黄金茶CsDAM2基因及启动子克隆与序列分析

发布时间：2018-02-12 03:15

本文关键词： 黄金茶发芽早 CsDAM基因 cDNA 启动子克隆序列分析　出处：《湖南农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：茶树(Camellia sinensis)是一种季节性很强的多年生经济作物,其茶芽解除休眠的早晚,直接影响到茶叶生产的经济效益。低温和短日照是影响茶树芽休眠的两个主要因素,两者中任何一个低于临界值就会启动芽的休眠。茶树芽的休眠与解除,是体内相关基因表达调控变化的结果。启动子是基因转录、蛋白表达调控的关键,直接影响茶树生长发育进程以及茶叶的产量和质量。因此从基因转录、蛋白表达水平研究茶树休眠与解除机制,对打破茶树休眠、提高茶叶产量与质量、培育出芽早的茶树新品种具有重要科学意义。黄金茶是一个原产于湖南湘西州保靖县的优异茶树品种资源,发芽早是其主要优异性状之一,一般较其它茶树品种出芽早10-15天,但其机理尚不清楚。前期实验以当地品种湘妃翠为参照,分离到可能与黄金茶发芽早相关的候选基因Dormancy Associated MADS-box (CsDAM2)。为此,本研究采用现代分子生物学技术,对CsDAM2基因全长cDNA及其启动子进行了克隆与序列分析,以期为该基因后续的功能分析及转基因植株的建立提供科学依据。主要研究结果如下：(1)以改良Tri-Reagent法提取黄金茶的RNA为原料,成功克隆了CsDAM2基因全长cDNA序列,序列分析表明,该序列长度为1386 bp,共编码218个氨基酸,包含一个完整的编码区657 bp；CsDAM2基因编码蛋白的分子量为24.88 KDa,分子式为C1075H1779N323O341S6,理论等电点PI值为8.96,Leu、Glu、Gln、Ser、Arg、Ala为其中含量较多的氨基酸,与毛白杨、可可树、咖啡树、甘薯、棉花相似性分别为71%、68%、61%、57%、49%,与茶树MADS-box蛋白相似性为35%；通过软件预测分析表明,CsDAM2蛋白具有1个磷酸化位点,属于亲水性的非分泌蛋白,蛋白肽链中螺旋占53%,无规则卷曲占39%,折叠占8%。(2)以黄金茶一号幼嫩叶为原料,采用改进的CTAB法提取总DNA,根据CsDAM2基因cDNA序列设计特异性引物,利用染色体步移技术对黄金茶基因启动子进行克隆扩增,通过与pMDTM19-T Vector构建真核表达载体及E.coli DH5a大肠杆菌的转化,获得一条长度为478 bp的CsDAM2启动子片段序列。生物信息学分析表明,该序列A/T含量为71.13%,符合植物启动子高A/T含量的特征。PlantCare和PLACE在线软件分析表明,CsDAM2基因启动子含有多种顺式作用元件,其中包括：G-box、I-box、 GAG-motif、MNF1等与光响应相关的作用元件,脱落酸响应元件ABREs赤霉素响应元件P-box等激素应激相关元件,热应激调控相关元件HSE,防御与胁迫响应元件TC-rich repeats以及部分未命名、功能特异或未知的其他顺式作用元件等。
[Abstract]:Camellia sinensis (Camellia sinensis) is a kind of perennial cash crop with strong seasonality, its tea bud is released from dormancy in the morning and evening, which directly affects the economic benefit of tea production. Low temperature and short sunshine are the two main factors that affect the dormancy of tea bud. The dormancy and release of tea bud is the result of changes of gene expression and regulation in vivo. Promoter is the key of gene transcription and protein expression regulation. Therefore, to study the mechanism of dormancy and release of tea plant from gene transcription and protein expression level, to break the dormancy of tea plant and improve the yield and quality of tea, is a direct influence on the growth and development process of tea plant and the yield and quality of tea. It is of great scientific significance to cultivate new tea varieties with early budding. Golden tea is an excellent tea variety resource originating from Baojing County, Xiangxi Prefecture, Hunan Province. Germination early is one of its main excellent characters, and it is usually 10-15 days earlier than that of other tea varieties. However, its mechanism is not clear. The candidate gene Dormancy Associated MADS-box CsDAM2, which may be related to the early germination of golden tea, was isolated from the local variety Xiangfeicui. Therefore, modern molecular biological techniques were used in this study. The full-length cDNA and its promoter of CsDAM2 gene were cloned and sequenced in order to provide scientific basis for the subsequent functional analysis of the gene and the establishment of transgenic plants. The main results are as follows: (1) RNA extracted from gold tea by modified Tri-Reagent method was used as raw material. The full-length cDNA sequence of CsDAM2 gene was cloned successfully. The sequence analysis showed that the sequence length was 1386 BP, encoding 218 amino acids. The molecular weight of the protein encoding a complete coding region of 657bpNCsDAM2 gene is 24.88 KDa.The molecular formula is C1075H1779N323O341S6, and the theoretical isoelectric point Pi value is 8.96% Leugnon Glnsern Sernserine Argendron Ala as the amino acids, and the amino acids of Populus tomentosa, Cocoa, Coffee Tree, Sweet Potato, Sweet Potato, Sweet Potato, Cocoa Tree, Coffee Tree, Sweet Potato, etc. The similarity between cotton and tea MADS-box protein is 35 and 35, respectively. The software prediction analysis shows that the CsDAM2 protein has a phosphorylation site and belongs to hydrophilic non-secretory protein. In the protein peptide chain, 53% were helical, 39% were irregular curl, and 8% were folding. The total CsDAM2 was extracted by modified CTAB method. Specific primers were designed according to the cDNA sequence of CsDAM2 gene. The promoter of golden tea gene was cloned and amplified by chromosome step technique. The eukaryotic expression vector and E. coli DH5a Escherichia coli were constructed with pMDTM19-T Vector. A 478bp CsDAM2 promoter sequence was obtained. Bioinformatics analysis showed that, The A- / T content of the sequence is 71.13, which is consistent with the plant promoter's high A / T content. Plant Care and PLACE online software analysis show that the CsDAM2 gene promoter contains a variety of cis-acting elements, including those related to photoresponse, such as: G-box I-box, GAG-motif MNF1, and so on. Abscisic acid response element ABREs gibberellin response element P-box, heat stress regulation element, defense and stress response element TC-rich repeats, and some unnamed, functionally specific or unknown cis-acting elements, etc.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S571.1

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