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 CkWRKY1Gene

作　　者：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] 

机构地区：[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

出　　处：《林业科学》2013年第7期62-68,共7页Scientia Silvae Sinicae

基　　金：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)

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1286条高质量的EST序列,平均长度475bp,拼接得到Unigenes645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CkWRKY1,GenBank登录号为JX987095。CkWRKY1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,CkWRKY1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。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 CkWRKY1 （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 CkWRKY1 expression at the transcriptional level, indicating that CkWRKY1 might be involved in the drought resistance mechanisms of C. korshinskii at molecular level.

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 

分 类 号：S718.46[农业科学—林学]