茶树CsCYP79A1基因克隆及表达分析  

Cloning and expression analysis of CsCYP79A1 gene in tea plant

作　　者：何荟如 林杰[1] 吴叶蝶 潘泓静 彭志松 苏祝成[1] HE Hui-ru;LIN Jie;WU Ye-die;PAN Hong-jing;PENG Zhi-song;SU Zhu-cheng(College of Agriculture and Food Science,Zhejiang Agriculture and Forestry University,Lin’an,Zhejiang 311300,China)

机构地区：[1]浙江农林大学农业与食品科学学院,浙江临安311300

出　　处：《南方农业学报》2021年第3期641-650,共10页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(31800582);浙江农林大学学生科研训练项目(113-2013200155)。

摘　　要：【目的】克隆茶树细胞色素P450(CYP)酶系的CYP79酶基因CsCYP79A1,并进行表达分析,为探究Cs-CYP79A1基因参与茶树防御的响应机制及在乌龙茶制作过程中的调控功能提供理论依据。【方法】克隆CsCYP79A1基因,通过生物信息学软件对其进行分析,用实时荧光定量PCR(qRT-PCR)检测其在不同茶树品种、不同嫩度叶片以及不同摇青次数叶片中的表达特性,并采用气相色谱—质谱联用仪(GC-MS)测定不同处理叶片中的苯乙腈释放量,以分析其与CsCYP79A1基因表达量的相关性。【结果】克隆获得的茶树CsCYP79A1基因编码区(CDS)序列长度为1617 bp,编码538个氨基酸残基,相对分子量为61.07 kD,理论等电点(pI)7.64,为不稳定的脂溶性亲水蛋白。CsCYP79A1蛋白与中华猕猴桃TXG46886.1的氨基酸序列相似性最高,达87%。CsCYP79A1蛋白存在2个跨膜结构,无信号肽,共有40个磷酸化位点,二级结构中α-螺旋、β转角、延伸链和无规则卷曲分别占氨基酸序列的49.07%、4.28%、11.71%和34.94%,三级结构与铁锈醇合酶的晶体结构(5ylw.1.A)相似度为26.61%。在4次摇青处理后,金萱和黄观音第3叶片中的CsCYP79A1基因表达量高于浙农113、白叶1号和福鼎大白茶,说明CsCYP79A1基因在适制乌龙茶的茶树品种中的表达量较高;CsCYP79A1基因在第3叶中的表达量显著高于第1叶和顶芽(P<0.05),且随着摇青次数的增加呈先升高后降低的变化趋势。4次摇青处理后,第3叶会释放大量苯乙腈,而第1叶和顶芽释放少量或不释放苯乙腈。苯乙腈的释放量随摇青处理次数的增加呈逐渐升高趋势。【结论】CsCYP79A1基因表达量与茶树品种、叶片嫩度和摇青次数密切相关,且其表达量和苯乙腈释放量均为第3叶最高,故推测CsCYP79A1基因是苯乙腈合成途径中的关键调控基因。【Objective】The CYP79 enzyme gene CsCYP79A1 of tea plant cytochrome P450(CYP)enzyme system was cloned,and its bioinformatics analysis and expression analysis were performed to provide a theoretical basis for indepth understanding of the mechanism of CsCYP79A1 gene involved in the defense response of tea plant and its function in the production process of oolong tea.【Method】The CsCYP79A1 gene was cloned using Huangguanyin leaves as materials,and the gene was analyzed by analysis softwares.Real-time fluorescent quantitative PCR(qRT-PCR)was used to detect its expression in different tea varieties,different tenderness leaves and different shaking times to analyze its expression characteristics.Gas chromatography-mass spectrometry(GC-MS)was used to determine the volatilization of phenylacetonitrile in leaves of different treatments to analyze its relationship with the expression of CsCYP79A1 gene.【Result】The cloned tea plant CsCYP79A1 gene coding region(CDS)was 1617 bp in length,encoded 538 amino acids,had a relative molecular mass of 61.07 kD and a theoretical isoelectric point(pI)of 7.64,and it was an unstable liposoluble hydrophilic protein.The amino acid sequence of CsCYP79A1 protein had the highest similarity with TXG46886.1(Actinidia chinensis),reaching 87%.The CsCYP79A1 protein had two transmembrane structures,no signal peptide,and had 40 phosphorylation sites.In the secondary structure,α-helix,βturn,extended chain and random coil accounted for 49.07%,4.28%,11.71%,and 34.94%of the amino acid sequence,respectively.The similarity between the tertiary structure and the crystal structure of rust alcohol synthase(5ylw.1.A)was 26.61%.After four times shaking treatments,the expression of CsCYP79A1 gene in the third leaves of Jinxuan and Huangguanyin was significantly higher than that of Zhenong 113,Baiye No.1 and Fuding Dabaicha,indicating that the expression level of CsCYP79A1 gene was higher in varieties which were suitable for making oolong tea.The expression level in the third leaves was significantly

关 键 词：茶树 CsCYP79A1 基因克隆 苯乙腈 生物信息学 表达分析 

分 类 号：S571.103.53[农业科学—茶叶生产加工]