本文關(guān)鍵詞：茶樹bZIP家族基因的非生物脅迫響應(yīng)及C亞家族CsbZIP6和CsbZIP4的功能初步分析　出處：《中國農(nóng)業(yè)科學(xué)院》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： CsbZIP轉(zhuǎn)錄因子 非生物脅迫 表達譜芯片 茶樹 擬南芥轉(zhuǎn)基因

【摘要】：研究植物如何響應(yīng)逆境脅迫一直是植物學(xué)研究領(lǐng)域的重要課題。堿性亮氨酸拉鏈b ZIP蛋白是真核生物中分布最廣泛且最保守的一類蛋白,已有研究表明其在植物的生長發(fā)育及逆境脅迫響應(yīng)中具有重要的作用。本論文在前期克隆11個Csb ZIP基因的基礎(chǔ)上開展,繼續(xù)對茶樹b ZIP基因進行克隆,采用熒光定量PCR檢測它們的組織表達特異性以及不同非生物脅迫下在茶樹葉片和根中的表達模式,并對Csb ZIP6和Csb ZIP4的抗逆功能進行了擬南芥過表達驗證研究。并本論文主要結(jié)果有以下幾方面:1、茶樹Csb ZIP基因的的克隆與生物信息學(xué)分析再次克隆了7個茶樹b ZIP基因,分別命名為Csb ZIP12-Csb ZIP18,分析結(jié)果表明,18個Csb ZIPs蛋白都含有典型的b ZIP結(jié)構(gòu)域,通過與擬南芥b ZIP家族75個成員氨基酸序列構(gòu)建發(fā)育樹及MEME預(yù)測保守結(jié)構(gòu)域分析,將18個Csb ZIP蛋白歸類為10個亞家族(A、B、C、D、E、F、H、I、S和K亞家族)。2、組織特異性表達和非生物脅迫響應(yīng)分析1)組織特異性表達結(jié)果顯示,在四種組織中,Csb ZIP18的表達量是所有基因中最低的;在不同組織間,Csb ZIP12、Csb ZIP14和Csb ZIP18這3個基因在根中的表達量相對較高,另外三種組織中的表達較低;相反,Csb ZIP16在根中的表達量極低;其余基因在這四種組織間的表達量相差不大。2)茶樹水培苗在低溫、ABA、鹽和干旱四種脅迫處理下的time-course實驗,檢測18個Csb ZIP基因在0,1,3,9,24,72和120 h 7個采樣點的葉片和根的定量表達。結(jié)果顯示,絕大部分Csb ZIP基因都對這幾種脅迫有響應(yīng),從誘導(dǎo)強度來看,Csb ZIP16、Csb ZIP17和Csb ZIP18在這幾種脅迫下表達量相對較大。3、Csb ZIP6負調(diào)控擬南芥的低溫響應(yīng)1)亞細胞定位顯示Csb ZIP6蛋白定位于細胞核。克隆獲得啟動子序列2 201 bp,發(fā)現(xiàn)含多個低溫脅迫響應(yīng)元件如ABRE、CBFHV、LTRE等。2)ABA敏感性:過表達Csb ZIP6增強轉(zhuǎn)基因擬南芥萌發(fā)時期對ABA的敏感性。3)低溫響應(yīng):過表達Csb ZIP6轉(zhuǎn)基因擬南芥幼苗凍傷情況比野生型嚴重,轉(zhuǎn)基因株系的存活率降低,相對電導(dǎo)率增大,可溶性糖含量降低,丙二醛含量升高,這些指標都間接說明了過表達Csb ZIP6降低擬南芥轉(zhuǎn)基因植株的抗凍性�；蛐酒治霭l(fā)現(xiàn),GO通路主要富集在響應(yīng)低溫、水分脅迫、糖代謝等過程;參與低溫和滲透相關(guān)的基因大多都是下調(diào)表達,如COR413IM1、DREB1A/CBF3、KIN1和LEA4-5等,而低溫響應(yīng)途徑中起負調(diào)控作用的MYB15以及對低溫敏感的基因BT5和DIN10上調(diào)表達,以及糖相關(guān)差異表達基因APL3、APL4和SBE2.2下調(diào)。4、Csb ZIP4正調(diào)控擬南芥的耐鹽響應(yīng)亞細胞定位顯示Csb ZIP4蛋白定位于細胞核。過表達Csb ZIP4提高了轉(zhuǎn)基因擬南芥株系在種子萌發(fā)階段的耐鹽性,降低了ABA敏感性。對Csb ZIP4轉(zhuǎn)基因幼苗進行鹽脅迫處理,轉(zhuǎn)基因株系的SPAD值顯著比野生型高,鹽脅迫響應(yīng)基因SOS1在轉(zhuǎn)基因植株中的表達量上調(diào),轉(zhuǎn)基因擬南芥的耐鹽性提高。
[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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S571.1

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