Establishment Of TRV-mediated Transient Gene-silencing System And Its Application In Soybean

Virus-induced gene silencing(VIGS) technology has been widely applied in the field of functional gene research in plants. Tobacco rattle virus-mediated gene silencing technology was used for gene function evaluation, and the TRV-mediated gene transient silence system was established using the Tobacco rattle virus-mediated gene silencing technology in soybean. And we used the system to conduct the functional analysis of callose synthesis of callose synthase gene(Gm Cal S7) and a salt-related genes(Gm NHX1/Gm NHX5). The main results of the test are as follows:1.The phytoene desaturase(PDS) was selected as a marker gene, and then was used to construct the gene silencing vector p TRV: RNA2: Gm PDS. The soybeans’ true leaves were injected with Agrobacterium suspension( carrying p TRV: RNA1 and p TRV: RNA2: Gm PDS). At 25 d after inculation, the upper leaves showed whitening phenotype and Gm PDS gene was confirmed effencienctly silenced by the semi-quantitative RT-PCR analysis.2.The soybean leaves were injectied with Agrobacterium carrying TRV and then with SMV,We found that the TRV has no influence on the performance of soybean’s resistance to SMV.3.The Gm CalS7 silencing vector TRV2: Gm Cal S7 was constructed. The soybean leaves were injected with Agrobacterium carrying TRV: Gm Cal S7,injected with Agrobacterium carrying TRV as control, 25 days later after injection:1) RT-PCR analysis the second round fronds,GmCalS7 gene was confirmed effencienctly silenced in Gm Cal S7 gene silenced plants. 2) Transgenic plants were inoculated with soybean mosaic virus(SMV) strains N3, after inoculation SMV 96h。 Detection of Transgenic plants callus under a fluorescence microscope. Gm Cal S7 silence plants Callose significantly reduced compared with the control plants.3) When transgenic plants were inoculated with SMV strains N3,the leaves inoculated could be detected the coat protein gene of SMV CP. In uninoculated upper leaves Gm Cal S7 gene silencing plants can be detected a trace amount of CP, but,the plants were not detected CP in the control group. The results showed that after Gm Cal S7 gene was silenced, callose synthesis decreased, and virus N3 spread to upper leaves.4.The gene silencing vector TRV2: Gm NHX1 and TRV2: Gm NHX5 were constructed. culturing in the hydroponic culture,soybean root were inoculated Agrobacterium carrying TRV: Gm NHX1 or TRV2: Gm NHX5,with TRV as controls.,7 days later after injection:1) RT-PCR analysis the first true leaf, Gm NHX1 and GmNHX5 gene were confirmed effencienctly silenced.. 2) Concentration of 200 mmol/L Na Cl were added to the transgenic plants.,1 hour later afer treatment, Gm NHX1 or Gm NHX5 silence plants wilting obviously than the control.In this study, TRV as vector, soybean PDS as an indicator gene, We establishmed the TRV-mediated transient Gene-silencing System, and proved that the system can be used for functional studies of soybean genes.