Homologous Sequence Cloning Of TaFUS3 Gene In A,B,D Genomes Related To Pre-Harvest Sprouting Resistance And Its Functional Study In Wheat

The wheat pre-harvest sprouting is a kind of worldwide natural disaster,which affects the yield and quality of wheatseriously.In this study,we used the pre-harvest sprouting resistant variety ‘huaimai 0360' and susceptible variety ‘zhongyou 9507'as the experimental materials and cloned the homologous sequence of TaFUS3 gene in A,B,D genomes related to pre-harvest sprouting resisitance,Meanwhile we analyzedtheir structure and expression patterns systematically.;the VIGS(Vrius-induced gene silencing,VIGS)method was used to study the functions of TaFUS3 genes;at the same time,we constructed the overexpression vector of TaFUS3 and transferred into the wheat variety of ‘Zhengmai9023'with the agrobacterium-mediated method,creating transgenic wheat.The main research results are as follows:1.The sequence of TaFUS3 three homologous genes were obtained by RT-PCR,named TaFUS3-3A?TaFUS3-3B and TaFUS3-3D,and the sequence analysis showed that the lengths of the three homoeologous genes were3478bp?3536bp?and 3502 bp,all containing seven exons and six introns,the first intron was different significantly.Between the resistant and susceptible varieties,no polymorphism was detected among sequences of TaFUS3-3A ? TaFUS3-3B and TaFUS3-3D.Except for 19 SNPs,TaFUS3-3Aand TaFUS3-3D c DNA had adeletion of 3 bp fragment and TaFUS3-3Band TaFUS3-3A had a deletion of 5 bp fragment.The amino acid sequences deduced from three homologous genes had 9 loci differences.Expression analysis showed that TaFUS3-3D gene was mainly expressed in seeds of 15 d after flowering,while the genes TaFUS3-3A and TaFUS3-3B had no transcription products.2.The stable and efficient VIGS system with barley stripe mosaic virus(BSMV)vector was established in our laboratory.We silencedthe wheat reporter gene PDSin the seedlings and panicles ofthe pre-harvest sprouting resistant variety ‘huaimai 0360' successfully,and there were obvious photobleachingphenotype in leaves of wheat seedlings and panicles of wheat.The expression analysis showed that the Ta PDS gene was significantly reducedin the leaves of the seedlings and panicles,and the chlorophyll content of the seedlings were significantly lower than the control,so the high success rate and stable system will be benefit to the function verification of the TaFUS3 gene in the wheat.3.The BSMV-?: TaFUS3 and BSMV-?:GFPtransgenic wheat plants were successfully obtained by using the VIGS method and the expression analysis showed that the expression of TaFUS3 was reduced significantly in the BSMV-?: TaFUS3 transgenic wheat plants than the BSMV-?:GFP transgenic wheat plants.The whole panicle germination and seed germination experimentsof mature wheat after harvest 5 d and 10 dshowed that VIGS-TaFUS3 plants seed germination rate,coleoptile length and root length were significantly higher than the control(BSMV-?:GFP),and the differences were significant.Further studies on its regulatory mechanism showed that the expression of seed dormancy related marker gene ?-amy1 was significantly up-regulated during seed development,while the expression level of Em1 and Em6 were significantly down-regulated in VIGS-TaFUS3 plants;In the seed germination process,the ABA metabolism gene CYP21 and GA synthesis gene GA20 ox were all significantly up-regulated.4.The overexpression of TaFUS3 was transferred into wheat variety ‘Zhengmai 9023' with agrobacterium-mediated method,200 T0 transgenic plants with overexoression TaFUS3 were obtained,and it provides important germplasm resources forwheatbreeding with the pre-harvest sprouting resistant variety.