茶叶CHS基因表达特性及其与儿茶素积累的关系  

Expression Characteristics of CHS Gene in Tea and Its Relationship with Catechins Accumulation

作　　者：施婧 张雪 张媛媛[1] 林晓蓉[1] 李斌[1,2] 陈忠正[1,2] SHI Jing;ZHANG Xue;ZHANG Yuan-yuan;LIN Xiao-rong;LI Bin;CHEN Zhong-zheng(College of Food Science,South China Agricultural University,Guangdong,Guangzhou 510642,China;Guangdong Provincial Key Laboratory of Nutraceuticals and Functional Foods,South China Agricultural University,Guangzhou 510642,China)

机构地区：[1]华南农业大学食話学院,广东广州510642 [2]广东省功能食品活性物重点实验室,华南农业大学,广东广州510642

出　　处：《食品工业科技》2020年第5期101-106,112,共7页Science and Technology of Food Industry

基　　金：广东省科技计划项目(2015A030302065);国家自然科学基金项目(31770727,31270726);现代茶叶产业技术体系(CARS-19);广州市科技计划项目(201607010139)

摘　　要：本研究用高效液相色谱法(HPLC)测定两种茶叶的儿茶素含量,通过RT-PCR克隆英红9号(YH)和南昆山毛叶茶(MY)的CHS基因进行比对分析,借助烟草瞬时表达技术对基因表达蛋白进行亚细胞定位;结合生物信息学分析选取特定肽段经人工合成后作为抗原制备CHS基因特异抗体;用荧光定量PCR(qRT-PCR)技术和Western blot技术测定比较两种茶叶CHS基因在转录、翻译水平的差异,并对比分析了两种茶叶间儿茶素含量差异与相应CHS基因表达的关系。结果表明:YH和MY中的儿茶素含量分别为181.51 mg/g和106.02 mg/g,前者显著高于后者并达到1.71倍;两种茶叶中所克隆的CHS基因具有单核苷酸差异但编码相同的氨基酸序列,其表达蛋白亚细胞定位于细胞质、细胞膜和细胞核;制备的CHS蛋白兔源抗体效价为1∶80000;CHS基因在YH中的表达无论在转录水平还是在翻译水平均显著高于MY,前者分别是后者的3.9倍和1.7倍,两种茶叶中CHS合成酶基因在转录、翻译水平的表达均与其对应的儿茶素积累水平一致。实验研究证明,茶叶儿茶素积累水平与其合成通路关键酶基因CHS的表达呈正相关。In this study,the content of catechins in two kinds of tea were detected with high performance liquid chromatography(HPLC),and the open reading frame(ORF)of CHS genes were cloned by RT-PCR from the Yinghong NO.9(YH)and Camellia ptilophylla Chang(MY)and presented comparison analysis. The subcellular localization of CHS gene expressed protein was carried out by tobacco transient expression technique. The CHS specific antibody was obtained with synthetic peptide segment on the basis of bioinformatics analysis. The expression characteristics of CHS conducted by fluorescence quantitative real-time PCR(qRT-PCR)at the level of transcription and by Western blot assays at the level of translation,respectively. Moreover,and the relationship of CHS expression with catechins contents was analyzed. Results showed that the content of catechins in YH and MY was 181.51 mg/g and 106.02 mg/g,the former was significantly higher than the latter and reached 1.71-fold.The CHS genes isolated from the two tea materials exhibited only one nucleotide difference but shared the same amino acid sequence. The protein encoded by CHS gene localized in cytoplasm,cell membrane,and nucleus based on tobacco transient expression system. The titer of the developed CHS antibody reached 1∶80000,and the CHS expression at transcription and translation level in YH were both higher than that in MY which reached 3.9 and 1.7-fold,the expression of CHS gene both at transcription and translation level consistent with the accumulation of tea catechins. Results verified the accumulation of catechins was positively correlated with the expression of CHS gene.

关 键 词：茶叶 儿茶素 查尔酮合成酶 基因表达 蛋白表达 

分 类 号：TS201.3[轻工技术与工程—食品科学]