桑氏链霉菌几丁质酶ChiKJ40基因的克隆表达及其抑菌作用

Cloning, expression and antibacterial functions of ChiKJ40, a chitinase gene from Streptomyces sampsonii

摘要:

【背景】目前关于桑氏链霉菌(Streptomyces sampsonii)生防基因的研究不多，仅从其基因组中克隆了2个几丁质酶基因片段，其单个几丁质酶的完整基因序列相关研究未见报道。【目的】克隆S. sampsonii KJ40的几丁质酶基因ChiKJ40并进行原核表达，纯化重组蛋白并研究其抑菌作用。【方法】采用PCR扩增法从S. sampsonii KJ40中克隆几丁质酶基因ChiKJ40，连接到表达载体pET-32a，导入Escherichia coli BL21(DE3)进行诱导表达。使用His标记蛋白质微量纯化试剂盒对重组几丁质酶进行纯化，Bradford蛋白浓度测定试剂盒测定粗酶液和纯化酶液的浓度，几丁质酶试剂盒测定粗酶液和纯化酶液的几丁质酶活性。观察重组几丁质酶对桉树焦枯病菌(Cylindrocladium scoparium)、栗疫病菌(Cryphonectria parasitica)、链格孢菌(Alternaria alternate)、紫丝核菌(Rhizoctonia violacea)几种致病真菌的抑菌作用。【结果】ChiKJ40基因(登录号为MF434484)在E. coli中经IPTG诱导表达，获得42 kD的重组几丁质酶，不同浓度IPTG 在37 °C诱导3 h，蛋白产量无明显变化。0.2 mmol/L IPTG 16 °C诱导过夜，重组几丁质酶主要以可溶性形式存在于上清，小部分以包涵体存在于沉淀中。粗酶液几丁质酶活性为0.080 U/mL，酶比活力为0.041 U/mg，纯化酶液几丁质酶活性为0.046 U/mL，酶比活力为0.115 U/mg，纯化倍数为2.8，酶活回收率为57.5%。重组几丁质酶处理后，C. scoparium、C. parasitica和A. alternata菌丝细胞出现分节、膨胀，R. violacea菌丝溶解且部分被破坏成碎片。【结论】ChiKJ40基因的研究补充了S. sampsonii的生防背景，为几丁质酶基因找到了新的来源，并为其应用奠定了理论基础。

Abstract:

[Background] There are very rare researches on the biocontrol genes of Streptornyces sampsonii until now. Only two chitinase gene fragments were cloned, and there is no relevant study in the complete gene sequence of the chitinase. [Objective] To clone and prokaryotic expression of the chitinase gene ChiKJ40 of S. sampsonii KJ40, then purify recombinant protein and investigating antibacterial characters. [Methods] Firstly we cloned the chitinase gene ChiKJ40 from S. sampsonii KJ40 by PCR amplification, then ligated it into vector pET-32a and expressed in Escherichia coli BL21(DE3). We purified the recombinant chitinase using the His-tagged protein microscopy kit, determined the concentration of the crude enzyme solution and the purified enzyme solution by the Bradford protein concentration assay kit, measured the chitinase activity of the crude enzyme solution and the purified enzyme solution using the chitinase kit. Finally also checked antibacterial characters of the recombinant chitinase on the Cylindrocladium scoparium, Cryphonectria parasitica, Alternaria alternata and Rhizoctonia violacea. [Results] We induced expression of the ChiKJ40 gene (accession number: MF434484) through IPTG in E. coli, the recombinant chitinase size is 42 kD. There are no significant differences in production of protein using different concentrations of IPTG at 37 °C inducing 3 h. 0.2 mmol/L IPTG inducing 16 °C overnight, recombinant chitinase mainly existed in the form of soluble supernatant, small existed in inclusions precipitation. The chitinase activities of crude protein and purified protein were 0.080 U/mL and 0.046 U/mL, respectively. The specific activity of crude protein and purified protein were 0.041 U/mg and 0.115 U/mg, the purification ratio was 2.8, the rate of 57.5%. After treating with the purified protein, mycelium cells of C. scoparium, C. parasitica, A. alternata were segmented with inflating the mycelia, and myceliums of R. violacea were broken. [Conclusion] This study of ChiKJ40 provides biocontrol background of S. sampsonii and finds a new source for the chitinase genes, and lays a theoretical foundation for its application.