茶树CsPNPO基因的功能鉴定与表达分析
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引用本文:陈星星,夏 莹,黄龙全,张剑韵.茶树CsPNPO基因的功能鉴定与表达分析[J].西北植物学报,2019,39(4):580~587
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作者单位
陈星星1,夏 莹1,黄龙全1*,张剑韵2 (1 安徽农业大学 茶与食品科技学院合肥 2300362 安徽农业大学 外国语学院合肥 230036) 
基金项目:国家自然科学基金面上项目(No. 31670297)
中文摘要:PNP氧化酶是VB6代谢途径中一个重要的转化酶。该研究以茶树‘龙井43’为材料,采用RT PCR方法克隆PNP氧化酶基因,以pET22b(+)为载体构建原核表达载体,通过IPTG进行诱导表达并进行功能鉴定;采用荧光定量PCR方法分析茶树不同组织中CsPNPO基因的表达差异以及在低温和干旱胁迫下的表达特征,为进一步解析茶树VB6的生理生化功能奠定基础。结果表明:(1)茶树CsPNPO编码框长度为1 503 bp,编码501个氨基酸,分子量为48.5 kD,理论等电点5.82,不含信号肽,属于亲水性的非分泌蛋白,定位于叶绿体;氨基酸序列分析结果显示,其有叶绿体转运肽区域、YjeF N功能域和PNP氧化酶功能域。(2)成功构建pET22b(+) CsPNPO原核表达载体,并在pH 8.5、37 ℃时测定重组蛋白有较强的PNP氧化酶活性。(3)荧光定量PCR检测结果显示,CsPNPO基因在茶树叶中的表达量最高,其次是茎,根的表达量最低仅为叶的十分之一,表明CsPNPO基因具有组织表达特异性;在低温和干旱条件下CsPNPO基因的表达量下降明显,推测CsPNPO基因可能参与了茶树对低温和干旱的逆境应答。
中文关键词:茶树  功能鉴定  逆境胁迫  组织特异性
 
Functional Identification and Expression Analysis of CsPNPO Gene in Tea Plant
Abstract:PNP oxidase is an important invertase in the VB6 metabolic pathway. In this study, the tea tree ‘Longjing 43’ was used as the material, and the PNP oxidase gene was cloned by RT PCR. The prokaryotic expression vector was constructed by using pET22b(+) as a vector, and induced expression by IPTG for functional identification; Fluorescence quantitative PCR was used to analyze the differential expression of CsPNPO gene in different tissues of tea plants and its expression under low temperature and drought stresses, which laid a foundation for further analysis of physiological and biochemical functions of tea tree VB6. The results showed that: (1) the coding frame length of CsPNPO was 1 503 bp, and it was predicted to encode a 501 amino acid protein. The molecular weight of CsPNPO protein was 48.5 kD, and the theoretical isoelectric was 5.82. The CsPNPO protein contains no signal peptide and is a non secreted hydrophilic protein, and localized to chloroplasts. Amino acid sequence analysis revealed that it has a chloroplast transit peptide region, a YjeF N functional domain, and a PNP oxidase functional domain. (2) The prokaryotic expression vector pET22b(+) CsPNPO was successfully constructed, and the recombinant protein had strong PNP oxidase activity at pH 8.5 and 37 ℃. (3) Fluorescence quantitative PCR detection shows that the expression level of CsPNPO gene in tea leaves was the highest, followed by stems, and the expression in roots was only one tenth of that of leaves, indicating that CsPNPO gene has tissue expression specificity; The expression level of CsPNPO gene decreased significantly under low temperature and drought conditions. It is speculated that CsPNPO gene may be involved in the stress response of tea tree to low temperature and drought.
keywords:tea plant  functional identification  abiotic stress  tissue specificity
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