桃全基因组数据库筛选出７个ＮＨＸ反向转运体，并对其序列特征、基因结构、进化关系、亚细胞定位、组织表达谱和盐胁迫响应性进行综合分析。
结果表明，７个桃ＮＨＸ具有清晰的亲缘远近关系；５个桃ＮＨＸ（ＮＨＸ１，ＮＨＸ４～ＮＨＸ７）基因在结构等方面具有相似性，亲缘关系较近，而与ＮＨＸ２和ＮＨＸ３亲缘关系较远。
７个桃ＮＨＸ蛋白在Ｎ端具有明显的跨膜结构区域。
顺式元件分析表明，桃ＮＨＸ基因家族上游启动子区域包含与光信号、激素、逆境胁迫及分生组织表达相关的顺式作用元件。
ｑＲＴ－ＰＣＲ分析结果显示，大多数桃ＮＨＸ家族成员在茎中表达较多；所有桃ＮＨＸ成员对外源盐处理具有响应性。
关键词：桃；ＮＨＸ基因家族；全基因组；基因表达中图分类号：Ｓ６６２．１：Ｑ７８１ 文献标识号：Ａ 文章编号：１００１－４９４２（２０２１）１２－０００８－０９Ｇｅｎｏｍｅ ＷｉｄｅＩｄｅｎｔｉｆｉｃａｔｉｏｎａｎｄＥｘｐｒｅｓｓｉｏｎＡｎａｌｙｓｉｓｏｆＮＨＸＧｅｎｅＦａｍｉｌｙｉｎＰｅａｃｈＬｉＭｉａｏ１，ＬｉＧｕｉｘｉａｎｇ１，ＬｉｕＷｅｉ１，ＧａｏＸｉａｏｌａｎ１，ＷａｎｇＸｉａｏｙｏｕ２，ＺｈａｎｇＡｎ’ｎｉｎｇ１（１．ＳｈａｎｄｏｎｇＩｎｓｔｉｔｕｔｅｏｆＰｏｍｏｌｏｇｙ，Ｔａｉａｎ２７１０００，Ｃｈｉｎａ；２．ＦｒｕｉｔＩｎｄｕｓｔｒｙＤｅｖｅｌｏｐｍｅｎｔＳｅｒｖｉｃｅＣｅｎｔｅｒｏｆＭｅｎｇｙｉｎＣｏｕｎｔｙ，Ｍｅｎｇｙｉｎ２７６５００，Ｃｈｉｎａ）Ａｂｓｔｒａｃｔ ＳｅｖｅｎＮＨＸｔｒａｎｓｐｏｒｔｅｒｓｗｅｒｅｓｃｒｅｅｎｅｄｏｕｔｆｒｏｍｐｅａｃｈｇｅｎｏｍｅ ｗｉｄｅｄａｔａｂａｓｅｂｙｂｉｏｉｎｆｏｒｍａｔ ｉｃｓｍｅｔｈｏｄ，ａｎｄｔｈｅｉｒｓｅｑｕｅｎｃｅｃｈａｒａｃｔｅｒｉｓｔｉｃｓ，ｇｅｎｅｓｔｒｕｃｔｕｒｅ，ｅｖｏｌｕｔｉｏｎａｒｙｒｅｌａｔｉｏｎｓｈｉｐ，ｓｕｂｃｅｌｌｕｌａｒｌｏｃａｌｉｚａ ｔｉｏｎ，ｔｉｓｓｕｅｅｘｐｒｅｓｓｉｏｎｐｒｏｆｉｌｅａｎｄｓａｌｔｓｔｒｅｓｓｒｅｓｐｏｎｓｅｗｅｒｅａｎａｌｙｚｅｄ．ＴｈｅｒｅｓｕｌｔｓｓｈｏｗｅｄｔｈａｔｔｈｅｓｅｖｅｎｐｅａｃｈＮＨＸｓｈａｄｃｌｅａｒｇｅｎｅｔｉｃｒｅｌａｔｉｏｎｓｈｉｐｓ，ａｎｄｔｈｅｇｅｎｅｔｉｃｒｅｌａｔｉｏｎｓｈｉｐｓｏｆｆｉｖｅＮＨＸｓ（ＮＨＸ１，ＮＨＸ４～ＮＨＸ７）ｗｅｒｅｃｌｏｓｅｒｗｉｔｈｓｉｍｉｌａｒｇｅｎｅｓｔｒｕｃｔｕｒｅ，ｂｕｔｔｈｏｓｅｗｅｒｅｆａｒｔｈｅｒｗｉｔｈＮＨＸ２ａｎｄＮＨＸ３．ＴｈｅｒｅｗｅｒｅｏｂｖｉｏｕｓｔｒａｎｓｍｅｍｂｒａｎｅｄｏｍａｉｎｓａｔｔｈｅＮｔｅｒｍｉｎａｌｏｆｓｅｖｅｎｐｅａｃｈＮＨＸｐｒｏｔｅｉｎｓ．Ｃｉｓ ｅｌｅｍｅｎｔａｎａｌｙｓｉｓｓｈｏｗｅｄｔｈａｔｔｈｅｕｐｓｔｒｅａｍｐｒｏｍｏｔｅｒｒｅｇｉｏｎｏｆｐｅａｃｈＮＨＸｆａｍｉｌｙｇｅｎｅｓｃｏｎｔａｉｎｅｄｃｉｓ ｅｌｅｍｅｎｔｓｒｅｌａｔｅｄｔｏｌｉｇｈｔｓｉｇｎａｌ，ｈｏｒｍｏｎｅｓ，ａｄｖｅｒｓｉｔｙｓｔｒｅｓｓａｎｄｍｅｒｉｓｔｅｍｅｘｐｒｅｓｓｉｏｎ．ＴｈｅｒｅｓｕｌｔｓｏｆｑＲＴ ＰＣＲｉｎｄｉｃａｔｅｄｔｈａｔｍｏｓｔｍｅｍｂｅｒｓｏｆＮＨＸｆａｍｉｌｙｍａｉｎｌｙｅｘｐｒｅｓｓｅｄｉｎｓｔｅｍｓ，ａｎｄａｌｌｍｅｍｂｅｒｓｗｅｒｅｒｅｓｐｏｎｓｉｖｅｔｏｅｘｏｇｅｎｏｕｓｓａｌｔｔｒｅａｔｍｅｎｔ．Ｋｅｙｗｏｒｄｓ Ｐｅａｃｈ；ＮＨＸｇｅｎｅｆａｍｉｌｙ；Ｗｈｏｌｅｇｅｎｏｍｅ；Ｇｅｎｅｅｘｐｒｅｓｓｉｏｎ Ｎａ＋／Ｈ＋反向转运体（Ｎａ＋／Ｈ＋ｔｒａｎｓｐｏｒｔｅｒｓ，ＮＨＸ）在植物耐盐性方面发挥着重要作用［１］。
甜叶菊RA苷合成关键基因SrUGT76G1对水分胁迫响应分析
第43卷第1期2021年1月Vol.43,No.1Jan.,2021中国糖料Sugar Crops of Chinadoi：10.13570/ki.scc.2021.01.001甜叶菊RA苷合成关键基因SrUGT76G1对水分胁迫响应分析张婷，杨永恒，孙玉明，王银杰，原海燕，张永侠，徐晓洋（江苏省中国科学院植物研究所/南京中山植物园，南京210014）摘要：RA苷是甜叶菊叶片中最主要的组分之一，其合成受到水分胁迫的影响。
为了探究其调控的分子机制，本研究首先分析了RA苷合成关键基因SrUGT76G1在水分胁迫下的表达模式，并对SrUGT76G1瞬时转化烟草和转基因拟南芥进行干旱处理及GUS染色，多方面阐释其对水分胁迫的响应。
研究结果表明，一定程度的水分胁迫可以促进SrUGT76G1的表达。
此外，对SrUGT76G1的启动子序列进行分析，发现存在多个茉莉酸响应元件及MYB转录因子结合位点。
本研究不但为后续深入开展甜叶菊栽培的高效水分利用与管理措施提供理论支持，也为进一步寻找RA苷上游水分胁迫响应调控因子并通过转基因手段提高RA苷含量奠定基础。
关键词：甜叶菊；RA苷；基因；SrUGT76G1启动子；水分胁迫中图分类号：S566.9文献标识码：A文章编号：1007-2624（2021）01-0001-06张婷，杨永恒，孙玉明，等.甜叶菊RA苷合成关键基因SrUGT76G1对水分胁迫响应分析[J].中国糖料，2021，43(1)：1-6.ZHANG Ting,YANG Yongheng,SUN Yuming,et al.Water stress response of the SrUGT76G1gene for rebaudioside A gly‐coside synthesis in Stevia rebaudiana Bertoni[J].Sugar Crops of China,2021,43(1):1-6.0引言甜菊糖苷（Steviol glycosides，SGs）是富含于菊科多年生草本植物甜叶菊（Stevia rebaudiana Bertoni）叶片中的多种四环二萜类化合物的总称。
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Planta (2010) 232:187–195DOI 10.1007/s00425-010-1160-7
123ORIGINAL ARTICLE
The sugar beet gene encoding the sodium/proton exchanger 1 (BvNHX1) is regulated by a MYB transcription factor
Guy Adler · Eduardo Blumwald · Dudy Bar-Zvi
Received: 14 January 2010 / Accepted: 24 March 2010 / Published online: 14 April 2010© The Author(s) 2010. This article is published with open access at Springerlink.com
AbstractSodium/proton exchangers (NHX) are keyplayers in the plant response to salinity and have a centralrole in establishing ion homeostasis. NHXs can be local-ized in the tonoplast or plasma membranes, where theyexchange sodium ions for protons, resulting in sodium ionsbeing removed from the cytosol into the vacuole or extra-cellular space. The expression of most plant NHX genes ismodulated by exposure of the organisms to salt stress orwater stress. We explored the regulation of the vacuolarNHX1 gene from the salt-tolerant sugar beet plant(BvNHX1) using Arabidopsis plants transformed with anarray of constructs of BvHNX1::GUS, and the expressionpatterns were characterized using histological and quantita-tive assays. The 5Ј UTR of BvNHX1, including its intron,does not modulate the activity of the promoter. Serial dele-tions show that a 337bp promoter fragment suYced fordriving activity that indistinguishable from that of the full-length (2,464bp) promoter. Mutating four putative cis-act-ing elements within the 337bp promoter fragment revealedthat MYB transcription factor(s) are involved in the activa-tion of the expression of BvNHX1 upon exposure to salt andwater stresses. Gel mobility shift assay conWrmed that theWT but not the mutated MYB binding site is bound bynuclear protein extracted from salt-stressed Beta vulgarisleaves.
KeywordsAbiotic stress · Arabidopsis · Beta vulgaris · MYB · NHX · Promoter activity · Sodium/proton exchanger · Sugar beet
AbbreviationsAtNHX1Arabidopsis NHX1BvNHX1Sugar beet NHX1EMSAElectrophoretic mobility shift assayGUS-GlucoronidaseMSMurashige and Skoog basal plant mediumMYBMyeloblastosis viral oncogene homologMYCMyelocytomatosis viral oncogene homologNHX1Sodium/proton exchanger 1
IntroductionSalinity and drought are the major abiotic stresses limitingthe production of crop plants worldwide (Zhu 2001; Munns2002). Plants exposed to these stress conditions respond inbiochemical, physiological and molecular levels to estab-lish a new homeostasis that would enable them to survivethe stress condition (Hasegawa etal. 2000; Serrano andRodriguez-Navarro 2001; Zhu 2001, 2003; Bohnert etal.2006; Pardo etal. 2006; Sahi etal. 2006). Sodium/protonexchangers (NHX) are major players in maintaining ionhomeostasis (Blumwald 2000; Serrano and Rodriguez-Navarro 2001; Pardo etal. 2006; Sahi etal. 2006). Thesemembrane-bound antiporters utilize electrochemical gradi-ent of protons across the tonoplast or plasma membrane totransport sodium or potassium ions into the vacuole orsodium ions outside the cell, respectively (Blumwald 2000;Serrano and Rodriguez-Navarro 2001; Zhu 2003). Theoverexpression of tonoplast NHXs was shown to improveplant salt tolerance (Yamaguchi and Blumwald 2005; Apse
G. Adler · D. Bar-Zvi (&)Department of Life Sciences, The Doris and Bertie Black Center for Bioenergetics in Life Sciences, Ben-Gurion University of the Negev, 84105 Beer-Sheva, Israele-mail: barzvi@bgu.ac.il
E. BlumwaldDepartment of Plant Sciences, University of California, Davis, CA 95616, USA188Planta (2010) 232:187–195123and Blumwald 2007). In agreement with this Wnding,decreased activity of tonoplast NHX resulted in increasedsalt sensitivity (Apse etal. 2003; Sottosanto etal. 2007).Vacuolar NHXs were cloned from a number of glyco-philic and halophilic plant species (Blumwald and Poole1985; Barkla etal. 1995; Fukuda etal. 1998; Hamada etal.2001; Yamaguchi etal. 2001; Xia etal. 2002; Aharon etal.2003; Wu etal. 2004; Kagami and Suzuki 2005; Vasekinaetal. 2005; Yang etal. 2005; Jiu etal. 2007; Zhang etal.2008). It was suggested that the activity of vacuolar Na+/H+antiporters diVer in salt-tolerant and salt-sensitive plants. Inagreement with this notion, following salt stress the induc-tion of NHX genes is higher in salt-tolerant plants than insalt-sensitive plants (Chauhan etal. 2000; Hamada etal.2001; Shi and Zhu 2002; Xia etal. 2002; Yokoi etal. 2002;Zhang etal. 2008). In addition, comparison of the tran-scriptomes of salt-sensitive Arabidopsis thaliana with thatof the related tolerant species Thellungiella halophila (sal-suginea) (Taji etal. 2004; Gong etal. 2005; Wong etal.2006) suggested that these plants mainly diVer in theexpression levels and expression patterns of stress-relatedgenes and ion transporters.A large number of regulatory genes are involved in sig-naling pathways that are responsive to abiotic stresses(Chen and Zhu 2004). Salt and water stresses involve anumber of signaling pathways, some of which are ABAdependent (Hasegawa etal. 2000; Zhu 2002; Chen and Zhu2004; Yamaguchi-Shinozaki and Shinozaki 2006). A largenumber of transcription factors have been shown to beassociated in the regulation of expression of target stress-inducible genes. Osmotic stress signaling pathway involvesthe AREB/ABF and DREB2 transcription factors, whichbind the ABRE/CE and DRE/CRT cis-acting elements,respectively (reviewed by Zhu 2002; Yamaguchi-Shinozakiand Shinozaki 2006). Other transcription factors such asMYB, MYC, NAC and AP2/ERF were also shown to beinvolved in the regulation of genes activity by these abioticstresses (Zhu 2002; Yamaguchi-Shinozaki and Shinozaki2006).Tonoplasts of the salt-tolerant sugar beet (Beta vulgaris)plant were shown to possess high Na+/H+-exchange activity(Blumwald etal. 1987; Blumwald and Poole 1987; Pantojaetal. 1990). NHX activity was correlated with the salinitytolerance of B. vulgaris cell cultures (Blumwald and Poole1987). A cDNA encoding BvNHX1 was isolated (Xia etal.2002). The BvNHX1 transcript levels in both the suspen-sion cell culture and whole plants increased following salttreatment and this increase was concomitant with elevatedBvNHX1 protein and vacuolar Na+/H+ antiporter activity.Here, the promoter of BvNHX1 was cloned and its activ-ity was studied in transgenic Arabidopsis expressing theBvNHX1::GUS construct. Interestingly, the promoter ofBvNHX1 does not contain ABRE or DRE cis-acting ele-ments, common in promoters of osmotic stress regulatedgenes. The minimal 337bp promoter sequence that was up-regulated by salt treatment and osmotic stress was identiWedby constructs containing serial deletions of the promoter.Anumber of potential cis-acting element sequences weremutated to evaluate their possible role in stress-modulatedexpression. Of these mutations only that in the binding sitefor the MYB transcription factor abolished the salt stressand water stress response.