铁观音茶树SOD家族成员基因克隆及其低温胁迫表达分析

发布时间：2018-03-03 05:17

本文选题：铁观音茶树　切入点：SOD基因克隆　出处：《福建农林大学》2016年硕士论文　论文类型：学位论文

【摘要】：茶树(Camellia sinensis (L.)O.Kuntze)是我国重要的经济作物,性喜温,其在我国种植分布主要受温度的制约。温度影响茶树的种植范围,异常的温度变化往往还严重影响现有茶园的生产秩序和经济效益,因此开展茶树的抗寒性研究和进行茶树的抗寒育种具有重要的理论意义和现实价值。本研究在鉴定铁观音茶树抗寒性的基础上,对茶树SOD基因家族6个成员进行cDNA克隆和部分启动子分离,并对其编码的氨基酸序列进行了生物信息学分析和功能预测。并进一步对SOD基因在不同时长低温胁迫下和不同温度低温胁迫下的表达情况进行分析,为揭示茶树SOD基因低温胁迫响应机制提供理论依据。本文主要研究结果如下：1铁观音茶树低温半致死温度为-5.8℃通过使用叶绿素荧光参数Fv/Fm结合Logistic方程鉴定铁观音茶树的低温半致死温度为-5.8℃。2茶树SOD基因克隆及基因结构克隆得到茶树SOD基因家族6个成员基因,CsCSD1、CsCSD2、CsCSD3、 CsFSD1、CsFSD3和CsMSD1基因,各个成员ORF全长分别为459bp、636bp、 471bp、903bp、813bp和693bp,分别编码152、211、156、300、270、230个氨基酸。基因结构分析发现,CsCSD1基因含有5个内含子,CsCSD2基因不含内含子,CsCSD3基因含有6个内含子,CsFSD3基因含有7个内含子。不同成员间基因结构的差异,可能影响逆境胁迫下各自的表达调控机制。3茶树SOD蛋白生物信息学分析生物信息学分析结果表明,CsCSD1、CsCSD2、CsCSD3、CsFSD1、CsFSD3和CsMSD1分别定位于细胞质、叶绿体、过氧化物酶体、叶绿体、叶绿体和线粒体中。茶树CsCSD二级结构具有极低的α-螺旋和较高的无规则卷曲结构的特征,CsFSD和CsMSD二级结构中均含有较高程度的α-螺旋和较低程度的β-转角。进化树分为2个大的分支,CuZnSOD和FeMnSOD分别在不同的分支,各个成员进一步分在不同的6个小分支中。含不同辅基金属离子的SOD进化距离较远,含相同辅基金属离子的SOD进化距离较近。不同SOD成员可能通过不同亚细胞定位进行分工清除各细胞器内多余的活性氧,并共同协调维持细胞内的活性氧平衡。4茶树SOD基因在低温胁迫下的表达分析茶树SOD基因在低温胁迫下分成三种类型的表达模式。CsCSD1为一类,在低温胁迫1d和6d时出现两个表达高峰,低温胁迫2d、4d时,其表达不断下调；CsCSD2和CsFSD3为一类,在逆境胁迫1d时,表达量迅速上调,胁迫继续持续到2d、4d、6d时,表达出现大幅度的持续下调；CsCSD3、CsFSD1和CsMSD1为一类,在低温胁迫1d时,其表达量均上调,低温胁迫持续到2d、4d时,表达出现缓慢下调,当低温胁迫继续进行时,其表达再次出现上调,但相对表达量仍低于2d时的表达量。5茶树CsCSD1基因启动子克隆及分析茶树CsCSD1基因的启动子作用元件中存在低温信号转导ABA依赖途径相关的脱落酸响应元件ABRE,而不存在低温信号转导CBF依赖途径所需要的CRT/DRE元件。表明茶树CsCSD1基因在低温胁迫下的低温信号转导是通过ABA依赖途径完成。CsCSD1启动子区域存在CpG岛,低温胁迫后期CsCSD1基因出现大幅度上调表达可能是通过CpG岛去甲基化进行调控。
[Abstract]:Tea plant (Camellia sinensis (L.) O.Kuntze) is an important economic crop in China, thermophilic, restricting the cultivated in China mainly by the distribution of temperature. Temperature affected the tea planting range, abnormal temperature change often has a serious impact on the tea production order and economic benefits, so to carry out cold tea breeding study and tea has important theoretical significance and practical value. Based on the identification of cold resistance of Tieguanyin tea, tea of 6 members of SOD gene family of partial cDNA cloning and isolation of promoter, and its encoding amino acid sequence by bioinformatics analysis and function prediction and further on. Expression of SOD gene in different time and different temperature under low temperature stress under low temperature stress were analyzed to reveal the tea SOD gene in response to cold stress mechanism to provide the theoretical basis for the The main results are as follows: 1 Tieguanyin tea semilethal temperature of -5.8 by using chlorophyll fluorescence parameters of Fv/Fm combined with Logistic equation and identification of Tieguanyin tea plants semilethal temperature of -5.8.2 tea SOD gene cloning and gene structure of cloned tea SOD gene family 6 member gene, CsCSD1, CsCSD2, CsCSD3, CsFSD1, CsFSD3 each member of the ORF and CsMSD1 genes, the full-length 459bp respectively, 636bp, 471bp, 903bp, 813bp and 693bp respectively, encoding 152211156300270230 amino acids. The analysis showed that the gene structure of CsCSD1 gene contains 5 introns, CsCSD2 gene intron of CsCSD3 gene contains 6 introns. The CsFSD3 gene contains 7 introns. The difference of the genetic structure of different members, may affect the analysis of bioinformatics analysis of node stress their expression and regulation mechanism of tea.3 SOD protein by Bioinformatics The results show that CsCSD1, CsCSD2, CsCSD3, CsFSD1, CsFSD3 and CsMSD1 were located in the cytoplasm, chloroplast, chloroplast, chloroplast peroxisomes and mitochondria. The random coil structure characteristics of tea CsCSD two grade structure has a very low alpha helix and higher, with high degree of alpha helix were CsFSD and CsMSD two in the primary structure and the low degree of p-turns. Phylogenetic tree is divided into 2 major branches, CuZnSOD and FeMnSOD respectively in different branches, each member is further divided in 6 different branches. SOD evolutionary distance with different metal ion cofactor is SOD cofactor containing the same evolutionary distance metal ions are close to different members of the SOD division may remove excess ROS in various organelles through different subcellular localization, and coordinate to maintain the intracellular expression of.4 SOD gene of tea plant active oxygen balance under low temperature stress points Analysis of SOD gene in tea plant under low temperature stress is divided into.CsCSD1 expression patterns of three types for a class, appear in the low temperature stress for 1D and 6D two expression peaks, 4D 2D, low temperature stress, the expression of CsCSD2 and CsFSD3 decreased continuously; as a group, under the stress of 1D, the expression of the rapid rise, stress continue to 2D, 4D, 6D, the expression of large amplitude continues to decline; CsCSD3, CsFSD1 and CsMSD1 as a class of low temperature stress on 1D, its expression increased, low temperature stress lasted for 2D, 4D, expression of slow down, when the low temperature stress to proceed, the expression again hike, but the relative expression level of.5 gene promoter CsCSD1 tea cold signal transduction ABA dependent pathways related to abscisic acid responsive element ABRE promoter element sub cloning and analysis of CsCSD1 gene expression in tea is still lower than 2D, and there is no signal transduction at low temperature CB The CRT/DRE element of the F dependent pathway is needed. Low temperature signal transduction of CsCSD1 gene in tea plant under low temperature stress is through ABA dependent pathway to complete the.CsCSD1 promoter CpG Island promoter region, low temperature stress of late CsCSD1 gene expression may be a big increase is regulated through CpG island to a base.

【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S571.1

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