【作者】 朱军；

【导师】 李平；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)是目前世界上应用范围最广的生防微生物,它在农作物、园艺植物、森林以及卫生害虫的防治方面得到广‘泛的应用。由于Bt杀虫基因的巨大应用价值,世界许多国家都将Bt新资源的收集、鉴定与开发作为重点,开展了大规模资源采集、分析的基础性工作。深入发掘我国丰富的Bt菌株和基因资源,筛选高毒力及特异性菌株,分离克隆新型杀虫基因,其结果不但可以为生产高效的Bt菌制剂和生产菌株的轮换提供后备菌株,也可以为构建高效广谱工程微生物和培育转基因抗虫植物带来基因资源。本研究立足具有独特生态环境和丰富生物多样性的四川盆地,对其Bt资源进行更为系统的调查研究,结果如下：1.采集四川盆地具有代表性的生态区土壤样品3834份,利用醋酸钠-抗生素法分离Bt菌1172株,平均分离率为13.9%。菌株的伴胞晶体有长(短)菱形、大(小)菱形、方形、球形和不定形等7种形状,充分显示了四川盆地Bt资源的多样性。采用PCR-RFLP鉴定方法对1172株菌株进行了cry和cyt基因的鉴定分析,共发现了cry1、cry2、cry3、cry9、cry8、cry4/10、cry30、cry40和cyt2等9种基因型；cryl和cry2型基因最丰富,分别占64%和39.4%; cry3、cry4/10、cry9、cry30和cyt2型含量较少;cry8和cry40类基因含量最少。对未鉴定出基因型的Bt菌株杀虫晶体蛋白进行了SDS-PAGE分析,结果表明这些菌株杀虫晶体蛋白类型存在多样性,可以推测这些菌株极有可能含不同的基因及基因组合。在PCR-RFLP鉴定过程中,发现菌株JF19-2、BtMc28、Hs18-1、Ywc2-8和Bm59-2含有新型模式cry基因。另外,本研究对部分Bt菌株进行了杀虫活性的测定,发现它们具有较宽的杀虫谱,对鳞翅目及双翅目害虫具有很好的杀虫活性。以上结果表明：四川生态区蕴藏着丰富而特别的Bt资源,为害虫的防治提供了基因及菌株资源。2.与Cryl类蛋白相比,Cry2A和Cyt类蛋白具有独特的结构和致病机制；另外,Cry2A和Cyt分别兼具较广的杀虫谱和对Cry类蛋白的协同作用等特性。因此本节首先对含cry2类基因的菌株的杀虫晶体蛋白进行分析,同时在鉴定四川盆地cry2和cyt2类基因基础之上,进一步对其基因的分布、基因的分型、以及新型基因的克隆和表达进行研究,结果表明：1)含cry2型基因的462个菌株主要分布在农田和森林之中,它们产生不规则形、棱形、方形和球状等4种伴胞晶体；通过SDS-PAGE分析可以看出,这些菌株主要表达4种不同的蛋白质,分子量为60—130 kDa之间；这些结果充分说明了四川盆地此类Bt菌株伴胞晶体蛋白具有多样性。2)PCR-RFLP分析表明,四川盆地含有3种不同的cry2基因类型：cry2Aa、cry2Ab和一个新型的cry2A类型(JF19-2菌株之中)；3)本地区仅在森林土壤之中发现11个含cyt2类基因的Bt菌,通过PCR-RFLP鉴定表明,这些基因属于cyt2Aa基因类型;4)以JF19-2及BtMc28(含cyt2Aa基因)为出发菌,采用Tail-PCR和PCR技术分别获得新型的cry2A类和cyt2Aa基因的全长序列,并被国际Bt杀虫晶体蛋白基因命名委员会正式命名为cry2Ag1和cyt2Aa3基因；5) cry2Ag1和cyt2Aa3基因在大肠杆菌中得到了表达,其中cry2Ag1基因表达产物对小菜蛾,棉铃虫及伊蚊都具有较好的杀虫活性,因此Cry2Ag1蛋白可以做为一个轮换或替代蛋白应用于双翅目、鳞翅目害虫的控制以及在转基因植物等领域；而cyt2Aa3的基因表达产物仅对伊蚊具有杀虫活性。本节研究明确了四川盆地cry2和cyt2类基因的分布、类型,同时发现了两个最有较好杀虫活性的杀虫晶蛋白,其研究成果在农业生产上具有重要意义和应用前景。3.含有新型的杀虫基因的BtMc28、Bm59-2、Hs18-1和Ywc2-8分别来自沐川原始森林、碧峰峡自然保护区、海螺沟冰川原始森林。本节对这4株Bt菌进行了生物学特性鉴定、新型模式基因克隆与表达研究。1)通过扫描电镜观察,这些菌株均产生球状晶体；2) SDS-PAGE分析结果显示,BtMc28主要表达3种分子量约为130、70和28 kDa的蛋白质,Ywc2-8与Hs18-1菌株均主要表达分子量为130和70 kDa的2种蛋白质,而Bm59-2只产生一种约70kDa的蛋白质;3)这4个菌株的质粒图谱在带型和大小上都存在很大差异(Hs18-1与Ywc2-8除外),这们的质粒图谱都与对照菌不一样;4)这4个菌株的鞭毛生长情况、生理生化特性和碳源利用,同所报道的Bt菌类似;H血清型鉴定结果显示,BtMc28、HS18-1和Ywc2-8菌株为新的血清型亚种,命名为：Bt serovar sichuansis, Bt serovar muchuansis, Bt serovar yaansis;Bm59-2属于血清型为H22的B.thuringiensis serovar shandongiensis亚种。5)运用SON-PCR. Tail-PCR以及PCR技术分别从BtMc28、Hs18-1、Ywc2-8和Bm59-2中克隆了11个新型基因(其中8个为新型模式基因),并获正式命名；这几个菌株在杀虫基因组成上存在差异(BtMc28含cry54Aa1、cry53Ab1、cry30Fa1、cry4Cc1、cyt2Aa3、Hs18-1含cry54Ba类,cry30Ga1、cry4Cb1;Ywc2-8含cry56Aa1、cry4Cb2、cry30Ea2、Bm59-2含cry52Ba1); 6) cry56Aa1、cry54Aa、cry52Ba1、cry30Fa1、cry30Ga1和cry4Cb1基因在大肠杆菌中得到了表达；生测结果表明,Cry56Aa1、Cry54Aa1、Cry52Ba1、Cry30Fa1和Cry30Ga1具有较宽的杀虫谱,它们对鳞翅目及双翅目害虫具有杀虫活性,其中Cry56Aa1的杀虫谱最宽,它对小菜蛾、棉铃虫和伊蚊都具很好的活性。综上所述,通过对这4个菌株的生物学特性、杀虫蛋白基因的分析和表达研究,其研究结果可以为进一步利用这几株菌,提供一定技术支持和理论依据；新菌株、新型杀虫蛋白基因的分离和克隆在降低害虫抗性风险、提升我国的害虫生物防治和转基因抗虫植物在国际上的竞争力等方面,具有重要的意义。更多还原

【Abstract】 Bacillus thuringiensis (Bt) is the most widely used insecticidal microorganism, which has been successfully used to control the pests in the areas of crop, horticultural crop, forest, and sanitation. Therefore, many countries were interesting to dig the resources of Bt strains and insecticidal crystal proteins. To further dig abundant resource of Bt in our country, screen high toxicity and specific strains, isolate and clone novel pesticidal genes will have important meanings in developing microbial insecticides, constructing high toxicity and broad-spectrum engineering strains and breeding insect resistant transgenic plants. This study described a systematic study of Bt resources in different ecological regions in Sichuan Basin. The concrete results are as follows:1. Sichuan Basin situated in the west of China, the fourth-largest basin of China, is a special area with complicated geomorphology (mountain, pasture, gorge, virgin forest, highland, hurst, glacier, and plain), and contains a rich and unique biodiversity. In total,8418 aerobic sporeformers strains have been isolated from 3834 soil samples collected from different typically ecological regions in Sichuan Basin.13.9% of the isolates were identified as Bt basing on the production of parasporal crystals. A total of 1172 Bt strains have been screened from 3834 soil samples. These Bt strains produced bipyramidal, square, round, and irregular crystal inclusions observed under scanning electron microscopy, indicating the diversity of Bt strains harboured in Sichuan Basin. The analysis of the cry and cyt genes was based on the method of PCR-restriction fragment length polymorphism (PCR-RFLP). cry1, cry2, cry3, cry4/10, cry9, cry8, cry30, cry40, and cyt2-type genes were found in this basin. Strains containing cry1 genes were the most abundant in our collection (66%). Bt strains harboring cry2 genes were the second most abundant (39.4%), and the strains containing cry3, cry4/10, cry9, cry30, and cyt2 genes were found in 1.8,4.1,4.2,3.2, and 1.2%, respectively. The cry8 and cry40 were the least in our collection. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) assay of 80 strains which did not produce any PCR products indicated that these strains may harbor potentially novel Cry proteins. Further more, several novel holotype cry genes were found in these strains (JF19-2, BtMc28, Hs18-1, Ywc2-8, and Bm59-2) by the PCR-RFLP and sequencing analysis. In addition, the results of insecticidal activity of some Bt strains showed that strains exhibited a wide range of insecticidal spectrum toxic to dipteran and lepidopteran pests. All these researches mentioned above revealed that the diversity and particularity of gene resources from Bt in Sichuan Basin.2. Cry2-type proteins were different from Cry 1-type not only in structure, but also in pesticidal mechanism. In addition, Cry2 and Cyt protein have the feature of wide range of insecticidal spectrum and enhancing the toxin activity of Cry protein, respectively. First of all, we studied the insecticidal crystal proteins of Bt strains which harbored cry2-type genes. Base on the results of identification of cry 2 and cyt2 gene of all collections in Sichuan Basin, we further analyzed the distribution and gene types of cry2 and cyt2. In addition, two novel pesticidal protein genes were cloned and expressed. The results are as follows:1) 462 Bt strains harbored cry2-type genes, which produced erose, spherical, bipyramidal, and square crystal. The SDS-PAGE analysis of their spore-crystal suspensions revealed that there were four different protein profiles, which had one or two major protein bands with the molecular weights ranged from about 60 to 130 kDa. All these researches mentioned above revealed the diversity and particularity of cry2-type gene resources from Bt strains in Sichuan basin.2) The result of PCR-RFLP showed that Sichuan Basin has three different cry2A-type genes, such as cry2Aa, cry2Ab, a novel cry2A-type gene:the combination of cry2Aa/cry2Ab genes was the most frequent (75.5%), followed by cry2Aa (21.2%) and cry2Ab (3.3%) alone, and one novel type of cry2 gene was cloned from one isolate (JF19-2).3) Only 11 Bt strains contained cyt genes, which was mainly distributed in the soil of forest, and the results of PCR-RFLP indicated that these cyt genes belong to cyt2Aa; 4) The full-length of this novel cry 2A-type gene from JF19-2 and cyt2Aa from BtMc28 was obtained by the method of thermal asymmetric interlaced PCR (Tail-PCR) and PCR, respectively, which were designated as cry2Agl and cyt2Aa3 by the Bt Pesticide Crystal Protein Nomenclature Committee, respectively.5) cry2Ag1 was expressed in E. coli BL21(DE3)pLysS cells, and the results of insecticidal activity assays showed that Cry2Ag1 was toxic to both Dipteran (Aedes aegypti) and Lepidopteran (Plutella xylostella and Helicoverpa armigera) pests. These results strongly suggest that the cloning of the cry2Ag1 gene is not only of interest to researchers studying insecticidal crystal genes, but may ultimately serve as a solution to the increasing resistance of pests to currently used insecticides. However, Cyt2Aa3 was only toxic to Aedes aegypti. These researches are useful for understanding the distribution of cry2-type genes, the features of Bt strains containing cry2-type genes in Sichuan basin, and the insecticidal of the two novel proteins, which may have important meanings in theories and practices.3. Four Bt strains (BtMc28, Bm59-2, Hs18-1 and Ywc2-8) harbored holotype cry genes were isolated from Mu Chuan virgin forest, Bi Feng Gorge natural protection area, and Hai Luo Glacier virgin forest, respectively. In this part, the biological characteristics of these four strains were identified, and the holotype cry genes from these strains were also cloned and successfully expressed in E. coli BL21 (DE3) pLysS cells. All the results are as follows:1) These four strains produced round crystal inclusions observed under scanning electron microscopy; 2) the results of SDS-PAGE analysis revealed that BtMc28 had three major proteins of about 28,70 and 130 kDa, Hsl8-1 and Ywc2-8 both had two major proteins of about 70 and 130 kDa, and Bm59-2 only had about 70 kDa protein.3) The plasmid patterns of these four strains were different from the reference Bt, and the plasmid patterns of Hs 18-1 and Ywc2-8 were similar to each other.4) the results of the identification of flagellum, the physiology and biochemistry test, and the Biolog test of these four strains conformed to the reference Bt strains, but the result of serotype test showed that H antigen of BtMc28, Hs18-1, and Ywc2-8 did not have agglutination reaction with antiserum of reference Bt strains, which indicated that BtMc28, HS18-1, and Ywc2-8 are new H serotypes. In addition, serotype test also showed that Bm59-2 was B. thuringiensis serovar shandongiensis.5) By the methods of SON-PCR, Tail-PCR, and PCR, the full length of 11 novel cry genes were obtained, which were designated as cry56Aa1, cry54Aa, cry54Ba-type, cry53Ab1, cry52Ba1, cry30Fa1, cry30Ea2, cry30Ga1, cry4Cb1, cry4Cb2, and cry4Cc1, respectively.6) cry56Aa1, cty54Aa1, cry52Ba1, cry30Fa1, cry30Ga1, and cry4Cb1 were expressed in E. coli BL21(DE3)pLysS cells. The results of insecticidal activity assays showed that Cry56Aa1, Cry54Aa, Cry52Ba1, Cry30Fa1, and Cry30Ga1 have a wide range of insecticidal spectrum toxic to dipteran and lepidopteran pests. Especially, Cry56Aa1 has the widest insecticidal spectrum, which was toxic to Aedes aegypti, Plutella xylostella, and Helicoverpa armigera. In conclusion, the studying of the biological characteristics of these strains, the analyzing of insecticidal crystal protein genes, and the expression of the holotype cry genes will have important meanings in theories and practices for further using these strains. Our results strongly suggest that the gene is not only a novel resource in the research of insecticidal crystal genes, but it may also serve as an alternative toxin for potential problems associated with insect resistance. The isolation of new Bt strains and toxins continues to benefit the further development of Cry proteins as competitive biological insecticides and breed insect resistant transgenic plants.更多还原