本文關(guān)鍵詞： CDPK F-box 表達(dá)特性 原生質(zhì)體轉(zhuǎn)化 耐鹽性　出處：《西北農(nóng)林科技大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：胡楊(Populus euphratica)是我國西北沙漠唯一能成林的高大喬木樹種,對(duì)鹽堿、干旱、極端溫度等抵抗能力較強(qiáng),是研究林木抗逆機(jī)理的候選模式樹種。因此,通過研究胡楊耐鹽基因的功能對(duì)于揭示樹木耐鹽性分子機(jī)理具有重要意義。其中CDPK基因作為第二信使的鈣離子是高等植物細(xì)胞中普遍存在的信號(hào)分子,幾乎介導(dǎo)了植物生長發(fā)育和逆境信號(hào)轉(zhuǎn)導(dǎo)的所有反應(yīng)。F-box蛋白在泛素-蛋白酶體途徑中能夠特異識(shí)別底物蛋白,調(diào)節(jié)多種激素的信號(hào)轉(zhuǎn)導(dǎo)途徑,并響應(yīng)多種非生物脅迫,參與植物抗逆途徑。本論文通過對(duì)收集的胡楊天然種質(zhì)資源全基因組深度重測序并進(jìn)行耐鹽性狀的GWAS分析,篩選出耐鹽候選基因CDPK、F-box及未知功能新基因,并通過克隆、表達(dá)特性分析、亞細(xì)胞定位、轉(zhuǎn)化擬南芥和轉(zhuǎn)化胡楊原生質(zhì)體的耐鹽性測定,探究胡楊CDPK、F-box及未知功能新基因的功能特性,為深入解析胡楊的耐鹽分子機(jī)理奠定了基礎(chǔ)。主要研究結(jié)果如下:胡楊CDPK基因全長822bp,編碼273個(gè)氨基酸。qRT-PCR結(jié)果表明胡楊CDPK基因在各組織中均有表達(dá),無組織特異性,其表達(dá)受鹽、高溫、低溫、干旱和ABA脅迫誘導(dǎo)。CDPK啟動(dòng)子表達(dá)模式分析表明,兩周的擬南芥幼苗,其葉脈,下胚軸與根部連接處和根尖處表達(dá)量高。PEG介導(dǎo)的胡楊原生質(zhì)體瞬時(shí)表達(dá)發(fā)現(xiàn)CDPK蛋白在細(xì)胞質(zhì)和細(xì)胞核中都表達(dá)。擬南芥種子發(fā)芽率實(shí)驗(yàn)證實(shí)超量表達(dá)CDPK基因能夠提高擬南芥種子的耐鹽性。胡楊F-box基因全長1086 bp,編碼361個(gè)氨基酸,含有一個(gè)F-box domain和一個(gè)F-box associated domain。qRT-PCR結(jié)果表明胡楊F-box基因在各組織中均有表達(dá),無組織特異性,其表達(dá)受鹽、高溫、低溫、干旱和ABA脅迫誘導(dǎo)。PEG介導(dǎo)的胡楊原生質(zhì)體瞬時(shí)表達(dá)發(fā)現(xiàn)F-box蛋白定位于細(xì)胞核上。流式細(xì)胞分析技術(shù)證實(shí)超量表達(dá)F-box基因能夠提高胡楊原生質(zhì)體的耐鹽性。胡楊未知新基因全長519 bp,編碼172個(gè)氨基酸;qRT-PCR結(jié)果表明胡楊未知新基因在各組織中均有表達(dá),無組織特異性,其表達(dá)受鹽、高溫、低溫、干旱和ABA脅迫誘導(dǎo)。PEG介導(dǎo)的胡楊原生質(zhì)體瞬時(shí)表達(dá)發(fā)現(xiàn)未知新蛋白定位于細(xì)胞質(zhì)上。擬南芥種子發(fā)芽率實(shí)驗(yàn)證實(shí)超量表達(dá)未知新基因能夠提高擬南芥種子的耐鹽性。研究結(jié)果初步揭示了胡楊CDPK、F-box及未知新基因的表達(dá)特性和耐鹽性,為進(jìn)一步解析胡楊CDPK、F-box及未知新基因的功能及胡楊抗逆分子機(jī)理奠定基礎(chǔ)。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S792.11

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