本文關(guān)鍵詞： 棉花 GhPP2C60 高溫病害脅迫 脫落酸 JA途徑　出處：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：蛋白質(zhì)的可逆磷酸化過程是蛋白磷酸酶與蛋白激酶共同合作完成的,通過修飾底物分子實現(xiàn)信號傳遞,從而對生物體的生長發(fā)育及生理生化過程進(jìn)行調(diào)控。大量研究表明,蛋白激酶廣泛參與到植物逆境脅迫過程并起到正調(diào)控的作用,蛋白磷酸酶與蛋白激酶一樣,在植物生理代謝中起到重要作用,但研究相對較少。PP2C是一類絲氨酸/蘇氨酸蛋白磷酸酶,在植物中參與逆境脅迫觸發(fā)的信號傳導(dǎo)途徑,同時與細(xì)胞生長發(fā)育、生長周期的調(diào)節(jié)等過程也有一定相關(guān)性。目前對于PP2C家族的報道,大多局限于研究A、B亞族在ABA信號途徑中發(fā)揮的作用,而對其他亞族中的成員則研究較少。我國是棉花生產(chǎn)大國,在實際生產(chǎn)中,不利的氣候條件和病菌的感染會對棉花產(chǎn)量和質(zhì)量造成嚴(yán)重影響。因此,利用分子生物學(xué)手段研究棉花抗逆機(jī)制,能夠為育種工作奠定堅實的理論基礎(chǔ)。在棉花中研究PP2C基因的抗逆功能,可以使棉花更好的適應(yīng)逆境脅迫,創(chuàng)造更高的經(jīng)濟(jì)價值。本研究中,我們從棉花中克隆得到一個PP2C基因GhPP2C60,并對其不同脅迫下的表達(dá)模式和生物學(xué)功能進(jìn)行了研究,結(jié)果如下:(1)GhPP2C60 cDNA的開放閱讀框(ORF)1086bp,推導(dǎo)出一個含362個氨基酸的蛋白序列,DANman預(yù)測其分子量及pI值分別為39.8KDa和4.68。氨基酸序列比對結(jié)果發(fā)現(xiàn),GhPP2C60基因在進(jìn)化過程中保守,并且擁有PP2C家族中11個典型的保守催化結(jié)構(gòu)域。聚類分析表明GhPP2C60歸屬于PP2C家族中I亞族。(2)亞細(xì)胞定位實驗發(fā)現(xiàn)GhPP2C60定位于細(xì)胞質(zhì)中,說明GhPP2C60在細(xì)胞質(zhì)中發(fā)揮作用。(3)對GhPP2C60在轉(zhuǎn)錄水平與蛋白水平上的表達(dá)特性進(jìn)行研究,發(fā)現(xiàn)低溫、高溫及Pst DC3000病原細(xì)菌可以誘導(dǎo)GhPP2C60的表達(dá),脫落酸(abscisic acid,ABA)及茉莉酸甲酯(methyl jasmonate,MeJA)植物激素可以不同程度地抑制該基因的表達(dá),同時受SA激素處理影響較小,表明GhPP2C60參與多種信號分子介導(dǎo)的抗逆信號轉(zhuǎn)導(dǎo)途徑,在植物抵御外界脅迫的防衛(wèi)反應(yīng)中發(fā)揮作用。(4)利用農(nóng)桿菌介導(dǎo)的葉盤法轉(zhuǎn)化本生煙,獲得GhPP2C60超表達(dá)煙草植株。繼而進(jìn)行在ABA處理條件下的種子萌發(fā)實驗,結(jié)果發(fā)現(xiàn)GhPP2C60可能參與ABA介導(dǎo)的信號轉(zhuǎn)導(dǎo)途徑,促進(jìn)種子的休眠,增強(qiáng)了種子萌發(fā)期對ABA的敏感程度,且影響了氣孔的開閉程度。(5)將GhPP2C60超表達(dá)煙草植株進(jìn)行高溫及Pst DC3000病原菌脅迫處理,發(fā)現(xiàn)超表達(dá)GhPP2C60明顯增強(qiáng)了植株對高溫和病原菌脅迫的敏感性,改變了防衛(wèi)反應(yīng)相關(guān)基因在處理前后的表達(dá)量。進(jìn)一步實驗證明,超表達(dá)葉片在處理后積累了更多的活性氧物質(zhì),其體內(nèi)抗氧化酶相關(guān)基因的表達(dá)量、抗氧化物酶活較野生型有所下降,意味著GhPP2C60參與調(diào)節(jié)體內(nèi)ROS的變化。同時,GhPP2C60沉默棉花植株則對高溫及Pst DC3000病原細(xì)菌脅迫表現(xiàn)為抗性,說明該基因作為負(fù)調(diào)控因子,參與到植物應(yīng)對高溫及Pst DC3000脅迫的過程。(6)Pst DC3000病原細(xì)菌處理后,利用qRT-PCR檢測JA途徑相關(guān)基因的表達(dá)模式,發(fā)現(xiàn)在野生型與超表達(dá)植株中存在顯著性差異,這表明GhPP2C60是通過JA途徑來調(diào)節(jié)抗病反應(yīng)。同時也檢測了高溫處理后ABA途徑相關(guān)基因的變化,綜合考慮GhPP2C60是通過調(diào)控ABA和JA介導(dǎo)的信號轉(zhuǎn)導(dǎo)途徑,行使抵御逆境脅迫的功能。
[Abstract]:Reversible phosphorylation of proteins is protein phosphatase and protein kinase cooperate to complete the transfer, to achieve signal by modifying the substrate molecule, thus the biological growth and physiological and biochemical process control. A number of studies have shown that protein kinase is widely involved in plant stress and play a positive regulatory role, protein phosphatase and protein kinase, plays an important role in physiological metabolism, but relatively few studies of.PP2C is a kind of serine / threonine protein phosphatase, in plants involved in stress triggering signal pathway, and cell growth and development, growth regulation process cycle but also has some relevance. The report on the PP2C family. Most of which focused on A, B subfamily play in ABA signaling, and other members of the subfamily is researched less. China is cotton Producing countries, in the actual production, adverse weather conditions and pathogen infection can have serious effects on the yield and quality of cotton. Therefore, the mechanism of resistance of cotton by means of molecular biology, to lay a solid theoretical basis for the breeding work. Research on the inverse function of anti PP2C gene in cotton, cotton can make better to adapt to stress, create greater economic value. In this study, we cloned a PP2C gene GhPP2C60 from cotton, and the different stress expression pattern and biological function were studied. The results are as follows: (1) GhPP2C60 cDNA open reading frame (ORF) of 1086bp, is derived a protein containing 362 amino acids sequence, DANman predicted the molecular weight and the pI value were 39.8KDa and 4.68. amino acid sequence analysis showed that GhPP2C60 gene conserved during evolution, and the PP2C family has 11. Conserved catalytic domain type. Cluster analysis showed that GhPP2C60 belongs to the PP2C family of I subfamily. (2) the subcellular localization experiment showed that GhPP2C60 was localized in cytoplasm, indicating the role of GhPP2C60 in the cytoplasm. (3) study on expression characteristics at the transcriptional level and the protein level of GhPP2C60 was low expression high temperature Pst DC3000 and pathogenic bacteria can induce GhPP2C60, abscisic acid (abscisic, acid, ABA) and methyl jasmonate (methyl jasmonate MeJA) plant hormone can inhibit the gene expression of SA at the same time, affected by the hormone treatment, suggesting that GhPP2C60 is involved in the resistance signal transduction pathway mediated by various signaling molecules. Play a role in the defense response against external stress in plants. (4) the transformation of nicotianabenthamiana using Agrobacterium mediated leaf disc method, GhPP2C60 over expression in tobacco plants. Then under ABA The experiment of seed germination, the results showed that GhPP2C60 may be involved in ABA mediated signal transduction pathway, promote seed dormancy, enhance the sensitivity of seed germination of ABA, and the influence of the degree of stomatal opening and closing. (5) the over expression of GhPP2C60 in tobacco plants to pathogens in DC3000 high temperature and Pst stress treatment, found that super the expression of GhPP2C60 significantly enhanced the sensitivity of plants to high temperature and pathogen stress, changes in the expression of defense related genes before and after treatment. Further experiments show that over expression of ROS accumulation in leaves more in the treatment, the expression of antioxidant enzymes related genes, antioxidant enzyme activity than the wild type to drop means that GhPP2C60 is involved in the regulation of changes in ROS. At the same time, GhPP2C60 is silent cotton plant pathogenic bacteria DC3000 and Pst on the high temperature stress showed that the resistance gene as Negative regulation factor involved in the response to high temperature stress. DC3000 and Pst of plant pathogenic bacteria DC3000 (6) Pst after the treatment, the expression pattern of qRT-PCR by detection of JA gene, found in the wild type and over expression differences of plants, indicating that GhPP2C60 is via JA pathway to regulate the change of resistance response. At the same time to detect the genes related to ABA pathway after high temperature treatment, comprehensive consideration of GhPP2C60 through the signal transduction pathway mediated by ABA and JA, the exercise of resist stress function.

【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S562

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