【作者】 杨梅；

【导师】 关雄；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 植物致病性病原菌最主要为革兰氏阴性细菌,其自身代谢产物N-酰基高丝氨酸内酯(N-acyl-homoserine lactones, AHL),是一类革兰氏阴性细菌群体致病性应答系统中的关键信号分子。当菌群中AHL累计到一定浓度后,就能激活病原菌致病基因表达,产生致病性并出现各种相关的病症。因此降低病原菌AHL的浓度将成为防治这些病害的关键。目前已从芽胞杆菌等克隆出aiiA基因,并发现其表达的产物可以降解AHL,从而大大减弱致病菌的危害。本研究克隆了来自苔藓、土壤和植物叶片中分离的苏云金芽胞杆菌中aiiA基因,即aiiA-B15、aiiA-B19、aiiA-S2、aiiA-S9、aiiA-P4、aiiA-P28,并对其编码的AHL-Lactonase蛋白进行了生物信息学分析。结果表明这些蛋白均为弱亲水性蛋白,含有3个典型的保守结构域,均属于β-内酰胺酶超家族的结构域。从计算机模拟的三级结构可以看出,AHL-Lactonase蛋白中的His-104、His-106、Asp-108、His-109、His-169、Asp-191、Tyr-194、His-235在空间结构上相互靠近,形成一个较为致密的与甘油结合的结构域,是重要的活性中心。6个α螺旋位于外侧,形成活性中心的表面保护层。通过比较16S rRNA基因和AHL-Lactonase蛋白的进化树,表明aiiA基因编码的AHL-Lactonase蛋白与Bt物种的进化并不同步,在该物种的进化过程中aiiA基因可能早于物种的分化;aiiA基因不能作为Bt物种进化的遗传标记。这些预测结果的综合应用不仅为aiiA基因表达、蛋白纯化等后续研究提供了大量宝贵的信息,并为改造AHL-Lactonase酶学特性,提高AHL- Lactonase的抗病能力等工作提供有价值的参考。本研究首先选用了pGEX-4T-3表达载体,构建重组质粒pGEXaiiA-B15,获得BL21(DE3)- pGEXaiiA-B15工程菌。表达条件的优化表明,在0.6 mmol/L IPTG、30℃下诱导3 h,蛋白的表达量最大,但绝大多数为包涵体。接着利用pET-29a构建了pET29a-aiiA融合表达载体,制备E.coli BL21 (DE3)-pET29a-aiiA工程菌,在0.8 mmol/L IPTG、20℃,25 h下诱导表达,获得了54.4μg/mL可溶性AHL- lactonase-B15蛋白,并采用亲和层析的方法纯化AHL-lactonase-B15。AHL- lactonase-B15对胡萝卜软腐欧文氏杆菌抗病性实验表明,AHL-lactonase-B15具有较好的抗病性,明显抑制了马铃薯的腐烂。值得注意的是,BL21(DE3)-pET29a-aiiA工程菌诱导表达后超声破壁的上清液样品也具有很好的水解AHL的活性和抗病性。这表明AHL-lactonase-B15在浓度较低并存在很多杂蛋白的情况下仍然有较强的活性。这也提示了AHL-lactonase-B15具备作为新型高效无公害的生物农药的潜能,具有进一步深入研究价值。AHL-lactonase-B15酶学特性表明,AHL- lactonase-B15酶促反应最适温度在40℃左右,最适pH约为8。以上研究结果为AHL-Lactonase蛋白质工程研究、微生物抗细菌病害基因工程育种、大规模发酵生产及其实际生产应用提供理论依据,对提高果蔬品质和产量、减少化学农药污染,实现经济效益、社会效益和生态效益的同步增长,具有重要的理论意义和实际应用价值。更多还原

【Abstract】 Plant pathogens are mostly gram-negative bacteria, their metabolites, N-acyl-homoserine lactones (AHL), are critical signaling molecules in bacterial quorum-sensing systems. When AHL reach a threshold concentration, they can activate the expression of pathogenic genes and induce diseases. Therefore reducing the concentration of AHLs is a key point of the diseases’control in plants.In this study, aiiA genes, designated as aiiA-B15, aiiA-B19, aiiA-S2, aiiA-S9, aiiA-P4 and aiiA-P28, were cloned respectively from six different strains of Bacillus thuringiensis from Bryophyte, soil and lamina. Bioinfromatical methods were utilized to analyze AHL-Lactonase, the products of these genes. The deduced AHL-Lactonase were weak hydrophilic proteins and all included three typical conserved domains belonging to theβ-lactamase superfamily. It was found that His-104, His-106, Asp-108, His-109, His-169, Asp-191, Tyr-194 and His-235 in AHL-Lactonase closed up on the spatial structure and formed an important activity center according to the simulative three-dimensional structure of AHL-Lactonase. Sixα-helices surrounded the activity center to keep its stabilization. By comparison of the phylogenetic tree of the cloned aiiA genes with that of 16S rRNA genes from the cloning stains, it was suggested that evolution of Bt species and that of aiiA genes are asynchronous. The differentiation of aiiA gene may be earlier than that of Bt species . Thus aiiA gene could not be used as a hereditary marker for Bt species evolution. These data provide important information for further study on aiiA gene expression and protein purification and also provide the valuable reference for the study of the enzyme character and antiviral ability of the AHL-Lactonase.pGEX-4T-3, an expression vector of AHL-Lactonase, was constructed and transformed into bacteria strain of E.coli BL21(DE3) for construction of genetically engineeringed bacterium strain. After 3h induction with 0.6 mmol/L IPTG at 30℃in the shaking flask culture, the expression of AHL-Lactonase protein reached to maximal level in the form of inclusion bodies. In order to obtain soluble expression of AHL-Lactonase protein, the expression vector of pET29a-aiiA was further constructed and transformed into E.coli BL21(DE3) for construction of soluble expression strain of BL21(DE3)-pET29a-aiiA. After 25h induction with 0.8 mmol/L IPTG at 20℃in the shaking flask culture, the AHL-Lactonase protein expressed in soluble form by this strain reached about 54.4μg/mL. The recombinant protein was purified with Ni-affinity chromatography. Biological activity analysis showed that AHL-Lactonase protein had a capability to hydrolyze AHL, and strong antimicrobial activity for Eriwinia carotovora. It was interesting to note that the supernatant of BL21(DE3)-pET29a-aiiA after breaking-up cell wall also had an AHL-hydrolyzing activity. This meant that rencombinant AHL-lactonase-B15 was able to hydrolyze AHL at low concentration under the contition of presenting many other native proteins and implied the potential for recombinant AHL-lactonase as a prospective biologic pesticide.The results of enzyme dynamic study demonstrated that the optimum temperature of AHL-lactonase was 40℃and the optimum pH value was 8.This study might provide theoretical support for protein engineering, large-scale production and application of AHL-Lactonase. Development of biological pesticides will reduce the pollution of chemical pesticides and be of economic,social and ecological benefits to our society.更多还原