摘要： 为明确几丁质酶在鸭梨抗病过程中的作用，以鸭梨为试材，提取总RNA，采用RT-PCR方法克隆几丁质酶基因PbChi Ⅱ ，分析PbChi Ⅱ 在梨黑星病菌诱导下的表达模式，构建原核表达载体pET30a-PbChi Ⅱ ，在大肠杆菌BL21（DE3）菌株中表达PbChiⅡ蛋白，分析重组蛋白在非生物胁迫下的生长能力。结果表明：PbChi Ⅱ 基因全长（基因登录号：KP876485）969 bp，实时定量PCR（Quantitative Real-time PCR，qRT-PCR）分析显示，PbChi Ⅱ 基因的表达受病原菌的调控，在供试的96 h内，梨黑星病菌可诱导该基因表达，且表达量在48 h达到最高。将PbChi Ⅱ 基因成功克隆到原核表达载体pET30a上，SDS-PAGE分析表明，该重组蛋白在37℃，1.0 mmol/L IPTG诱导2 h，表达量最大。转pET30a-PbChi Ⅱ载体的大肠杆菌BL21（DE3）菌株表达了分子量约35.53 kDa的重组蛋白。诱导的蛋白可增强菌体在NaCl、CuCl₂、CdCl₂和ZnSO₄等非生物胁迫下的生长能力。为进一步探索PbChi Ⅱ 基因的功能提供了基础资料。

关键词: 鸭梨, 几丁质酶基因, 原核表达, 非生物胁迫, 表达模式

Abstract: To clarify the role of chitinase in the process of pear resistance disease, the total RNA of Yali was extracted and specifi c primers were designed to amplify the coding region of PbChi Ⅱ protein by RT-PCR,analysed the expression pattern of PbChi Ⅱ after treatment with pathogens. The prokaryotic expression vector pET30a-PbChi Ⅱ was constructed and the recombinant protein was expressed in E.coli BL21 (DE3). The recombinant protein growth ability was analyzed under abiotic stress. The results showed that the length of PbChi Ⅱ (GenBank accession Number:KP876485) gene was 969 bp,Quantitative Real-time PCR (qRT-PCR) analysis revealed that the expression of PbChi Ⅱ was regulated by pathogens,and enhanced up to the peak at 48 h after treatment with Venturia nashicola during 96 h. The PbChi Ⅱ gene was successfully subcloned into the expression vector pET30a. SDS-PAGE analysis showed that a specific recombinant protein of approximately 35.53 kDa was produced in the BL21 (DE3) with the prokaryotic expression vector pET30a-PbChi Ⅱ in 37℃ with 1.0 mmol/L IPTG for 2 hours. This protein enhanced the stress of isolate with NaCl,CuCl₂,CdCl₂ and ZnSO₄. This research provided basic references for further study the function of PbChi Ⅱ.

Key words: Pyrus bretschneideri Yali, Chitinase gene, Prokaryotic expression, Abiotic stresses, Expression pattern

中图分类号: 

• Q78