Cry1Ac基因载体构建及其在枯草芽孢杆菌中的杀虫活性表达

摘要/Abstract

摘要：

根据苏云金芽孢杆菌Bacillus thuringiensis HD-73基因Cry1Ac和枯草芽孢杆菌Bacillus subtilis木糖诱导型启动子PxylR序列, 分别设计2对特异引物Cry1Ac F/R和Pxy F/R，扩增获得了完整的启动子PxylR和Cry1Ac基因序列，进一步以上述产物混合物为模板，以Pxy F/Cry1Ac R作引物进行重迭PCR，获得了载体PxylR-Cry1Ac，经SphⅠ和BamHⅠ完全酶切后，将PxylR-Cry1Ac插入大肠杆菌-苏云金芽孢杆菌穿梭载体pHT315，重组表达质粒pCry1Ac315转化枯草芽孢杆菌感受态细胞。工程菌株质粒酶切电泳分析、SDS-PAGE电泳分析和杀虫生物活性测定结果证实了Cry1Ac基因的导入及其在枯草芽孢杆菌JAAS01D中的有效表达。

关键词: 苏云金芽孢杆菌, 枯草芽孢杆菌, 重迭PCR, Cry1Ac基因, 杀虫活性

Abstract:

The full length sequence of the promoter and Cry1Ac gene were obtained by PCR with two pairs of unique primers Cry1Ac F/R and Pxy F/R respectively, which were designed according to the Cry1Ac gene and promoter sequence of xylase operon from Bacillus subtilis 168 Then, the fused translational expression vector PxylR-Cry1Ac was constructed using overlapping PCR technique with the primers pair PxyF/Cry1AcR and the mixture of above PCR production. After being digested by SphⅠ and BamHⅠ, PxylR-Cry1Ac expression vector was inserted into E. coli-B. thuringiensis shuttle vector pHT315, and the resulted recombinant plasmids were named as pCry1Ac315 The recombinant plasmids were transferred into B. subtilis laboratory strain JAAS01D. Efficient expression of the Cry1Ac gene in the engineered JAAS01D-1Ac was proved with restriction enzyme analysis, SDS-PAGE electrophoresis analysis and insecticidal activity assay.

Key words: Bacillus thuringiensis, Bacillus subtilis, overlapping PCR, Cry1Acgene, insecticidal activity

刘济宁^1,2，刘贤进¹，余向阳¹，彭正强². Cry1Ac基因载体构建及其在枯草芽孢杆菌中的杀虫活性表达[J]. , 2005, 48(3): 342-346.

LIU Ji-Ning^1,2, LIU Xian-Jin^1*, YU Xiang-Yang¹, PENG Zheng-Qiang². Construction of recombined plasmid carrying Cry1Ac gene and expression of insecticidal activities in Bacillus subtilis [J]. , 2005, 48(3): 342-346.

[1]	李而涛, 曹雅忠, 张帅, 李克斌, 李建一, 王庆雷, 刘春琴, 尹姣. 昆虫病原线虫Heterorhabditis beicherriana LF品系与Bt HBF-18菌株混用对华北大黑鳃金龟幼虫的防治效果[J]. 昆虫学报, 2019, 62(5): 602-614.
[2]	芦小鹏, 吴琼, 李毛龙, 李三兴, 马志卿, 张兴. 126种植物精油或精油主要成分对玉米黄呆蓟马的熏杀活性筛选[J]. , 2018, 61(8): 941-949.
[3]	张敏, 刘佳, 傅伟杰, 代光辉. 姜黄根提取物对苜蓿蚜成蚜有杀虫活性化合物的分离与鉴定[J]. , 2018, 61(6): 698-703.
[4]	赵静茹, 袁守丽, 高斌, 朱顺义. 抑制剂胱氨酸结多肽的抗菌和杀虫活性及进化[J]. , 2018, 61(10): 1212-1221.
[5]	李振, 韩兰芝, 刘玉娣, 侯茂林. Bt杀虫蛋白处理后二化螟幼虫中肠细菌群落的变化[J]. 昆虫学报, 2016, 59(3): 292-300.
[6]	付盈盈, 马玉堃, 任淑文, 杨立军, 刘春光, 付海燕, 侯跃莹, 杨峰山. 革兰氏阳性菌苏云金芽孢杆菌和革兰氏阴性菌大肠杆菌诱导的柞蚕蛹生理指标变化[J]. 昆虫学报, 2016, 59(2): 192-199.
[7]	Ali MEHRVAR. 荧光增白剂对伊朗核型多角体病毒分离株对棉铃虫幼虫的杀虫活性的增效作用（英文）[J]. , 2013, 56(6): 708-714.
[8]	黄继光, 赵欢欢, 苗辉, 徐汉虹. 羽裂蟹甲草对家蝇和白纹伊蚊的杀虫活性和活性成分(英文)[J]. , 2012, 55(12): 1355-1361.
[9]	李有志, 李冠华, 魏孝义, 刘仲华, 徐汉虹. 灰毛豆树皮中的杀虫成分及其杀虫活性（英文）[J]. , 2011, 54(12): 1368-1376.
[10]	邱思鑫，范晓静，洪鹏翔，关雄，胡方平. 苏云金芽胞杆菌营养期杀虫蛋白基因vip3A在枯草芽胞杆菌中的表达[J]. , 2010, 53(5): 508-516.
[11]	王学贵，沈丽淘，田永清，徐汉虹. 岩木瓜化学成分的分离、结构鉴定与杀虫活性[J]. , 2010, 53(5): 525-530.
[12]	李秀梅, 方继朝. 18种中草药提取物对褐飞虱和甜菜夜蛾的杀虫活性分析[J]. , 2010, 53(3): 298-306.
[13]	何玲，赵娟，师宝君，胡兆农，吴文君. 杠柳杀虫组分F3-28对东方粘虫和小地老虎幼虫中肠消化酶活性的影响[J]. , 2010, 53(11): 1248-1255.
[14]	黄素青, 徐汉虹, 童松, 张志祥. Interruptin B对小菜蛾和亚洲玉米螟幼虫的拒食活性及对斜纹夜蛾卵巢细胞的毒力[J]. , 2010, 53(10): 1104-1110.
[15]	张云, 彭映辉, 陈飞飞, 曾冬琴, 黄谊, 钟海雁. 椿叶花椒果实精油对两种蚊虫的生物活性及成分分析[J]. , 2009, 52(9): 1028-1033.

Cry1Ac基因载体构建及其在枯草芽孢杆菌中的杀虫活性表达

Construction of recombined plasmid carrying Cry1Ac gene and expression of insecticidal activities in Bacillus subtilis 

PDF (PC)

赞

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

Metrics

本文评价

推荐阅读 10