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

The vernalization characteristics of wheat greatly influenced the proceeding of spike differentiation and the susceptibility of freezing injury,and this should be carefully considered before the decision of planting area,seed introduction and even the utilization of cultivation techniques in the wheat producion.It is very important in theoretical and practical significance to study the regulatory mechanisms of vernalization in molecular level.In this paper,the growth habits and DNA compositions of three vernalization genes,VRN1,VRN2 and VRN3 were analyzed in nine representive wheat cultivars(Triticum aestivum L.) and their expression patterns in the developing leaves were systematically analyzed under different vernalization treatments.The main results were summarized as follows:1 The composition of vernalization gene VRN1 and growth habit of wheatSeedling-heading period(SH)and Seedling-double ridge of spike differentiation period(SDR) can been shortened by the vernalization treatments in some degree.The more springness of wheat cultivars,the less sensitiveness to low temperature.The more winterness,the more sensitiveness to low temperature.Under different vernalization treatments,SH variation coefficient(CV) of Liaochun No 10(Vrn-A1,Vrn-B1,Vrn-D1) and Xinchun No 2(Vrn-A1,vrn-B1,vrn-D1) were less than 5%and the two cultivars were springness habit.SH CV of Yumai18(vrn-A1,vrn-B1,Vrn-D1) and Zhengmai9023(vrn-A1,Vrn-B1,vrn-D1) were 5%～10%and the two cultivars were poor springness habit.SH CV of Zhoumai18(vrn-A1,vrn-B1,vrn-D1) and Yumai49-198(vrn-A1,vrn-B1,vrn-D1) were 20%～24%and they were semi-winterness habit.SH CV of Jing841(vrn-A1,vrn-B1,vrn-D1),Xindong18(vrn-A1,Vrn-B1,vrn-D1) and Feimai(vrn-Alvrn-Blvrn-D1) were 25%～32%,,they were winterness habit and the winterness of Feimai was highest among the cultivars.By the sequence-specific PCR amplification and sequence analysis,the growth habit of wheat cultivars were correlated with composition of vernalization genes.When three allelic genes VRN1 were all dominant,the growth habits of wheat cultivars were higher springness.When VRN1 alleles were all recessive,the growth habits of wheat cultivars were semi-winterness to winterness.When any one of VRN-A1,VRN-B1 and VRN-D1 was dominant,the growth habits of wheat cultivars were spring,and the springness of three allelic genes were Vrn-A1＞Vrn-B1＞Vrn-D1,agreeing with the springness growth habits of wheat cultivars..2 Cloning and Expression of Vernalization Gene VRN1 in WheatVRN1 was cloned by gene-specific PCR amplification technique.The nucleotide sequences of VRN-A1,VRN-B1 and VRN-D1 had some difference between A,B and D genomes.The difference was more between VRN-A1 and VRN-B1,VRN-D1 than between VRN-B1 and VRN-D1.However,the sequence difference of VRN-A1,VRN-B1 and VRN-D1 was less among the cultivars.The study of expression characteristics of vernalization gene VRN1 by specific RT-PCR showed that three VRN1 alleles of Liaochun No 10 initiated to express at first-leaf stage (conus stage),and the high level of expression keeped from three-leaf stage(double ridge stage) to flower.There was one dominant VRN1 alleles in Xinchun No 2,Yumai 18 and Zhengmai9023 (VRN-A1,VRN-D1 and VRN-B1 respectively).The express of these dominant Vrn1 allele initiated at first-leaf stage(Conus stage),whereas expression of other two recessive vrn1 alleles in these cultivars initiated later,and their expression characteristics agreed with composition of Vrn-A1vrn-B1vrn-D1,vrn-A1vrn-B1vrn-D1 and vrn-A1Vrn-B1vrn-D1 respectively;In semi-winterness wheat cultivars(Zhoumail 8 and Yumai49-198) and winterness(Jing841 and Feimai),three recessive vrn1 alleles were hypersensitive to low temperature,and their sensitivety were vrn-A1＞vrn-B1＞vrn-D1.The initiation of recessive vrn1 alleles expression was later and correlated with composition of vernalization genes.However,the vernalization treatments can induced the expression of recessive vrn1 alleles and the high level expression can be keeped to flower from the vernalization development stage.3 Cloning and Expression of Vernalization Gene VRN2 in WheatVernalization gene VRN2,including two tandemly duplicated genes designated ZCCT1 and ZCCT2 and coding a protine including CCT domain were cloned by gene-specific PCR amplification technique.VRN2 has one 1249 bp～1449 bp intron.The expression of VRN2 was not detected in Liaochun No 10 and detected only at first-leaf stage in Xinchun No 2,Yumai18 and Zhengmai9023.In winterness cultivars,VRN2 was expressed only in the vernalization stage and interrupted after the vernalization stage.The time of interruption was agree with the double ridge of winterness cultivars.So the VRN2 expressing was repressed by vernalization treatment.4 Cloning and Expression of Vernalization Gene VRN3 in WheatThe cloning and sequence analysis of VRN3 showed that VRN3 may include three members at least in common wheat(Triticum aestivum L.) and codes a domain with phosphatidylethanolamine-binding protein(PBP).It has two introns with different size in different cultivars.By semiquantitative PCR analysis,there were three patterns of VRN3 expression.First, the initiation of expression of VRN3 was earlier than initiation of spike differentiation in Liaochun No 10 and Yumai18.Second,the expression were initiated at elongation stage or single ridge in Xinchun No 2,Zhengmai9023,Jing841 and Xindong18;Third,the initial expressions were detected after double ridge stage in Zhoumai18,Yumai49-198 and Feimai.The VRN3 was constantly expressed in leaves till to the flower stage in all detected cultivars.5 Expressions of Three Vernalization Genes in different organsThe expression characteristics of Chinese spring and Luohan No 2were studied by RT-PCR technique.The results showed that VRN1 was expressed at various level in root,stem,leaf,flag leaf,anther,ovule and developping seed,but was not expressed at dry and sprouting seeds.The vernalization effected expression characteristics of VRN1 in winterness cultivars.The expressions of VRN2 was closely correlated with proceeding of wheat developing,and was only in leaf before double ridge stage and embryo bud of seed germination,whereas the expresses was not in leaf at other developing stage and other tissues.The expressions of VRN3 was the same patternas as VRN1 excluding no expression in root.According to expression characteristics of three vernalization genes,VRN1 is expressed with proceeding of wheat developing and induced by low temperature treatment.The more winterness the cultivars,the longer induction time the low temperature.The expression of VRN1 can keep on to flower.The expression of VRN3 is little later than VRN1,and has same pattemas as VRN1. VRN2 doesn't express in strong spring wheat cultivar,and only expresses before double ridge stage regardless of spring or winter.Meanwhile,VRN1 and VRN3 keep higher level of expression. So,we conclude VRN1 and VRN3 are imperative for generative growth or flower and have an important contribution to generative growth of wheat.The new ideas in this study were as follows:(1) The composition of vernalization gene VRN1 in the cultivars from different wheat ecotope, and the relation of expression of VRN1,VRN2 and VRN3 with growth habits were identified firstly. The additive effect of three domainant Vrn1 alleles and the potency of springness, Vrn-A1＞Vrn-B1＞Vrn-D1,were discovered and these were the molecular basis of controling growth habit.(2) The disparity characteristics of VRN1,VRN2 and VRN3 sequences and the conservatism of coding region among the cultivars were clarified.The diversity of VRN1sequences was in introns regions,whose polymorphism is basis of exploiting molecular marker for identitying growth habit and variety trait.(3) A further clarification for interaction mechanism of three vernalization genes was as follow:The domainant Vrn1 allele control spring growth habit,and recessive vrn1 allele control winter growth habit,VRN1 and VRN3 are imperative for generative growth or flower,their expression product play a important role in generative growth or flower.The expression of domainant Vrn1 alleles cann't inhibited by VRN2,however the expression of recessive vrn1 alleles can be repressed by VRN2,VRN2 can down-regulate negatively VRN1 and VRN3,VRN3 can up-regulate VRN1.The expression of VRN1 and VRN3 can be induced and that of VRN2 repressed by low temperature,then wheat present the response to vernalization.