Expression Characteristics And Functional Study Of Ta14S Genes In Regulating Preharvest Sprouting

Pre-harvest sprouting(PHS)is worldwide natural disasters that seriously affects wheat yield and quality of the grain.In this study,the PHS-resistant variety ‘Huaimai 0360’ and PHS-susceptible variety ‘Zhongyou 9507’ were used as the experimental materials.The genome sequences of three Ta14 S homologous genes,Ta14S-2A,Ta14S-2B and Ta14S-2D,were cloned,and their structure and sequence characteristics in different resistant materials were compared using bioinformatics software.Expression patterns of the Ta14 S homoeologs were analyzed in seed developing,seed germinating process and,vitro embryo germinating process under ABA treatment.The overexpression and RNAi vectors of Ta14S-2B were constructed and then transferred into wheat variety ‘Zhengmai 9023’ by agrobacterium-mediated method,and the seed germinating characteristics in transgenic wheat lines were preliminarily analyzed.The main research results are as follows:1.The genome sequences of three Ta14 S homologous genes,Ta14S-2A,Ta14S-2B and Ta14S-2D,were cloned from PHS-resistant variety ‘huaimai 0360’ and PHS-susceptible variety ‘zhongyou 9507’.Sequence analysis showed that the genome sequence of Ta14S-2Aand Ta14S-2D l are 2424 bp and 2337 bp in lengths,respectively.The both genes contain five exons and four introns.But Ta14S-2B is 3205 bp in length and contains five exons and six introns.Among of three homologous genes,sequences in code region are relatively conservative,only single-base insertion or deletion,but there were many difference in the 5 ’UTR and 3’ UTR.However,no sequence differences were detected between PHS-resistant and PHS-susceptible varieties.2.The expression patterns of three Ta14 S homologous in seed developing and germinating process were analyzed by real time RT-PCR.The results indicated:(1)During seed development,three Ta14 S homologous genes mainly expressed in the early stage of the seed development,and the relative expression amount of each gene in PHS-susceptible varieties were higher than that in PHS-resistant varieties.The expression trends of three homologous genes were same in embryo and endosperm 25,30 and 35 d after anthesis(DAA)and the highest expression was observed in 35 DAA.In embryo 35 DAA,the expression amounts of three Ta14 S homologous genes in PHS-susceptible varieties were significantly higher than that in PHS-resistant varieties.The expression levels were successively Ta14S-2A>Ta14S-2B>Ta14S-2D.In endosperm 35 DAA,the expression levels of Ta14S-2A and Ta14S-2B in PHS-susceptible varieties were higher than that in PHS-resistant varieties.The up-regulation range was Ta14S-2B > Ta14S-2A.While,the expression amount of Ta14S-2D in PHS-resistant varieties was higher than that in PHS-susceptible varieties.(2)During seed germination,the expression trends of three homologous genes in seed and isolated embryos were similar.In the period of 6–36 h after seed soaking,the expression levels of Ta14S-2B and Ta14S-2D in PHS-susceptible variety were significantly higher than that in PHS-resistant variety.The up-regulating range was Ta14S-2B>Ta14S-2D.While,Ta14S-2A just showed difference after soaking at 24–36 h.In isolated embryos,the transcript levels of three Ta14 S homologous genes in PHS-susceptible varieties were significantly higher than that in PHS-resistant varieties under 1–4 d treatments,and the expression range was Ta14S-2B> Ta14S-2A > Ta14S-2D.3.Expression profiles of three homologous genes in mature isolated embryos were investigated under ABA treatment.Compared with untreated controls,the expression levels of three homologous genes were decreased quickly after 1d treatment,and then expression levels basicly remained no change.But the decline range was different between two varieties.The down-regulating ranges of the three Ta14 S homologous gene was lower in PHS-susceptible variety than in the PHS-resistant variety,and the degree of variation was Ta14S-2B > Ta14S-2A> Ta14S-2D.These results suggested that the sensitivity of three homologous genes to ABA was different.4.The overexpression and RNAi inhibition expression vectors of Ta14S-2B were constructed and introduced into wheat variety ‘zhengmai 9023’ via agrobacterium-mediated transformation method,and 19 overexpression and 13 RNAi transgenic lines were obtained,respectively.PPT test combined PCR analysis showed that 1139 positive plants were obtainted by detecting T1 and T2 generation seedlings,including 147 T1 plants,among which 74 overexpression,73 RNAi and 992 T2 plants,among which 339 overexpression and 653 RNAi.5.Semi-quantitative RT-PCR analysis showed that the expression levels of Ta14S-2B gene in T2 transgenic lines was higher in 8 over-expression lines than in wild type plants.While,the expression levels of Ta14S-2B gene had different degrees of inhibition in 5 RNAi lines,indicating that exogenous gene in transgenic wheat could express normally.6.Analysis of seed germination characteristics showed that the germination rates in over-expression transgenic lines were significantly higher than in the non-transgenic control,but the germination rates in RNAi transgenic lines was lower than in the control.Above these result showed that Ta14 S gene plays an important regulating role in seed germinating process.