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作物学报 ›› 2010, Vol. 36 ›› Issue (12): 2035-2044.doi: 10.3724/SP.J.1006.2010.02035

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

干旱胁迫下棉花SSH文库构建及其抗旱相关基因分析

王德龙,叶武威*,王俊娟,宋丽艳,樊伟丽,崔宇鹏   

  1. 中国农业科学院棉花研究所 / 农业部棉花遗传改良重点开放实验室，河南安阳 455000
  • 收稿日期:2010-05-18 修回日期:2010-08-01 出版日期:2010-12-12 网络出版日期:2010-10-09
  • 通讯作者: 叶武威，E-mail:yeww@cricaas.com.cn;Tel:0372-2562283
  • 基金资助:

    本研究由国家农作物转基因生物新品种培育重大专项(2008ZX08005-004)和中央级公益性科研院所基本科研业务专项。

Construction of SSH Library and Its Analyses of Cotton Drought Associated Genes under Drought Stress

WANG De-Long,YE Wu-Wei*,WANG Jun-Juan,SONG Li-Yan,FAN Wei-Li,CUI Yu-Peng   

  1. Cotton Research Institute, Chinese Academy of Agricultural Sciences / Key Laboratory for Cotton Genetic Improvement, Ministry of Agriculture, Anyang 455000, China
  • Received:2010-05-18 Revised:2010-08-01 Published:2010-12-12 Published online:2010-10-09
  • Contact: YE Wu-Wei，E-mail:yeww@cricaas.com.cn;Tel:0372-2562283

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被引次数

39

摘要： 以耐旱自交系邯郸177为材料，利用抑制性差减杂交技术(SSH)，构建棉花苗期叶片的正向差减文库。挑取300个阳性克隆进行PCR验证，并对验证后的单克隆进行测序和分析，共获得284个有效序列。聚类后得到202条uniESTs序列，其中174条singlets，28条contigs。经过BlastN分析，156个unigene可以在GenBank中找到同源序列，46个unigene未能找到同源匹配。经BlastX分析，40个unigene与未知功能蛋白或假定蛋白有较高相似性，116条unigene与已知功能蛋白有较高同源性。用KOBAS系统将33个unigene定位到55个Pathways中，其中P值小于0.5的Pathway有23条。初步分析发现, 丙酮酸盐代谢(pyruvate metabolism)途径、乙醛酸和二羧酸代谢(glyoxylate and dicarboxylate metabolism)途径与棉花抗旱相关性较大。这些unigene基因涉及信号传导、能量代谢、蛋白质代谢、核酸代谢、光合作用及膜运输等代谢过程。发现了苹果酸合成酶基因(Ms1, 001_B03; Ms2, 003_E04)、苹果酸脱氢酶基因(Md1, 001_C12; Md2, 002_F01)；NAC(001_C08)、锌指蛋白(zfp, 003_C06)、BZR1/BES1(003_G04)等转录调节因子，以及翻译控制肿瘤蛋白基因(TCTP，002_C04)等耐旱相关基因。

关键词: 干旱胁迫, SSH, unigene, 耐旱

Abstract: A forward cDNA-SSH library was established by suppression subtractive hybridization using seedling leaf of Handan 177, a drought-tolerant cotton (Gossypium hirsuutm L.) inbred line, among which 300 positive clones were selected for sequencing. After detection by PCR for each clone, each single clone was sequenced. Totally 284 available sequences and 202 uniESTs which 28 were contigs and 174 were singlets were obtained by cluster analyses of the ESTs sequencing. The results of BlastN showed that 156 uniESTs had homologous sequences in GenBank database while the other 46 had no protein homologous. The BlastX results indicated that 116 uniESTs had significant protein homology and 40 uniESTs were unknown proteins and putative proteins. KOBAS mapped 33 ESTs of the 202 uniESTs to 55 KEGG pathways, in which there were 23 pathways at P-value<0.05. This study suggested that there were closely relationships with cotton drought tolerance among pyruvate metabolism, glyoxylate and dicarboxylate metabolism. A large group of drought stress-induced genes were found in the cDNA library, which involved in many metabolism pathways such as signal transduction, energy metabolism, protein metabolism, nucleic acid metabolism, photosynthesis, transmembran. And some genes related to drought tolerance were found, such as malate synthase genes (MS1,0001_C12; MS2, 0002_F01) and malate dehydrogenase genes(Md1, 001_C12; Md2, 002_F01), some transcription factors like NAC(001_C08), BZR1/BES1(003_G04) , zinc finger protein genes (zfp, 003_C06), and the translationally controlled tumor protein gene(TCTP，002_C04).

Key words: Drought-stress, Suppression subtractive hybridization (SSH), unigene, Drought tolerance

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