茶树丝氨酸羧肽酶基因的克隆及表达分析

doi:10.13305/j.cnki.jts.2013.03.011

茶叶科学 ›› 2013, Vol. 33 ›› Issue (3): 202-211.doi: 10.13305/j.cnki.jts.2013.03.011

茶树丝氨酸羧肽酶基因的克隆及表达分析

仇传慧¹, 李伟伟², 王云生², 李明卓¹, 骆洋², 刘亚军², 高丽萍^2,*, 夏涛^1,*

1. 安徽农业大学教育部茶叶生物化学与生物技术重点实验室,安徽合肥 230036;
2. 安徽农业大学生命科学学院,安徽合肥 230036

收稿日期:2012-12-14 修回日期:2013-01-30 出版日期:2013-06-30 发布日期:2019-09-04
通讯作者: *。
作者简介:仇传慧（1988— ）女,安徽芜湖人,硕士,主要从事茶树次生代谢及分子生物学研究。
基金资助:
国家自然科学基金（30972401、31170647和31170282）、安徽省自然科学基金（11040606M73）、安徽省高校自然科学基金（KJ2012A110）

The Gene Cloning and Expression Analysis of SCPL in Tea Plant (Camellia sinensis)

QIU Chuan-hui¹, LI Wei-wei², WANG Yun-sheng², LI Ming-zhuo¹, LUO Yang², LIU Ya-jun², GAO Li-ping^2,*, XIA Tao^1,*

1. Key Lab of Tea Biochemistry and Biotechnology, Ministry of Education, Anhui Agricultural University, Hefei 230036, China;
2. School of Biology Science, Anhui Agricultural University, Hefei 230036, China

Received:2012-12-14 Revised:2013-01-30 Online:2013-06-30 Published:2019-09-04

摘要/Abstract

摘要： 近年来,人们发现丝氨酸羧肽酶类蛋白（SCPL）参与植物次生代谢产物的酰基转移过程,即具有转酰基功能。本研究采用RT-PCR技术,获得了3个茶树丝氨酸羧肽酶基因的全长序列;生物信息学分析表明,3个CsSCPL蛋白均包含了1个底物结合位点、3个催化作用保守区和多个N-糖基化位点,及其Ser-Asp-His三联体催化中心等SCPL家族的典型特征;进化树分析表明,3个CsSCPL可能具有酰基转移酶的功能。实时荧光定量PCR结果表明3个基因在芽叶茎根中都有表达,其中,CsSCPL1和CsSCPL3在叶中的相对表达量明显高于茎和根,而CsSCPL2则在根中高表达。本研究成功地将CsSCPL重组到表达载体pET32a(+)上进行原核表达,并对诱导时间及诱导温度进行了优化;经IPTG诱导、SDS-PAGE检测,目标蛋白条带分子量为70βkD,与预测大小相符。

关键词: 茶树, 丝氨酸羧肽酶, 生物信息学分析, 原核表达

Abstract: In recent years, serine carboxypeptidase-like proteins (SCPL) have been found that they are involved in plant secondary metabolites with the function of transferring acyl. The full-length cDNA of three CsSCPL were cloned from Camellia sinensis by RT-PCR technology. Bioinformatics analysis showed that the three CsSCPL proteins contained SCPL family's characteristic structures, such as one substrate binding and three catalysis conserved regions, a number of N-glycosylation sites and a conserved catalytic triad Ser-Asp-His amino acid active catalytic site and so on. The phylogeny analysis showed that CsSCPL probably possessed acyltransferase function. Quantitative RT-PCR analysis showed that the CsSCPL genes expressed in bud, leaf, stem and root. The relative expression of CsSCPL1 and CsSCPL3 in the leaves was significantly higher than that in stems and roots, while CsSCPL2 was highly expressed in the root. The CsSCPL genes were constructed into expression vector pET-32a(+) for over expression in prokaryotic cells and optimal inducing conditions including time, temperature were studied. The SDS-PAGE showed that recombinant proteins with formula weight 70βkD were induced successfully by IPTG, which coincided with the prediction.

Key words: Camellia sinensis, serine carboxypeptidase-like proteins, bioinformatics analysis, prokaryotic expression

中图分类号:

仇传慧, 李伟伟, 王云生, 李明卓, 骆洋, 刘亚军, 高丽萍, 夏涛. 茶树丝氨酸羧肽酶基因的克隆及表达分析[J]. 茶叶科学, 2013, 33(3): 202-211. doi: 10.13305/j.cnki.jts.2013.03.011.

QIU Chuan-hui, LI Wei-wei, WANG Yun-sheng, LI Ming-zhuo, LUO Yang, LIU Ya-jun, GAO Li-ping, XIA Tao. The Gene Cloning and Expression Analysis of SCPL in Tea Plant (Camellia sinensis)[J]. Journal of Tea Science, 2013, 33(3): 202-211. doi: 10.13305/j.cnki.jts.2013.03.011.

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茶树丝氨酸羧肽酶基因的克隆及表达分析

The Gene Cloning and Expression Analysis of SCPL in Tea Plant (Camellia sinensis)

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