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植物研究 ›› 2011, Vol. 31 ›› Issue (1): 95-99.doi: 10.7525/j.issn.1673-5102.2011.01.017

• 论文 • 上一篇    下一篇

疫霉侵染下辣椒幼苗消减文库的构建与初步分析

贺俐;吴杨*;许东风   

  1. 井冈山大学生命科学学院,吉安 343009
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2011-01-20 发布日期:2011-01-20
  • 通讯作者: 吴杨
  • 基金资助:
     

Construction and Primary Analysis of Subtractive cDNA Library of Capsicum annuum L. Under Phytophthora capsici Stress

HE Li;WU Yang*;XU Dong-Feng   

  1. School of Life Science,Jinggangshan University,Ji’an 343009
  • Received:1900-01-01 Revised:1900-01-01 Online:2011-01-20 Published:2011-01-20
  • Contact: WU Yang
  • Supported by:
     

摘要: 为了分离和鉴定辣椒中疫霉诱导基因,以高抗疫霉病辣椒品种L11为材料,以接种辣椒疫霉菌的幼嫩叶片为处理(tester),以未接种自然生长的幼嫩叶片为对照(driver),利用抑制性消减杂交技术(suppression subtractive hybridization,SSH)构建了疫霉侵染下辣椒幼苗的消减文库。从消减文库中随机挑取30个阳性克隆,提取质粒进行PCR鉴定,显示插入片段大小大部分集中在200~1 000 bp之间,文库质量良好。随机挑取40个克隆进行测序,共获得35个有效EST序列。经Blastx分析表明:有30个EST与GenBank中其他序列有同源性,5个EST为未知功能序列。已知功能的EST序列分别编码NAC转录因子、丝氨酸/苏氨酸蛋白激酶、P450单加氧酶、叶绿素a/b结合蛋白、谷胱甘肽转移酶、几丁质酶等,这些蛋白涉及抗病信号传递、抗氧化作用、转录调控及光合作用等多种生理过程。本研究为抗病基因克隆和系统研究疫霉侵染下辣椒基因的表达奠定了重要的理论基础。

关键词: 疫霉, 抑制性消减杂交, 抗病, 辣椒, EST

Abstract: To identify the Phytophthora capsici stress induced genes of pepper, a high P.capsici resistant pepper cultivar was used as main material in this study. With cDNA from pepper seedlings treated with P.capsici as the tester and cDNA from this plant in normal growth as the driver, we constructed subtracted library using suppression subtractive hybridization (SSH). Thirty positive clones were picked out randomly from SSH library, plasmid PCR suggested that the inserts were between 200-1 000 bp and library was suitable for the following work. Forty clones were randomly selected and sequenced, 35 differential gene fragments were obtained. Blastx analysis showed that 30 ESTs have high homology with known genes in GenBank, 5 ESTs was unknown function genes. The obtained known function ESTs code NAC transcription factor protein, Serine/threonine protein kinase, cytochrome P450 monooxygenase, chlorophyll a b-binding protein, glutathione s-transferase, chitinase, respectively. These expressed genes are involved in disease-resistant signal transduction, antioxidative stress, transcriptional regulation, photosynthesis, physiological processes. The research established a basis for cloning stress resistance genes and further studying genes expression in Capsicum annuum L. seedlings under P.capsici stress.

Key words: Phytophthora capsici, suppression subtractive hybridization, disease-resistant, pepper, expressed sequence tags

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