一种基于TRV-VIGS的高通量大豆基因功能验证方法  

A TRV-VIGS-based Approach for High Throughput Gene Function Verification in Soybean(Glycine max)

作　　者：孙天杰 麻楠 孙立永 王梦璇 孙希哲 张洁 王冬梅 SUN Tian-Jie;MA Nan;SUN Li-Yong;WANG Meng-Xuan;SUN Xi-Zhe;ZHANG Jie;WANG Dong-Mei(State Key Laboratory of North China Crop Improvement and Regulation/Key Laboratory of Hebei Province for Plant Physiology and Molecular Pathology/College of Life Sciences,Hebei Agricultural University,Baoding 071001,China;Collaborative and Innovation Center of Hebei,Shijiazhuang 050000,China)

机构地区：[1]省部共建华北作物改良与调控国家重点实验室/河北农业大学生命科学学院/河北省植物生理与分子病理学重点实验室,保定071001 [2]河北省协同创新中心,石家庄050000

出　　处：《农业生物技术学报》2020年第11期2080-2090,共11页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(30971706,31471421);973计划前期研究专项课题(2014CB160318)。

摘　　要：病毒介导的基因沉默(virus-induced gene silencing,VIGS)是一种快速、高效、便捷的基因功能验证的反向遗传学手段。烟草脆裂病毒(Tobacco rattle virus,TRV)介导的VIGS技术可以在多种植物上实现基因沉默,但其应用却因由侵染操作引起的植物损伤与沉默效率低下受到限制。本研究以大豆(Glycine max)为材料,通过对农杆菌(Agrobacterium tumefaciens)侵染方法进行改良,使TRV从植物根部侵染,随病毒在植物体内的运输,实现对植物内源基因的系统性沉默。实验结果表明,将携带VIGS载体的农杆菌使用本方法从根部侵染大豆后,可以使同一处理批次内近全部植株产生有效基因沉默,基因沉默效率均在67%以上,最高达到98%;对处理植株不同位置的叶片进行检测,发现第1至第3轮三出复叶均有较为理想的沉默效果,其中第1轮三出复叶的沉默效率最高;本研究有效沉默的基因包括非特异性脂质转运蛋白(non-specific lipid-transfer protein,LTP)、病程相关蛋白1(pathogenesis-related protein 1,PR1)、索马甜类蛋白(thaumatin-like protein,TLP)、脱水素类蛋白(dehydrin-like protein,DHN)、木葡聚糖内糖基转移酶(xyloglucan endotransglucosylase,XTH)和富半胱氨酸跨膜蛋白(cysteine-rich transmembrane protein,CYSTM)。本方法简单易行,可以在短时间内获得大量的基因沉默植株,为基因功能的验证建立了高效的实验操作体系。Virus-induced gene silencing(VIGS)is an efficient and convenient reverse genetics method for rapid gene function study.The application of the VIGS is often restricted due to the incompatibility of viruses and plants.Tobacco rattle virus(TRV)-mediated VIGS technology achieves gene silencing on a variety of plant species,but its application is limited by plant damage and low silencing efficiency caused by conventional inoculation operation that applies agroinfiltration in plant leaves.In this study,soybean(Glycine max)was used as a material to validate a modified TRV-mediated VIGS protocol that used Agrobacterium tumefaciens to infect from the root after soil irrigation.The results showed that the A.tumefaciens carrying the VIGS plasmids that infected soybean seedlings implemented effectively silence in all plants tested in the same treatment batch,silencing efficiency of targeted genes were above 67%,and reached 98% of the highest.Further examination on the soybean ternately compound leaves at different positions of the treated plants revealed that the first to the third-round of ternately compound leaves had a satisfactory silencing effects,of which the first-round leaves had the highest silencing efficiency.The genes that were effectively silenced in this experiment included non-specific lipid-transfer protein(LTP),pathogenesis-related protein 1(PR1),thaumatin-like protein(TLP),dehydrin-like protein(DHN),xyloglucan endotransglucosylase(XTH),and cysteine-rich transmembrane protein(CYSTM).The method was proved to be simple and feasible,and a large number of gene silenced plants were obtained in a short period of time.This study established an efficient system for rapid gene function verification in soybean.

关 键 词：病毒介导的基因沉默(VIGS) 烟草脆裂病毒 沉默效率 大豆 系统性沉默 

分 类 号：S184[农业科学—农业基础科学]