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1、河北大学 硕士学位论文 大茂山苏云金芽孢杆菌的分离、鉴定及杀虫基因的克隆 姓名：宋健 申请学位级别：硕士 专业：农业昆虫与害虫防治 指导教师：冯书亮 2011-06 摘 要 I 摘 要 苏云金芽孢杆菌（Bacillus thuringiensis，Bt）是目前世界范围内应用最广泛，的一 种生防微生物，对人畜及非靶标昆虫无害，具有巨大的经济价值和生态价值。为了克服 害虫抗性发生、Bt 杀虫图谱窄等问题，筛选具有高效杀虫活性的新型 Bt 基因就具有了 非常重要的现实意义。 本研究选取河北省大茂山地区为土壤样品采集地区， 以地表以下 5 公分的腐殖土作 为采集对象，根据不同海拔高度，共采集土壤样品
2、784 份，利用温度-抗生素筛选法分 离得到 76 株分离菌株。显微观察结果表明这些菌株的伴孢晶体包括菱形、圆形、方形、 椭圆形、不规则形等多种形态，充分体现了大茂山地区 Bt 资源的丰富及多样性。 采用 PCR-RFLP 体系对分离获得的 76 株分离菌株进行基因鉴定，结果发现含有 cry1、cry2、cry3、cry8、cry9、cry26、cry1I 和 vip 共 8 种基因型，其中包含有 cry8 类 基因的菌株最多，有 34 株，另外 12 株包含有 cry1 类基因，5 株包含有 cry2 类基因、2 株包含有 cry3 类基因、7 株包含有 cry9 类基因、6 株包含有 cr
3、y26 类基因、11 株包含有 cry1I 类基因，3 株包含有 vip 基因。在基因组合方面，cry8+cry9 基因组合最多，有 7 株，cry3+cry8 基因组合为 1 株，cry1+cry2 基因组合为 5 株，cry8+cry26 基因组合为 6 株。此外有 26 株分离菌株未鉴定出基因型，可能含有未知类型的基因，需要借助其他 方法进一步研究。 SDS-PAGE 蛋白晶体分析结果表明， 包含有 cry1 类基因的菌株表达 130 kDa 大小的 蛋白，同时包含有 cry1 类和 cry2 类基因的菌株表达 130 kDa 和 60 kDa 大小的蛋白，包 含有 cry8 类基因的菌
4、株表达 130 kDa 大小的蛋白，同时包含有 cry8 类和 cry9 类基因的 菌株表达 130 kDa 和 80 kDa 大小的蛋白，同时包含有 cry3 类和 cry8 类的菌株表达 130 kDa 大小的蛋白，未鉴定出基因型的菌株表达蛋白大小多种多样。 测定了 76 株菌株对小菜蛾、棉铃虫、华北大黑鳃金龟、铜绿丽金龟、暗黑鳃金龟 幼虫的生物活性。 结果表明， 10 株对鳞翅目昆虫毒力较高， 4 d 校正死亡率可达到 100%， 4 株对华北大黑鳃金龟毒力较高 14 d 校正死亡率可达 70%以上。 菌株 TDM-19 在对铜绿 丽金龟的生物活性测定中表现出较高活性，14 d 校正死亡
5、率为 100%，其 LC50为 2.4943 108芽胞/ml 菌液，杀虫效果高于对照菌株 HBF-1。该菌株表达 97 kDa、66 kDa、 摘 要 II 45 kDa 的三条条带的蛋白，而并未表达一般对鞘翅目昆虫有活性的 130 kDa 蛋白。使用 PCR-RFLP 体系对其进行鉴定， 在 27 种通用引物范围内未获得预期片段。 利用 Bt cry 基 因的简并引物 OL1/OL5 对其保守序列进行扩增，得到 900 bp 左右基因片段，其测序结 果在基因序列比对中未发现与之具有较高相似性的已公布 cry 基因， 推测含有新型基因， 具有较高研究价值。 从菌株 FB-32 的总 DNA
6、中克隆得到了全长的 cry8Ca3 基因，该基因编码区为 3483 bp，包含 1160 个氨基酸，分子量为 130.32 kDa，等电点为 4.74，为弱酸性氨基酸。提 交 EMBL/GenBank 基因库注册，登录号为 EU625349，并由 Bt 毒蛋白基因国际命名委 员会命名为 cry8Ca3 基因，是第 4 等级新基因。 关键词 苏云金芽孢杆菌 PCR-RFLP 基因克隆 生物活性 Abstract III Abstract Bacillus thuringiensis (Bt) has been widely used in the world as a kind of bio-c
7、ontrol microbial. For harmless to persons, animals and the non-target insects, Bt possess huge economic value and ecological value. In order to resolve a series of problems just like pest resistances occurrence or narrow-spectrum of insecticide, screening of new Bt strains with high insecticidal act
8、ivity become very important. In this research, 76 Bt stains were isolated from total 784 soil samples which were collected from the Damaoshan Mountains of Hebei province by temperature- antibiotics screening method. Microscopic observation results showed that the spore crystal include diamond, spher
9、ical, square, ovoid, irregular shape, et al. The great diversity and richness of Bt strains had been proved in Damaoshan Mountains. In this research, 76 Bt stains were identified by using of PCR-RFLP. The results showed that all the stains contained 8 kinds of genotypes: cry1, cry2, cry3, cry8, cry9
10、, cry26, cry1I and vip, and the cry8 genes were found in 34 strains accounted for the biggest proportion, the cry1 gene were found in 12 strains, the cry2 gene were found in 5 strains, the cry3 gene were found in 2 strains, the cry9 gene were found in 7 strains, the cry26 gene were found in 6 strain
11、s, the cry1I gene were found in 11 strains and the vip gene were found in 3 strains. In the gene combination, 7 strains owned both cry8 and cry9, 5 strains owned both cry1 and cry2, and 6 strains owned cry8 and cry26. Besides, 134 stains which were not identified genetypes, possibly include the unkn
12、own genotypes, and need to draw supports from other methods to more research. SDS-PAGE protein crystals analysis results showed that the strains which contained cry1 genes mostly expressed 130kDa protein; the strains which contain cry1 genes and cry2 genes together expressed 130kDa and 60kDa protein
13、; the strains which contain cry8 genes expressed 130kDa protein; the strains which contain cry8 genes and cry9 genes mostly expressed 130kDa and 80kDa protein; the strains which contain cry3 genes and cry8 genes mostly expressed 130kDa protein. The size of protein which were expressed in the strains
14、 had Abstract IV not identified genotype were diverse. Bioassay of 76 Bt strains against larva of Holotrichia oblira, A.corpulenta, H.parallela, Plutella xylostella and Heliothis armigera was conducted. Results showed that 10 strains had insecticidal activity to lepidoptera insect with the mortality
15、 rate which was 100%, 4 strains were toxic to Holotrichia oblira insect with the mortality rate which was past 70%. The strain TDM-19 had high activity against A.corpulenta, and the 100% mortality was gained after 14 days. The LC50 was 2.4943 108 spores/ml to A.corpulenta, and the insect disinfestat
16、ion effect is higher than standard strain HBF-1. The strain expressed 97kDa, 66kDa, 45kDa protein, but not the 130kDa protein which had high activity against A.corpulenta. The expectations fragments had not been gained by using the general primer to identificate its genotype. The degenerate primers OL1/OL5 were used to amplificate its conservative sequences, and 900 bp genetic fragments were gained. The fragment of 928bp cloned from the strain TDM-19 had no high-comparability with Bt genes kno
