番茄斑萎病毒N基因的VIGS载体构建

目的研究番茄斑萎病毒(Tomato spotted wilt orthotospovirus，TSWV) 核衣壳蛋白基因(N)对辣椒抗病性的影响。方法以易感TSWV的辣椒湘研11为研究对象，采用同源序列克隆获得辣椒CaPDS基因和TSWV N基因；利用烟草脆裂病毒(Tobacco rattle virus，TRV)和病毒诱导的基因沉默(virus-induced gene silencing，VIGS)技术构建辣椒CaPDS基因沉默载体(pTRV2-CaPDS)和TSWV N基因沉默载体(pTRV2-N)，农杆菌GV3101注射接种辣椒，通过qRT-PCR检测重组载体沉默效率。结果湘研11接种含pTRV2-CaPDS载体菌液3周后，新叶出现白化现象，说明辣椒上的pTRV2-CaPDS沉默载体构建成功。qRT-PCR结果表明：pTRV2-N载体可高效沉默N基因，其表达量仅为6.79%±2.56%，说明沉默N基因后，湘研11不易感染TSWV。结论本研究成功构建了pTRV2-N沉默载体，沉默TSWV的N基因后辣椒对入侵的TSWV产生一定的抗性。

Additional changes totaxonomy ratified in a special vote by the International Committee on Taxonomy of Viruses (October 2018)

PurposeTo explore the impact of Tomato spotted wilt orthotospovirus (TSWV) nucleocapsid protein gene (N) on pepper disease resistance.MethodXiangyan 11, a pepper susceptible to TSWV, was used as the research object. Pepper phytoene desaturase (CaPDS) gene and TSWV N gene were cloned by homologous sequence. The CaPDS gene silencing vector (pTRV2-CaPDS) and TSWV N gene silencing vector (pTRV2-N) of pepper were constructed by using Tobacco rattle virus (TRV) and virus-induced gene silencing (VIGS) technology. Agrobacterium GV3101 was inoculated with pepper. The silencing efficiency of the recombinant vector was detected by qRT-PCR.ResultThree weeks after inoculation with pTRV2-CaPDS, the new leaves appeared albino, which indicated that the construction of pTRV2-CaPDS silencing vector was successful. The qRT-PCR results showed that the pTRV2-N vector can efficiently silence the N gene, and its expression is only 6.79%±2.56%, indicating that after silencing the N gene, Xiangyan 11 is not susceptible to TSWV.ConclusionpTRV2-N silencing vector was successfully constructed. After silencing the TSWV N gene, pepper had certain resistance to invading TSWV.