促冻杀虫基因工程菌的构建  

A Stably Transgenic INA Enterobacter cloacae forControl of Insect Pests

作　　者：张新建[1,2] 孙福在[1] 赵廷昌[1] 丁爱云[3] 唐朝荣[1] 

机构地区：[1]中国农业科学院植物保护研究所/植物病虫害生物学国家重点实验室 [2]山东农业大学植物保护学院,泰安271018 [3]山东农业大学植物保护学院

出　　处：《中国农业科学》2004年第2期227-232,共6页Scientia Agricultura Sinica

基　　金：国家自然科学基金资助项目(30170624)

摘　　要：利用转座酶基因位于转座单位以外的微转座子载体,将冰核基因iceA置于转座单位里,构建了携带iceA的自杀性工程质粒pTnice1, 并进一步利用该质粒的接合转移特性和Tn5转座功能,将iceA整合到阴沟肠杆菌染色体DNA上,成功地构建了在无选择压力下冰核基因仍稳定存在并有效表达冰核活性的促冻杀虫基因工程菌Enc1.2022ina。经应用试验证明,用该工程菌饲喂玉米螟幼虫,在饲菌后9 d内,虫体的过冷却点比不饲菌的对照提高10℃左右;在饲菌后第6天,于-5℃和-7℃低温下处理12 h,工程菌处理过的幼虫冻死率分别为85%和100%,而对照虫体的冻死率分别为0和5%。试验还证明,该工程菌可以在昆虫体内稳定定殖增生,在植物上附生定殖能力明显比野生型冰核细菌弱。因此,解决了阻碍冰核细菌促冻杀虫应用中的难题,为防治我国北方地区重要农林果树和仓储主要越冬害虫探索出了一条生防新途径。A suicide recombinant plasmid pTnice1 through the mini-transposon pMini-Tn5 with the ability of broad-host-range conjugal mobilization and integration of iceA into chromosomal DNA of many gram-negative bacteria by Tn5 transpositionwas constructed. We used this plasmid to integrate the iceA into chromosomal DNA of Ent. cloacae and got the transgantic strainEnc1.2022ina. In this transgenic Ent. cloacae, iceA will never be transferred elsewhere through transposition, and constantlyexpresses high ice nucleation activity even in the absence of antibiotic pressure. The transgenic strain was ingested by corn borerlarvae. Over the 9d after ingestion, the mean supercooling points (SCPs) of the larvae ingesting the transgenic strain was 10higher than that of larvae treated with distilled water (control). The maintenance of these high SCPs is related to the transgenicstrain having a stable gut colonization. On the 6th day after ingestion, the larva was exposed at -5℃ and -7℃ for 12 h, thepercentage of larvae frozen to death was 85% and 100% respectively, while that of control larvae was 0 and 5% respectively.Further studies demonstrated that this transgenic strain bears weakly plant-epiphytic ability. Therefore, this genetically engineeredstrain may be a promising candidate for control of insect pests in agricultural fields.

关 键 词：促冻杀虫基因工程菌 微转座子载体 冰核基因 生物防治 农业害虫 仓储害虫 越冬害虫 转基因 

分 类 号：S476[农业科学—农业昆虫与害虫防治] S188[农业科学—植物保护]