Studies On Agrobacterium-mediated Soybean Transformation With RNAi HC-Pro Gene Of Soybean Mosaic Virus

With its high protein and oil content,soybean[Glycine max(L.)Merr.],which originated in China,has been a vital part of the human diet.Soybean mosaic virus(SMV)disease is broadly distributed in the world and causes significant yield losses and seed quality deterioration.Traditional breeding program for soybean is the most common approach for controlling the SMV disease,while it is hard to introduce broad-spectrum SMV-resistance into adapted soybean cultivars in a short period of time due mainly to the resistance of the parents is specific to the SMV strains/isolates.Genetic transformation can break the barriers between the species and directionally reconstruct,recombine and transfer the excellent resistance genes,which can greatly shorten the process of breeding disease-resistant cultivars.In particular,the RNA interference(RNAi)technology develops rapidly and it has become the most popular approach in the researches of transgenic virus-resistant crops.Thus,present study is aiming to produce genetically engineered soybean plants by introducing the inverted repeat sequence(IRs)of SMV conserved fragment.Based on the RNAi mechanism,the RNA silencing,otherwise known as post-transcriptional gene silencing(PTGS)can be induced in the transgenic plants.Transgenic soybeans with broad-spectrum and persistent SMV-resistance can be achieved by conferring RNA-mediated resistance,namely Pathogen-derived resistance(PDR).Additionally,both breeding SMV-resistant cultivars and theoretical studies on resistance mechanism must base on the SMV-resistant genes with the clear function.However,the low transformation efficiency makes it hard to evaluate SMV-resistant candidate genes for their functions in the transgenic soybean plants.Nicotiana benthamiana,whose transformation method is simple and highly efficient,is an ideal model plant for the investigation of the molecular basis of virus-resistant genes.However,there are no detailed reports on SMV infecting N.benthamiana up till now.Thus,present study is aiming to infect the N.benthamiana plants by SMV and results from these studies will provide theoretical basis for SMV transgenic research in N.benthamiana.The main results were as follows:1.Construction of RNAi vectorThe conserved region of SMV HC-Pro gene was confirmed by nucleotide sequence alignment of 11 popular SMV strains.Then,the 268-bp(2043 bp-2310 bp)RNAi target fragment(HC-Proi)was cloned from the cDNA of SMV strain SC3.Using the GATEWAYTM system,the RNAi vector pB7GWIWG2(?)-HC-Proi suitable for Agrobacterium-mediated soybean transformation was constructed.The BP and LR products were sequenced and blasted in the National Center for Biotechnology Information(NCBI),where showing the 100%nucleotide(nt)identities to SC3.The expected 464-bp and 478-bp amplicons were obtained after PCR verification indicating that the HC-Proi formed the IR structure in the pB7GWIWG2(?)vector.The recombinant vector was digested into two bands of 783 bp and 10 818 bp by Xba?,and 2 256 bp and 9 346 bp by EcoR1 synergistical with Hind?,indicating two ccdB sites were all replaced by HC-Proi.The resulting recombinant pB7GWIWG2(?)-HC-Proi construct was introduced into the A.tumefaciens strain EHA105 by the freeze-thaw method.2.Optimization of soybean transformation system17 soybean cultivars were used for the cotyledonary node-Agrobacterium-mediated soybean transformation system and inoculated with A.tumefacines strain EHA105 harboring pCAMBIA3301 vector.Subsequently,the soybean genotype,chlorine sterilization time,explant type,activity of A.tumefacines,infection concentration,infection time and co-cultivation time were optimized through GUS experiments.The results showed that the best chlorine sterilization time was 14-18 h;the best explant type was 24 h-gemination explant;the best OD600nm of A.tumefacinesis was 0.8-1.0;the best OD600nm of infectious suspension was 0.6-0.8;the best infection time was 30 min;the best co-cultivation time was 4 d.Finally,genotypes including Tianlong 1,Huachun 3,Huachun 6,Williams 82 and Jack were proved to be with higher susceptibility to A.tumefacines strain EHA105 and regeneration rate than those of the other 12 soybean cultivars.3.Soybean transformation of SMV HC-Pro gene24 h-gemination explants from five soybean cultivars including Tianlong 1,Huachun 3,Huachun 6,Williams 82 and Jack were used for the optimized cotyledonary node-Agrobacterium-mediated soybean transformation system using the bar gene as a selectable marker.233 glufosinate-resistant seedlings were obtained which generated from the tissue culture containing germination,explant preparation,A.tumefacines suspension preparation,inoculation,co-cultivation,shoot induction,shoot elongation,rooting,acclimatization,domestication and transplant.The leaf-painting assay was used to estimate herbicide tolerance in the plants;PCR was performed to screen for the presence of the 449-bp fragment of 35S promoter,the 435-bp fragment of 35S terminator,and the 402-bp fragment of bar gene;Putative transformants with two red lines on a LibertyLink(?)strip were considered positive.As a result,105 positive T0 plants were obtained and the average transformation efficiency of the five genotypes was 2.30%(ranging from 0.70%to 3.07%).4.SMV-resistance assessment in the Ti and T2 generationsA total of 1605 Ti plants derived from 105 positive T0 plants of five genotypes were obtained,and 1059 T1 plants were confirmed to be positive.Southern blotting confirmed insertion of the T-DNA into the genomic DNA and revealed a low-copy integration pattern in the transgenic plants.Chi-square(?2)analyses were conducted and most T0 lines transmitted the exogenous genes to their Ti progenies in 3:1 and 15:1 ratios;these ratios are suggestive of a single functional locus and two independent loci,respectively:Additionally,the P-values were indicative of a significant fit for a Mendelian pattern of inheritance(P>0.05).In the Ti generation,virus resistance was evaluated visually after inoculation with SMV(strain SC3)and 441 plants were highly resistant(HR)to SMV.SMV disease rating was classified at five(0-4)levels.In the positive Ti plants,the disease rating on average was 1.42(range 0.45-2.14)versus 3.2(range 2-4)for the nontransformed plants.Virus accumulation in the T1 plants was detected by quantitative real-time(qRT)-PCR analysis at 15 and 30 days post-inoculation(dpi)with SMV;the results revealed a gradual reduction over time in the viral content in the transgenic plants,while the viral content increased rapidly in the nontransformed ones.Double antibody sandwich enzyme-linked immunosorbent assays(DAS-ELISA)analysis of the Ti plants was performed at 2 months post-inoculation with SMV;the results revealed no virus was detectable in the transgenic plants.With the T2 generation,75 transgene-positive plants were inoculated with SMV(strain SC3),where 57 nonsusceptible HR plants and no susceptible ones were identified.DAS-ELISA analysis of the T2 plants was performed at 3 and 5 weeks post-inoculation(wpi)with SMV,where showed no virus or a gradual reduction over time in the viral content.Moreover,virus-induced seed coat mottling was eliminated in the T3 seeds harvested from the resistant lines.5.Preliminary exploration of SMV-resistance candidate gene function in transgenic Nicotiana benthamianaN.benthamiana plants were mechanically inoculated with 37 isolates from 21(SC1-SC21)SMV strains.Plants inoculated with SMV strain SC7(isolates S9 and S10)produced suspected SMV mosaic symptoms on the upper leaves and SMV CP gene(1058 bp)was detected by reverse transcriptase polymerase chain reaction(RT-PCR).Also,plants were identified to be positive for SMV by DAS-ELISA.Inocula prepared from symptomatic leaves of N.benthamiana plants were inoculated back to Nannong 1138-2 seedlings.Two weeks later,typical SMV mosaic symptoms were appeared on the leaves of Nannong 1138-2.Subsequently,RT-PCR and DAS-ELISA both revealed the presence of SMV.Finally,RT-PCR products of CP gene from SC7-S9 and SC7-S10 were sequenced and blasted in the NCBI,where showed that CP gene sequences(795 nucleotides in length)of SC7-S9 and SC7-S10 were the same and both of them had 97%nucleotide(nt)identities to SMV strain G3.These results above all indicated that N.benthamiana plants had been infected by SMV(SC7-S9 and SC7-S10)through artificial inoculation,which laid the foundation for identification of the function of SMV-resistance candidate genes in transgenic N.benthamiana.