Cloning And Expression Analysis Of Vernalization Genes VRN3 In Wheat (Triticum Aestivum L.)

In this study,in order to get a comprehensive understanding for VRN3 gene sequences characteristics and expression pattern in common wheat,we cloned three homoeologous VRN3 genes in A,B,D genomes from eight wheat varieties with different developmental characteristics using sequence-specific PCR amplification;we also cloned the full-length c DNA of VRN-B3,VRN-D3 in the spring varieties Liaochun NO.10 and winter varieties Jing 841 using Race and RT-PCR amplification technology,respectively;and the part of promoter sequences of the two homologous gene using site-finding PCR was cloned.At last,we analized the expression patterns of the three VRN genes during wheat growth process.The results were as follows:1.Using eight common wheat varieties with different vernalization characteristics,we amplified partial sequences of VRN3 in genome and c DNA levels by PCR technology.The results showed that there are three sequences for VRN3 in the genome for eight varieties.Combining VRN3 reference sequences on NCBI(http://www.ncbi.nlm.nih.gov/)(Gen Bank accession numbers EF428115.1 DQ890164.1 EF428113.1)and c DNA sequence,we concluded that they were VRN3 homologous genes coming from A,B,D genome,respectively.Each homologous gene contained two introns and three exons.Sequences alignment analysis showed that the differences of the three exons sequences were very small and the main differences were in the first and second intron.2.Two homoeologous full-length c DNA of VRN3 in spring variety Liaochun NO.10 were cloned.They were VRN-B3,VRN-D3 combining with VRN3 genome amplification sequence results.Sequence analysis showed that the full-length c DNA of VRN-B3 was 1253 bp,whose 5 ’UTR and 3’ UTR were 129 bp and 593 bp,respectively;the full-length c DNA of VRN-D3 was 1248 bp with its 5 ’UTR 123 bp,and 3’ UTR 591 bp,respectively.The ORF of both VRN-B3 and VRN-D3 were 534 bp,encoding 177 amino acids.Further analysis showed that there were 11 single base mutations of VRN-B3 relative to VRN-D3 in the coding region,and more obvious differences between VRN-B3 and VRN-D3 were in the 5’UTR and 3’UTR region.Based on the specific sequence difference between VRN-B3 and VRN-D3,two specific primers were designed to amplify VRN-B3 and VRN-D3 full-length c DNA from Jing841,Liaochun NO.10 and Xinchun NO.2.The sequence alignment results showed that VRN-B3 and VRN-D3 c DNA sequence in different varieties were highly conservative except one T base mutation into A of VRN-D3 in c DNA encoding box of J841.3.The amino acid sequences of VRN-A3,VRN-B3 and VRN-D3 of Liaochun10 alignment results showed VRN-A3 、VRN-B3 and VRN-D3 corresponding amino acid sequence were almost the same,except that there was one amino acid valine(Val)in VRN-B3 mutation into isoleucine(Ile)in VRN-D3 at locus 23,while this variation was not in PBP functional domain.4.Expression patterns of VRN3,VRN1 and VRN2 in different growth stages of wheat varieties were analyzed by real-time quantitative RT-PCR.The results showed that the expression trends of three VRN genes in spring and winter wheat were similar under vernalization conditions;vernalization could promote the expression of VRN1 and inhibit the expression of VRN2,the expression of VRN3 increased gradually under long-day condition and no depression of VRN2.Under non-vernalization treatment,the expressions of VRN1 and VRN3 in spring wheat variety increased gradually from the three-leaf stage,while the expression of VRN2 was suppressed;in winter wheat variety,there were nearly no expression for VRN1 and VRN3,while the expression of VRN2 exhibited high level.5.The relationship of the expression patterns of VRN-B3,VRN-D3 and VRN3 were analyzed by real-time quantitative RT-PCR.The results showed that the transcript levels of VRN-B3 was the main sourse of the expreeion for VRN3 both in winter and spring varieties,and the transcript level of VRN-D3 was less than that of VRN-B3.The expression levels of VRN-B3 and VRN-D3 were nearly approximate with that of VRN3 transcript level,suggesting that VRN-A3 gene might be silent.This conclusion needs further to be explorated.6.The promoter sequence of VRN3 of Liaochun10 was cloned by site-finding PCR,and got partial promoter sequences of VRN-B3 and VRN-D3 were gained enventually.There was big difference in promoter sequences between VRN-B3 and VRN-D3.Whether the difference caused the expression difference between VRN-B3 and VRN-D3 need further analysis.The promoter sequence of VRN-B3 was also very conservative among different wheat varities.All results of this study showed that VRN3 gene was highly conservative among different wheat varieties.