摘要
WRKY转录因子是植物中最大的转录因子家族之一,在各种生物和非生物胁迫响应及多种生长发育过程中发挥着关键作用。用PCR技术克隆McWRKY16基因并进行生物信息学分析,通过实时荧光定量PCR技术分析McWRKY16基因在白粉病菌胁迫下的表达方式。序列分析表明,McWRKY16基因开放阅读框1 007 bp,编码295个氨基酸,其编码蛋白分子量约为32 371.45 kD,理论等电点pl为8.92;蛋白序列中α-螺旋、β-转角、无规则卷曲、延伸链分别占32.2%、1.69%、55.25%、10.85%;McWRKY16蛋白不含信号肽序列,属非跨膜蛋白,预测其亚细胞定位于细胞核;启动子预测结果表明,该基因含有多个响应光照及与生长发育、激素以及胁迫反应相关的顺式调控元件;序列对比与系统进化树分析结果显示,蛋白序列中该基因蛋白序列中包含2个WRKY保守结构域,锌指结构为C2H2型,属于典型的WRKY基因家族中的Ⅱ类;McWRKY16氨基酸序列与拟南芥的AtWRKY18(NP_567882.1, Arabidopsis thaliana)同源性高达97%,表明McWRKY16蛋白氨基酸序列具有高度保守性,并与黄瓜蛋白CsWRKY33、甜瓜蛋白CmWRKY33等同科植物亲缘关系最近;与0 h(接种白粉菌前)相比,McWRKY16基因的相对表达量在接种白粉菌后均呈上调趋势。本次克隆的McWRKY16基因属于典型的WRKY家族成员,推测该基因在苦瓜中可能参与多种胁迫反应及激素应答,可能在应对白粉病菌侵染过程中发挥正调控作用,为苦瓜抗白粉病育种提供了潜在的基因资源。
WRKY transcription factor is one of the largest transcription factor families in plants. It plays a key role in various biological and abiotic stress responses and various growth and development processes. McWRKY16gene was cloned by PCR and analyzed by bioinformatics. The expression mode of McWRKY16 gene under powdery mildew stress was analyzed by real-time fluorescence quantitative PCR. Sequence analysis showed that the open reading frame of McWRKY16 gene was 1 007 bp and encoded 295 amino acids. The molecular weight of the encoded protein was about 32 371.45 kD and the theoretical isoelectric point pl was 8.92;in protein sequence α-β-turn, irregular curl and extended chain accounted for 32.2%, 1.69%, 55.25% and 10.85% respectively;McWRKY16protein does not contain signal peptide sequence and belongs to non transmembrane protein. It is predicted that its subcellular localization is in the nucleus. Promoter prediction showed that the gene contained multiple cis regulatory elements in response to light and related to growth and development, hormones and stress response;sequence comparison and phylogenetic tree analysis showed that the protein sequence of the gene contained two WRKY conserved domains, and the zinc finger structure was C2H2type, belonging to class II in the typical WRKY gene family.The amino acid sequence of McWRKY16 has 97% homology with AtWRKY18(NP_567882.1, Arabidopsis thaliana)of Arabidopsis thaliana, indicating that the amino acid sequence of McWRKY16 protein is highly conservative and has the closest genetic relationship with cucumber protein Cs WRKY33 and melon protein CmWRKY33;Compared with 0 h(before powdery mildew inoculation), the relative expression of McWRKY16 gene increased after powdery mildew inoculation. The cloned gene belongs to a typical WRKY family member. It is speculated that the gene may be involved in a variety of stress responses and hormone responses in balsam pear. Mcwrky16 gene expression was up-regulated after inoculation with powdery mildew, which may play a positive regulatory role in the process of dealing with powdery mildew, providing potential gene resources for improved varieties of Momordica charantia resistant to powdery mildew.
作者
马丽荣
商桑
田丽波
范晨伟
杨怡
Ma Lirong;Shang Sang;Tian Libo;Fan Chenwei;Yang Yi(Key Laboratory for Quality Regulation of Tropical Horticultural Crops of Hainan Province,Horticulture College of Hainan University,Haikou,570228;College of Life Sciences of Hainan University,Haikou,570228)
出处
《分子植物育种》
CAS
北大核心
2022年第24期8051-8063,共13页
Molecular Plant Breeding
基金
国家自然科学基金项目(32060682)
海南省自然科学基金高层次人才项目(2019RC045,2019RC150)
海南省重大科技计划项目(ZDKJ2021010)共同资助。
