Construction, Characterization, and Expressed Sequence

Construction, Characterization, and Expressed Sequence
Construction, Characterization, and Expressed Sequence

Construction,Characterization,and Expressed Sequence Tag(EST)Analysis of Normalized cDNA Library

of Thermo-Photoperiod-Sensitive Genic Male Sterile (TPGMS)Wheat from Spike Developmental Stages

D.Yang&Z.H.Tang&L.P.Zhang&C.P.Zhao&

Y.L.Zheng

Published online:17September2008

#Springer-Verlag2008

Abstract Thermo-photoperiod-sensitive genic male sterile (TPGMS)wheat is important in utilization of heterosis.To facilitate the use of such wheat line in agriculture,more knowledge about molecular mechanisms of TPGMS genes is required.In this study,we set up a normalized complementary DNA(cDNA)library based on the strategy of saturation hybridization with genomic DNA using TPGMS wheat line.This normalized cDNA library consists of cDNA from six directionally cloned cDNA libraries constructed with spike and anther tissues from spike developmental stages.From the normalized cDNA library, 3,264single-pass expressed sequence tag(EST)were obtained.Exclusion of sequences shorter than100bp resulted in3,223vector-trimmed ESTs with a mean length of926bp.Clustering and assembly analysis resulted in 2,175unique ESTs from423contigs and1,752singletons. Taking advantage of various tools and database,gene function classification showed that60%of the ESTs were predicted to have putative gene function.Of the2,175 unique ESTs,264(12%)displayed significant homology (BlastX E values<10?5)to genes previously reported to be involved in cold-response related processes.Among these,sequences encoding activities related to primary metabo-lism,signal transduction,and transcriptional regulation were observed.Finally,in the total EST sequences,108 potential SSRs were found.The unigene dataset will now be used to fabricate biochips carrying all identified genes for TPGMS wheat functional genomic research. Keywords EST.Gene ontology.Normalized cDNA library.TPGMS.Wheat

Abbreviations

CHA chemical hybridizing agents

CMS cytoplasmic male sterility

contigs contiguous consensus sequences CSRD cold-stress-related database

EST expressed sequence tag

GO gene ontology

IP3inositol-1,4,5-triphosphate

MAP kinase mitogen-activated protein kinase

nr non-redundant

ROS reactive oxygen species

SSRs simple sequence repeats

TPGMS thermo-photoperiod-sensitive genic

male sterile

TPGMSSUniGene TPGMS wheat Spike development

related UniGene

TPP trehalose-6-phosphate phosphatase

Introduction

Male sterility is of particular interest in agricultural crops because of its potential use in utilization of heterosis.The

Plant Mol Biol Rep(2009)27:117–125

DOI10.1007/s11105-008-0050-7

D.Yang

:Z.H.Tang:Y.L.Zheng(*)

National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University,

Wuhan430070,China

e-mail:zhyl@https://www.360docs.net/doc/3616253951.html,

D.Yang

e-mail:2002yangdi@https://www.360docs.net/doc/3616253951.html,

Z.H.Tang

:L.P.Zhang:C.P.Zhao(*)

Beijing Engineering and Technique Research Center of Hybrid Wheat,Beijing Academy of Agricultural and Forestry Sciences, Beijing100097,China

e-mail:cp_zhao@https://www.360docs.net/doc/3616253951.html,

utilization of heterosis in wheat production is facilitated by the use of chemical hybridizing agents(CHA)and the establishment of cytoplasmic male sterility(CMS)three-hybrid system(Zhao et al.1999).There are limits to use CHA,such as its cost and potential residue problem.The CMS system requires male sterile;however,maintainer and restorer lines are cumbersome.It restricts the choice of parents and makes hybrid seed production costly(Murai 2001;Zhao et al.1999).The thermo-photoperiod-sensitive genic male sterile(TPGMS)system has focused on its broad restoring ability,easy maintenance multiplication of the male sterile line by self-pollination,and low cost.It has been considered to be more efficient than CMS system in hybrid wheat seed production(Zhao et al.1999).

Male sterility expression in a TPGMS line is heritable but regulated by temperature and photoperiod.At certain temperature or photoperiod occurring after spike differen-tiation,the male sterility is altered into partial to complete fertility(Li et al.2006).Although mapping of over four major TPGMS genes and lots of quantitative trait loci has improved our knowledge regarding the genetic mechanisms of TPGMS(Cao et al.2004;Guo et al.2006),the molecular basis of the male sterility response to thermo/ photoperiod factors remains poorly understood.Recent research indicated that post-transcriptional regulation mech-anism is related with thermo/photoperiod-sensitive male sterility in rice(Jiang et al.2007;Chen et al.2007). Whether it was one of the major factors controlling fertility alteration still remained open.Therefore,it was necessary to further analyze the genome-wide regulatory network for this particular biological process.

EST,as a genomic approach for identification of expressed genes,has been widely used in genome-wide gene expression studies in various organisms(Adams et al. 1995;Nathalie et al.2005).It has been employed to identify the genes that are expressed in various tissues,cell types,or development stages(Ogihara et al.2003;Ronning et al.2003).The plentiful ESTs have accelerated further molecular characterization of interesting genes and high quality complementary DNA(cDNA)clones provided materials for cDNA microarray manufacture.

In this study,we used large-scale EST sequencing to identify expression genes in TPGMS wheat spikes at sterile and fertile conditions.We constructed six cDNA libraries using messenger RNA(mRNA)isolated from spikes and anthers tissues at sterile and fertile conditions and then normalized the six cDNA libraries by saturation hybridization with genomic DNA.The normalized cDNA library contains more than1.3×105clones.A total of3,264EST sequences from normalized library were generated,from which2,175 unique sequences were identified.We performed extensive analysis of the ESTs derived from the normalized cDNA library using multiform methods.The goal of this work is to establish a platform for functional genomics studies of TPGMS wheat.

Materials and Methods

Experimental Materials

TPGMS wheat,line BS20,was used in this study(Li et al. 2006).The temperature has a much stronger effect on fertility alteration than photoperiod,and the critical point of temperature was10–12°C.Below the critical temperature, BS20remained sterile and was not affected by photoperiod. BS20was cultivated in the fields in Haidian District, Beijing Territory(high-temperature and long-photoperiod zone,fertile condition)and Funan County,Anhui Province (low-temperature and short-photoperiod zone,sterile con-dition).Six spike and anther tissues of BS20,which cultivated in Beijing Territory and Anhui Province respec-tively,were used for construction of the individual cDNA library(Table1).

Construction of Individual cDNA Libraries

Total RNA was extracted using Trizol Reagent(Life Technologies,USA),and mRNA was isolated with Oligo-tex(Qiagen,Germany)according to the manufacture’s instruction.Individual cDNA libraries were constructed using CloneMiner cDNA Library Construction Kit(Invi-trogen,USA),and the whole procedure was performed according to the manufacturer’s recommendation.In par-ticular,every10ng double-strand cDNA were ligated with att B1adaptor.After the size fractionation of cDNA was optimized by column chromatography,the cDNAs contained adaptors recombinated with donor plasmids. Table1Tissues used for construction of six individual libraries

Tissue Development stage Line Ecological

condition

Spike Floret differentiation stage BS20Anhui Province a

Beijing Territory b Spike Anther development stage BS20Anhui Province

Beijing Territory Spike Microspore mother cell stage BS20Anhui Province

Beijing Territory Spike Tetrad stage BS20Anhui Province

Beijing Territory Anther Mono-nucleate microspores stage BS20Anhui Province

Beijing Territory Anther Mature pollen stage BS20Anhui Province

Beijing Territory

a Low-temperature and short-photoperiod zone,sterile condition

b High-temperature and long-photoperiod zone,fertile condition

The recombinant products were isolated and transformed into competent cells(Invitrogen)using an Escherichia coli Pulser(Bio-Rad,USA).The electroporated cells were mixed with super optimal broth with catabolite repression medium and incubated for1h at300rpm at37°C.Two hundred microliters of the cell mixture was used to count the number of recombinants by spreading on Luria–Bertani (LB)plates containing50μg/ml kanamycin.Thirty recombinants were randomly selected to identify average size of inserted fragments.

Construction of Normalized Spike Development Library Equal amounts(10μg)of plasmids isolated from each of the six individual libraries were mixed.It ensured that there was enough cDNA of rare class for saturation hybridization with genomic DNA during normalization(Chu et al.2003). The procedures of establishing fixed affinity systems with genomic DNA for normalization were modified from Chu Zhaohui(Chu et al.2003).Total DNA(100μg)isolated from BS20was digested with100U of restriction enzymes Eco RI and Bam HI,respectively,to prepare the two affinity systems for normalization.The digested DNA was purified with phenol/chloroform/isoamyl alcohol(25:24:1).The five overhangs of the digested DNA were filled by Klenow Fragment and were labeled with C-14biotin2′-deoxyur-idine5′-triphosphate(dUTP;Clontech,USA)in a200-μl volume containing100μl digested DNA,12μl of 0.2mmol/l2′-deoxycytidine5′-triphosphate(dCTP),2′-deoxyguanosine5′-triphosphate,and2′-deoxythymidine5′-triphosphate,respectively,7μl of0.4mmol/l C-14biotin dUTP,and10U Klenow Fragment for30min at37°C.The DNA was then recovered using one-tenth volume of sodium acetate(3mol/l)and two volumes of100%ethanol.The two DNA samples,digested with Eco RI and Bam HI, respectively,were further digested with the alternative enzyme Bam HI and Eco RI,respectively,to prevent reannealing of the double-strand DNA after denaturation in the following operations.The Dynabeads M-280(Dynal USA)were washed according to the manufacture’s instruc-tion and re-suspended in800μl of1×binding and washing buffer containing2mmol/l NaCl,10mmol/l Tris–Cl (pH7.5),and1mmol/l ethylenediaminetetraacetic acid. The two DNA samples each digested with two restriction enzymes were denatured at95°C for10min and then chilled on ice.Each of the denatured DNA samples were first mixed with400μl of1×binding and washing buffer and then400μl of washed Dynabeads.The mixture was incubated overnight with constant rotating(60rpm)at 42°C.The biotin-labeled single-strand DNA was specially combined with Dynabeads.The unlabeled DNA fragments were removed by washing the beads in sterile water at70°C for three times.The single-strand genomic DNA(5μg)fixed on Dynabeads was pre-hybridized with400μl hybridization buffer containing100μg/ml of herring sperm DNA,6×saline-sodium citrate(SSC),0.1%sodium dodecyl sulfate(SDS),and1×Denharts in a1.5ml tube for6h at 65°C.The mixed plasmids(150μg)from six individual cDNA libraries were denatured by keeping at100°C for 10min,diluted with400μl of1×hybridization buffer and then hybridized with the single-strand genomic DNA at65°C for at least20h.The tube containing the genomic DNA and plasmids was immediately put on a magnetic stand to separate Dynabeads from the hybridization buffer after hybridization.The Dynabeads attached with plasmid-ge-nomic DNA were washed three times with800ml buffer containing2×SSC and1%SDS for15min at65°C.The plasmids were captured from the hybridized genomic DNA by re-suspending the Dynabeads attached with plasmid-genomic DNA into50μl deionized water,incubating for 10min at72°C and collecting the supernatant.The plasmids in the supernatant were precipitated with one-tenth volume of sodium acetate and two volumes of100%ethanol and re-dissolved into30μl deionized water.The concentration of the plasmid was measured using spectrophotometer DU640 (Beckman,USA).Each50ng plasmids were mixed with 50μl competent E.coli cells for transformation through electroporation.The recombinant clones were picked out and stored at?70°C.Two hundred microliters of the cell mixture was used to count the number of recombinants by spreading on LB plates containing50μg/ml kanamycin. Two hundred clones were randomly selected to identify average size of inserted fragments.

Sequence Processing and Analysis

Transformed bacteria were randomly selected,and plasmid DNAs were extracted.Inserted cDNA were sequenced from 5′end by conventional procedure using the big-dye terminators on the ABI3730xl DNA analyzer(Chinese National Human Genome Center,China).This procedure included base calling and quality control by PHRED(Brent and Phil1998).The EST sequences were clustered and assembled into contiguous consensus sequences(contigs) using the Essamble web service(http://egassembler.hgc.jp/; Ali et al.2006).Clusters that contained only one sequence were classified as singletons.Altogether,contigs and singletons made up the dataset of putative unique ESTs.

All similarity searches were batch executed locally using the BlastN,BlastX,and TBlastX tools(Stephen et al. 1990).InterProScan was used to scan translated ESTs for protein signatures in the InterPro member databases(http:// https://www.360docs.net/doc/3616253951.html,/InterProScan;Evgeni and Rolf2001; Apweiler et al.2001).Gene ontology(GO)annotation was performed using the Blast2go software package(Ana et al.2005).

Results

Construction of Individual cDNA Libraries and Normalized Spike Development Library

Six individual cDNA libraries were constructed with typical tissues from spike developmental stages representing the special characters of spike of TPGMS wheat.The spike developmental stages include floret differentiation stage,anther development stage,microspore mother cell stage,tetrad stage,mono-nucleate microspores stage,and mature pollen stage.Except for one library constructed with microspore mother cell stage,others owned 100%recombi-nant ratio.The number of recombinant of individual libraries ranges from 1.6×106to 2.0×107.Twenty-four clones were random selected from each individual libraries to analyze insert fragment size.The insert fragment size was large than 0.85kb (Table 2).

The normalized library consisted of 4.0×105clones.Two hundred clones were randomly selected from normal-ized library to analyze the insert fragment size.The clones had different insert lengths ranging from 0.6to 4.6kb and an average insert length of 1.3kb (Fig.1).Thus,this normalized cDNA library is presumed to have an average insert length of 1.3kb.

Generation and Assembly of ESTs Derived from Normalized cDNA Library

A total of 3,264clones randomly selected from normalized spike development cDNA library were sequenced,which

generated 3,223vector-trimmed EST sequences with an average sequencing read length of 926bp.To identify overlapping EST sequences and to produce non-redundant (nr)EST data for further functional annotation and comparative analysis,3,223ESTs were assembled into clusters through EGassembler online bioinformatics service (Ali et al.2006).Based upon regions of nucleotide identity,EST sequences were merged into contigs.A total of 2,175nr EST clusters,putatively regarded as unigenes,were generated,consisting of 423contigs and 1,752singletons.This final dataset was denoted as the TPGMS wheat Spike develop-ment-related UniGene (TPGMSSUniGene)set.The cluster size varied from one to 17copies of any given EST (Fig.2).In the distribution of the EST number in each cluster,the majority of ESTs are present in low copy number.Most of the reads were singletons (1,752sequences,80.55%),and the biggest number of clusters (227)contained only two sequences.The largest cluster contained 17ESTs,and this was followed by the next largest clusters of 15,14,and 12,respectively.The number of these largest clusters is one or two.Overall,the distribution of EST number per cluster shows that there was a low redundancy in our dataset.It makes our normalization approach favorable for functional genomics analysis.

Comparison our ESTs to Common Wheat Spike Development Related EST Sequences

To investigate the number of our ESTs highly homologous to common wheat spike development-related ESTs in the

Table 2Qualifying the individual libraries Tissue Development stage

Number of recombinant

Percent recombinant

Average insert size (kb)

Spike Floret differentiation stage 1.2×107100%0.85Spike Anther development stage 8.0×106100% 1.1Spike Pollen mother cell stage 2.0×10796% 1.18Spike Tetrad stage

3.6×106100% 1.5Anther Mono-nucleate microspores stage 1.6×106100%0.94Anther

Mature pollen stage

4.0×106

100%

1.1

F r e q u e n c y d i f f e r e n t s i z e f r a g m e n t (%)

0.6

0.8

1.0

1.2

1.4

1.8

2.0

2.4

Size inserted fragment (Kb)

Fig.1Size distribution of inserted fragments of normalized cDNA library

502

3

456789101112141517

Cluster size

F r e q u e n c y o f E S T s

Fig.2Distribution of ESTs by cluster size.ESTs were clustered into putative unigene sets,and the number of cluster members of each size category was plotted relative to their abundance within the EST collection

public databases,we performed a comparative matching analysis of our ESTs to six EST datasets derived form common wheat spike tissue(http://www.shigen.nig.ac.jp/ wheat/komugi/ests/tissueBrowse.jsp),Affymetrix wheat 61K genechip(https://www.360docs.net/doc/3616253951.html,/modules/PD_data Selection/blast2expression.php),and the entire collection of wheat ESTs(Triticum aestivum;http://www.ncbi.nlm. https://www.360docs.net/doc/3616253951.html,/blast/Blast.cgi).In general,TPGMSSUniGene set showed a moderate similarity(E value<10?5)to ESTs of common wheat spike tissues(Table3).The percentage of TPGMSSUniGene set matched to Affymetrix wheat61K genechip dataset,and all wheat ESTs were84%and89%. Although224EST sequences(11%)showed no similarity in all wheat EST dataset,158EST sequences had significant BlastX hits(E value<10?5)in the nr database. The comparative analysis indicated the specific gene expression pattern in spike tissues of TPGMS line. Annotation and Functional Categorization of ESTs

One of the most important aspects in mining genomics data is to associate individual sequences and related expression information with biological function.Functional annotation allows categorization of genes in functional classes;it is useful to understand the physiological meaning of large amount of genes and to assess functional differences between subgroups of sequences.The annotation of the TPGMSSUniGene set is based on homology.Each gene in the TPGMSSUniGene set inherited the annotation from the best match found after a BlastX search against nr protein database at NCBI.The E value threshold is10?5,and minimal value of alignment length(in amino acids)is33 were used in BlastX research.A total of460(21%of total UingGenes)were annotated as unknown with E value above or minimal value of alignment length below these thresholds.

To functionally categorize ESTs of TPGMSSUniGene set using Blast2GO bioinformatics tool(Ana et al.2005),the all unigenes were translated in six reading frames and imported into nr database of NCBI,1,554(72%)unigenes had significant BlastX hits(E value<10?5)in the nr database,while the remaining621(28%)had hits with E values greater than10?5or no hit.Mapping of significant hits to GO assigned1,329out of1,554unigenes with6,825 GO accession numbers.Finally,the program extracts the GO terms associated to each of the obtained hits and returns5,784 evaluated GO annotations for1,170query unigenes.One drawback with the Blast2go classification system is that it is somewhat crude because many of the functional classes were assigned automatically.Therefore,in addition to the automat-ed process,GO annotation was also conducted for all unigenes with the InterPro(Apweiler et al.2001)search.We manually inspected the functional classification assignment for all proteins and re-assigned them if necessary.In this way, InterPro annotation results confirmed86%GO terms of Blast2go.Merging Blast2go and InterPro annotation results, 1,301unigenes had annotated with GO terms.In the Table3, we summarized the GO assignment of ESTs in terms of biological processes,molecular functions,and cellular components,covering a broad range of the GO functional categories.

In the biological processes classification,sequences belonged to seven categories:cellular process,metabolic process,localization,biological regulation,response to stimulus,developmental process,and multicellular organ-ismal process.Cellular process category included a largest number of sequences,followed by metabolic process category.These two categories represented964entries in the TPGMSSUniGene set,corresponding to more than75% of all GO annotated genes.To increase the resolution of GO annotation analysis and to improve the identification of putative cold-regulated(low temperature,sterile condition) genes in spikes of TPGMS wheat,we establish a specific database with genes previously reported in the articles involved in cold-stress-related process(Seki et al.2002;

Table3Comparative matching of the ESTs isolated in this study to the spike tissues related ESTs,Affymetrix wheat61K genechip dataset,and all wheat ESTs

Source Tissue Total

sequence No.of

homologues

Percentage of

homologues

Wh library Spike at meiosis14,7701,39864% Wh_f library Spike at flowering date12,3001,51270% Wh_h library Spike at heading date11,2851,51970% Wh_yd library Spikelet at late flowering12,0301,41865% Wh_yf library Spikelet at early flowering11,0721,42065% Wh_FL library Spikelet full length cDNA24,5681,59974% Affymetrix wheat61K genechip dataset55,0521,82584% The entire collection of wheat(Triticum aestivum)

ESTs a

1,051,1751,95089% a Dataset released at December14,2007

Fowler and Thomashow2002;Keiichi et al.2006).This cold-stress-related database(CSRD)presently comprises 775entries,which include315entries from common wheat (Keiichi et al.2006).Using the BlastX program to search the CSRD,we identified264sequences in the TPGMSSU-niGene set that showed significant homology(E<10?5)to genes in the CSRD.Thus,12%of the Unigene set seem to be cold-stress related in spike tissues.There are two classes, primary metabolism and cellular communication and signal transduction,which include almost53%of all sequences that were homologous to potential cold-stress-related proteins of CSRD.

Genes involved in the primary metabolism pathway related to cold respond of TPGMS line spikes have been identified in the TPGMSSUniGene set.One unigene encode trehalose-6-phosphate phosphatase(TPP),which transiently induced expression in response to chilling and other abiotic stresses(Ajay et al.2002).A recent study established that a maize TPP gene is involved in inflores-cence development(Satoh-Nagasawa et al.2006).Similarly,

Table4The GO categorization of ESTs by biological process,molecular function and cellular component

Gene ontology term Representation Percentage Biological process Cellular process93272%

Cellular metabolic process72956%

Establishment of localization30724%

Cell organization and biogenesis25319%

Regulation of cellular process1189%

Metabolic process77660%

Cellular metabolic process72956%

Primary metabolic process67252%

Macromolecule metabolic process59246%

Biosynthetic process22317%

Regulation of metabolic process1078%

Localization30824%

Biological regulation14111%

Response to stimulus14011%

Response to stress846%

Response to abiotic stimulus655%

Response to chemical stimulus615%

Developmental process736%

Multicellular organismal process645% Molecular function Binding68553%

Nucleic acid binding32625%

Nucleotide binding19515%

Protein binding14411%

Ion binding13510%

Cofactor binding212%

Catalytic activity4935%

Oxidoreductase activity1028%

Transferase activity17213%

Kinase activity776%

Hydrolase activity17913%

Isomerase activity20<1%

Lyase activity19<1%

Ligase activity262%

Structural molecule activity13811%

Transporter activity766%

Carrier activity282%

Ion transporter activity222%

Transcription regulator activity595%

Transcription factor activity413%

Translation regulator activity222% Cellular component Intracellular1,07783%

Membrane25327%

two unigenes encoded the enzymesδ1-pyrroline-5-carbox-ylate synthetase and ornithineδ-aminotransferase that are involved in praline biosynthesis,a metabolite that was found to increase during cold acclimation and salt stress(Xin and Browse1998;Roosens et al.1998;Hannah et al.2005; Chinnusamy et al.2006).Furthermore,several unigenes involved in the modulation of reactive oxygen species(ROS) have been identified.The high levels of ROS in tissues serve as the metabolic signal,which reduced cold-induction genes and decreased cold tolerance(Zhu et al.2007).

Signaling cascades-related proteins and transcriptional factors are most important for the cold signal transduction in spikes of TPGMS wheat.Unigenes encoding several proteins involved in signaling cascades were found such as calcium-binding protein,guanosine-triphosphate-binding protein,mitogen-activated protein kinase(MAP kinase), casein kinase,serine/threonine kinase,receptor-like pro-tein kinase,phosphatases2C,and inositol-1,4,5-triphos-phate(IP3).In the precise signaling of cold stress,the plasma membrane may be the primary cold-stress sensor, and the IP3level was regulated,which in turn control cytosolic Ca2+signatures.Cold-induced ROS may activate the MAP kinase cascade that regulates cold stress and other abiotic stresses(Hannah et al.2005;Viswanathan and Zhu2002).The potentially increased activity of the various signaling pathways is associated with the differ-ential expression of many families of transcription factor. Among the transcription regulator activity class(Table4), 41were found to be homologous to transcription factor.In the Table5,transcription factors include zinc finger,bZIP, WRKY,homeodomain leucine zipper,MADS box,MYB, bHLH,and AP2.Several members of these families were previously identified as being responsive to various stresses.Identification of Microsatellite Repeats

Using the SSRSCAN perl script and threshold values as default,the2,715-unigene set was searched for potential simple sequence repeats(SSRs).In total,108di-to pentanucleotide SSRs that fulfilled the criteria of the search were identified(Cardle et al.2000).In the sequence collection,tri-nucleotide repeats were the most commonly found,matches the results from Arabidopsis(Cardle et al. 2000).EST-SSRs are especially advantageous when defin-ing sequence-tagged sites for map-based gene cloning strategies and when there is a need to track desirable traits in molecular assistant breeding program.Work is now in progress to determine which of these SSRs can be reproducibly amplified by polymerase chain reaction,are polymorphic,and can be linked to a phenotypic marker.We expect that several of these will turn out to be useful markers for map-based TPGMS gene clone and molecular assistant breeding.

Discussion

Plant ESTs have been proven to be valuable tools in functional genomic research.A number of collections from many plant species and tissues are now available(Lazo et al.2004;Keiichi et al.2006).In contrast,few researches worked on molecular mechanism of TPGMS wheat,and there is no EST derived from development spikes of TPGMS wheat.To facilitate the research of TPGMS wheat, there is a great need for more ESTs from TPGMS lines.In this study,we constructed a normalized cDNA library of TPGMS wheat spikes in different fertility conditions and generated3,264ESTs.

The normalized cDNA library in this study was specifically designed to collect the maximum number of unique ESTs from spikes in different fertility conditions with a minimum investment in sequencing reaction. Although,there are several strategies that could be utilized for normalization(Ko1990;Soares et al.1994),a new method based on saturation hybridization with genomic DNA has been effectively used in rice and maize(Chu et al. 2003;Zhang et al.2005).In our study,this approach was followed to construct a normalized cDNA library of TPGMS wheat.The normalization method employed substantially reduced frequency of abundant cDNAs while increasing the frequency of typically more rare cDNAs encoding transcription factors,receptor-like proteins,and others.In addition,a high-level(21%)ESTs observed as “novel”ESTs in normalized cDNA library.These“novel”ESTs seem likely to be more rarely expressed genes on the basis of the rationale that highly and moderately expressed wheat genes were more likely to have been already

Table5Classification of transcription factors

Family No.of genes CSRD

AP242 bHLH10 bZIP85 MADS20 MYB21 Zinc finger138 C2H2zinc finger21 WRKY31 Homeodomain Leucine zipper protein30 Other transcrption factor52 Total4120

Distribution of the41transcription factors,identified in the PTSGMSSUniGene set,in different families classified according to https://www.360docs.net/doc/3616253951.html,/Structure/cdd/cdd.shtml.CSRD is set of cold-related genes collected from the public database.

identified as expressed genes in wheat(Triticum aestivum). In addition,the pre-mRNA splicing is of particular importance for cold stress in plants(Zhu et al.2007).It will derive novel ESTs from normalized cDNA library involving cold-stress spike tissues.In all,these results demonstrated that the method of saturation hybridization with genomic DNA is also suitable for wheat normalized cDNA library construction.

To reveal what types of genes are included in our EST collection,the Blast2go and InterProScan programs were used to predict the putative function of the encoded proteins.Among the unigene set of2,175ESTs,60%of the ESTs were predicated to have known functions and were classified into six functional categories,leaving a relatively large proportion of the genes(approximately 40%)outside of this classification.Perception of metabolic response,stress stimuli,and transduction of the stress signal are necessary if the spikes of TPGMS lines react to low temperature.In TPGMS wheat,however,very little is known about cold-stress response in spikes at this level, although the general mechanisms are probably similar in all plants.Based on sequence homology,at least26%ESTs of TPGMSSUniGene set were inferred to be unique in comparison with the ESTs derived from common wheat spikes.This result indicated that careful traces of these different expression patterns might provide information of novel gene-network systems in response to environmental stress.To better identify spike genes involved in the cold response,we created a specific database denoted CSRD (cold-stress-related database),in which all genes available from the public domain and classified as cold-stress responsive or cold-induced were collected.When the TPGMSSUniGene set was compared to the sequences in the CSRD,we observed that264sequences matched, indicating that at least12%of all the genes in the TPGMSSUniGene set involved in cold-stress response. Among these,sequences encoding activities related to primary metabolism,signal transduction,and transcription-al regulation were observed.

Our functional analysis of the TPGMSSUniGene set showed that transcription factor genes were represented by 41sequences,belonging to at least eight different families (Table5).Of these,20were homologous to cold-induced or cold-responsive genes from other organisms(Table5). Molecular analyses have showed that CBFs play a critical role in regulation of genes encoding late-embryogensis-abundant type cold-regulated proteins and osmoprotectant biosynthesis across plant species(Sarhan and Danyluk 1998;Stockinger et al.1997;Chinnusamy et al.2006). From the AP2family of transcription factor class,we identified two genes that are homologous to TaCBFIII-D3 and TaCBFIV-A22,respectively(Mohamed et al.2007). Recent research revealed that wheat species,T.aestivum and Triticum monococcum,may contain up to25different CBF genes and that Poaceae CBF s can be classified into ten groups.This indicates that CBF factors have intricate and different individual roles in inducing and maintaining cold acclimation in plant.Thus,a more detailed analysis of the structure and regulation of CBF genes in spike of TPGMS wheat may reveal new pathways of cold induction,not present in leaf tissues.

In conclusion,the method of saturation normalization appears to be a superior method for constructing a normalized cDNA library.This method was successfully used in this study to build a normalized wheat cDNA library representing expressed genes in multiple spike tissues.The Unigene set derived from normalized cDNA library enabled us to position several genes in their respective metabolic pathway,suggesting that these path-ways are involved in cold-stress responses.As it is beyond the scope of this report to cover all possible pathways involved,we highlight some of the key elements that likely contribute to the cold response of TPGMS wheat.Further-more,TPGMSSUniGene set will now be used to fabricate biochips carrying all identified genes for TPGMS wheat functional genomic research.

Acknowledgments This study was supported by the Program of Beijing Basic Research and Innovation Platform for Agricultural Breeding(No.YZPT01-04),National“863”Program(no. 2006AA100102),and Excellence Scholar Fostered Program of Beijing Government.

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recommendations for the characterization of immunogenicity

Review Recommendations for the characterization of immunogenicity response to multiple domain biotherapeutics Boris Gorovits a ,?,Eric Wakshull b ,Renuka Pillutla c ,Yuanxin Xu d ,Marta Starcevic Manning e ,Jaya Goyal f a P ?zer,United States b Genentech,United States c BMS,United States d Sano ?US,United States e Amgen,United States f Biogen Idec,United States a r t i c l e i n f o a b s t r a c t Article history: Received 13November 2013 Received in revised form 15May 2014Accepted 15May 2014 Available online 24May 2014 Many biotherapeutics currently in development have complex mechanisms of action and contain more than one domain,each with a specific role or function.Examples include antibody –drug conjugates (ADC),PEGylated,fusion proteins and bi-specific antibodies.As with any biotherapeutic molecule,a multi-domain biotherapeutic (MDB)can elicit immune responses resulting in the production of specific anti-drug antibodies (ADA)when administered to patients.As it is beneficial to align industry standards for evaluating immunogenicity of MDBs,this paper highlights pertinent immunogenicity risk factors and describes steps involved in the design of a testing strategy to detect and characterize binding (non-neutralizing and neutralizing,NAb)ADAs.In a common tier based approach,samples identified as ADA screen positive are confirmed for the binding specificity of the antibodies to the drug molecule via a confirmatory assay.The confirmation of specificity is generally considered as a critical step of the tier based approach in overall ADA response evaluation.Further characterization of domain specificity of polyclonal anti-MDB ADA response may be required based on the analysis of molecule specific risk factors.A risk based approach in evaluating the presence of NAbs for MDB is discussed in this article.Analysis of domain-specific neutralizing antibody reactivity should be based on the risk assessment as well as the information learned during binding ADA evaluation.Situations where additional characterization of NAb specificity is possible and justified are discussed.Case studies demonstrating applicability of the risk factor based approach are presented.In general,the presence of a domain with high immunogenicity risk or presence of a domain with high endogenous protein homology may result in an overall high immunogenicity risk level for the entire MDB and can benefit from domain specificity characterization of immune response.For low immunogenicity risk MDBs,domain specificity characterization could be re-considered at later clinical phases based on the need to explain specific clinical observations.Inclusion of domain specificity characterization in early phase clinical studies for MDBs with limited clinical immunogenicity experience may be considered to help understand its value in later clinical development.It is beneficial and is recommended to have a well-defined plan for the Keywords: Multi-domain biotherapeutic Immunogenicity response characterization Anti-drug antibody Journal of Immunological Methods 408(2014)1–12 ?Corresponding author at:Pfizer,1Burtt Rd,Andover,MA 01810,United States.E-mail address:boris.gorovits@p ?https://www.360docs.net/doc/3616253951.html, (B. Gorovits). https://www.360docs.net/doc/3616253951.html,/10.1016/j.jim.2014.05.010 0022-1759/?2014Elsevier B.V.All rights reserved. Contents lists available at ScienceDirect Journal of Immunological Methods j o u r n a l h o me p a g e :w ww.e l s e v i e r.c o m /l oc a te /j i m

Synthesisandcharacterizationofnovel

Synthesis and characterization of novel thermoresponsive ?uorescence complexes based on copolymers with rare earth ions Guihua Cui a ,b ,Shuiying Chen a ,Bao Jiang c ,Yan Zhang a ,Nannan Qiu a ,Toshifumi Satoh d ,Toyoji Kakuchi d ,Qian Duan a ,? a Department of Materials Science and Engineering,Changchun University of Science and Technology,Changchun 130022,China b Department of Chemistry,Jilin Medical College,Jilin 132013,China c New Technology Research and Development Co.,Ltd.,Dongguan 523087,China d Division of Biotechnology and Macromolecular Chemistry,Graduate School of Engineering,Hokkaido University,Sapporo 060-8628,Japan a r t i c l e i n f o Article history: Received 9April 2013 Received in revised form 4June 2013Accepted 5June 2013 Available online 4July 2013 Keywords: Poly(N -isopropylacrylamide)Terbium Europium Lower critical solution temperature (LCST)Atom transfer radical polymerization (ATRP) a b s t r a c t The thermo-sensitive and ?uorescent complexes containing Eu(III)or Tb(III)were synthesized and char-acterized,in which cholesterol-g-poly(N -isopropylacrylamide)(PNIPAM)copolymer was used as a poly-mer ligand.The results from the experiments indicated that Eu(III)or Tb(III)was bonded to nitrogen and oxygen atoms in the polymer chain.The ?uorescence lifetimes of the powdered Eu(III)and Tb(III)com-plexes was 11.48ms and 10.71ms,respectively.The maximum emission intensity of the PNIPAM–Eu(III)complex at 613nm and the PNIPAM–Tb(III)complex at 545nm were enhanced about 11.1and 11.3times compared with that of the corresponding rare earth ions,respectively.Additionally,the lower critical solution temperature (LCST)of complexes were slightly higher than those of the copolymers. ó2013Elsevier B.V.All rights reserved. 1.Introduction The rare earth ions in a ligand-free cationic state,have a lower absorption/emission ef?ciency in the visible region of the spectrum,but when they bound to organic ligands of high molar absorption coef?cients and form the rare earth complexes,their luminous intensity can be enhanced drastically [1–4].This phenomenon is mainly attributed to the organic ligands which absorb ultraviolet ray energy and transfer to central rare earth ions,so that the charac-teristic ?uorescence will be enhanced.Due to the unique long lumi-nescence lifetimes,sharp emission bands and photostability [5–10],the lanthanide-based complexes have generated much research interest as a new optical components,especially those containing europium (III)or terbium (III),which possess high color purity and produce red-emission (Eu 3+)and green-emission (Tb 3+)are often used as probes and labels in many ?elds such as materials science,biological technology and photoluminescent devices [11–15]. It is an attractive idea to functionalize lanthanide-based com-plexes with stimuli–responsive polymers,and the obtained mate-rials can be used to control the release of guest molecules in immunodiagnostic assays under external stimuli,such as temper-ature,pH,ionic strength [16–18].Perhaps the most extensively studied stimuli-responsive complexes are these modi?ed poly(N-isopropylacrylamide)(PNIPAM).PNIPAM is a well-known thermoresponsive polymer which that can change its appearance from a clear solution to a turbid suspension in water at a relatively lower critical solution temperature (LCST)of 32°C (near that of the human body)[19].Herein we synthesized a new class of composite complexes via conjugating the ?uorescent Eu 3+or Tb 3+with ther-mosensitive and biocompatible cholesterol-g-PNIPAM copolymers.Some polymers such as polymethylmethacrylate [20,21],styrene-co-butylmethacrylate [22],and polyvinylpyrrolidone (PVP)[23,24]were doped with Eu 3+and Tb 3+forming the complexes,in our previous papers we have reported a new composite materi-als from cellulose-g-PNIPAM polymers doped with Eu 3+ions [25],which had large Stokes shifts and long luminescence lifetimes.Once coupled to cholesterol-g-PNIPAM,the complexes should be-come a robust functional probe and is suitable for optical imaging applications.2.Experimental 2.1.Materials and instrumentation N -isopropylacrylamide (Aldrich,98%)was recrystallized twice from a hexane/benzene mixture (3/2,v/v).Tris(2-(dimethyl-amino)ethyl)amine (Me 6TREN)was synthesized from tris(2-ami- 0925-3467/$-see front matter ó2013Elsevier B.V.All rights reserved.https://www.360docs.net/doc/3616253951.html,/10.1016/j.optmat.2013.06.010 Corresponding author.Tel.:+8643185583105;fax:+8643185583105. E-mail address:duanqian88@https://www.360docs.net/doc/3616253951.html, (Q.Duan).

Synthesis and Characterization of

This article was downloaded by: [University of Ottawa] On: 09 April 2014, At: 19:25 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Molecular Crystals and Liquid Crystals Publication details, including instructions for authors and subscription information: https://www.360docs.net/doc/3616253951.html,/loi/gmcl20 Synthesis and Characterization of Carbazole-based Copolymers Containing Benzothiadiazole Derivative for Polymer Light-Emitting Diodes Jin Su Park a , Sung-Ho Jin a , Yeong-Soon Gal b , Jun Hee Lee c & Jae Wook Lee d a Department of Chemistry Education, Interdisciplinary Program of Advanced Information and Display Materials , Institute for Plastic Information and Energy Materials , Busan , 609-735 , Korea b Polymer Chemistry Lab , Kyungil University , Hayang , 712-701 , Korea c Department of Advance d Materials Engineering , Dong-A University , Busan , 604-714 , Korea d Department of Chemistry , Dong-A University , Busan , 604-714 , Korea Published online: 17 Sep 2012. PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.

20.Characterization theme of Tess

Characterization of Tess Character---Tess Durbeyfield The heroine The eldest child in family The wife of Angel The mistress of Alec Tess Durbeyfield A simple country girl with some education ?As a country girl, she is pure, and noble; industrious and diligent. ?As a family member, she is kind, responsible and dedicated. ?As a friend, she is sympathetic and sincere. ?As a lover and wife, she is honest and loyal, and with high-standarded moral. ?Facing all the sufferings, she is strong and fortitudinous. She is self-respecting and self-dependent.

Character---Angel Clare The youngest son in his family Tess’s husband Angel Clare ?Gentleman from the appearence ?Physically progressive and rebellious ?Independent from his family ?Mentally deep-rooted with conventional mores ?Double-standarded morality ?Hypocritical and self-centered ?Be cruel to Tess ?Stubborn and snobbish ?Idealist ?Mixture of modern thoughts and conventional ideas ?Stands for masculine reason Character---Alec ?The son of a arriviste ?The man who ruined Tess Character---Alec ?A typical villain ?Libertine(sensual): have affairs with many women ?Satanic character: wicked and devil ?Hypocritical in religion ?Selfish, rude and arrogant ?Seduce and trouble Tess all the time ?Stands for masculine sexual desire Other characters ?John Durbeyfield: father of Tess ?He is poor,ignorant higgler. He is a weak man, to a considerable extent under the infulence of his wife, and is also largely a comic character. His weakness and lack of guidance have bad effects on his family and especially on Tess. Like all the rest of Hardy’s country people, he is fatalistic, accepting whatever happens to him. Other characters

Characterization of supramolecular polymers

Cite this:Chem.Soc.Rev .,2012,41,5922–5932Characterization of supramolecular polymers w Yiliu Liu,Zhiqiang Wang and Xi Zhang* Received 20th March 2012DOI:10.1039/c2cs35084j Supramolecular polymers are made of monomers that are held together by noncovalent interactions.This is the reason for the wide range of novel properties,such as reversibility and responses to stimuli,exhibited by supramolecular polymers.A range of supramolecular polymerization methods have been developed leading to a number of novel supramolecular materials.However,standard techniques for the characterization of supramolecular polymers have yet to be established.The dynamic nature of supramolecular polymers makes them di?cult to be fully characterized using conventional polymer techniques.This tutorial review summarizes various methods for characterizing supramolecular polymers,including theoretical estimation,size exclusion chromatography,viscometry,light scattering,vapor pressure osmometry,mass spectrometry,NMR spectroscopy,scanning probe microscopy,electron microscopy,and atomic force microscopy-based single molecule force spectroscopy.Each of these methods has its own particular advantages and disadvantages.Most of the methods are used to characterize the supramolecular polymer chain itself.However,some of the methods can be used to study the self-assembled state formed by supramolecular polymers.The characterization of a supramolecular polymer cannot be realized with a single method;a convincing conclusion relies on the combination of several di?erent techniques. Introduction Supramolecular polymer chemistry originated from a close integration of polymer science and supramolecular chemistry,and now stands as a popular and independent research area.1–3In contrast to conventional polymers,the connection between monomers of supramolecular polymers is noncovalent.4–8 The dynamic nature of noncovalent interactions gives supramo-lecular polymers many novel properties,which can be comple-mentary to conventional polymers.9For example,supramolecular polymers possess very sensitive thermal responsiveness.A small change in temperature can lead to a large variation in viscosity,which makes supramolecular polymers much easier to process than conventional polymers.In addition,the reversibility derived from the noncovalent interactions gives supramolecular polymers the potential to be recyclable and self-healing.10–11 The evolution of supramolecular polymers has resulted from two streams of e?ort.One is to develop new mechanisms for supramolecular polymerization,such as hydrogen bonding, Key Lab of Organic Optoelectronics and Molecular Engineering,Department of Chemistry,Tsinghua University,Beijing,100084,China.E-mail:xi@https://www.360docs.net/doc/3616253951.html, w Part of a themed issue on supramolecular polymers. Yiliu Liu Yiliu Liu got his BA in the Department of Environmental Engineering,Xi’an Jiaotong University.In 2008,he joined Prof.Xi Zhang’s group as a PhD student in the Depart-ment of Chemistry at Tsinghua University.Currently,he is working on supramolecular polymerization based on host-enhanced noncovalent in-teractions. Zhiqiang Wang Zhiqiang Wang is a full pro-fessor of the Department of Chemistry,Tsinghua Univer-sity.His research interests are focused on supramolecular self-assembly and organic thin ?lms. Chem Soc Rev Dynamic Article Links https://www.360docs.net/doc/3616253951.html,/csr TUTORIAL REVIEW P u b l i s h e d o n 06 J u n e 2012. D o w n l o a d e d b y B e i j i n g U n i v e r s i t y o f C h e m i c a l T e c h n o l o g y o n 04/04/2015 01:57:32. View Article Online / Journal Homepage / Table of Contents for this issue

Characterization of aroma compounds of Chinese

Characterization of Aroma Compounds of Chinese“Wuliangye” and“Jiannanchun”Liquors by Aroma Extract Dilution Analysis W ENLAI F AN AND M ICHAEL C.Q IAN* Department of Food Science and Technology,Oregon State University,Corvallis,Oregon97331 Aroma compounds in Chinese“Wuliangye”liquor were identified by gas chromatography-olfactometry (GC-O)after fractionation.A total of132odorants were detected by GC-O in Wuliangye liquor on DB-wax and DB-5columns.Of these,126aromas were identified by GC-mass spectrometry(MS). Aroma extract dilution analysis(AEDA)was further employed to identify the most important aroma compounds in“Wuliangye”and“Jiannanchun”liquors.The results showed that esters could be the most important class,especially ethyl esters.Various alcohols,aldehydes,acetals,alkylpyrazines, furan derivatives,lactones,and sulfur-containing and phenolic compounds were also found to be important.On the basis of flavor dilution(FD)values,the most important aroma compounds in Wuliangye and Jiannanchun liquors could be ethyl butanoate,ethyl pentanoate,ethyl hexanoate, ethyl octanoate,butyl hexanoate,ethyl3-methylbutanoate,hexanoic acid,and1,1-diethoxy-3-methylbutane(FD g1024).These compounds contributed to fruity,floral,and apple-and pineapple-like aromas with the exception of hexanoic acid,which imparts a sweaty note.Several pyrazines, including2,5-dimethyl-3-ethylpyrazine,2-ethyl-6-methylpyrazine,2,6-dimethylpyrazine,2,3,5-tri-methylpyrazine,and3,5-dimethyl-2-pentylpyrazine,were identified in these two liquors.Although further quantitative analysis is required,it seems that most of these pyrazine compounds had higher FD values in Wuliangye than in Jiannanchun liquor,thus imparting stronger nutty,baked,and roasted notes in Wuliangye liquor. KEYWORDS:GC-olfactometry;AEDA;Wuliangye;Jiannanchun;Chinese liquors;distillate;aroma compounds;pyrazines INTRODUCTION Chinese liquor is a traditional distillate fermented from grains. After the fermentation,the fresh spirit is distilled out and then aged under controlled conditions.The aged distillate is adjusted to the designated ethanol concentration and blended to ensure the quality of finished product(1).Chinese liquor has an annual consumption of approximately4million kiloliters,creating a sales revenue of500billion Chinese Yuan.There is no standard procedure to make Chinese liquor.The traditional manufacturing is more of an art than a science.The raw materials for making Chinese liquor can be quite different depending upon availability and the economics of the raw materials.In general,Chinese liquor is made from sorghum or a mixture of sorghum,wheat, corn,rice,and sticky rice.Rice hull is typically used as the fermentation aide(1,2). The saccharifying and fermentation cultures used for Chinese liquor are Daqu,Xiaoqu,or other enzyme preparations.Daqu is the most widely used culture and is made from wheat or a mixture of wheat,barley,and pea.The raw materials of Daqu are typically milled and pressed into a mould of different sizes depending upon the manufacturer.The Daqu is then incubated under controlled conditions.On the basis of the maximum temperature at which the Daqu is incubated,the types of Daqu can be classified into low-temperature Daqu(<45°C),moder-ate-temperature Daqu(45-60°C),and high-temperature Daqu (>60°C).Daqu is rich in various microorganisms including bacteria,yeast,and fungi(1).In addition,complex enzyme systems are accumulated in the finished Daqu(3). The grains used for liquor fermentation are first cooked and then mixed with Daqu powder.The fermentation is typically carried out at28-32°C for60days under anaerobic conditions in a solid state.After fermentation,the liquor is distilled out with steam and aged in sealed pottery jars to develop the balanced aroma.While most of the liquors are aged for about 1year,some of them are aged for more than3years.The aged liquor is diluted with water and blended to yield an ethanol content of40-55%(v/v)for constant quality in the finished product. Because of differences in manufacturing practices,the aroma profiles of various Chinese Daqu liquors are quite different.On the basis of aroma characteristics,Chinese liquor can be classified into strong aroma style,light aroma style,soy sauce aroma style,sweet honey style,and miscellaneous style.Of *To whom correspondence should be addressed:Department of Food Science and Technology,100Wiegand Hall,Oregon State University, Corvallis,OR97331-6602.Telephone:541-737-9114.Fax:541-737-1877. E-mail:michael.qian@https://www.360docs.net/doc/3616253951.html,. J.Agric.Food Chem.2006,54,2695?27042695 10.1021/jf052635t CCC:$33.50?2006American Chemical Society Published on Web03/03/2006

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