【作者】 陈中义；

【导师】 黄大昉； Didier Lereclus；

【作者基本信息】 中国农业科学院 ， 植物病理学， 2002， 博士

【摘要】 本论文针对小菜蛾、甜菜夜蛾、水稻二化螟等重要的农业害虫，以高效Bt C002菌株、可应用于植物病害生物防治的荧光假单胞菌P303、枯草芽孢杆菌B916和棉花叶面天然分离株B918等为试验材料，系统开展了多功能载体构建、Bt 质粒复制区与杀虫蛋白基因克隆、组合基因工程菌研制和Mini-Tn10微转座子转座研究等多项研究，主要研究结果总结如下： 1 构建了两个新的质粒载体pSpcα-lac和pSpcPα-lac。无选择压条件下连续培养50小时穿梭载体pSpcPα-lac在P303中稳定性为100％，与假单胞菌穿梭质粒pJMS6α-lac亲和稳定性不低于99.94％／代。生物安全广宿主表达的壮观霉素抗性标记、pBlueScript KS(-)多克隆位点与α互补功能赋予了两种质粒多功能载体的优点，为基因克隆、测序、细菌复制区分离和复制机制研究的提供了易于操作的载体工具。 2 利用载体pSpcPα-lac和枯草芽孢杆菌168研究系统，从Bt C002菌株克隆了一个6909 bps的新的质粒复制区片段，GenBank注册为AY126018。序列分析表明克隆DNA至少编码ORF145、ORF190和ORF200三个蛋白。ORF190为新的蛋白质，仅与细菌重组酶／整合酶有40％以下的一致性：ORF145和ORF200与Bt复制蛋白Rep9741两端保守核心序列有97％一致性，但大小不同。37℃连续培养55.5h复制区质粒在168菌中稳定性达98％，为建立拥有自主知识产权的Bt载体系统和进一步构建高效稳定工程菌株创造了条件。 3 以pSpcP α-lac为载体构建了携带Bt crylAb基因的重组表达质粒pZY311和pZY313，分别与携带crylAc和cry2Aa双价基因的pJMS6α-lac衍生质粒共转化荧光假单胞菌P303，获得了三价Bt荧光假单胞菌工程菌BioP6和BioP8。连续培养和连续稀释培养72小时，BioP6和BioP8中两种质粒亲和稳定性分别为83.5％、89.1％和80.0％、84.8％。 4 室内生测表明BioP6对小菜蛾杀虫活性与双价工程菌BioP202相当，BioP8则明显高于BioP202。2000～2001连续两年田间试验显示BioP8的10倍稀释液96h对小菜蛾田间虫口减退率分别为98.3％和94.8％，100倍稀释液虫口减退率为84.8％，BioP8田间防效与市售Bt制剂相当(86.1％)。越冬前后对2000年试验地土壤样品追踪检测没有发现工程菌株的残留与扩散，目前BioP8已获准进入环境释放试验研究。利用不同亲和群质粒稳定共存的特点研制的三价基因荧光假单胞菌工 中国农业科学院博士学位论文 中文摘要 程菌与国外转单价基因工程菌株混配策略明显不同，显示了本研究的特色，进一步 促进了我国异源细菌转Bt杀虫蛋白基因微生物杀虫剂研究的产业化进程。 5 以穿梭载体pHT315为载体自 Bt C002菌株分离克隆了 Cry］Cas和 CryZAb3基 因，Genbank注册为 AF362020和 AF164666，并获 Bt cry基因国际命名委员会正 式命名。进一步构建了一系列含不同Cry基因或基因组合的大肠杆菌一枯草芽胞杆 菌穿棱和染色体整合重组质粒。遗传转化枯草芽抱秆菌168，获得了BS－IAb。 Bs．IAb．ZAa、Bs．ZAa、BS．IAc和 Bs－IAc－ZAa单价或双价穿梭质粒工程菌株和 BDs．IAbZAa、BDs．IAb、BDs．ICa和 BDs．ICa．ZAa染色体整合工程菌株。 6 室内生测结果显示含CryjAb基因的工程菌对二化螟、亚洲玉米螟和欧洲玉米 螟活性最强，含 CIy］Cas基因的工程菌株对二化螟、棉贪夜蛾具有高毒力；双价基 因 cry1Ac＋cryZAa和 cry1Ab＋clyZAal程菌对供试害虫没有明显的协同增效或抑 制作用，而cry］Cas化 双价基因工程菌株对甜菜夜蛾和棉贪夜蛾杀虫活性明 显高于CryjCas单基因工程菌，校正死亡率分别为70％、37．5％和 100％、50％， 可能存在增效作用。 7 以水稻纹枯病菌生防菌Bgl6为受体，构建了单价和双价杀虫防病工程菌株 Bgl6—IAb和 Bgl6—IAc－ZAa。研究表明工程菌4天对水稻二化螟初孵幼虫校正 死亡率为 100％，7天对亚洲玉米螟和棉铃虫初孵幼虫体重抑制率分别为 97石7％和 98％。连续培养 42小时工程菌株稳定性高于 79．5％，并保持了出发菌株对水稻纹 枯病菌良好的抑菌活性，显示了杀虫防病枯草芽抱杆菌工程菌株的良好应用前景。 吕 研究发现枯草芽抱秆菌Bgls不仅能形成结实的生物膜（Biofilms），而且具有 类似子实体特征的立体结构。遗传转化研究表明电击转化适用于Bgl6和B918，转 化效率为 10＼fu／PgDNA。微转座于质粒 pHV1249转化 Bgls，构建了 Bgls微转 座子插入突变库，诱导转座效率为 2．66X 10＼ 为进一步分离克隆 Bgls抗酉相关 基因（簇）和阐明类子实体结构形成的信号传导与分化发育机制奠定了基础。更多还原

【Abstract】 Aiming at biocontrol of important agricultural insect pests such as Plutella xylostella, Spodotera exigua and Chilo suppresalis, the commercialized biocontrol bacteria Pseudomonas flurescens P303, Bacillus subtilis B916, a natural isolates B. subtilis B918 from cotton leaves and Bt C002 with high insecticidal toxicity were selected and the construction of versatile plasmid vectors, cloning of Bt cry genes and plasmid replicon, transposition of mini-TnlO transposon and development of genetically modified P. flurescens and B. subtilis were systematically studied. The major research results are summarized as follows.1 Novel E. coli and E. coli-Pseudomonas shuttle plasmid vectors pSpca-/ac and pSpcPoc-/flc were constructed. After continuous culturing for 5 Oh without antibiotic selecting press, the stability of P. flurescens P303 carrying pSpcPa-lac was 100% stability. No deletion, rearrangement and a -complementary function altering could be detected in pSpcPa -lac. Research proved that plasmid pSpcPot -lac and pJMS6a -lac are well compatible with a 99.94% co-existing stability per generation in P303o The biosafety and expressing in different bacteria of spectinomycin resistant gene, the Multi-Cloning-Sites (MCS) and the a-complementation function of plasmid pBlueScript KS(-) contributed to two versatile plasmids, which could be useful and easily manipulating for gene cloning, sequencing and isolation of bacterial plasmid replicon and research on replicating mechanism,2 With plasmid pSpcPa-/ac and B. subtilis 168 research system, a 6909 bps DNA fragment carrying a novel plasmid replicon was isolated from Bt C002 and registered in GenBank as AY1260180 Sequence analysis showed that there are at least three ORF (Open Reading Frame) in the cloned DNA, i.e. ORF145> ORF190 and ORF200 respectively. ORF 190 is a novel protein which only has less than 40% identities with Integrase/recombinase family from bacteria; Both ORF 145 and ORF200 have 97% identities with the N-and C-conserve core sequence of Bt Replicating related protein Rep9741, but with different molecular weighto After continuous culturing for 55.5h at 37℃ without antibiotic selecting press, the stability of plasmid carrying cloned replicon in B. subtilis 168 was 98%. This work laid a foundation for further construction of stable vectors system and genetically modified Bt strains.3 Recombinant shuttle plasmids pZY311 and pZY313 were constructed by inserting full length Bt crylAb gene in the MCS of pSpcP a-lac. Plasmids pZY311 and pZY313 with pJBT2008 a derivative of pJMS6cc-/ac carried crylA and cry2Aa were co-transferred into P. flurescens P303 respectively, the engineered strains with three Bt cry genes were designed as BioP6 and BioPS . After continuous and successive diluting culture for 72h, the plasmids compatible stabilities in BioP6 and BioPS were 83.5%, 89.1% and 80.0%, 84.8%, respectively.4 Results of lab bioassay showed that BioP6 and BioP202(P303 with pJBT2008) have the same insecticidal activities against P. xylostella, while BioPS is much more toxic than BioP202. In year 2000 and 2001, BioPS was tested in the fields for biocontrol of P. xylostella and the larvae decrease rate ( LDR ) after treating 96h using its 10 times diluting ferment were 98.3% and 94.8%, and the LDR for 100 times diluting ferment was 84.8% which is equal to commercialized Bt product (86.1%). No remnants and diffuse could be detected in the soil samples from tested field in year 2000. BioPS has been granted to release in field trial. It is just the characteristic of this work that using the compatibility between plasmids from different computable groups to construct genetically modified bacteria carrying three different Bt cry genes, which is quite different from the strategy combining different modified bacteria carrying one cry gene, e.g. the Cellcap system. It greatly promoted the commercializing proceeding of inter-genus recombinant bacteria by transferring Bt cry genes.5 Bt crylCaS and cry2Ab3 gene were cloned from the strain C002更多还原