Molecular cloning and characterization of an anti-bolting

Molecular cloning and characterization of an anti-bolting

related gene(BrpFLC)from Brassica rapa ssp.Pekinensis

Zhugang Li a,1,Lingxia Zhao a,1,Chongshi Cui b,Guoyin Kai a,

Lida Zhang a,Xiaofen Sun c,Kexuan Tang a,c,*

a Plant Biotechnology Research Center,Fudan-SJTU-Nottingham Plant Biotechnology R&D Center,

School of Agriculture and Biology,Shanghai Jiao Tong University,1954Huashan Road,Shanghai200030,China

b College of Horticulture,Northeast Agricultural University,Harbin150030,China

c State Key Laboratory of Genetic Engineering,School of Life Sciences,Morgan-Tan International Center for Life Sciences,

Fudan-SJTU-Nottingham Plant Biotechnology R&D Center,Fudan University,Shanghai200433,China

Received16April2003;received in revised form13August2004;accepted31August2004

Available online25September2004

Abstract

Bolting reduces the commercial value of Chinese cabbage,so inhibiting Chinese cabbage from bolting is important.Flowering locus C (FLC),a repressor of?owering,encodes a MADS-domain transcription factor in Arabidopsis thaliana.Here we report on the cloning and characterization of a FLC homologous gene from Chinese cabbage(designated as BrpFLC,GenBank accession number:AY364013)using RACE-PCR.The full-length cDNA of BrpFLC was851base pair(bp)and contained a591bp open reading frame(ORF)encoding a protein with197amino acid residues.The BrpFLC gene had a high degree of similarity to Brassica napus MADS-box protein mRNA(AY036890) and Brassica rapa cultivar samjin?owering locus C(FLC)mRNA(AY273162).Southern blot analysis indicated that the FLC gene belonged to a multi-gene family when the coding sequence of BrpFLC was used as a probe,and there was a single copy when a30UTR region was used as a probe.Northern blot analysis using the30UTR of BrpFLC as the probe revealed that the transcript level of BrpFLC of different anti-bolting Chinese cabbage cultivars was reduced by vernalization at different degrees,and the BrpFLC transcript levels of anti-bolting cultivars remained higher than those of easy bolting cultivars after vernalization,implying that the degree of vernalization sensitivity of the BrpFLC gene was related to the level of bolting of Chinese cabbage.

#2004Elsevier Ireland Ltd.All rights reserved.

Keywords:Bolting;Brassica rapa ssp.Pekinensis;BrpFLC;Chinese cabbage;Molecular cloning;RACE

1.Introduction

Chinese cabbage(Brassica rapa ssp.Pekinensis), originated in China[1,2],is widely cultivated in Northeast Asia as one of the most widely cultivated crops,and it can be dried,pickled,or cooked for eating[3].Exposing the germinating seed or plantlet to a range of low temperatures (vernalization)will accelerate?owering of the plant. Flowering reduces the commercial value of Chinese cabbage,so it is desirable to use late-?owering Chinese cabbage in agricultural production[4].Bolting of Chinese cabbage is an early signal of?owering,which is accelerated by vernalization and following?ower induction Chinese cabbage cannot be harvested.The temperature in spring in China is usually low,which often reduces Chinese cabbage production.

The study of genes controlling?owering time has been conducted predominantly in Arabidopsis.The FRI,FLC, CO,FT and FCA have been isolated by the generation of mutants with alteration to?owering time[5–7].FRI and FLC are major loci determining?owering time in late-and early-?owering Arabidopsis,synergistically causing late ?owering[8–11].The level of FLC gene expression correlates with the time of?owering in Arabidopsis,and

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Plant Science168(2005)407–413

*Corresponding author.Tel.:+862162932002;fax:+862162824073.

E-mail address:kxtang1@https://www.360docs.net/doc/a32584370.html,,kxtang1@https://www.360docs.net/doc/a32584370.html,(K.Tang).

1Co-?rst authors.

0168-9452/$–see front matter#2004Elsevier Ireland Ltd.All rights reserved.

doi:10.1016/j.plantsci.2004.08.012

the FLC gene encodes a dosage dependent repressor of ?owering[10,12].Flowering is repressed when the FLC gene is overexpressed in transgenic plants[8,12–17]. Expression analyses of?ve FLC-related genes from Brassica napus suggest that BnFLC1–5could delay?ower-ing by3weeks to more than7months in Arabidopsis,and the expression of Arabidopsis FLC could delay?owering time by2–6weeks in early?owering of B.napus cultivar [12].In the present paper,we described the cloning of the anti-bolting gene(BrpFLC)from Chinese cabbage. The expression pro?les of BrpFLC in Chinese cabbage cultivars(B.rapa ssp.Pekinensis)with different bolting following vernalization and without vernalization are also presented.

2.Materials and methods

2.1.Plant materials

Five different bolting Chinese cabbage cultivars,pro-vided by Professor Chongshi Cui of Northeast Agricultural University in China were used in the study.The?ve Chinese cabbage cultivars were as following:Ricaze No.1(the high anti-bolting,strong winter cultivar),Dongchun No.2(the high anti-bolting,strong winter cultivar),Yangchun(the anti-bolting,medium winter cultivar),Dongnong901(the easy bolting,frail winter cultivar)and Jingxiawang(the easier bolting,frailer winter cultivar).The cultivar Chundawang,purchased from seed market in Shanghai is a strong winter as well as anti-bolting cultivar.All unvernalized Chinese cabbages were planted in a green-house with a14-h photoperiod at258C.The seeds were vernalized at1–28C in dark for28,35,49,and63days and then sown in the same greenhouse[10].

2.2.Cloning and sequencing of the full-length cDNA of BrpFLC

Total RNA was extracted from leaves of anti-bolting Chinese cabbage‘‘Chundawang’’with TRIzol reagents (GIBCO BRL,USA)according to the manufacturer’s instruction.The?rst strand cDNA was synthesized from 5m g total RNA according to the protocol of the30RACE System for rapid ampli?cation of cDNA ends(RACE)using the adapter primer(AP,Table1)(GIBCO BRL,USA).The following primers were designed based on the coding sequence of Arabidopsis FLC protein mRNA(GenBank accession number:AF537203):the forward primer(primer 1)and the reverse primer(primer2,Table1),in which Xba I and Sac I enzymatic sites were incorporated.All the primers used in cloning of the BrpFLC full-length cDNA,except the primers provided in the kit(AP,AAP,AUAP),were synthesized by Shanghai Sangon Biotechnological Com-pany in China.The sequences of all the primers used in this experiment were given in Table1.PCR was carried out in a total volume of25m l containing1m l cDNA,10pmol of primer1,10pmol of primer2,10m mol dNTPs,1?PCR reaction buffer and 2.5U Taq polymerase.PCR was performed under the following condition:the template was denatured at948C for3min followed by35cycles of ampli?cation(948C for1min,568C for1min,728C for 1min)and by8min at728C.The PCR product was puri?ed and cloned into pGEM-T easy vector(Promega,Madison, WI,USA)followed by sequencing.The ampli?ed FLC coding sequence from Chinese cabbage‘‘Chundawang’’was con?rmed by NCBI search.The30RACE primer (primer3,Table1)was designed according to this coding sequence and30RACE-PCR was performed as described above.

Based on the30RACE sequence,the gene speci?c primers4and5(Table1)were designed to amplify the50 end of BrpFLC by the cDNA tailed with oligoC.The?rst cycle of PCR was performed with primer4and abridged anchor primer(AAP)as described above.The second round of ampli?cation was performed with primer5and AUAP using the?rst-cycled PCR product diluted50-fold. By aligning30and50product and coding sequence region, the sequence of the full-length cDNA was deduced.To amplify the full-length cDNA of BrpFLC,PCR ampli?ca-tion was carried out using primer6and AUAP in a total volume of50m l reaction solution containing5m l of10?pfu buffer(plus Mg2+),1m l of10mM dNTP mixture,1m l of10m M primer6,1m l of10m M AUAP,2m l of cDNA and10U of pfu DNA polymerase using the following protocol:948C for3min followed by35cycles of ampli?cation(948C for1min,588C for1min,728C for 90s)and by728C for10min.The ampli?ed blunt-ended DNA fragment was dA-tailed using dA-tailing Kit (Sangon,China),ligated with pGEM-T easy vector followed by sequencing.

2.3.Sequence analysis

Sequencing of BrpFLC gene was performed on ABI377 Sequencer(Perkin-Elmer,USA).Amino acid sequence analysis and multiple alignments were conducted on internet network(http://www.expasy.ch).The phylogenetic tree was constructed by software CLUSTAL W1.81and MEGA2 using neighbor-joining method[18–19].

Z.Li et al./Plant Science168(2005)407–413 408

Table1

Primers used in the cloning of the full-length cDNA of BrpFLC by RACE

AP50-GGCCACGCGTCGACTAGTAC(T)17-30

AAP50-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-30

AUAP50-GGCCACGCGTCGACTAGTAC-30

Primer150-CGCTCTAGAATGGGAAGAAAAAAACTAGAA-30

Primer250-ATAGAGCTCCTAATTAAGTAGTGGGAGAG-30

Primer350-GCTGCTGAAAGAAGAGAATCAG-30

Primer450-AGAGCTTTTGACTGAAGATTCAGG-30

Primer550-CGGAGAAGCTGTAGAGCTT-30

Primer650-CGCTCTAGAATGGGAAGAAAAAAACTAGAA-30

2.4.Southern blot analysis

Genomic DNA was extracted from young leaves of plants at three-to?ve-leaf stage from unvernalized anti-bolting Chinese cabbage‘‘Chundawang’’using CTAB method[20]. Aliquots of DNA(13m g)were digested overnight at378C with Hin dIII,Xba I,Bam HI and Eco RI,respectively,which did not cut the coding sequence region,fractionated by1.0% agarose gel,transferred to a positively charged Hybond-N+ nylon membrane(Amersham Pharmacia,Sweden)and hybridized with BrpFLC coding sequence as the probe.In order to further identify the copy number of BrpFLC, aliquots of Chinese cabbage genomic DNA(15m g)were digested overnight at378C with Kpn I,Eco RI and Sna BI, respectively,fractionated by1.0%agarose gel transferred to a positively charged Hybond-N+nylon membrane and hybridized with another gene speci?c probe of BrpFLC(30 UTR).The probes were labeled with biotin-dUTP on the basis of gene images random prime labeling module (Amersham Pharmacia,Sweden)and Southern blot analyses were performed according to the manufacturer’s instruc-tions.Hybridizing bands were detected using CDP-star detection system(Amersham Pharmacia)and signals were visualized by exposure to Fuji X-ray?lm at room temperature for2–3h.

2.5.Northern blot analysis

Total RNA was extracted from leaves of the?ve different bolting Chinese cabbage cultivars(Jingxiawang,Dongnong 901,Yangchun,Dongchun No.2and Ricaze No.1)after treated with vernalization or unvernalization using TRIzol reagents(GIBCO BRL,USA)according to the manufac-turer’s instruction.Aliquots of total RNA(20m g)were separated in1.0%formaldehyde-denatured(w/v)agarose gel,transferred to a Hybond-N+nylon membrane and hybridised with the BrpFLC30UTR and its coding sequence probes.Hybridization and?uorescence exposure were the same as mentioned in Southern blot analysis except that Northern blot was performed at658C.In order to reveal the differentiation among different cultivars after vernalization treatments more clearly,the Northern blot?gure was captured and analyzed by using a UVP transilluminator and the labworks(UVP Inc.,Cambridge,UK).

3.Results and discussion

3.1.Cloning of the full-length cDNA of BrpFLC and sequence analysis

Based on the conserved region of FLC in Arabidopsis (AF537203),speci?c primers(primer1and primer2)were designed to amplify the coding region of FLC from Chinese cabbage‘‘Chundawang’’.A speci?c612bp band was ampli?ed and sequence analysis with ORF?nder(https://www.360docs.net/doc/a32584370.html,)indicated that this fragment con-tained a591bp ORF,which was highly homologous to MADS genes.A30primer was designed for30RACE-PCR and a speci?c342bp band was ampli?ed with an overlap of 157bp.Two speci?c reverse primers designed according to the coding sequence(primers4and5)were used for the ampli?cation of the50end of BrpFLC and a500bp fragment was obtained which contained a50untranslated region of 54bp.By aligning and comparing the coding sequence region,30and50RACE product,the full-length cDNA of BrpFLC was deduced and con?rmed by RT-PCR using primers6and AUAP.The cloned full-length cDNA of BrpFLC gene was851bp with a591bp ORF encoding197 amino acids(Fig.1).NCBI search revealed that BrpFLC belonged to MADS gene family.Sequence alignment revealed that the amino acid sequence of BrpFLC was 98%identical to B.rapa cultivar samjin FLC(AAP31678) and81%identical to FLC in Arabidopsis(AAN04056).At the nucleotide level,the BrpFLC was99%identical to B. rapa cultivar samjin FLC mRNA(AY273162)and93% identical to Arabidopsis FLC mRNA(AF537203).As B. napus MADS protein(AAK70217)and Arabidopsis FLC (AAN04056)were demonstrated to delay?owering[10,12], BrpFLC might also have the function of repressing ?owering.Multiple sequence alignments revealed that BrpFLC also contained typical MADS-box,a conserved domain with about55amino acids in all the reported MADS-box proteins(Fig.2(A)).There was an I-box(from 58th to84th aa,Fig.2(A))made up of27-hydrophilic amino acid residues(indicated by the arrowhead,Fig.3)between MADS-box(from1st to55th aa,Fig.2(A))and K-box(from 86th to150th aa,Fig.2(A)),which was not a conserved region[16],and C-terminus was downstream from the K-box,which was not a conserved region made up of more 10-hydrophobic amino acid residues(indicated by the arrowhead,Fig.3).The ORF encoded a197aa protein with a calculated molecular weight of21.64kDa and an isoelectric point of9.25,revealed by Expasy Molecular Biology Server Analysis(https://www.360docs.net/doc/a32584370.html,).

3.2.Phylogenetic tree analysis

Phylogenetic tree analysis revealed that BrpFLC,three FLCs from B.rapa(AAP31680,AAP31681and AAP31678),FLC from B.oleracea(AAP31677)and FLC from B.napus(AAK70217)were grouped into one cluster with nearest distance as bootstrap value of the subgroup was100%which was in good agreement with previous molecular evolution results that AA genome was much more related with AACC genome than with CC genome in Brassica[21–23].FLC from B.napus(AAK70215)and FLC(AAN04056)from Arabidopsis were grouped into the other cluster,and the two clusters joined together to form a higher subgroup1.MAF4(AAO65315),MADS-box protein (NP_201311),MAF2(AAO65307)and AGL3(AAG37904) from Arabidopsis were grouped into subgroup2.Subgroup1

Z.Li et al./Plant Science168(2005)407–413409

and 2were derived from a common ancestor in evolution (Fig.2(B)).

3.3.Southern blot analysis

In order to test whether BrpFLC belonged to a multi-gene family,Southern blot analysis was performed using BrpFLC coding sequence as the probe.The result revealed that there were several hybridization bands (Fig.4(A)),indicating that the BrpFLC belonged to multiple-gene family which was in agreement with previous reports about FLC genes in B.napus [12]and B.rapa [24].In order to further investigate the copy number of the BrpFLC gene,Southern blot analysis was carried out using another speci ?c probe (30UTR of BrpFLC )and the result showed that there was only one hybridization band (Fig.4(B)),suggesting that BrpFLC was a single-copy gene.

3.4.Expression pro?le of BrpFLC in different bolting Chinese cabbage cultivars with and without vernalization Total RNA extracted from the ?ve different bolting Chinese cabbage cultivars under vernalization or unverna-lization treatment,was used to investigate the expression pro ?les of BrpFLC by Northern blot analysis.Northern blot analysis was performed using the full coding sequence of BrpFLC as the probe and the result revealed that the transcript level of the BrpFLC gene was greatly reduced by vernalization in all cultivars and there was no obvious difference among the ?ve Chinese cabbage cultivars without vernalization after 4weeks (Fig.5(A)).There were two hybridization bands in the Northern blot picture (Fig.5(A)),implying that there was at least another homologue related to vernalization with high similarity to the BrpFLC .

When the BrpFLC 30UTR was used as speci ?c probe in Northern blot,the results revealed that the transcript level of BrpFLC was obviously reduced by vernalization in all the ?ve cultivars,whereas there was no obvious difference among the ?ve Chinese cabbage cultivars without vernaliza-tion (Fig.5(B)).All the ?ve cultivars still remained certain levels of BrpFLC transcripts 4or 5weeks post-treatment (vernalization),but BrpFLC transcript levels of the two high anti-bolting cultivars (Ricaze No.1and Dongchun No.2)were reduced much less than those of the other three cultivars,as a whole the transcript levels of BrpFLC were much higher after 4weeks of treatment than those after 5weeks of treatment.The transcript levels of BrpFLC were reduced much more by vernalization treatments after 7weeks than those after 5weeks.However,the BrpFLC transcripts were almost not detectable in Yangchun,

Z.Li et al./Plant Science 168(2005)407–413

410Fig.1.The full-length cDNA sequence and deduced amino acid sequence of BrpFLC .The start codon (ATG)was underlined and the stop codon (TAA)was underlined italically.

Z.Li et al./Plant Science168(2005)407–413411

Fig.2.Multiple alignment and phylogenetic tree analyses of BrpFLC.(A)Multiple alignment of BrpFLC with other MADS proteins.The identical amino acid residues were indicated with black background and the different amino acid residues were indicated with white background.Gray shade indicated60%or more conservation among all the aligned sequences.AAP31678:?owering locus C of B.rapa cultivar samjin(BrsFLC);AAP31681:?owering locus C(B.rapa); AAP31680:?owering locus C(B.rapa);AAK70217:MADS-box protein(B.napus);AAP31677:?owering locus C(B.oleracea var.capitata);AAK70215: MADS-box protein(B.napus);AAO65307:MADS affecting?owering2variant I(A.thaliana);NP_201311:MADS-box protein(A.thaliana);AAO65315: MADS affecting?owering4variant I(A.thaliana);AAG37904:MADS-box protein AGL31(A.thaliana);AAN04056:?owering locus C protein(A.thaliana).

(B)Neighbor-joining phylogenetic tree analysis with bootstrap values.

Dongnong 901and Jingxiawang,faint in Dongchun No.2and comparatively stronger in Ricaze No.1.After 9weeks of vernalization treatments,the BrpFLC transcripts were almost undetectable among all the ?ve cultivars.As also could be summarized by Fig.5(B),the transcript levels of the BrpFLC reduced gradually along with vernalization treatment in all ?ve cultivars.During 4weeks,all the BrpFLC transcripts reduced remarkably but the BrpFLC transcripts reduced gradually from 4to 9weeks in the cultivars Yangchun,Dongchun No.2and Ricaze No.1,while those of cultivars Jingxiawang and Dongnong 901reduced much less than the other three cultivars.

Our results indicated that BrpFLC transcripts in high anti-bolting cultivars (Dongchun No.2and Ricaze No.1)remained somewhat higher levels than the other three cultivars in a comparatively long period of vernalization

Z.Li et al./Plant Science 168(2005)407–413

412Fig.3.Analysis of hydrophilic and hydrophobic domains of deduced amino acid sequence of

BrpFLC.

Fig.4.Southern blot analysis.(A)Genomic DNA was isolated from leaves of Chinese cabbage ‘‘Chundawang ’’(unvernalized)and digested with Hin dIII,Xba I,Bam HI and Eco RI,respectively,followed by hybridization with the biotin-dUTP-labeled BrpFLC coding sequence as the probe.(B)Genome DNA was digested with Kpn I,Eco RI and Sna BI,respectively,followed by hybridization with the biotin-dUTP-labeled BrpFLC 30UTR as the

probe.

Fig.5.Northern blot analysis for the expression of BrpFLC in different bolting Chinese cabbage cultivars after vernalization or non-vernalization treatment.Total RNA was isolated from leaves followed by hybridization with the biotin-dUTP-labeled BrpFLC 30UTR or the coding sequence as the probe.The rRNA was used as internal control paralleling in Northern blot (Upper panel).Lane 1:Jingxiawang was not vernalized;Lane 2:Jingxiawang was treated by vernalization;Lane 3:Dongnong 901was not vernalized;Lane 4:Dongnong 901was treated by vernalization;Lane 5:Yangchun was not vernalized;Lane 6:Yangchun was treated by vernalization;Lane 7:Dongchun No.2was not vernalized;Lane 8:Dongchun No.2was treated by vernalization;Lane 9:Ricaze No.1was not vernalized;and Lane 10:Ricaze No.1was treated by vernalization.The 4,5,7and 9weeks represented 28,35,49and 63days of venalization treatment,respectively.BrFLC denotes BrpFLC .(A)Northern blot analysis was conducted using the BrpFLC coding sequence as the probe for materials treated by 4weeks of vernalization.(B)Left:Northern blot analysis was conducted using the 30UTR as the probe for materials treated by 4,5,7and 9weeks of vernalization,respectively.Right:the UVP scanning result.Data represents the mean values ?S.E.of three replicates.

treatment(at least within7weeks),implying that BrpFLC was related to bolting in B.rapa ssp.Pekinensis.

Bolting means the transformation of vegetative growth to ?owering,which in?uences Chinese cabbage production in China and other regions of high latitude in autumn[25]. Bolting is a signal of?owering and is usually accelerated by vernalization.In the present study,we have demonstrated that BrpFLC is related with both vernalization and bolting. The anti-bolting cultivars still have a certain level of BrpFLC transcripts after vernalization,suggesting that BrpFLC encodes the dosage dependent repressor of ?owering[10,12,24].FLC represses?owering when over-expressed in transgenic plants,both in early and late ?owering B.napus[10,12,16,26,27].The cloning of the BrpFLC will enable us to study the role of BrpFLC gene on anti-bolting in B.rapa ssp.Pekinensis by genetic manipulation in the future.

Acknowledgements

This research was supported by China National‘‘863’’High-Tech Program,China Ministry of Education,Shanghai Science and Technology Committee and UK/CHINA Science and Technology Collaboration Fund.We are grateful to Youfang Cao and Shi Liu for their assistance with computer analysis.

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