芽胞外壁基质组成蛋白的编码基因启动子PexsY指导的cry1Ac基因表达

Expression of cry1Ac gene directed by PexsYpromoter of the exsY gene encoding component protein of exosporium basal layer in Bacillus thuringiensis

摘要:

摘要:【目的】利用非cry基因启动子PexsY(芽胞外壁基质组成蛋白编码基因启动子)表达Cry1Ac晶体蛋白，发现可用于cry基因表达的新元件，为高效工程菌的构建奠定基础。【方法】采用启动子融合lacZ技术，通过β-半乳糖苷酶活性分析了PexsY启动子和截短的PexsY启动子的转录活性;利用该启动子在苏云金芽胞杆菌(Bacillus thuringiensis，Bt)HD73菌株中表达了cry1Ac基因，通过透射电子显微镜观察晶体形态;蛋白定量、SDS-PAGE比较蛋白产量;生物活性测定进行功能验证。【结果】PexsY启动子在芽胞晚期转录活性很高，透射电镜观察到利用该启动子表达的cry1Ac基因形成了菱形晶体，SDS-PAGE分析可以检测到133kDa的Cry1Ac蛋白，且与cry3A启动子指导表达的蛋白产量相近，少于cry8E启动子指导表达的蛋白产量;生物活性测定表明PexsY指导表达Cry1Ac蛋白对玉米螟(Ostrinia furnacalis)具有杀虫活性。【结论】在Bt无晶 体突变体中，非cry基因启动子PexsY可以正常表达133kDa的Cry1Ac蛋白，并形成晶体，具有在芽胞形成晚期表达cry基因的能力，该类启动子将在Bt工程菌构建中发挥重要作用。

Abstract:

Abstract:［Objective］To discover new elements for cry gene expression，PexsY，which is the promoter of the exosporium basal layer structural gene exsY，was used to express cry1Ac gene in Bacillus thuringiensis．［Methods］We used be tagalactosidase assays by promoter-lacZ fusion to analyze the transcriptional activity of exsY promoter and truncated exsY promoter．The cry1Ac gene was directed by the non-cry gene promoter PexsY and was then expressed in Bacillus thuringiensis HD73．Transmission electron microscope (TEM) was used to observe the formation of crystal inclusion．The Cry1Ac yieldswere evaluated by protein quantification and SDS-PAGE analysis．Bioassays against Ostrinia furnacalis were used for the functional verification．［Results］ Beta-galactosidase assays showed that the exsY promoter had a strong transcriptional activity in the acrystalliferous mutant strain HD73－on the late sporulation phase．Cry1Ac expression products directed by the PexsY could form diamond crystals．SDS-PAGE analysis showed that the cry1Ac gene directed by the cry8E promoter has the highest protein yield among the four promoters while the cry1Ac gene under the direction of PexsYorcry3A promoters showed similar protein yields．The bioassay results showed that the Cry1Ac protein directed by the PexsY promoter was toxic against Ostrinia furnacalis．［Conclusion］The cry1Ac gene under the direction ofthe non-cry gene promoter PexsY was able to express the Cry proteins at the late sporulation phase and could form crystal inclusion in a B．thuringiensis strain．Our finding provides applicationpotential for the genetically modification of engineered Bt strains．