农杆菌介导法获得辣木PKM-1转双价抗虫基因(sck+Cry1-Ac)愈伤组织  

Obtaining Transgenic-callus of Moringa oleifera PKM-1 with In-sect-resistant Gene(sck+Cry1-Ac)via Agrobacterium-mediated Transformation

作　　者：杨钺戈 李跃 张慧[1] 阮明菊 马丽宣 曾千春[1] YANG Yuege;LI Yue;ZHANG Hui;RUAN Mingju;MA Lixuan;ZENG Qianchun(College of Agronomy and Biotechnology,Yunnan Agricultural University,Kunming 650201,China)

机构地区：[1]云南农业大学农学与生物技术学院,云南昆明650201

出　　处：《云南农业大学学报（自然科学版）》2023年第1期27-33,共7页Journal of Yunnan Agricultural University:Natural Science

基　　金：国家自然科学基金项目(31860414);国家现代农业产业技术体系岗位科学家(CARS-11-YNZQC);云南省院士专家工作站(2019IC007);云南省基础研究面上项目(202101AT070214)。

摘　　要：[目的]辣木生产中鳞翅目害虫辣木瑙螟危害日益严重,为提高辣木抗虫性,使用农杆菌介导法将抗虫基因导入辣木,以期获得抗虫转基因转化子。[方法]利用内质网定位修饰的豇豆胰蛋白酶抑制剂基因(sck)和苏云金芽孢杆菌基因(Cry1-Ac)筛选标记基因nptⅡ;利用农杆菌EHA105介导遗传转化辣木PKM-1的叶片和茎段,以卡那霉素筛选获得抗性植株;采用PCR和NPTⅡImmunoStrip检测确定nptⅡ基因的整合与表达。[结果]辣木叶片和茎段无需预培养可直接进行农杆菌转化,最佳转化条件为:工程菌液OD_(600)值为0.2,侵染时间为2 min,共培养时间为2 d;乙酰丁香酮添加与否对获得抗性愈伤率的影响不明显;200 mg/L提门叮可有效抑制农杆菌生长;卡那霉素抗性愈伤组织PCR检测表明:标记基因nptⅡ已成功整合到辣木PKM-1基因组中;NPTⅡImmunoStrip检测表明NPTⅡ蛋白水平得以表达。[结论]获得了辣木PKM-1卡那霉素抗性愈伤组织,为辣木抗虫分子育种提供了材料。[Purpose]The Lepidopteran pest,Noorda blitealis,has become increasingly harmful in the production of Moringa oleifera.In order to improve the insect resistance of M.oleifera,the present study was performed to introduce insect-resistant genes into M.oleifera by Agrobacterium tumefaciens-mediated transformation method,so as to obtain transformants.[Methods]The marker gene nptⅡwas screened by using the modified cowpea trypsin inhibitor-derived gene signal-CpTI-KDEL(sck)and Cry1-Ac gene from Bacillus thuringiensis,which used by endoplasmic reticulum localization.The leaves and stems of M.oleifera PKM-1 were subject to genetic transformation mediated by EHA105.Resistant plants were obtained by kanamycin-resistant selection.Meanwhile,PCR and NPTⅡ ImmunoStrip detection were used to confirm the integration and expression of the nptⅡ gene. [Result]The leaves and stems of M. oleifera could be directly transformed through mediation us-ing A. tumefaciens without pre-culture, the optimal transformation condition was: the OD_(600) value of the fermentation broth of genetically engineered bacteria was 0.2, with two minutes infection time and two days appropriate co-culture time. The addition of acetosyringone or not had no obvious effect on the resistant callus rate, and the use of 200 mg/L Timentin could effectively inhibit the growth of A. tumefaciens. PCR detection of kanamycin-resistant callus showed that the marker gene nptⅡ had been successfully integrated into the genome of M. oleifera PKM-1;the NPTⅡ ImmunoStrip test showed that NPTⅡ protein was expressed. [Conclusions]M. oleifera PKM-1 kanamycin-resistant callus is obtained in this study, which provides reference material for the molecular breeding of M. oleifera.

关 键 词：辣木 抗虫转基因 农杆菌 愈伤组织 

分 类 号：S794[农业科学—林木遗传育种]