摘要 【目的】以杜仲种仁为试材,克隆EuCPI基因全长序列并分析其结构特征,探究该基因的抗逆功能及对黄粉虫生长发育的影响,为今后深入研究其抗逆机制提供理论基础。【方法】提取杜仲种仁的RNA并反转录成cDNA,采用RACE技术扩增EuCPI基因的全长cDNA序列;利用生物信息学网站对其所编码蛋白的理化性质及结构功能进行分析预测;利用pET28a质粒构建原核表达重组载体,对重组蛋白进行SDS-PAGE、Western Blot及蛋白纯化试验;利用重组的原核表达系统探究在盐胁迫和高温胁迫下含有EuCPI基因的大肠杆菌和对照组之间生长曲线的不同,初步鉴定该基因对大肠杆菌的影响;此外,利用诱导后的重组菌液饲喂黄粉虫,探究EuCPI基因对黄粉虫生长发育的影响。【结果】EuCPI基因具有547 bp的全长序列(GenBank登录号:MT702592),开放阅读框(ORF)为309 bp,编码蛋白由102个氨基酸组成,理论等电点(pI)为6.41,分子质量为11.17 kDa,不稳定指数为27.73,属于稳定蛋白,总平均亲水性为-0.416,属于亲水蛋白。氨基酸序列分析结果表明,EuCPI蛋白无跨膜结构,无信号肽,主要由50%的α-螺旋、36.27%的无规则卷曲、11.76%的延伸链和1.96%的β-折叠组成,属于半胱氨酸蛋白酶抑制剂家族。系统进化树分析结果表明,该蛋白与欧洲栓皮栎CPI编码蛋白同源性较高。通过构建的原核表达载体pET28a-EuCPI,诱导表达并纯化出15.29 kDa大小的重组蛋白。高温胁迫试验生长曲线结果表明,在37℃条件下前10 h含有EuCPI基因的BL21(DE3)大肠杆菌与对照组长势基本一致,而在42℃和50℃条件下前10 h含有EuCPI基因的菌生长速度明显大于对照组,且对照组出现明显的生长抑制情况。固体培养基盐胁迫试验结果表明,随着培养基中NaCl浓度增加,试验组和对照组菌落均出现生长抑制现象,但含有EuCPI基因的菌落数量明显多于对照组,尤其在0.75 mol·L-1时,前者有较多� 【Objective】The full length sequence of EuCPI gene was cloned from Eucommia ulmoides seed kernel and its structural characteristics were analyzed.The stress resistance function of EuCPI and its impact on the growth and development of Tenebrio molitor were explored.The results can provide a theoretical basis for further study of the stress-resistance mechanism in the future.【Method】The RNA of E.ulmoides kernels was extracted and reverse transcribed into cDNA,the full-length cDNA sequence of EuCPI gene was amplified by RACE technology;the physicochemical properties and structural functions of the encoded protein were analyzed and predicted by using bioinformatics websites;the fragment containing EuCPI gene was inserted into pET28a to construct a prokaryotic expression recombinant vector,and the recombinant protein was investigated by SDS-PAGE,Western Blot,and protein purification tests;to preliminarily identify the effect of EuCPI gene on Escherichia coli,the differences in growth curves under salt and high temperature stress between E.coli containing EuCPI gene and control group were explored using recombinant prokaryotic expression system;in addition,the effect of EuCPI gene on the growth and development of T.molitor was studied by feeding T.molitor with the induced E.coli containing EuCPI gene.【Result】The full-length sequence of EuCPI gene was 547 bp(GenBank accession number:MT702592),the gene contained a 309 bp open reading frame(ORF),encoding 102 amino acid residues.The predicted molecular weight of the putative protein was 11.17 kDa with the isoelectric point(pI)of 6.41.The protein was a stable and hydrophilic protein,the instability index was 27.73 and the total average hydrophilicity was-0.416.The amino acid sequence analysis showed that EuCPI protein had no transmembrane structure and no signal peptide,mainly composed of 50%α-helix,36.27%random coil,11.76%extended chain and 1.96%β-sheet,which belonged to the cysteine proteinase inhibitor family.Phylogenetic tree analysis showed that EuCPI prot
出处 《林业科学》 EI CAS CSCD 北大核心 2021年第3期29-38,共10页 Scientia Silvae Sinicae
基金 国家自然科学基金项目(31000305) 河北省高等学校科学技术研究项目(QN2015182)。
关键词 杜仲 EuCPI 基因克隆 耐盐 耐高温 抗虫 Eucommia ulmoides EuCPI gene cloning salt resistance high temperature resistance insect resistance
