棉花脱水素GhDHN1的克隆及其表达  

Molecular Clone and Expression of GhDHN1 Gene in Cotton(Gossypium hirsutum L.)

作　　者：王俊娟[1] 穆敏[1] 王帅[1] 陆许可[1] 陈修贵[1] 王德龙[1] 樊伟丽[1] 阴祖军[1] 郭丽雪[1] 叶武威[1] 喻树迅[1] 

机构地区：[1]中国农业科学院棉花研究所/棉花生物学国家重点实验室/农业部棉花遗传改良重点开放实验室,河南安阳455000

出　　处：《中国农业科学》2016年第15期2867-2878,共12页Scientia Agricultura Sinica

基　　金：河南省基础与前沿技术研究计划项目(142300413232)

摘　　要：【目的】通过对棉花脱水素基因结构特征及其在低温胁迫下表达模式进行分析,探讨脱水素在棉花响应低温过程中的功能,为棉花抗冷育种提供理论基础。【方法】以棉花抗冷品种豫2067为试验材料,根据棉花陆地棉基因组序列查找已知脱水素(dehydrin,Dhn)基因的CDS序列,利用Primer5软件设计引物,克隆该基因,并命名为GhDHN1;采用生物信息学方法分析其蛋白质性质、氨基酸含量特征、功能结构域、系统进化树;选择XbaⅠ和SmaⅠ酶切位点对植物表达载体p BI121::GFP进行双酶切,采用In-Fusion连接技术构建融合蛋白瞬时表达载体p BI121-GhDHN1::GFP;分析其在洋葱表皮细胞中的瞬时表达,进行亚细胞定位;利用抗冷材料豫2067在三叶期对低温处理(4℃,24 h)前后的叶片和根系进行转录组测序,筛选差异表达基因;在三叶期时分别对豫2067(抗冷品种)和衡棉3号(冷敏感品种)进行低温(4℃,24 h)处理,利用实时荧光定量方法分别比较根、茎、叶中GhDHN1表达量,对该基因在2个抗冷差异材料叶中的表达量进行比较;对豫2067进行不同时间低温(4℃)处理,分析GhDHN1在叶片和根中的动态表达模式。【结果】该基因全长为726 bp,开放阅读编码框为636 bp,编码211个氨基酸,预测分子量为23.79 k D,等电点为5.04,富含谷氨酸(26.10%)和赖氨酸(19.40%),不含色氨酸,半衰期为30 h,蛋白呈酸性,带负带荷,带负电荷的残基总数为60%;GhDHN1的二级结构α螺旋(Alpha helix)包含116个氨基酸残基,占54.98%,组成该蛋白的主体结构,无规则卷曲(Random coil)的氨基酸残基有87个;GhDHN1位于陆地棉D亚组第9染色体(Dt_chr9)上,在cDNA的259—348位置上含有一个长度为90 bp的内含子,2个外显子长度分别为258和378 bp;SMART和CDD分析表明该氨基酸序列含有2个保守的富含赖氨酸的K片段和1个保守的富含丝氨酸S片段,具有亲水素蛋白结构域pfam00257,表明该蛋白为K_2S型脱水素;�【Objective】In order to explore functional genes related to low temperature stress tolerance of cotton, the characteristics of cotton dehydrin gene and its expression patterns responsed to low temperature stress in cotton were analyzed, thus providing a theoretical basis for the application of dehydrin gene in cotton chilling tolerant breeding. 【Method】 In this study, based on the upland cotton genome sequence, specific primers were designed by Primer 5 software and the dehydrin gene was cloned from the upland cotton variety Yu2067, named GhDHN1. Bioinformatics analysis was conducted to analyze the properties, amino acids content, functional domains and evolutionary relationships of the gene. Plant expression vector p BI121：：GFP at XbaⅠand SmaⅠrestriction sites was constructed with double enzyme digestions and the transient expression vector p BI121-GhDHN1：：GFP was constructed by In-Fusion connection technology. And the subcellular localization of GhDHN1 was studied by transient expression analysis of onion epidermal cells. Combined with the transcriptome sequencing data of chilling-resistant cotton variety Yu2067, real-time fluorescent quantitative PCR expression of leaves, stems, and roots in chilling-resistant Yu2067 and chilling-sensitive variety Hengmian No.3 under low temperature stress treatments（4℃, 24 h） at trefoil stage was performed to study the function of GhDHN1. The expression of the gene in the leaves of two different cold resistant varieties was compared. The dynamic expression of GhDHN1 gene in leaves and roots of Yu2067 was detected under 4 low temperature treatment. ℃ 【Result】 A cotton dehydrin gene was cloned and the sequencing analysis showed that the cDNA of the dehydrin gene was 726 bp, and the gene encoded 211 amino acids with a predicted molecular weight of about 23.79 k D and the isoelectric point was 5.04. Amino acid sequence analysis indicated that the GhDHN1 was rich in glutamic acid content（26.10%） and lysine amino acid content（19.40%） with a ha

关 键 词：棉花 脱水素 亚细胞定位 低温胁迫 转录组分析 实时荧光定量PCR 

分 类 号：S562[农业科学—作物学]