茶树休眠芽与萌动芽抑制消减杂交文库的构建与初步分析

doi:10.13305/j.cnki.jts.2010.02.009

茶叶科学 ›› 2010, Vol. 30 ›› Issue (2): 129-135.doi: 10.13305/j.cnki.jts.2010.02.009

茶树休眠芽与萌动芽抑制消减杂交文库的构建与初步分析

王新超^1,2, 杨亚军^1,*, 陈亮^1,*, 马春雷¹, 姚明哲¹

1. 中国农业科学院茶叶研究所茶树资源和改良研究中心/国家茶树改良中心,浙江杭州 310008;
2. 中国农业科学院研究生院,北京 100081

收稿日期:2009-11-04 修回日期:2009-12-30 发布日期:2019-09-10
作者简介:王新超（1975— ）,男,安徽霍邱人,助研,主要从事茶树资源、育种与分子生物学研究。
基金资助:
国家自然科学基金项目（30872059）,现代农业（茶叶）产业技术体系项目

Construction and Preliminary Analysis of the Suppression Subtractive Hybridization cDNA Libraries between Dormant and Sprouting Buds of Tea Plant (Camellia sinensis)

WANG Xin-chao^1,2, YANG Ya-jun^1,*, CHEN Liang^1,*, MA Chun-lei¹, YAO Ming-zhe¹

1. Research Center for Tea Germplasm and Improvement, Tea Research Institute of Chinese Academy of Agricultural Sciences; National Center for Tea Improvement, Hangzhou 310008, China;
2. Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081, China

Received:2009-11-04 Revised:2009-12-30 Published:2019-09-10

摘要/Abstract

摘要： 为了解茶树越冬芽休眠及其解除的分子机理,以休眠芽和萌动芽为材料,构建了茶树休眠芽与萌发芽的正、反向抑制消减杂交文库,并从两个文库各随机挑选96个阳性克隆进行序列测定和生物信息学分析。建库质量分析结果表明,所构建的正、反向消减文库有效富集了差异基因,消减效率达到要求,插入片段集中于250~750 bp之间,文库质量较好。对正、反向文库随机挑选的阳性克隆测序和分析结果表明,正、反向文库在基因表达谱上存在一定的差异,萌动芽文库在亚细胞器形成、转运载体等方面的基因与休眠芽文库有较大差异,而休眠芽文库在抗氧化、翻译调控以及胁迫响应等方面的基因与萌动芽文库有较大差异。这两个文库的建立为进一步了解茶树芽休眠与解除的分子机制奠定了基础。

关键词: 茶树(Camellia sinensis), 芽休眠, 表达序列标签, 抑制消减杂交

Abstract: Bud dormancy and its release are important biological phenomena in tea plant (Camellia sinensis). In order to understand the molecular mechanism of tea bud dormancy and its release, 2 SSH-cDNA libraries were constructed using dormant and sprouting buds. The forward library contained genes preferentially expressed in sprouting buds and the reverse library contained genes preferentially expressed in dormant buds. One hundred and ninety two positive clones were randomly selected for sequencing and bioinformatics analyzing from the two libraries. The results showed that differential express genes were enriched effectively in the forward and reverse libraries, and subtraction efficiency reached the demand. Most of the length of inserted fragments was 250~750 bp. It implied that the quality of the SSH-cDNA libraries was satisfied. The bioinformatics analysis results indicated that there were differences between forward and reverse libraries on gene expression profiles. In forward library, the percent of genes related with organelle part and transporter was higher than reverse one. And in the reverse library, the percent of genes related with antioxidant, translation regulator and response to stimulus was higher than the forward. The 2 SSH-cDNA libraries can be further used for gene isolation and provide a foundation for further investigation to explore the molecular mechanism of bud dormancy and its release in tea plant.

Key words: tea plant (Camellia sinensis), bud dormancy, expressed sequence tag (EST), suppression subtractive hybridization (SSH)

中图分类号:

S571.1
Q507

王新超, 杨亚军, 陈亮, 马春雷, 姚明哲. 茶树休眠芽与萌动芽抑制消减杂交文库的构建与初步分析[J]. 茶叶科学, 2010, 30(2): 129-135. doi: 10.13305/j.cnki.jts.2010.02.009.

WANG Xin-chao, YANG Ya-jun, CHEN Liang, MA Chun-lei, YAO Ming-zhe. Construction and Preliminary Analysis of the Suppression Subtractive Hybridization cDNA Libraries between Dormant and Sprouting Buds of Tea Plant (Camellia sinensis)[J]. Journal of Tea Science, 2010, 30(2): 129-135. doi: 10.13305/j.cnki.jts.2010.02.009.

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茶树休眠芽与萌动芽抑制消减杂交文库的构建与初步分析

Construction and Preliminary Analysis of the Suppression Subtractive Hybridization cDNA Libraries between Dormant and Sprouting Buds of Tea Plant (Camellia sinensis)

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