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茶叶科学 ›› 2005, Vol. 25 ›› Issue (3): 177-182.doi: 10.13305/j.cnki.jts.2005.03.004

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茶树巯基蛋白酶抑制剂基因的cDNA克隆与序列分析

王朝霞1, 2, 李叶云1, 江昌俊1, *, 余有本3   

  1. 1. 安徽农业大学农业部茶叶生物化学与生物技术重点开放实验室,安徽 合肥 230062;
    2. 安徽教育学院生物系,安徽 合肥 230061;
    3. 西北农林科技大学茶叶研究所,陕西 杨陵 712100
  • 出版日期:2005-09-25 发布日期:2019-09-16
  • 通讯作者: Jangcj@ahau.edu.cn
  • 作者简介:王朝霞(1974— ),女,讲师,博士研究生,主要从事茶树种质资源与分子生物学研究。
  • 基金资助:
    安徽省教育厅自然科学研究重点项目(2004kjl37zd), 安徽农大农业部茶叶生物化学与生物技术重点开放实验室基金项目

Molecular Cloning and Sequence Analysis on cDNA of Cystatin Gene from Tea Leaves

WANG Zhao-xia1, 2, LI Ye-yun1, JIANG Chang-jun1, YU You-ben3   

  1. 1. Key Laboratory of Tea Biochemistry &Biotechnology, Ministry of Agriculture, Anhui Agriculture University, Hefei 230036, China;
    2. Department of Biology, Anhui Institute of Education, Hefei 230061,China;
    3. Tea Research Insititute , Northweast A&F University, Yanglin, 712100, China.;
  • Online:2005-09-25 Published:2019-09-16

摘要: 对多种已知植物巯基蛋白酶抑制剂(cystatin)基因的氨基酸序列进行比对分析,根据其高度保守的氨基酸序列设计一对简并引物,并从茶树品种龙井43鲜叶中提取总RNA,用RT-PCR法扩增出一204βbp的cDNA特异片段,然后通过3’/5’RACE的方法,分别扩增出3’端和5’端的序列,从而获得茶树巯基蛋白酶抑制剂基因的cDNA全长序列,所得序列全长627βbp,编码101个氨基酸,分子量约11.062βKDa。该基因在推测的氨基酸序列中含有巯基蛋白酶抑制剂家族中高度保守的、与其活性有关的QXVXG结构,且经Blast分析表明,该基因序列与其他植物巯基蛋白酶抑制剂基因的氨基酸序列同源性为54% ~ 77%。

关键词: 茶树, 巯基蛋白酶抑制剂基因, cDNA, 克隆

Abstract: Two degenerate primers were designed according to the conserved region among the known plant cystatins. A cDNA fragment of 204βbp was amplified by RT-PCR (reverse transcription polymerase chain reaction) of total RNA extracted from fresh leaves of Tea plant (Camellia sinensis cv Longjing43). A full-length cDNA of the cystatin gene was obtained by 3’/5’RACE (rapid amplification of cDNA ends). The cDNA sequence of this 627βbp clone contained an open reading frame encoding a polypeptide of 101 amino acid residues with a predicable molecular mass of 11.026βKDa. The deduced amino acid sequence contained the motif QXVXG conserved among most members of the cystatin superfamily. By using the program of Blast on GenBank database, the sequence presented a high match with the cystatin genes from other plants, such as European chestnut, Cassava, Cowpea, Tomato, Soybean et al. All researched out sequences were all cystatins, so we can conclude that the cloned sequence is a member of cystatin gene from Tea plant.

Key words: Camellia sinensis, cystatin, cDNA, clonging, sequence

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