薰衣草醇酰基转移酶基因(AAT)的克隆和表达分析  

Cloning and expression analysis of lavender alcohol acyltransferase gene(AAT)

作　　者：廖燕 尹松松 龚林涛 闫博文 孙明辉 苏秀娟[1,2] 王爱凡 LIAO Yan;YIN Song-song;GONG Lin-tao;YAN Bo-wen;SUN Ming-hui;SU Xiu-juan;WANG Ai-fan(College of Agronomy,Xinjiang Agricultural University,Urumqi 830052,China;Lavender Research Institute of Xinjiang Agricultural University/Xinjiang Key Laboratory of Crop Improvement&Germplasm Enhancement,Urumqi 830052,China)

机构地区：[1]新疆农业大学农学院,新疆乌鲁木齐830052 [2]新疆农业大学薰衣草研究所/新疆作物遗传改良与种质创新重点实验室,新疆乌鲁木齐830052

出　　处：《江苏农业学报》2024年第4期599-606,共8页Jiangsu Journal of Agricultural Sciences

基　　金：新疆维吾尔自治区重大科技专项(2022B02036-1);国家自然科学基金地区科学基金项目(31760429);新疆维吾尔自治区科学技术厅“天山青年计划”优秀青年科技人才培养项目(2020Q016);国家级大学生创新训练计划项目(201810758003);作物学重点学科发展基金项目(XNCDKY2021015)。

摘　　要：醇酰基转移酶(AAT)可以催化乙酰辅酶A与萜烯醇类物质的结合,生成各类乙酸酯类化合物。为探明薰衣草AAT基因在酯类化合物合成中的调控作用,本研究以薰衣草品系杂花叶片为供试材料,利用反转录PCR技术克隆薰衣草AAT基因的cDNA序列,该cDNA序列长度为1344 bp,其编码蛋白质由447个氨基酸组成,是非跨膜亲水性蛋白质,属于PLN02481超级家族;薰衣草AAT与黄色猴面花EgAAT亲缘关系最近,相似度为60.87%。AAT基因在苞叶中的表达量最低,在花瓣中的表达量最高;在花冠不同发育时期,该基因在盛开期表达量到达峰值。构建原核表达载体pET-28a-AAT,筛选了重组蛋白在大肠杆菌Transetta(DE3)中的最适诱导表达条件,诱导后获得的重组蛋白质相对分子质量为5.026×10^(4),重组蛋白以包涵体形式存在,具有将芳樟醇催化合成乙酸芳樟酯的生物活性。本研究结果为进一步验证薰衣草AAT基因在酯类化合物合成过程中的功能提供了基础。Alcohol acyltransferase(AAT)can catalyze the combination of acetyl coenzyme A and terpene alcohols to produce various types of acetate compounds.In order to explore the regulatory role of lavender AAT gene in the synthesis of ester compounds,the cDNA sequence of lavender AAT gene was cloned by reverse transcription PCR using the leaves of lavender strain Zahua as the test material.The length of the cDNA sequence was 1344 bp,and the encoded protein was composed of 447 amino acids,which was a non-transmembrane hydrophilic protein and belonged to the PLN02481 superfamily.The AAT in lavender was closely related to the EgAAT in yellow monkey-faced flower,with a similarity of 60.87%.The expression level of AAT gene was lowest in bracts and highest in petals.At different developmental stages of corolla,the expression of this gene reached the peak at the full-bloom stage.Prokaryotic expression vector pET-28a-AAT was constructed.The optimal expression conditions of the recombinant protein in Escherichia coli Transetta(DE3)were screened.The relative molecular weight of the recombinant protein was about 5.026×10^(4).The recombinant protein existed in the form of inclusion bodies and had the biological activity of catalyzing the synthesis of linalyl acetate from linalool.The results of this study provide a basis for further verifying the function of lavender AAT gene in the synthesis of ester compounds.

关 键 词：薰衣草 醇酰基转移酶基因(AAT) 克隆 表达模式 酶活性鉴定 

分 类 号：S184[农业科学—农业基础科学]