【作者】 张睿；

【导师】 孙明；

【作者基本信息】 华中农业大学 ， 微生物学， 2005， 硕士

【摘要】 苏云金芽胞杆菌幕虫亚种(Bacillus thuringiensis subsp. finitimus)能形成两种伴胞晶体。当芽胞形成细胞裂解后,一种晶体被释放出来,形成自由的伴胞晶体,另一种则始终和芽胞粘连在一起。电镜结果显示该晶体在芽胞外壁内侧形成,成熟后被包裹在芽胞外壁以内不脱落。我室分离的对北方根结线虫有毒力的苏云金杆菌020菌株也具有这一特殊现象。脉冲电泳和蛋白质SDS-PAGAE电泳显示020菌株和幕虫亚种标准血清型菌株T02有相同的质粒组成,伴胞晶体蛋白大小亦相同。 苏云金杆菌幕虫亚种不但具有品胞粘连的特殊现象,还具有独特的杀虫谱带。可能是因为芽胞在昆虫肠道内再次萌发,产生了其他致病因子,如肠毒素、溶血素等,与杀虫晶体蛋白协同作用;也可能是因为伴胞晶体受到疏水的芽胞外壁层保护而能在水中等一般伴胞晶体不容易存在的地方被易感昆虫摄食。因此,根据苏云金杆菌晶胞粘连原理构建“生物囊”杀虫制剂具有很高的实际应用价值。将它和其他杀虫晶体蛋白结合起来,不但可以构建广谱高效的杀虫工程菌,还可以利用芽胞保护伴胞晶体,减少辐射、干旱或生产对晶体毒力的影响。此外研究cry基因的起源和调控也是目前国际上的热点领域。 本实验根据已发表的cry26Aal和cry28Aal基因序列设计引物,从苏云金芽胞杆菌020菌株中克隆到cry26Aa和cry28Aa基因,通过穿梭载体将这两个基因分别和同时转化到苏云金芽胞杆菌无晶体突变株BMB171中。透射电镜下观察到cry26和cry28基因同时表达在芽胞外壁内侧和外侧都可形成内含体。而当两个cry基因单独存在时,cry26基因表达合成的内含体位置不确定,大部分在芽胞外壁外侧形成,少数在芽胞外壁内侧形成;而cry28基因则只在芽胞外壁外侧表达合成内含体。同时PAGE胶检测和镜检结果显示两个基因表达的蛋白都不稳定,极易降解。 同时苏云金杆菌020菌株形成的自由伴胞晶体也很不稳定,难以检测。对020菌株总DNA进行PCR扩增,显示该菌株含有S-层蛋白N-端保守区和S-层蛋白基因上游csaAB操纵子区。由苏云金杆菌幕虫亚种CTC菌株的伴胞晶体是由S-层蛋白过量表达累积而成且该伴胞晶体不稳定推测020菌株办含有S-层蛋白。 综上结果显示,苏云金芽胞杆菌幕虫亚种的伴胞晶体组成成分复杂。cry26和cry28基因的共表达有可能导致晶胞粘连的产生,但其稳定性及表达量可能还受到其它因子的影响。更多还原

【Abstract】 Bacillus thuringiensis subsp. finitimus produced at least two parasporal inclusions. One inclusion was formed within the exosporium and remained with the spore after mother cell lysis. A second inclusion formed somewhat later exterior to the exosporium. Bt 020 isolated by our lab also has this phenomenon. And these two strains have the same constitutions of plasmid and parasporal crystal protein based on PFGE and SDS-PAGE.Strains of finitimus not only have the distinguished location of parasporal crystal protein, but also the unique spectrum of susceptible larvae. This may partially attribute to packaging an inclusion within the hydrophobic exosporium so as to keep the inclusion in the water where target larvae maybe actively feeding. Or spore has some synergic effects to the insecticidal protein by releasing some pathogenic factors during the process of germination. So it must have some important and pratical value to construct a "bio-capsule" insecticide by using spore-associated proteins, such as increasing the exposure resistance of insecticidal protein under the UV light or drought. In addition, understanding the origin of cry genes and how their regulation became integrated with sporulation is also the hot spot of research.According to the crystal protein gene sequence in finitimus, cry26 and cry28 were cloned from strain 020 and transformed into the crystal negative Bt strain BMB171 by shuttle vectors, alone or together. Crystals can be observed both inside and outside of the exosporium when co-expression of these two genes. But when they expressed alone, cry26 gene leaded inclusion body formation within or outside of the exosporium, whereas cry28 gene only induced crystal formation outside of exosporium. All of the proteins could not stably exist.On the other hand, the free parasporal protein degraded easily as well. PCRamplification suggested that strain 020 has slh fragment of s-layer gene and csaAB operon.Along with the previous result that strain CTC of finitimus could form parasporal crystals. by s-layer protein, a putative s-layer protein may also exist in strain 020 and has somecooperative effect to spore-associated crystal formation.In above, it suggests cooperation between cry26 and cry28 gene could lead crystal forming inside of the exosporium. But other help proteins maybe also involved in enclosure.更多还原