A Preliminary Study On The Factors Of Wheat Endogenous 1Dx2 Gene Silencing Induced By Transfer 1Dx5 Gene

In this study, the tolerant winter wheat varieties Xindong 26 receptor material were transformed the 1Dx5 gene by using pollen tube pathway method, gain the transgenic wheat of T0 generation, in the detection of T1 generation seeds,found some seeds 1Dx2 endogenous gene is not expressed. To this end we construct three barley streak mosaic virus(BSMV) recombinant vector carrying the exogenous 1Dx5 gene partial fragments to infect Chinese spring wheat containing 1Dx2 genes, to explore whether exogenous gene can be caused the endogenous gene silencing by the action of RNA interference. Provide a theoretical basis for the transgenic gene breeding silencing phenomenon, and thus speed up the process of transgenic breeding.The main findings are as follows:(1)Optimization of transgenic conditions by pollen tube pathway.During Pollen Tube Pathway Transgenic, control time interval between pollination and gene transformation,to 1.0h, 2.0h, 3.0h, 4.0h for the treatment,proved the processing time was in 2.0h the gene transfer efficiency is highest to 2.78%,the average conversion rate is about 0.94%, transgenic wheat with high quality 1Dx5 HMW-GS gene was obtained.(2)Analysis of HMW-GS components in the T1 generation seeds of Transgenic positive Wheat.Eventually bred 37 transgenic lines were harvested 4686 T1 generation seeds, 1/4 in which randomly selected seeds(total 1178) SDS-PAGE electrophoresis, the protein level T1 generation seeds of HMW-GS composition appears 3 phenotypes: phenotypeâ… , 1Dx5 exogenous gene is not expressed; phenotype â…¡,foreign 1Dx5 genes was expressed; phenotype â…¢, endogenous 1Dx2 gene is not expressed, the three phenotypes in different lines and different single spike are prevalent, the emergence of the probability is about 45.9%, 34.3% and 19.8%.(3)Establish BSMV virus-induced gene silencing system.The eight hydrogen tomato red pigment dehydrogenase(PDS) gene was the indicator gene, when wheat heading in Ear infection vaccination BSMV: PDS, while vaccination BSMV: 00 as the control,found:appeared light bleaching of wheat ears was after vaccinating BSMV:PDS 5-8dpi, more obvious in 15 dpi, in 23-25 dpi light bleaching are the greatest degree, in the control group, there was no such phenomenon as the spike of Wheat.The qRT-PCR analysis showed that compared with BMSV:00, the relative expression of PDS gene was obviously down-regulated in photo-bleaching spikes that infected with BMSV:PDS.(4)BSMV virus-induced 1Dx2 gene silencing.Three target gene fragments was cloned from 1Dx5 gene, were designated as KL-1, KL-2, KL-3, constructed VIGS recombinant vector. After Wheat flowering 12 days, the ears of wheat were friction inoculated with BSMV:KL-1, BSMV:KL-2, BSMV:KL-3, inoculated BSMV: 00 as the control. RT- PCR analysis of The relative expression of 1Dx2 in inoculated wheat, showed that: in the wheat of inoculated three VIGS recombinant vector,the relative expression of 1Dx2 gene decreased.Prove that: in plants transferred to homologous genes, may cause endogenous gene silencing by RNA interference, to provide theoretical basis for the study of transgene silencing.