棉花蛋白磷酸酶2C60基因（GhPP2C60）的鉴定及生物学功能分析

发布时间：2018-02-08 10:17

本文关键词： 棉花 GhPP2C60 高温病害胁迫脱落酸 JA途径　出处：《山东农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：蛋白质的可逆磷酸化过程是蛋白磷酸酶与蛋白激酶共同合作完成的,通过修饰底物分子实现信号传递,从而对生物体的生长发育及生理生化过程进行调控。大量研究表明,蛋白激酶广泛参与到植物逆境胁迫过程并起到正调控的作用,蛋白磷酸酶与蛋白激酶一样,在植物生理代谢中起到重要作用,但研究相对较少。PP2C是一类丝氨酸/苏氨酸蛋白磷酸酶,在植物中参与逆境胁迫触发的信号传导途径,同时与细胞生长发育、生长周期的调节等过程也有一定相关性。目前对于PP2C家族的报道,大多局限于研究A、B亚族在ABA信号途径中发挥的作用,而对其他亚族中的成员则研究较少。我国是棉花生产大国,在实际生产中,不利的气候条件和病菌的感染会对棉花产量和质量造成严重影响。因此,利用分子生物学手段研究棉花抗逆机制,能够为育种工作奠定坚实的理论基础。在棉花中研究PP2C基因的抗逆功能,可以使棉花更好的适应逆境胁迫,创造更高的经济价值。本研究中,我们从棉花中克隆得到一个PP2C基因GhPP2C60,并对其不同胁迫下的表达模式和生物学功能进行了研究,结果如下:(1)GhPP2C60 cDNA的开放阅读框(ORF)1086bp,推导出一个含362个氨基酸的蛋白序列,DANman预测其分子量及pI值分别为39.8KDa和4.68。氨基酸序列比对结果发现,GhPP2C60基因在进化过程中保守,并且拥有PP2C家族中11个典型的保守催化结构域。聚类分析表明GhPP2C60归属于PP2C家族中I亚族。(2)亚细胞定位实验发现GhPP2C60定位于细胞质中,说明GhPP2C60在细胞质中发挥作用。(3)对GhPP2C60在转录水平与蛋白水平上的表达特性进行研究,发现低温、高温及Pst DC3000病原细菌可以诱导GhPP2C60的表达,脱落酸(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该基因的表达,同时受SA激素处理影响较小,表明GhPP2C60参与多种信号分子介导的抗逆信号转导途径,在植物抵御外界胁迫的防卫反应中发挥作用。(4)利用农杆菌介导的叶盘法转化本生烟,获得GhPP2C60超表达烟草植株。继而进行在ABA处理条件下的种子萌发实验,结果发现GhPP2C60可能参与ABA介导的信号转导途径,促进种子的休眠,增强了种子萌发期对ABA的敏感程度,且影响了气孔的开闭程度。(5)将GhPP2C60超表达烟草植株进行高温及Pst DC3000病原菌胁迫处理,发现超表达GhPP2C60明显增强了植株对高温和病原菌胁迫的敏感性,改变了防卫反应相关基因在处理前后的表达量。进一步实验证明,超表达叶片在处理后积累了更多的活性氧物质,其体内抗氧化酶相关基因的表达量、抗氧化物酶活较野生型有所下降,意味着GhPP2C60参与调节体内ROS的变化。同时,GhPP2C60沉默棉花植株则对高温及Pst DC3000病原细菌胁迫表现为抗性,说明该基因作为负调控因子,参与到植物应对高温及Pst DC3000胁迫的过程。(6)Pst DC3000病原细菌处理后,利用qRT-PCR检测JA途径相关基因的表达模式,发现在野生型与超表达植株中存在显著性差异,这表明GhPP2C60是通过JA途径来调节抗病反应。同时也检测了高温处理后ABA途径相关基因的变化,综合考虑GhPP2C60是通过调控ABA和JA介导的信号转导途径,行使抵御逆境胁迫的功能。
[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.

【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S562

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