發(fā)布時(shí)間：2018-04-15 23:19

  本文選題：茶樹 + CsICE1��； 參考：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：茶樹是我國(guó)重要的經(jīng)濟(jì)作物之一,持續(xù)的干旱、低溫等非生物脅迫都會(huì)影響茶葉的產(chǎn)量和品質(zhì),為了篩選茶樹與抗旱性相關(guān)的重要基因并進(jìn)一步解析失水逆境信號(hào)轉(zhuǎn)導(dǎo)調(diào)控的分子網(wǎng)絡(luò),本研究通過(guò)茶樹鮮葉離體失水轉(zhuǎn)錄組測(cè)序分析,鎖定了ABA信號(hào)轉(zhuǎn)導(dǎo)通路中的重要蛋白激酶SnRK2s以及多胺代謝關(guān)鍵酶精氨酸脫羧酶(ADC),并進(jìn)一步通過(guò)酵母雙雜交檢測(cè)二者分別與轉(zhuǎn)錄因子CsICE1的互作關(guān)系,同時(shí)采用qRT-PCR測(cè)定了離體失水條件下,不同茶樹品種中以上基因的表達(dá)情況,得到的主要研究結(jié)果如下:1、通過(guò)茶樹鮮葉離體失水轉(zhuǎn)錄組數(shù)據(jù)分析發(fā)現(xiàn),獲得的測(cè)序數(shù)據(jù)拼接質(zhì)量較高,clean data共計(jì)104.12 G,unigene共計(jì)233131個(gè)。其中值得關(guān)注的是,差異基因KEGG富集分析發(fā)現(xiàn):ABA信號(hào)轉(zhuǎn)導(dǎo)通路相關(guān)基因在茶樹鮮葉離體失水3 h、12 h和24 h時(shí)均顯著上調(diào)表達(dá)。2、在“鄂茶1號(hào)”茶樹品種中,克隆獲得CsICE1、CsSnRK2.1、CsSnRK2.6、CsSnRK2.8和CsADC 5個(gè)基因的cDNA序列,生物信息學(xué)分析結(jié)果表明:5個(gè)基因的ORF分別為1587 bp、1077 bp、1027 bp、1035 bp和2163 bp;編碼氨基酸數(shù)目分別為:528、358、342、344和720;疏水性和跨模性分析結(jié)果表明:這5個(gè)基因編碼的蛋白質(zhì)全部都為親水性蛋白質(zhì),且均不跨膜;系統(tǒng)進(jìn)化樹表明,CsSnRK2的3個(gè)成員分別屬于CsSnRK2亞家族的三組;CsADC與番木瓜CpADC的同源性較高;多序列比對(duì)分析和蛋白質(zhì)結(jié)構(gòu)域預(yù)測(cè)分析結(jié)果表明:CsICE1包含bHLH結(jié)構(gòu)域,CsSnRK2s包含Ser/Thr結(jié)構(gòu)域,CsADC包含Orn-DAP-Arg-deC結(jié)構(gòu)域和Orn-DAP-Arg-2吡哆醛-脯氨酸結(jié)合位點(diǎn);蛋白質(zhì)二級(jí)結(jié)構(gòu)的結(jié)果表明:CsICE1、CsSnRK2s及CsADC均由ɑ-螺旋、延伸鏈、β-轉(zhuǎn)角和無(wú)規(guī)則卷曲構(gòu)成;磷酸化位點(diǎn)分析表明:CsICE1、CsSnRK2s及Cs ADC均具有多處Ser、Thr和Tyr磷酸化位點(diǎn);亞細(xì)胞定位在線預(yù)測(cè)分析結(jié)果表明:CsSnRK2s主要存在于細(xì)胞質(zhì)和細(xì)胞核,CsICE1主要存在于細(xì)胞核,CsADC主要存在于細(xì)胞質(zhì)。3、酵母雙雜交實(shí)驗(yàn)結(jié)果表明:轉(zhuǎn)錄因子CsICE1全長(zhǎng)具有自激活性,短截片段CsICE1-73和CsICE1-146均不具有自激活性,且與CsSnRK2.6和CsADC均不發(fā)生互作。4、相對(duì)表達(dá)量分析結(jié)果表明:CsSnRK2.1、CsSnRK2.8、CsICE1和CsADC在4個(gè)品種中經(jīng)脫水脅迫處理后,其上調(diào)基本不超過(guò)3倍,下調(diào)不低于原來(lái)的1/3。但CsSnRK2.6的相對(duì)表達(dá)量在黔輻4號(hào)中上調(diào)最為明顯,脫水脅迫處理3 h后,CsSnRK2.6的表達(dá)量就上調(diào)超過(guò)了5倍,脫水脅迫處理9 h時(shí),其表達(dá)量就上調(diào)超過(guò)了16倍,但在脫水脅迫處理12 h后,其表達(dá)量呈下調(diào)趨勢(shì),且下調(diào)量不明顯,而在鄂茶10號(hào)、紫娟和鄂茶1號(hào)中,CsSnRK2.6的上下調(diào)表達(dá)量基本不太明顯。
[Abstract]:Tea is one of the most important cash crops in China. Continuous drought, low temperature and other abiotic stresses will affect the yield and quality of tea.In order to screen important genes related to drought resistance of tea plants and to further analyze the molecular network of signal transduction regulation of water loss stress, in this study, in vitro water loss transcriptome sequencing of fresh tea leaves was carried out.The important protein kinase SnRK2s and arginine decarboxylase of polyamine metabolism were locked in the ABA signal transduction pathway, and the interaction between them and transcription factor CsICE1 was detected by yeast two-hybrid.At the same time, qRT-PCR was used to determine the expression of the above genes in different tea varieties under the condition of water loss in vitro. The main results were as follows: 1.The sequence data obtained were of high quality and clean data, the total number of which was 233131, the total number of 104.12 GfU Unigene was 233131.Among them, the KEGG enrichment analysis of differentially expressed genes showed that the genes related to the signal transduction pathway of KEGG were significantly up-regulated at the time of 3 h and 24 h of fresh leaf water loss in tea plant, and were significantly up-regulated in "Echa 1" tea varieties.The cDNA sequences of CsSnRK2.1, CsSnRK2.6, CsSnRK2.8 and CsADC were cloned.The results of bioinformatics analysis showed that the ORF of the five genes were 1587 BP, 1077 BP, 1027 BP and 2163 BP, respectively, the number of amino acids encoded were 528358342344 and 720, respectively. The results of hydrophobic and transmodal analysis showed that all the proteins encoded by the five genes were hydrophilic proteins.The phylogenetic tree showed that the three groups of CsSnRK2 belonging to CsSnRK2 subfamily had high homology with papaya CpADC.The results of multiple sequence alignment analysis and protein domain prediction analysis showed that: CsICE1 contains bHLH domain CsSnRK2s including Ser/Thr domain CsADC contains Orn-DAP-Arg-deC domain and Orn-DAP-Arg-2 pyridoxal-proline binding site.The results of protein secondary structure showed that both CsADC and CsSnRK2s were composed of helix, extension chain, 尾 -rotation angle and irregular curl, and the phosphorylation site analysis showed that the phosphorylation sites of CsICE1CsSnRK2s and Cs ADC were both Serr-Thr and Tyr phosphorylation sites.The results of on-line prediction of subcellular localization showed that: CsSnRK2s mainly existed in cytoplasm and nucleus, CsICE1 mainly existed in cytoplasm, CsADC mainly existed in cytoplasm. Yeast two-hybrid experiment showed that the full length of transcription factor CsICE1 was self-activating.The results of relative expression analysis showed that CsSnRK2.1CsSnRK2.8CsSnRK2.8CsICE1 and CsADC could not be increased by more than three times and down-regulated by less than 1 / 3 after dehydration stress.However, the relative expression of CsSnRK2.6 was up-regulated in Qianfu 4. After 3 h of dehydration stress, the expression of CsSnRK2.6 was increased by more than 5 times, and that of CsSnRK2.6 was more than 16 times at 9 h of dehydration stress.However, after 12 h of dehydration stress, the expression of CsSnRK2.6 was down-regulated, and the down-regulation of CsSnRK2.6 was not obvious in Echa 10, Zijuan and Echa 1.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S571.1

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本文編號(hào)：1756269