胡杨CDPK等3个耐盐基因的克隆与功能分析

发布时间：2018-02-27 16:23

本文关键词： CDPK F-box 表达特性原生质体转化耐盐性　出处：《西北农林科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：胡杨(Populus euphratica)是我国西北沙漠唯一能成林的高大乔木树种,对盐碱、干旱、极端温度等抵抗能力较强,是研究林木抗逆机理的候选模式树种。因此,通过研究胡杨耐盐基因的功能对于揭示树木耐盐性分子机理具有重要意义。其中CDPK基因作为第二信使的钙离子是高等植物细胞中普遍存在的信号分子,几乎介导了植物生长发育和逆境信号转导的所有反应。F-box蛋白在泛素-蛋白酶体途径中能够特异识别底物蛋白,调节多种激素的信号转导途径,并响应多种非生物胁迫,参与植物抗逆途径。本论文通过对收集的胡杨天然种质资源全基因组深度重测序并进行耐盐性状的GWAS分析,筛选出耐盐候选基因CDPK、F-box及未知功能新基因,并通过克隆、表达特性分析、亚细胞定位、转化拟南芥和转化胡杨原生质体的耐盐性测定,探究胡杨CDPK、F-box及未知功能新基因的功能特性,为深入解析胡杨的耐盐分子机理奠定了基础。主要研究结果如下:胡杨CDPK基因全长822bp,编码273个氨基酸。qRT-PCR结果表明胡杨CDPK基因在各组织中均有表达,无组织特异性,其表达受盐、高温、低温、干旱和ABA胁迫诱导。CDPK启动子表达模式分析表明,两周的拟南芥幼苗,其叶脉,下胚轴与根部连接处和根尖处表达量高。PEG介导的胡杨原生质体瞬时表达发现CDPK蛋白在细胞质和细胞核中都表达。拟南芥种子发芽率实验证实超量表达CDPK基因能够提高拟南芥种子的耐盐性。胡杨F-box基因全长1086 bp,编码361个氨基酸,含有一个F-box domain和一个F-box associated domain。qRT-PCR结果表明胡杨F-box基因在各组织中均有表达,无组织特异性,其表达受盐、高温、低温、干旱和ABA胁迫诱导。PEG介导的胡杨原生质体瞬时表达发现F-box蛋白定位于细胞核上。流式细胞分析技术证实超量表达F-box基因能够提高胡杨原生质体的耐盐性。胡杨未知新基因全长519 bp,编码172个氨基酸;qRT-PCR结果表明胡杨未知新基因在各组织中均有表达,无组织特异性,其表达受盐、高温、低温、干旱和ABA胁迫诱导。PEG介导的胡杨原生质体瞬时表达发现未知新蛋白定位于细胞质上。拟南芥种子发芽率实验证实超量表达未知新基因能够提高拟南芥种子的耐盐性。研究结果初步揭示了胡杨CDPK、F-box及未知新基因的表达特性和耐盐性,为进一步解析胡杨CDPK、F-box及未知新基因的功能及胡杨抗逆分子机理奠定基础。
[Abstract]:Populus euphratica is the only tall tree species in the northwest desert of China, which has strong resistance to salt and alkali, drought and extreme temperature, and is a candidate tree species for studying the mechanism of forest stress resistance. It is important to study the function of salt-tolerant genes in Populus euphratica to reveal the molecular mechanism of salt tolerance in trees. The calcium ion of CDPK gene, as the second messenger, is a common signal molecule in higher plant cells. Almost all responses to plant growth and development and stress signal transduction. F-box proteins can specifically recognize substrate proteins in the ubiquitin proteasome pathway, regulate signal transduction pathways of many hormones, and respond to many abiotic stresses. This paper screened the candidate gene CDPKF box and new genes of unknown function by deep genome resequencing and GWAS analysis of salt-tolerant traits in collected natural resources of Populus euphratica. Expression characteristic analysis, subcellular localization, salt-tolerance test of transformed Arabidopsis thaliana and transformed Populus euphratica protoplasts, to explore the functional characteristics of CDPKK F-box and new genes of unknown function of Populus euphratica. The main results are as follows: the CDPK gene of Populus euphratica is 822 BP in length and encodes 273 amino acids. QRT-PCR results show that the CDPK gene of Populus euphratica is expressed in all tissues and has no tissue specificity, and its expression is salt-tolerant. The expression pattern of CDPK promoter in Arabidopsis thaliana seedlings induced by high temperature, low temperature, drought and ABA stress was analyzed. The transient expression of CDPK protein in cytoplasm and nucleus of Populus euphratica was found to be high in Hypocotyl and root junction and root tip. The results of seed germination rate test of Arabidopsis thaliana showed that the overexpression of CDPK gene was able to express CDPK gene in the cytoplasm and nucleus of Populus euphratica. To improve the salt tolerance of Arabidopsis thaliana seeds, the F-box gene of Populus euphratica was 1086 BP in length, encoding 361 amino acids. The results of F-box domain and F-box associated domain.qRT-PCR showed that the F-box gene of Populus euphratica was expressed in all tissues and had no tissue specificity. It was expressed in salt, high temperature and low temperature. The transient expression of P.euphratica protoplasts mediated by drought and ABA stress showed that the F-box protein was located on the nucleus. Flow cytometry showed that the overexpression of F-box gene could improve the salt-tolerance of P. euphratica protoplasts. The total length of the novel gene was 519 BP, encoding 172 amino acids. The results of qRT-PCR showed that the unknown gene of Populus euphratica was expressed in all tissues. No tissue specificity, its expression is salt, high temperature, low temperature, Drought and ABA stress induced. PEG- mediated transient expression of protoplasts of Populus euphratica revealed that unknown new proteins were located in cytoplasm. The germination rate experiment of Arabidopsis thaliana seeds proved that overexpression of unknown new genes could improve salt tolerance of Arabidopsis thaliana seeds. The results revealed the expression and salt tolerance of CDPKF box and unknown genes of Populus euphratica. The results provided a basis for further analysis of the function of CDPKN F-box and new unknown genes and the molecular mechanism of stress resistance of Populus euphratica.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S792.11

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