Functional Verification Of The Soybean Antiviral Gene GmNH23 Via TasiRNA-mediated Silencing System

Plants are capable of triggering defense responses to prevent pathogen invasion by using biological,chemical,and physical systemic defenses.The biological systemic defense relies on a class of important gene called R(Resistance)gene,which encodes an R protein that specifically recognizes a specific pathogen to stimulate the plant's defense response.R gene can be divided into several classes,among which the NBSLRR class is one of the largest gene families in the plant genome,and the TIR-NBSLRR class of disease resistance gene is the major type,such as tobacco N gene and Arabidopsis RPS4 gene,etc.Soybean mosaic virus(SMV)is a serious disease of soybean,but the research on resistance genes toward SMV is still lagging behind.The soybean GmNH23 gene belongs to the TIR-NBS-LRR class and it is homologous to the tobacco N gene which is resistant to tobacco mosaic virus(TMV).In the previous study,our laboratory has demonstrated that GmNH23 gene were resistant to both TMV and SMV by using transient overexpression system in N.benthamianah.However,the antiviral activity of the GmNH23 gene in stable overexpressing plants and its antiviral function in soybeans have not been studied.Therefore,we conducted in-depth research on the function of the GmNH23 gene in this thesis from two aspects.First,we obtained GmNH23 overexpressing(GmNH23-Ox)transgenic N.benthamiana and verified that it is resistant to both TMV and SMV,further confirming the resistance of the GmNH23 gene to these two viruses.Trans-acting siRNA(tasiRNA)is a class of small interfering RNA(siRNA)whose biogenesis is induced by specific MicroRNA and it represses target gene expression through post-transcriptional gene silencing.Therefore,the antiviral function of GmNH23 against SMV and TMV was verified by tasiRNA interfering with the expression of the GmNH23 gene in soybean.The binary silencing vector p KGWRRtasiRNA-GmNH23 was constructed by using the target sequence of miR1514 a which produces endogenous tasiRNA.First,the silencing effect of tasiRNA-GmNH23 on the target gene was verified by transient expression in 17 wt N.benthamiana and rdr6 silence N.benthamiana.Then,tasiRNA-GmNH23 was transiently expressed in GmNH23-Ox N.benthamiana,medium-resistant soybean strain Hefeng No.55,highlyresistant soybean strain Kefeng No.1,and the resistance activity of GmNH23 was confirmed by combining with virus infection experiments.Agrobacterium rhizogenes-mediated hairy root transformation is widely used in legume species.As the siRNA can be transported from the roots to the leaf tissue of plants through the phloem vascular tissue,this study used hairy root transformation to express tasiRNA-GmNH23 in the roots of soybean.The tasiRNA-GmNH23 expressed in the root tissue of the highly-resistant soybean strain Kefeng No.1 not only silenced the expression of GmNH23 gene in root tissues,but also decreased the GmNH23 mRNA level in leaves.Silencing of the GmNH23 gene resulted in an increase in the accumulation of SMV virus in the leaves,and therefore confirmed that GmNH23 was resistant to SMV in soybean.