本文關(guān)鍵詞： 黃金茶 發(fā)芽早 CsDAM基因 cDNA 啟動子 克隆 序列分析　出處：《湖南農(nóng)業(yè)大學》2016年碩士論文　論文類型：學位論文

【摘要】：茶樹(Camellia sinensis)是一種季節(jié)性很強的多年生經(jīng)濟作物,其茶芽解除休眠的早晚,直接影響到茶葉生產(chǎn)的經(jīng)濟效益。低溫和短日照是影響茶樹芽休眠的兩個主要因素,兩者中任何一個低于臨界值就會啟動芽的休眠。茶樹芽的休眠與解除,是體內(nèi)相關(guān)基因表達調(diào)控變化的結(jié)果。啟動子是基因轉(zhuǎn)錄、蛋白表達調(diào)控的關(guān)鍵,直接影響茶樹生長發(fā)育進程以及茶葉的產(chǎn)量和質(zhì)量。因此從基因轉(zhuǎn)錄、蛋白表達水平研究茶樹休眠與解除機制,對打破茶樹休眠、提高茶葉產(chǎn)量與質(zhì)量、培育出芽早的茶樹新品種具有重要科學意義。黃金茶是一個原產(chǎn)于湖南湘西州保靖縣的優(yōu)異茶樹品種資源,發(fā)芽早是其主要優(yōu)異性狀之一,一般較其它茶樹品種出芽早10-15天,但其機理尚不清楚。前期實驗以當?shù)仄贩N湘妃翠為參照,分離到可能與黃金茶發(fā)芽早相關(guān)的候選基因Dormancy Associated MADS-box (CsDAM2)。為此,本研究采用現(xiàn)代分子生物學技術(shù),對CsDAM2基因全長cDNA及其啟動子進行了克隆與序列分析,以期為該基因后續(xù)的功能分析及轉(zhuǎn)基因植株的建立提供科學依據(jù)。主要研究結(jié)果如下：(1)以改良Tri-Reagent法提取黃金茶的RNA為原料,成功克隆了CsDAM2基因全長cDNA序列,序列分析表明,該序列長度為1386 bp,共編碼218個氨基酸,包含一個完整的編碼區(qū)657 bp；CsDAM2基因編碼蛋白的分子量為24.88 KDa,分子式為C1075H1779N323O341S6,理論等電點PI值為8.96,Leu、Glu、Gln、Ser、Arg、Ala為其中含量較多的氨基酸,與毛白楊、可可樹、咖啡樹、甘薯、棉花相似性分別為71%、68%、61%、57%、49%,與茶樹MADS-box蛋白相似性為35%；通過軟件預測分析表明,CsDAM2蛋白具有1個磷酸化位點,屬于親水性的非分泌蛋白,蛋白肽鏈中螺旋占53%,無規(guī)則卷曲占39%,折疊占8%。(2)以黃金茶一號幼嫩葉為原料,采用改進的CTAB法提取總DNA,根據(jù)CsDAM2基因cDNA序列設(shè)計特異性引物,利用染色體步移技術(shù)對黃金茶基因啟動子進行克隆擴增,通過與pMDTM19-T Vector構(gòu)建真核表達載體及E.coli DH5a大腸桿菌的轉(zhuǎn)化,獲得一條長度為478 bp的CsDAM2啟動子片段序列。生物信息學分析表明,該序列A/T含量為71.13%,符合植物啟動子高A/T含量的特征。PlantCare和PLACE在線軟件分析表明,CsDAM2基因啟動子含有多種順式作用元件,其中包括：G-box、I-box、 GAG-motif、MNF1等與光響應相關(guān)的作用元件,脫落酸響應元件ABREs赤霉素響應元件P-box等激素應激相關(guān)元件,熱應激調(diào)控相關(guān)元件HSE,防御與脅迫響應元件TC-rich repeats以及部分未命名、功能特異或未知的其他順式作用元件等。
[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.
【學位授予單位】：湖南農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S571.1

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