基于茶树鲜叶离体失水转录组的CsSnRK2s和CsADC基因鉴定与功能分析

发布时间：2018-04-15 23:19

本文选题：茶树 + CsICE1　；参考：《华中农业大学》2017年硕士论文

【摘要】：茶树是我国重要的经济作物之一,持续的干旱、低温等非生物胁迫都会影响茶叶的产量和品质,为了筛选茶树与抗旱性相关的重要基因并进一步解析失水逆境信号转导调控的分子网络,本研究通过茶树鲜叶离体失水转录组测序分析,锁定了ABA信号转导通路中的重要蛋白激酶SnRK2s以及多胺代谢关键酶精氨酸脱羧酶(ADC),并进一步通过酵母双杂交检测二者分别与转录因子CsICE1的互作关系,同时采用qRT-PCR测定了离体失水条件下,不同茶树品种中以上基因的表达情况,得到的主要研究结果如下:1、通过茶树鲜叶离体失水转录组数据分析发现,获得的测序数据拼接质量较高,clean data共计104.12 G,unigene共计233131个。其中值得关注的是,差异基因KEGG富集分析发现:ABA信号转导通路相关基因在茶树鲜叶离体失水3 h、12 h和24 h时均显著上调表达。2、在“鄂茶1号”茶树品种中,克隆获得CsICE1、CsSnRK2.1、CsSnRK2.6、CsSnRK2.8和CsADC 5个基因的cDNA序列,生物信息学分析结果表明:5个基因的ORF分别为1587 bp、1077 bp、1027 bp、1035 bp和2163 bp;编码氨基酸数目分别为:528、358、342、344和720;疏水性和跨模性分析结果表明:这5个基因编码的蛋白质全部都为亲水性蛋白质,且均不跨膜;系统进化树表明,CsSnRK2的3个成员分别属于CsSnRK2亚家族的三组;CsADC与番木瓜CpADC的同源性较高;多序列比对分析和蛋白质结构域预测分析结果表明:CsICE1包含bHLH结构域,CsSnRK2s包含Ser/Thr结构域,CsADC包含Orn-DAP-Arg-deC结构域和Orn-DAP-Arg-2吡哆醛-脯氨酸结合位点;蛋白质二级结构的结果表明:CsICE1、CsSnRK2s及CsADC均由ɑ-螺旋、延伸链、β-转角和无规则卷曲构成;磷酸化位点分析表明:CsICE1、CsSnRK2s及Cs ADC均具有多处Ser、Thr和Tyr磷酸化位点;亚细胞定位在线预测分析结果表明:CsSnRK2s主要存在于细胞质和细胞核,CsICE1主要存在于细胞核,CsADC主要存在于细胞质。3、酵母双杂交实验结果表明:转录因子CsICE1全长具有自激活性,短截片段CsICE1-73和CsICE1-146均不具有自激活性,且与CsSnRK2.6和CsADC均不发生互作。4、相对表达量分析结果表明:CsSnRK2.1、CsSnRK2.8、CsICE1和CsADC在4个品种中经脱水胁迫处理后,其上调基本不超过3倍,下调不低于原来的1/3。但CsSnRK2.6的相对表达量在黔辐4号中上调最为明显,脱水胁迫处理3 h后,CsSnRK2.6的表达量就上调超过了5倍,脱水胁迫处理9 h时,其表达量就上调超过了16倍,但在脱水胁迫处理12 h后,其表达量呈下调趋势,且下调量不明显,而在鄂茶10号、紫娟和鄂茶1号中,CsSnRK2.6的上下调表达量基本不太明显。
[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.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S571.1

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