苏云金芽孢杆菌的cry2A芽孢期启动子和分子伴侣ORF1-ORF2对Cry11Aa蛋白表达的影响  

Influence of cry2A sporulation-dependent promoter and molecular chaperone ORF1-ORF2 from Bacillus thuringiensis on Cry11Aa protein

作　　者：师永霞[1] 曾少灵[1] 袁美妗[1] 孙钒[1] 庞义[1] 

机构地区：[1]中山大学有害生物控制与资源利用国家重点实验室

出　　处：《微生物学报》2008年第5期672-676,共5页Acta Microbiologica Sinica

基　　金：国家“973项目”(G2000016209);广东省自然科学基金~~

摘　　要：【目的】分析苏云金芽孢杆菌的cry2A型芽孢期启动子对晶体蛋白Cry11Aa的协调作用和分子伴侣ORF1-ORF2对Cry11Aa表达的促进功能。【方法】3个包括cry11Aa编码区的重组质粒pHcy1、pHcy2和pHcy4被构建并电激转化到苏云金芽孢杆菌晶体缺陷株4Q7中,其中pHcy1质粒携带cry11Aa基因自身启动子和分子伴侣p19基因,pHcy2携带cry2A型芽孢期启动子和分子伴侣orf1-orf2基因,pHcy4质粒在pHcy1的上游插入了cry2A型芽孢期启动子和分子伴侣orf1-orf2基因。SDS-PAGE分析了Cry11Aa蛋白在各重组苏云金菌株中的表达情况,并通过生物测定确定了其对蚊虫的生物活性。【结果】SDS-PAGE结果表明,Cry11Aa蛋白在4Q7(pHcy1)和4Q7(pHcy4)均获得了表达,在4Q7(pHcy2)中未检测到Cry11Aa蛋白,推测晶体蛋白Cry11A不能利用cry2A型启动子进行表达调控;Cry11Aa蛋白在等体积4Q7(pHcy4)培养液中的表达量是4Q7(pHcy1)菌株的1.25倍,暗示着分子伴侣ORF1-ORF2在某种程度上能提高Cry11Aa的蛋白表达量。4Q7(pHcy1)和4Q7(pHcy4)形成的Cry11Aa蛋白晶体的形状和大小相似,两者对致倦库蚊的生物活性没有明显差异,LC50s分别为59.33ng/mL和66.21ng/mL。【结论】推测晶体蛋白Cry11A能否成功表达与其使用启动子的类型和两者的协调配合有关。分子伴侣ORF1-ORF2虽然在某种程度上能提高Cry11Aa的蛋白表达量,但对提高Cry11Aa蛋白的杀蚊毒力没有显著性帮助。[Objective] To analyze the coordination function of cry2A sporulation-dependent promoter and the enhanced expression of molecular chaperone ORF1-ORF2 to crystal protein Cry11Aa. [Methods] We constructed three recombinant plasmids pHcyl, pHcy2 and pHcy4 containing cryllAa gene. pHcyl carried cryllAa own promoter and p19 gene, and pHcy2 carried cry2A sporulation-dependent promoter and orf1-orf2 gene. pHcy4 inserted cry2A promoter and orf1-orf2 gene upstream pHcyl plasmid. The recombinant plasmids were introduced into an acrystalliferous mutant 4Q7 of Bacillus thuringiensis sub sp. israelensis. We performed SDS-PAGE to analyze CryllAa protein expression in the recombinant Bt strains and carried out the mosquitocidal bioassay. [Results] SDS-PAGE showed that CryllAa protein was detected in 4Q7（pHcyl） and 4Q7（pHcy4）, but not in 4Q7（pHcy2）. The cryllAa gene could not be regulated under cry2A promoter. CryllAa protein had a 1.25 fold expression amount in the equal volume culture of 4Q7（pHcy4） to that of 4Q7（pHcyl）, which indicated that molecular chaperone ORF1-ORF2 could enhance CryllAa expression amount to a certain extent. Both 4Q7（pHcyl） and 4Q7（pHcy4） formed CryllAa crystals in a similar size and shape during sporulation under the transmission electron microscope. Their LC50s against 3rd-instar Culex quinquefasciatus were 59.33 ng/mL and 66.21 ng/mL respectively. [Conclusion] Whether crystal protein from B. thuringiensis could successfully express might relate to the type of the used promoter and their coordination. Molecular chaperone ORF1-ORF2 could enhance Cryl 1Aa expression amount to a certain extent with an unknown mechanism, but did not have an effect on high mosquitocidal toxicity of CryllAa protein. This research might play an important role to search the best collocation between ICP promoter or chaperone gene and ICP gene and to construct high-toxic Bacillus thuringiensis engineering strain by chaperone gene.

关 键 词：苏云金芽孢杆菌 分子伴侣 p19基因 orf1-off2基因 晶体蛋白Cry11Aa 

分 类 号：Q78[生物学—分子生物学] S476.11[农业科学—农业昆虫与害虫防治]