Construction Of Soybean Mosaic Virus CP Gene RNAi Vectors And Genetic Transformation

Soybean mosaic virus(SMV),a worldwide disease,causes mosaic phenomenon on the leaves and affects photosynthesis after infection,which can cause severe dieback and necrosis and result in the whole plant to death.Annually,SMV will cause 15-30 percent reduction of China's soybean production,which has seriously affected the yield and quality of soybean.The use of transgenic technology to cultivate the resistant varieties for all SMV quickly has become an important task in resistance breeding areas which impacts the China's soybean prospects.Coat protein gene(CP)is a key gene SMV virus synthesis of viral shell in plants.It can hinder viral replication in plants if the CP gene was interfered,so that we can limit the virus in a certain part to control the spread of the virus.In this study,we transferred the CP gene into interference expression vectors pB7GWIWG2(?)using GATEWAY technology which according to RNA interference principle and putting Bar genes for resistance screening.PCR results showed that the size and the direction of the target gene inserted into the vector are right and digestion results indicated our target gene had inserted the correct position.In summary,pB7GWIWG2(?)-CPi has successfully constructed and CPi gene in the form of an inverted repeat took the place of two ccdB loci in pB7GWIWG2(?)which can form hairpin structures.In the present study,we optimized the key methods in Agrobacterium-mediated soybean cotyledon nodes including seed sterilization methods,vitality of bacterial strain,concentration of infection liquid,infection time and co-cultivation time.As a result,chlorine sterilization method was superior to ethanol sterilization method which the average germination rate was 86.10%and the average contamination rate was 7.37%;The highest GUS transient rate was achieved when the bacterial concentration at OD600nm was 0.8-1.0 and the infection liquid at OD600nm was 0.6-0.8.We also found that the best infection and co-cultivation time were 30 min and 4 d,respectively.For the comprehensive evaluation about the transient GUS expression under different treatments and the shoot induction rate of 17 soybean varieties,HC-6 was the ideal genotype for transformation-Based on the above optimized research,we used the prebuilt interference expression vector pB7GWIWG2(?)-CPi to infect HC-6 and then we ditected the T0 transgenic plants with polymerase chain reaction(PCR),herbicides smear and LibertyLink strips.The results show that the target gene has been successfully integrated into the soybean cultivar HC-6 genome and expressed at the protein level.The highest transformation efficiency in this transformation System is 3.76%,these results provide a foundation for the modified soybean varieties research of Agrobacterium-mediated genetic transformation of soybean cotyledon cultivation.