枸杞WRKY2基因的克隆及组织表达分析  

Cloning and Tissue Expression Analysis of WRKY2 Gene in Lycium barbarum L.

作　　者：徐惠娟[1,2,3] 杨亚珺 石晶[1,2,3] 郑蕊 王婷婷[1,2,3] 高楚 陈任[1,2,3] 王丽娟 Xu Huijuan;Yang Yajun;Shi Jing;Zheng Rui;Wang Tingting;Gao Chu;Chen Ren;Wang Lijuan(College of Life Science,Ningxia University,Yinchuan,75002 1;WBRPU Lab of National Education Ministry,Yinchuan,75002 l;Key Lab of Modem Molecular Breeding for Dominant and Special Crops in Ningxia,Yinchuan,750021)

机构地区：[1]宁夏大学生命科学学院,银川750021 [2]西部特色生物资源保护与利用教育部重点实验室,银川750021 [3]宁夏优势特色作物现代分子育种重点实验室,银川750021

出　　处：《基因组学与应用生物学》2018年第10期4398-4406,共9页Genomics and Applied Biology

基　　金：国家自然科学基金(31260065,31360361,31401444);宁夏大学自然科学基金(ZR16011)项目共同资助

摘　　要：WRKY是调控果实发育的一类重要转录因子,在植物的生长发育调节、物质代谢调节等生理活动中发挥重要的作用。以宁夏枸杞果实为材料,利用RACE技术克隆枸杞WRKY基因,采用DNAMAN软件分析核酸和氨基酸序列,构建进化树;通过BLASTn和BLASTp进行相似性分析;采用ExPaSy的SOPMA和Phyre 2软件进行蛋白质二级结构预测与3D结构建模;利用qPCR方法检测Lb WRKY2在果实发育不同时期、不同器官及不同组织的表达特性。结果显示：Lb WRKY2基因（GenBank登录号为KU955318）开放阅读框1 055 bp,编码335个氨基酸。Lb WRKY2蛋白二级结构预测发现不规则卷曲（random coil）结构所占比例最大,为43.28%;α螺旋（alpha helix）次之,比例为32.24%;延伸链结构（extended strand） 19.2%;β-转角（beta turn）最少,仅为5.28%。生物信息学分析显示Lb WRKY2蛋白具有70个氨基酸残基组成的信号肽,与烟草（Nicotiana sylvestris XP 009798860.1）相似度74%、马铃薯（Solanum tuberosum XP 006339686.1）相似度74%、芝麻（Sesamum indicum XP 011093555.1）相似度48%。Real time QPCR结果显示,Lb WRKY2在枸杞不同的器官中,表达存在差异,在花中的表达量达到最高值,且枸杞果实形成过程的每一阶段Lb WRKY2都有表达,在果实发育第四阶段表达量最高;Lb WRKY2在果实的不同组织中的表达量趋势为：果皮〉种子〉果肉,以上结果表明枸杞Lb WRKY2调控枸杞果实生长发育。WRKY is an important transcription factor for regulating fruit development, it plays an important role in the physiological and development activities such as plant growth, development and metabolism regulation. RACE technique was used to clone WRKY gene in wolfoerry （Lycium barbarum L.）. The sequencing data were assembled by DNAMAN software. BLASTn and BLASTp in NCBI were used to conduct the similarity analysis. DNAMAN was used to analyze the amino acid sequences and to create the phylogenetic tree. The protein secondary structure and the structure of 3D modeling was predicted by SOPMA and Phyre2 programs. Quantitativereal-time PCR （qRT-PCR） was performed to determine the expression pattern ofLb WRKY2 in different organs, different developmental stages of fruit and different fruit tissue. A full-length cDNA sequence of the homologous WRKY gene was cloned from woltberry. Sequence analysis showed that the Lb WRKY2 gene contains a 1 055 bp open reading frame （ORF） encoding 335 amino acids. The sequence was submitted to GenBank, and the registration number is KU955318. The predictive secondary structure showed that Lb WRKY2 protein was made up of 32.24% alpha-helix, 5.28% beta-turn, 19.2% extended strand and 43.28% random coil. The amino acid sequences shared 48%-74% in homology compared with tabacco （XP 009798860.1）, potato （XP 006339686.1）, sesame （XP 011093555.1）. Subcellular localization assays showed that the Lb 1VRKY2 protein was located in the cytoplasm. The qRT-PCR results suggested that Lb WRKY2 gene showed a high transcription level in flower. The Lb WRKY2 gene could be detected during the whole period of fruit development. Interestingly, Lb WRKY2 gene showed a high transcription level in. stage 4. The expression level in the peel was higher than that in the seed and pulp. The Lb WRKY2 gene takes part in fruit development and growth in wolfoerry. The above results indicate that the Lb WRKY2 gene takes part in fruit development and growth in wolfberry.

关 键 词：枸杞 WRKY 果实发育 表达模式 

分 类 号：S567.19[农业科学—中草药栽培] Q943.2[农业科学—作物学]