Cloning And Functional Analysis Of Vernaliaztion-Related Gene In Wheat (Triticum Aestivum L.)

Vernalization is one of important regulatory processes of growth and development in wheat.In this study,two highly expressed ESTs CL13519 and Unigene 1312 and two down-regulated ESTs Unigene 4460 and Unigene 20 were studied,which selected from high-throughput sequencing results from wheat varieties with different vernalization characteristics.Two wheat varieties liaochun 10?spring?and jing 841?winter?were used as the experimental materials,and each of the four ESTs was cloned and analyzed as well as expression patterns.BSMV-VIGS was used to study the function of the four ESTs.The over-expression vector of CL13519 was constructed and introduced into Arabidopsis.The positive transgenic Arabidopsis was obtained and the growth and development were studied.The results were as follows:1.Two wheat varieties liaochun 10?spring?and jing 841?winter?were used as the experimental materials to clone the four ESTs.The results showed that the length of CL13519 sequence is 1010 bp,with a c DNA open reading frame?ORF?of 681 bp,and CL13519 encodes a protein with 266 amino acids.In comparison with the sequence of Ta HOX1,the nucleotide sequence homologies of CL13519 is 87.49 %.The length of Unigene1312 sequence is 479 bp,with a c DNA open reading frame?ORF?of 282 bp,including 169 bp in the 5? untranslated region and 26 bp in the 3? untranslated region.Unigene1312 encodes a protein with 93 amino acids.In comparison with the sequence of dehydrin?Triticum turgidum subsp,durum?,the nucleotide sequence homologies of Unigene1312 is 85 %.The length of Unigene4460 is 361 bp,and it maybe related to the methylation of H3 protein with blast in TAIR.The length of Unigene20 is 632 bp,nucleotide sequence homologies with a sequence,which comes from Chinese Spring located in homologous group 3B,is 83.91 %.Unigene4460 shares high homology with hypothetical protein F775₃2582?Aegilops?with the sequence similarity of 77.15 %.2.The expression patterns of the four ESTs in the vernalization stage were analyzed by RT-PCR in two varieties.The results showed that CL13519 and Unigene1312 are parabolic relation with the vernalization treatment time in liaochun10,and reach the maximum in 5 week.There is a parabola change direction between the CL13519 and Unigene1312 expresses and the vernalization treatment time in jing841,and the maximum is in 5 week.Unigene4460 down-regulated expresses in liaochun 10,but arises suddenly and intensely at the first week in jing841.Unigene20 expresses undulately in two varieties,and reached the lowest in 5 week.3.Using VIGS technology and taking BSMV as a carrier to establish technology system of VIGS gene function verification,liaochun 10 was taken as experimental material.The results indicated that the wheat infected by BSMV recombinant vectors which were constructed in this paper,the process of young spike development were influenced differently.Differences among blank,positive and negative control were not significant,but there were significant differences between control and the target genes.Development of the young spike of the plants infected by BSMV:CL13519,BSMV:Unigene1312 and BSMV:Unigene20 were laterer than negative control.But there was earlier than negative control in plants infected by BSMV:Unigene4460.4.The over-expression of CL13519 was constructed and introduced into arabidopsis by Agrobacterium-mediated transformation.The positive transgenic arabidopsis was obtained by Glufosinate and PCR,vegetative growth and reproductive growth of which were better than wild type.