| Wheat pre-harvest sprouting is a kind of worldwide natural disaster,which not only affect the wheat yields,but also affect its qualities.In this study,the pre-harvest sprouting resistant varieties 'huaimai 0360' and susceptible varieties to pre-harvest sprouting 'zhongyou 9507' were used as the experimental materials,and the full length of both TaLECl and TaFUS3 genes which are related to the resistant pre-harvest sprouting were cloned and their expression patterns in different organization,different seed development stages,vitro embryo germination in maturation period as well as under high salt,mannitol,high temperature and ABA treatment were studied.Besides,to explore the functions of the genes,the overexpression and RNAi inhibition expression vectors of TaFUS3 and TaLEC1 were constructed and were transferred into wheat by agrobacterium-mediated menthod.The main research results are as follows:1.Two dormancy correlated genes were cloned by Reverse transcription-Polymerase Chain Reaction(RT-PCR)and rapid amplification of cDNA ends(RACE)technique.Sequence analysis showed that the full-length cDNA sequence of TaFUS3 gene was 1019 bp,containing a 906 bp open reading frame(ORF),with 47 bp in the 5' UTR and 65 bp in the 3' UTR,and predicted the molecular weight of its encoding protein which consists of 301 amino acids is 33.76kD.Genome sequence analysis revealed that TaFUS3 gene contained seven exons and six introns.The full-length cDNA sequence of TaLEC1 gene was 1074 bp,containing a 741 bp open reading frame(ORF),with 23 bp in the 5' UTR and 370 bp in the 3' UTR.The predicted protein consists of 246 amino acids with the molecular weight of 26.22 kD.In addition,TaLECl gene had no introns by the alignment of genome and cDNA sequence.2.The expression patterns of cloned gene in different tissues,different seed development and germination stages by real time RT-PCR were studied.The results showed that the TaFUS3 gene was mainly expressed in embryo of 25 d after anthesis,while TaLECl gene was mainly expressed in the leaf of 10 d germinating.The expression trends of TaFUS3 gene in anti/sense pre-harvest sprouting wheat varieties during seed development process were basically the same and the expression contents were changed with a single peak curve.During seed development process,the relative expression content of TaFUS3 genes in pre-harvest sprouting-resistant varieties 'huaimai 0360' were all higher than that in pre-harvest Sprouting-susceptible varieties 'zhongyou 9507',but the relative expression c ontent of TaLECl gene in susceptible varieties to pre-harvest sprouting 'zhongyou 9507'were all higher than that in resistant varieties to pre-harvest sprouting 'huaimai 0360',and the expression of TaLEC1 gene was mainly at the early stage of the seed development,but its expression level declined before seeds maturing.The analysis of TaFUS3 expression characteristics in the process of vitro embryo germination 35 d after anthesis showed that the expression quantity of TaFUS3 gene in anti/sense pre-harvest sprouting wheat varieties increased significantly compared to control(0 d).The expression quantity of TaFUS3 gene in pre-harvest sprouting resistant varieties 'huaimai 0360' was significantly higher than that in susceptible varieties to pre-harvest Sprouting 'zhongyou 9507' after 2 d treatment.The expression level of TaLEC1 gene in pre-harvest sprouting resistant varieties 'huaimai 0360'was up-regulated but the differences were not significant after 2 d treatment.3.The expression patterns of the marker gene ?-AMY1,Eml and Em6 related to seed germination during seed development process were studied.The results showed that the expression levels of three genes at the early stage of the seed development were all low and there were no differences in anti/sense pre-harvest sprouting varieties.The differences mainly reflected at the later seed development stages(20?35 d after anthesis),and the expression quantity of ?-AMY1 in pre-harvest sprouting susceptible varieties 'zhongyou 9507' was significantly higher than that in pre-harvest sprouting resistant varieties 'huaimai 0360'.While,the expression levels of Eml and Em6 in pre-harvest sprouting-resistant varieties 'huaimai 0360' were significantly higher than that of in susceptible varieties to pre-harvest sprouting 'zhongyou 9507'.Thus,it was inferred that TaFUS3 genes might involve in seed dormancy regulating process through positivly regulating the expression of Eml and Em6 and negativly regulating expression of ?-AMY1.4.The expression patterns of cloned gene under the high salt(NaCl),mannitol,high temperature and ABA stress were studied.The results revealed that under the treatments with all kinds of stress,the expression level of TaFUS3 gene in both 'zhongyou 9507' and'huaimai 0360' were up-regulated.TaLEC1 gene was up-regulated expressed at the early stages of the stress treaments,but down-regulated quickly,and there was difference between two varieties in expression patterns of Ta LEC1 gene.5.The pMAL-c2x-TaFUS3 and pMAL c2x-TaLEC1 fusion protein expression vectors were constructed and expressed were detected in E.coli BL21 cell.And their expression product total proteins were confirmed that the two fusion genes could express the corresponding fusion proteins in prokaryotic system by SDS-PAGE,in which formula weight of TaFUS3 gene fusion protein was about 75 KD and formula weight of TaLEC1 gene fusion protein was about 70 KD.6.The overexpression and RNAi plant expression vectors of TaFUS3 and TaLECl were constructed and they were used in the transformation of wheat plant via agrobacterium mediated transformation method.The identification of transgenic plants showed that 10 transgenic plants with overexpression TaFUS3,12 transgenic plants with RNAi TaFUS3,9 transgenict plants with sense TaLEC1 and 9 transgenict plants with RNAi TaLEC1 transgenic wheat plants were obtained by detecting T1 generation seedlings.These research results laid an important foundation for TaFUS3 and TaLEC1 functional verification in wheat. |
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