Cry1Ia蛋白N端信号肽对其蛋白表达量的影响  

Effect of N-terminal Signal Peptide of Cry1Ia Protein on Its Protein Expression

作　　者：郭俊佩 刘璇 韩阜志 高建华[1] 史宗勇[1] GUO Junpei;LIU Xuan;HAN Fuzhi;GAO Jianhua;SHI Zongyong(College of Life Sciences,Shanxi Agricultural University,Taigu 030801,China)

机构地区：[1]山西农业大学生命科学学院,山西太谷030801

出　　处：《山西农业科学》2021年第6期694-698,共5页Journal of Shanxi Agricultural Sciences

基　　金：国家转基因重大专项(2018ZX08012001-009)。

摘　　要：Cry1Ia是苏云金芽孢杆菌表达的分泌型Cry杀虫蛋白。为了研究Cry1Ia蛋白N端信号肽(Iasp)区域对其蛋白表达量的影响,梯度删除了N端氨基酸序列,将这些缺失突变体基因接入p ET28a Del表达载体,并转化大肠杆菌BL21 Star(DE3)感受态细胞,得到一系列缺失突变体,即Cry1Ia D5、Cry1Ia D10、Cry1Ia D13、Cry1Ia D16、Cry1Ia D24、Cry1Ia D32、Cry1Ia D44和Cry1Ia D73,分析其蛋白表达和m RNA热稳定性。结果显示,8种Cry1Ia蛋白突变体均成功表达,其中,Cry1Ia D32和Cry1Ia D44的表达量高于Cry1Ia,分别为Cry1Ia的1.43、1.12倍,Cry1Ia D13和Cry1Ia D16表达量与Cry1Ia基本持平,其余4种蛋白的表达量低于Cry1Ia;对m RNA起始密码子附近的核苷酸序列的二级结构进行预测发现,不同突变体该区域能够形成不同的茎环结构,且其热稳定性能具有一定的差异;进一步对蛋白相对表达量和m RNA结构热稳定性进行相关性分析表明,2个变量间无显著相关关系。研究结果可为揭示Iasp的功能奠定一定理论基础。Cry1Ia is a type of secreted Cry insecticidal protein expressed by Bacillus thuringiensis.To investigate the effect of the signal peptide of Cry1Ia(Iasp)on its expression,a series of deletion mutants,including Cry1IaD5,Cry1IaD10,Cry1IaD13,Cry1IaD16,Cry1IaD24,Cry1IaD32,Cry1IaD44 and Cry1IaD73,were obtained by gradient deleted the N-terminal amino acid sequence,cloned those into pET28aDel expression vector and transformed into Escherichia coli BL21 Star(DE3)strains.The expression level of proteins and the mRNA stability were analyzed.The results showed that all eight types of Cry1Ia protein were successfully expressed.The expression level of Cry1IaD32 and Cry1IaD44 was 1.43 and 1.12-fold higher than that of Cry1Ia.The expression levels of Cry1IaD13 and Cry1IaD16 were basically same as Cry1Ia.The expression level of the other proteins was lower than that of Cry1Ia.The results of secondary-structure stability near the start codon of mRNA indicated that different domains were able to form different stem-loop structures,and their thermal stability properties were somewhat different.Further correlation analysis of their relative expression level of proteins and secondary-structure stability showed that there was no significant correlation between the two variables.The above findings will lay the foundation of the study to reveal the function of Iasp.

关 键 词：苏云金芽孢杆菌 大肠杆菌 Cry1Ia蛋白 N端信号肽 原核表达 

分 类 号：Q78[生物学—分子生物学]