茶树bZIP家族基因的非生物胁迫响应及C亚家族CsbZIP6和CsbZIP4的功能初步分析

发布时间：2018-01-17 14:04

本文关键词：茶树bZIP家族基因的非生物胁迫响应及C亚家族CsbZIP6和CsbZIP4的功能初步分析　出处：《中国农业科学院》2016年博士论文　论文类型：学位论文

【摘要】：研究植物如何响应逆境胁迫一直是植物学研究领域的重要课题。碱性亮氨酸拉链b ZIP蛋白是真核生物中分布最广泛且最保守的一类蛋白,已有研究表明其在植物的生长发育及逆境胁迫响应中具有重要的作用。本论文在前期克隆11个Csb ZIP基因的基础上开展,继续对茶树b ZIP基因进行克隆,采用荧光定量PCR检测它们的组织表达特异性以及不同非生物胁迫下在茶树叶片和根中的表达模式,并对Csb ZIP6和Csb ZIP4的抗逆功能进行了拟南芥过表达验证研究。并本论文主要结果有以下几方面:1、茶树Csb ZIP基因的的克隆与生物信息学分析再次克隆了7个茶树b ZIP基因,分别命名为Csb ZIP12-Csb ZIP18,分析结果表明,18个Csb ZIPs蛋白都含有典型的b ZIP结构域,通过与拟南芥b ZIP家族75个成员氨基酸序列构建发育树及MEME预测保守结构域分析,将18个Csb ZIP蛋白归类为10个亚家族(A、B、C、D、E、F、H、I、S和K亚家族)。2、组织特异性表达和非生物胁迫响应分析1)组织特异性表达结果显示,在四种组织中,Csb ZIP18的表达量是所有基因中最低的;在不同组织间,Csb ZIP12、Csb ZIP14和Csb ZIP18这3个基因在根中的表达量相对较高,另外三种组织中的表达较低;相反,Csb ZIP16在根中的表达量极低;其余基因在这四种组织间的表达量相差不大。2)茶树水培苗在低温、ABA、盐和干旱四种胁迫处理下的time-course实验,检测18个Csb ZIP基因在0,1,3,9,24,72和120 h 7个采样点的叶片和根的定量表达。结果显示,绝大部分Csb ZIP基因都对这几种胁迫有响应,从诱导强度来看,Csb ZIP16、Csb ZIP17和Csb ZIP18在这几种胁迫下表达量相对较大。3、Csb ZIP6负调控拟南芥的低温响应1)亚细胞定位显示Csb ZIP6蛋白定位于细胞核。克隆获得启动子序列2 201 bp,发现含多个低温胁迫响应元件如ABRE、CBFHV、LTRE等。2)ABA敏感性:过表达Csb ZIP6增强转基因拟南芥萌发时期对ABA的敏感性。3)低温响应:过表达Csb ZIP6转基因拟南芥幼苗冻伤情况比野生型严重,转基因株系的存活率降低,相对电导率增大,可溶性糖含量降低,丙二醛含量升高,这些指标都间接说明了过表达Csb ZIP6降低拟南芥转基因植株的抗冻性。基因芯片分析发现,GO通路主要富集在响应低温、水分胁迫、糖代谢等过程;参与低温和渗透相关的基因大多都是下调表达,如COR413IM1、DREB1A/CBF3、KIN1和LEA4-5等,而低温响应途径中起负调控作用的MYB15以及对低温敏感的基因BT5和DIN10上调表达,以及糖相关差异表达基因APL3、APL4和SBE2.2下调。4、Csb ZIP4正调控拟南芥的耐盐响应亚细胞定位显示Csb ZIP4蛋白定位于细胞核。过表达Csb ZIP4提高了转基因拟南芥株系在种子萌发阶段的耐盐性,降低了ABA敏感性。对Csb ZIP4转基因幼苗进行盐胁迫处理,转基因株系的SPAD值显著比野生型高,盐胁迫响应基因SOS1在转基因植株中的表达量上调,转基因拟南芥的耐盐性提高。
[Abstract]:It has been an important subject in botany to study how plants respond to stress. The basic leucine zipper b ZIP protein is the most widely distributed and conserved class of proteins in eukaryotes. It has been shown that it plays an important role in plant growth and stress response. In this paper, 11 Csb ZIP genes were cloned. The b ZIP gene of tea plant was cloned and its expression specificity was detected by fluorescence quantitative PCR. The expression patterns in leaves and roots of tea plants under different abiotic stress were detected. The overexpression of Arabidopsis thaliana (Arabidopsis thaliana) was verified by Csb ZIP6 and Csb ZIP4. The main results of this paper are as follows: 1. The cloning and bioinformatics analysis of the Csb ZIP gene of tea plant re-cloned 7 b ZIP genes of tea tree, respectively named Csb ZIP12-Csb ZIP18. The results showed that 18 Csb ZIPs proteins contained a typical b ZIP domain. By constructing developmental tree with 75 amino acid sequences of b ZIP family of Arabidopsis thaliana and analyzing the conserved domain of MEME prediction, 18 Csb ZIP proteins were classified into 10 subfamilies. The results of tissue specific expression and abiotic stress response analysis showed that there were four kinds of tissues. The expression of Csb ZIP18 was the lowest among all genes. The expression of CSB ZIP12CSB ZIP14 and Csb ZIP18 in different tissues was relatively high in root, and low in the other three tissues. On the contrary, the expression of CSB ZIP16 in root was very low. The expression of other genes in the four tissues was not different. 2) the time-course experiment of tea seedlings under four stress treatments: low temperature, salt and drought. The quantitative expression of 18 Csb ZIP genes in leaves and roots of 7 sampling sites were detected. Most of the Csb ZIP genes were responsive to these stresses, and CSB ZIP16 could be seen from the induction intensity. The expression of Csb ZIP17 and Csb ZIP18 was relatively large under these stresses. The subcellular localization of Csb ZIP6 negatively regulated Arabidopsis thaliana showed that the Csb ZIP6 protein was located in the nucleus. The promoter sequence was 2201 BP. It was found that there were many low temperature stress response elements such as ABREE CBFHV. LTRE et al. 2ABA sensitivity: overexpression of Csb ZIP6 enhances sensitivity to ABA in transgenic Arabidopsis thaliana during germination. 3). Response to low temperature: frostbite of transgenic Arabidopsis thaliana seedlings with overexpression of Csb ZIP6 was more serious than that of wild type. The survival rate of transgenic lines decreased, the relative conductivity increased, the content of soluble sugar decreased, and the content of malondialdehyde increased. These results indicated that overexpression of Csb ZIP6 could reduce the freezing resistance of Arabidopsis thaliana transgenic plants. Gene chip analysis showed that the GGO pathway was mainly enriched in response to low temperature and water stress. Glucose metabolism; Most of the genes involved in hypothermia and osmosis are down-regulated, such as Cor413IM1 / DREB1A / CBF3KIN1 and LEA4-5. However, MYB15, which plays a negative role in the response to hypothermia, and BT5 and DIN10, which are sensitive to hypothermia, can up-regulate the expression, as well as the differentially expressed gene APL3, which is related to glucose. APL4 and SBE2.2 down-regulated 4. 4. Csb ZIP4 is regulating salt-tolerant subcellular localization of Arabidopsis thaliana. Csb ZIP4 protein is located in nucleus. Overexpression of Csb. ZIP4 improved the salt tolerance of transgenic Arabidopsis during seed germination. The sensitivity of ABA was reduced. The SPAD value of transgenic lines of Csb ZIP4 was significantly higher than that of wild-type seedlings under salt stress. The expression of salt stress response gene SOS1 was up-regulated in transgenic plants and salt tolerance of transgenic Arabidopsis thaliana was improved.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S571.1

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