烟草脆裂病毒介导的茶树VIGS体系的构建  

Construction of Virus-Mediated Genetic Transformation System of Camellia sinensis

作　　者：胡锦瑜 刘桂芝 陈兰 黄梦迪 苏芹 谭月萍 刘硕谦[1,2] 田娜[1,2] HU Jinyu;LIU Guizhi;CHEN Lan;HUANG Mengdi;SU Qin;TAN Yueping;LIU Shuoqian;TIAN Na(Key Laboratory of Tea Science of Ministry of Education,College of Horticulture,Hunan Agricultural University,Changsha 410128,China;Department of Tea Science,College of Horticulture,Hunan Agricultural University,Changsha 410128,China;Hunan Tea Group Co.,Ltd.,Changsha 410126,China)

机构地区：[1]湖南农业大学园艺学院茶学教育部重点实验室,长沙410128 [2]湖南农业大学园艺学院茶学系,长沙410128 [3]湖南省茶业集团股份有限公司,长沙410126

出　　处：《园艺学报》2024年第11期2710-2724,共15页Acta Horticulturae Sinica

基　　金：湖南省种业创新项目(2021NK1008);国家自然科学基金项目(U22A20500,32172629);全国茶树育种联合攻关项目(GJCSYZLHGG-12)。

摘　　要：以茶树(Camellia sinensis)八氢番茄红素脱氢酶(phytoene desaturase)基因CsPDS和咖啡碱合成酶1(caffeine synthase 1)基因TCS1为指示基因,本氏烟草(Nicotiana benthamiana)为载体增殖积累烟草脆裂病毒,探究侵染液浓度、培养温度、不同载体和培养时间对增殖病毒的影响。并探究不同接种方式和不同茶树品种对构建VIGS(Virus-Induced Gene Silencing)体系的影响。结果显示,用乙酰丁香酮(Acetosyringone,AS)浓度为200μmol·L^(-1)、菌液浓度OD_(600)=1.0的侵染液接种烟草后,23℃培养有利于病毒在烟草中增殖。使用‘舒茶早’的叶片通过注射接种构建茶树的VIGS体系并成功利用该体系进行以GFP为指示基因探究外源基因表达的可能性。In this study,Camellia sinensis phytoene desaturase gene(CsPDS)and tea caffeine synthase gene(TCS1)as indicator genes and Nicotiana benthamiana as a vector were employed to enrich tobacco rattle virus.The effects of the concentration of infiltration solution,incubation temperature,different vectors and incubation time on the enriched viruses were investigated,with N.benthamiana used as the vector to enrich the viruses.The effects of different inoculation methods and cultivars on the construction of a virus-induced gene silencing(VIGS)system were also investigated in tea plant.The results demonstrated that inoculation of tobacco with acetosyringone(AS)at a concentration of 200μmol·L^(-1)bacteriophage with an optical density(OD_(600))of 1.0 was conducive to virus enrichment in tobacco when incubated at 23℃.The leaves of‘Shuchazao’were used to construct the VIGS system of tea plant by injection inoculation,and the system was successfully used to explore the possibility of foreign gene expression with GFP as the indicator gene.

关 键 词：茶树 病毒诱导的基因沉默 烟草脆裂病毒 八氢番茄红素脱氢酶基因 咖啡碱合成酶基因 绿色荧光蛋白基因 

分 类 号：S571.1[农业科学—茶叶生产加工]