干旱胁迫下柠条锦鸡儿叶片SSH文库构建及CkWRKY1基因克隆

doi:10.11707/j.1001-7488.20130709

林业科学 ›› 2013, Vol. 49 ›› Issue (7): 62-68.doi: 10.11707/j.1001-7488.20130709

干旱胁迫下柠条锦鸡儿叶片SSH文库构建及CkWRKY1基因克隆

杨杞¹, 张涛¹, 王颖¹, 李高^1,2, 尹佳佳¹, 韩晓敏¹, 齐力旺³, 李国婧¹, 王瑞刚¹

1. 内蒙古农业大学生命科学学院呼和浩特 010018;
2. 北京市颐和园管理处北京 100091;
3. 中国林业科学研究院林业研究所北京 100091

收稿日期:2012-10-09 修回日期:2013-02-21 出版日期:2013-07-25 发布日期:2013-07-23
通讯作者: 王瑞刚
基金资助:
教育部博士学科点基金项目(20111515110001);内蒙古农业大学科技创新团队培育计划(NDPYTD2010-3)。

Construction of a Suppression Subtractive Hybridization Library of Caragana korshinskii Under Drought Stress and Cloning of CkWRKY 1 Gene

Yang Qi¹, Zhang Tao¹, Wang Ying¹, Li Gao^1,2, Yin Jiajia¹, Han Xiaomin¹, Qi Liwang³, Li Guojing¹, Wang Ruigang¹

1. College of Life Sciences, Inner Mongolia Agricultural University Hohhot 010018;
2. Beijing Summer Palace Management Office Beijing 100091;
3. Research Institute of Forestry, CAF Beijing 100091

Received:2012-10-09 Revised:2013-02-21 Online:2013-07-25 Published:2013-07-23

摘要/Abstract

摘要：

为研究柠条锦鸡儿抗旱的分子机制,挖掘干旱响应相关基因,成功构建干旱胁迫下柠条锦鸡儿叶片的抑制性差减杂交(SSH)文库,获得1 286条高质量的EST序列,平均长度475 bp,拼接得到Unigenes 645条。对这些Unigenes进行Blast比对注释后发现很多抗旱相关基因,例如LEA蛋白、DHN蛋白编码基因,MYB、bZIP、WRKY、NAC和bHLH转录因子等。通过RACE技术克隆其中1个WRKY转录因子的cDNA全长,命名为CkWRKY 1,GenBank 登录号为JX987095。CkWRKY 1 编码330个氨基酸的蛋白质,氨基酸序列中有1个"WRKYGQK"的WRKY转录因子家族保守序列,属于第Ⅱ类WRKY转录因子。利用实时荧光定量PCR技术检测发现,CkWRKY 1 基因在干旱处理后mRNA的表达水平明显上升,预示该转录因子可能与柠条锦鸡儿抗旱的分子机制相关。

关键词: 柠条锦鸡儿, 干旱胁迫, 抑制性差减杂交(SSH)文库, WRKY, 基因克隆

Abstract:

Caragana korshinskii is an important cultivated shrub with high ecological and forage value, and widely distributes in Northwest China. C. korshinskii has a strong tolerance to drought, cold, heat, saline and poor soil. In order to study its resistance mechanisms to drought and explore for drought response related genes, a suppression subtractive hybridization (SSH) library of C. korshinskii under drought stress was constructed. Totally 1 286 ESTs from the SSH library were obtained and the average length was 475 bp. As many as 645 Unigenes were identified through the EST sequence assembly. Many drought-related genes were annotated by Blasting, such as the genes encoding dehydration related LEA and DHN proteins as well as transcription factors including MYB, bZIP, WRKY, NAC, and bHLH families. A candidate gene encoding a member of WRKY transcription factor family was cloned by rapid amplification of cDNA ends technique, and was named as CkWRKY 1 (GenBank accession No. JX987095). The protein deduced from the cDNA was composed from 330 amino acids with a conserved region "WRKYGQK". Real-time-quantitative PCR analysis showed that drought stress enhanced CkWRKY 1 expression at the transcriptional level, indicating that CkWRKY 1 might be involved in the drought resistance mechanisms of C. korshinskii at molecular level.

Key words: Caragana korshinskii, drought stress, suppression subtractive hybridization (SSH) library, WRKY, gene cloning

中图分类号:

S718.46

杨杞, 张涛, 王颖, 李高, 尹佳佳, 韩晓敏, 齐力旺, 李国婧, 王瑞刚. 干旱胁迫下柠条锦鸡儿叶片SSH文库构建及CkWRKY1基因克隆[J]. 林业科学, 2013, 49(7): 62-68.

Yang Qi, Zhang Tao, Wang Ying, Li Gao, Yin Jiajia, Han Xiaomin, Qi Liwang, Li Guojing, Wang Ruigang. Construction of a Suppression Subtractive Hybridization Library of Caragana korshinskii Under Drought Stress and Cloning of CkWRKY 1 Gene[J]. Scientia Silvae Sinicae, 2013, 49(7): 62-68.

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干旱胁迫下柠条锦鸡儿叶片SSH文库构建及CkWRKY1基因克隆

Construction of a Suppression Subtractive Hybridization Library of Caragana korshinskii Under Drought Stress and Cloning of CkWRKY 1 Gene

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