组成型表达chi基因的Bt工程菌株构建及其特性

Construction and characteristic of a constitutive expression chi of engineered Bt strains

【目的】通过构建能够组成型高效表达几丁质酶的苏云金芽胞杆菌工程菌株,以提高其抑真菌活性。【方法】首先通过PCR扩增获得Bti75菌株chiB全长及系列缺失启动子片段,与本室构建的启动子探测型载体pCB连接,转化Bti75菌株,通过β-半乳糖苷酶活性检测及β-半乳糖苷酶mRNA的Real time-PCR检测,确定了一段长度为190 bp的组成型高效表达启动子。将这一启动子分别与Bti75自身几丁质酶基因chiA以及地衣芽胞杆菌(Bacillus licheniformis)的几丁质酶基因chiMY连接构建了组成型高效表达的工程菌株Bti75(pDA)和Bti75(pDM)。应用几丁质酶酶活检测、SDS-PAGE及酶谱分析检测了工程菌几丁质酶的表达情况。最后,通过检测工程菌发酵粗酶液对真菌孢子萌发和菌丝生长的影响,评估了工程菌对3种植物病原真菌的抗真菌活性。【结果】在没有诱导物存在的情况下,Bti75(pBPΔ7)菌株表达的β-半乳糖苷酶酶活和β-半乳糖苷酶mRNA的转录量分别是Bti75(pBP)菌株的7倍和2.5倍左右。在没有几丁质诱导的情况下,与野生株Bti75相比,工程菌株Bti75(pDA)和Bti75(pDM)的几丁质酶活性均提高了3.5倍左右。SDS-PAGE及酶谱分析证明目的几丁质酶在非诱导条件下达到组成型高效表达,抑真菌实验显示工程菌Bti75(pDA)和Bti75(pDM)抑制3种植物病原真菌的活性明显提高。【结论】发现190 bp的缺失启动子能够组成型高效表达不同来源的几丁质酶,无需诱导物的诱导,工程菌株就能展现良好的抗真菌能力。

[Objective] We constructed Bti engineering strains that can highly express chitinase genes constitutively, so that the strains can inhibit the growth of fungi more effectively. [Methods] The full length and series of deletion promoter fragments of chitinase gene chiB from Bacillus thuringiensis were cloned into a shuttle promoter-probe vector pCB, and all of the constructed plasmids were transformed into Bti75 strains. A 190 bp-length high constitutive expression promoter was identificated based on the results of β-galactosidase activity and real time-PCR. By using this promoter, we constructed Bti75 engineering strains that express chitinase gene chiMY of Bacillus licheniformis and chiA gene of Bti75 itself. SDS-PAGE and zymogram analysis were used to examine chitinase expression. In order to evaluate the antifungal activity assay of engineering strains against 3 plant pathogenic fungi, mycelium growth and spore germination inhibition assay were done. [Results] Without induction, β-galactosidase activity and mRNA contents of β-galactosidase of Bti75（pBPA7） was about 7-fold and 2.5-fold high than that in Bti75（pBP）, respectively. Without chintin induction, the chitinase activity of Bti75（pDA） and Bti75（pDM） was up to 3.5-fold higher compared with the parent strain. The results of SDS-PAGE and zymogram analysis confirmed that chitinase could be highly expressed in engineered strains. The results of antifungal activity assay indicated that the two engineered strains achieved a significant improvement in inhibiting three plant pathogenic fungi species. [Conclusion] The 190 bp deletion mutant promoter could drive the expression of different chitinase genes constitutively and efficiently. Without induction, the two engineered strains with the deletion promoter exhibited high antifungal activity.