一步法快速构建长片段RNAi发夹的载体  

A one-step method for rapid construction of long-fragment RNA interference vector

作　　者：刘廷利[1] 郭冬姝 姚瑶 张保龙[1] LIU Ting-li;GUO Dong-shu;YAO Yao;ZHANG Bao-long(Excellence and Innovation Center,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China)

机构地区：[1]江苏省农业科学院卓越创新中心,江苏南京210014

出　　处：《江苏农业学报》2021年第5期1131-1136,共6页Jiangsu Journal of Agricultural Sciences

基　　金：江苏省自然科学基金项目(BK20180308);江苏省农业科技自主创新基金项目[CX(18)3012]。

摘　　要：利用RNA干扰(RNA interference,RNAi)技术可以进行基因功能研究以及作物遗传改良,但常规构建RNAi载体方法费时费力。本研究报道了一种基于USER酶一步法快速构建RNAi发夹结构的载体2301/RNAi/OS及其应用方法,操作简便,省时省力。与已报道的利用酶切连接、Gateway兼容的同源重组或PCR直接连接等构建RNAi载体相比,该载体不需要酶切片段,片段扩增完成后即可与制备好的线性化载体进行反应,缩短操作流程和时间,提高成功率;该载体对插入片段的酶切位点和长度没有要求,理论上任何位置和长度的片段均可进入2301/RNAi/OS载体中,尤其是长片段RNAi的构建。本试验以本氏烟(Nicotiana benthamiana)PDS(Phytoene desaturase)基因为例,利用农杆菌介导的瞬时表达体系验证了该RNAi能够在植物体内有效地实现基因沉默。该载体以新霉素磷酸转移酶基因(NPTII)作为筛选标记基因,可以利用卡那霉素(Kanamycin)和G418(Geneticin)等抗生素筛选转基因阳性植株,适合单子叶和双子叶植物的遗传转化。RNA interference(RNAi)technology can be used for gene function research and crop genetic improvement,but the conventional construction method of RNAi vector is time-consuming and laborious.In this research,we provide a one-step method for rapid construction of long-fragment RNAi vector using uracil-specific excision reagent(USER)enzyme.The method is simple,time-saving and labor-saving.Compared with the reported RNAi vectors constructed by restriction enzyme ligation method,Gateway compatible homologous recombination method and PCR direct ligation method,this vector dose not need enzyme digestion fragments.After the fragment amplification is completed,it can react with the prepared lineurized vector.Therefore,the operation process and time are shortened,and the success rate is improved.This vector has no requirements for the restriction site and length of the inserted fragment.Using neomycin phosphotransferase(NPTII)as selective marker gene in this vector,kanamycin and geneticin(G418)can be used to screen transgenic positive plants.It is suitable for genetic transformation of dicot and monocot plants.

关 键 词：RNA干扰(RNAI) USER酶 一步法 载体构建 基因沉默 

分 类 号：Q782[生物学—分子生物学]