Comparison Of Two VIGS Vectors For Gene-silencing In Soybean(Glycine Max)

Soybean(Glycine max),originated in China,is one of the main oil crops in the world.Virus-induced gene silencing(VIGS)technology has become one of the key technologies to study gene function in soybean due to the low efficiency of soybean transgenosis.Soybean mosaic virius(SMV)is one of the most prevalent pathogens affecting soybean production worldwide,seriously affecting the yield and quality of soybean.SMV often causes two symptoms:mosaic and necrosis on soybean leaves.Proteomic analysis showed that ascorbate peroxidase(APX)was up-regulated in cells with necrotic symptoms.In order to further determine the function of APX in SMV-induced symptoms on soybean leaves,we silenced the soybean endogenous APX gene by VIGS technology.The two VIGS vectors were constructed based on the Bean pod mottle virus(BPMV).r-BPMV is constructed on the basis of RNA,while p-BPMV is constructed on the basis of DNA.The main difference between the two vectors is r-BPMV require in vitro transcription.The gene fragments of conserved sequence of APX was cloned and linked into two BPMV vectors.By artificial friction inoculation,the recombinant vector was transferred into the cotyledon of soybean,the efficiency of gene silencing was analyzed,and the symptoms difference between the gene-silenced and wild type soybean plants inoculated with SMV was analysed.The results showed that r-BPMV vector was successfully constructed,but no mosaic virus was observed after recombinant vector inoculated into the soybean cotyledon.The reason may due to the unstable in vitro transcription of recombinant vector.p-BPMV was cloned,inoculated into two soybean varieties(Nannong 1138-2 and ZheA8901)leaves and showed mosaic symptoms,the expression of CP gene of SMV and APX gene in gene-silenced plants was detect by qRT-PCR.The results showed that the expression of APX in gene-silenced plants was lower than that in BPMV empty vector(positive control)and PBS buffer(negative control),indicated that the endogenous gene was silenced.There was no significant difference in symptoms between gene-silenced plants and positive control after SMV inoculated in two soybean varieties.The qRT-PCR and ELISA analysis of CP gene expression after virus inoculation showed that CP gene was up-regulated at individual time points.In summary,the results showed that r-BPMV vector could not effectively silence endogenous genes because of its unstable transcription in vitro,and p-BPMV silencing vector had certain effect on silencing endogenous genes.APX did not promote mosaic symptoms on soybean leaves induced by SMV.