Interference With The Interaction Between NS3 Of Rice Stripe Virus And OsIAA25 Of Rice For Cultivating Virus-Resistant Rice

Plant viral diseases cause serious damage to agricultural production.After the plant is infected with the virus,the plant is stunted due to stunted growth,the leaf is atrophic,even causes the decay,causes the crop quality to decline,the yield to decrease.According to statistics,the annual loss of crops caused by viral diseases around the world is as high as ten billion dollars.The protein interaction between plant virus and host is the key to determine whether the virus can invade the host and replicate and spread effectively in the host.The study of the interaction between plant viruses and their hosts will provide important basis for the formulation of effective virus control strategies.Rice,as an important food crop,suffers from serious viral diseases.Rice Stripe Virus(RSV)of the slender virus genus can carry out persistent transmission through egg transmission by means of the medium grey planthopper,seriously harming the yield and quality of Rice.In this study,the rsv-encoded NS3 was used as bait to screen rice cDNA library by yeast two-hybridization,and it was found that NS3 could interact with the auxin pathway inhibitor and co-receptor Aux/IAA family protein OsIAA25.On this basis,we constructed RNAi vectors of NS3,OsIAA25 single gene and NS3/OsIAA25 double gene,transformed rice,and obtained new rsv-resistant transgenic rice materials.The main research results and conclusions are as follows:(1)Interaction between NS3 and OsIAA25 of Aux/IAA family proteinThe NS3 as the bait protein to screen the cDNA library of rice,it was found that OsIAA25 could interact with NS3,and the interaction between NS3 and OsIAA25 was verified by in vitro immunoco-precipitation(pull-down)experiment and BIFC experiment.Later,we tested whether the NS3 interacted with other members of the Aux/IAA family by yeast double hybrid,and found that the NS3 interacted with OsIAA25,but not with other members of the Aux/IAA family.(2)The preparation of OsIAA25 antibodyFor the need of late protein detection,we prepared the antibody of OsIAA25 protein.Firstly,the prokaryotic expression vector of OsIAA25 gene was constructed and introduced into rossetta bacteria,where 0.5m IPTG was induced for 24 h at 16℃,and OsIAA25 recombinant protein(> 5mg)was induced and purified in large quantities.The antiserum of OsIAA25 was prepared by immunizing female rabbits with OsIAA25 recombinant protein as antigen,and OsIAA25 antibody was obtained after purification.ELISA showed that the antiserum value was 128 K and the antibody titer was 64 K.The WB test showed positive bands,indicating the successful preparation of OsIAA25 protein antibody.We also expressed OsIAA1 and OsIAA29 of Aux/IAA family proteins in prokaryotes.Western blot analysis showed that the prepared antibodies could only specifically bind to OsIAA25 protein,indicating that OsIAA25 antibody had the specificity of binding..(3)Construction of RNAi vector and acquisition of transgenic riceIn order to study the role of NS3 and OsIAA25,we constructed RNAi vectors p1300-NS3-RNAi,p1300-OsIAA25-RNAi and p1300-NS3/OsIAA25-RNAi.The RNAi vectors were introduced into agrobacterium EHA105 by freeze-thaw method,and the wild L10 callus were transformed by agrobacterium-mediated transformation method to obtain 17,22 and 19 of the T0 generation transgenic plants,respectively.(4)Gene expression analysis of transgenic rice plantsNorhtern blot analysis of p1300-NS3-RNAi transgenic plants showed that the NS3 gene could be normally transcribed into mRNA in the plants,and then the mRNA could be cut into siRNA.The qRT-PCR detection showed that the target gene OsIAA25 was significantly down-regulated at the transcription level of p1300-OsIAA25-RNAi transgenic plants.Western blot analysis showed that OsIAA25 protein expression in transgenic plants decreased significantly.In p1300-NS3/OsIAA25-RNAi transgenic plants,Norhtern blot analysis showed that NS3 gene could also be transcribed normally and siRNA was cut.qRT-PCR and western blot analysis showed that OsIAA25 gene was also significantly down-regulated and its protein expression was significantly reduced.(5)Detection of virus resistance in T1 transgenic riceThe T0 generation transgenic plants containing the target gene were self-cross-seeded to obtain T1 generation transgenic strains.50 strains of T1 generation transgenic rice were selected for each strain.When the seedlings were at the 5 leaf stage,the nymph of grey planthopper with RSV was inoculated,and wild L10 was used as the control.After 4 weeks,the results of virus resistance identification showed that 5 transgenic lines were transformed into p1300-NS3-RNAi.There were 5 transgenic lines in p1300-OsIAA25-RNAi transgenic lines.Among the transgenic lines transformed p1300-NS3/OsIAA25-RNAi,there were 7 lines,whose infection rate was less than 6%,showing high resistance.Other transgenic rice lines showed moderate resistance.Among the three transgenic lines,29.41%,22.72% and 36.84% were highly resistant to plants,and the chimeric lines of OsIAA25/NS3 genes were higher than those of isolated OsIAA25 and NS3 genes,indicating that the interaction between interfering NS3 of Rice Stripe Virus and OsIAA25 of rice could more effectively resist the infection of RSV.