鸭梨PbChiⅡ的克隆与表达分析

Cloning and Expression Analysis of PbChiⅡ from Pyrus bretschneideri Yali

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PbChiⅡ,分析PbChiⅡ在梨黑星病菌诱导下的表达模式,构建原核表达载体pET30a-PbChiⅡ,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:PbChiⅡ基因全长(基因登录号:KP876485)969bp,实时定量PCR(Quantitative Real-time PCR,qRT-PCR)分析显示,PbChiⅡ基因的表达受病原菌的调控,在供试的96h内,梨黑星病菌可诱导该基因表达,且表达量在48h达到最高。将PbChiⅡ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℃,1.0mmol/LIPTG诱导2h,表达量最大。转pET30a-PbChiⅡ载体的大肠杆菌BL21(DE3)菌株表达了分子量约35.53kDa的重组蛋白。诱导的蛋白可增强菌体在NaCl、CuCl_2、CdCl_2和ZnSO_4等非生物胁迫下的生长能力。为进一步探索PbChiⅡ基因的功能提供了基础资料。

To clarify the role of chitinase in the process of pear resistance disease,the total RNA of Yali was extracted and specific primers were designed to amplify the coding region of PbChiⅡprotein by RT-PCR,analysed the expression pattern of PbChiⅡ after treatment with pathogens. The prokaryotic expression vector pET30 a-PbChi Ⅱwas 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 PbChiⅡ（ Gen Bank 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 Pb Chi Ⅱ gene was successfully subcloned into the expression vector p ET30 a. SDSPAGE analysis showed that a specific recombinant protein of approximately 35. 53 k Da was produced in the BL21（ DE3） with the prokaryotic expression vector p ET30 a-PbChiⅡ in 37 ℃ with 1. 0 mmol/L IPTG for 2 hours. This protein enhanced the stress of isolate with NaCl,CuCl_2,CdCl_2 and ZnSO_4. This research provided basic references for further study the function of PbChiⅡ.