沙糖橘几丁质酶基因orange1.1t05124的克隆及原核表达  

Cloning and prokaryotic expression of the orange1.1t05124 gene encoding chitinase from Citrus reticulata var. Shatangju

作　　者：胡亚平[1] 陈聪 吉前华[1] 郭雁君[1] 郭丽英[1] 蒋惠[1] 杨凤梅 HU Yaping;CHEN Cong;JI Qianhua;GUO Yanjun;GUO Liying;JIANG Hui;YANG Fengmei(Fruit Research Institute,Zhaoqing University/School of Life Sciences,Zhaoqing University,Zhaoqing,Guangdong,526000,China)

机构地区：[1]肇庆学院果树研究所/肇庆学院生命科学学院,广东肇庆526061

出　　处：《中国南方果树》2025年第1期9-15,共7页South China Fruits

基　　金：“十四五”广东省农业科技创新十大主攻方向“揭榜挂帅”项目(2022SDZG)资助。

摘　　要：柑橘几丁质酶作为一种重要的抗真菌蛋白,可以降解真菌细胞壁中的几丁质。利用沙糖橘的cDNA文库克隆了沙糖橘几丁质酶基因orange1.1t05124。该基因位于第8染色体末端,包含3个外显子。将该基因克隆到pEASY-Blunt E1载体中,测序验证序列无误,随后转入大肠杆菌BL21(DE3)中,经异丙基-β-硫代半乳糖苷(IPTG)诱导生成了目的蛋白,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE电泳)验证成功表达,在产物的包涵体中纯化到了目标蛋白。通过生物信息学分析发现,该蛋白具有信号肽,定位在细胞外,是单体的球状蛋白质。通过进化树分析发现,该几丁质酶在植物中广泛存在同源蛋白质,且都含有GH19几丁质酶催化结构域。对柑橘几丁质酶基因进行克隆和表达,可为了解该基因家族的抗病机理和开发基于几丁质酶的新型生物农药、生物肥料提供基因资源。The chitinase of citrus,an important antifungal protein,could degrade the chitin in the fungal cell wall.The citrus chitinase gene orange1.1t05124 was cloned from the cDNA library of Shatangju(Citrus reticulata var.Shatangju).This gene is located at the end of the chromosome 8,with 3 exons.It was subsequently cloned into the pEASY-Blunt E1 vector,and the accuracy of the sequence was confirmed by sequencing,and then,transformed into Escherichia coli BL21(DE3).Production of target protein was induced by IPTG,and the successful expression was verified by SDS-PAGE electrophoresis.The target protein was further purified from the inclusion bodies of the products.Bioinformatic analysis revealed the presence of a signal peptide in this protein.The protein is an extracellular localization and monomeric globular protein.Phylogenetic tree analysis demonstrated that the homologous proteins of this chitinase widely exist in plants and all contain the GH19 chitinase catalytic domain.The cloning and expression of the citrus chitinase gene could provide genetic resources for understanding the disease resistance mechanisms of this gene family and development of novel biopesticides and biofertilizers based on chitinase.

关 键 词：沙糖橘 几丁质酶基因 原核表达 抗真菌蛋白质 

分 类 号：S666[农业科学—果树学]