棉花转录因子GhWRKY23基因的分离及功能分析

发布时间：2018-05-29 23:48

  本文选题：棉花 + GhWRKY23　； 参考：《山东农业大学》2016年硕士论文

【摘要】：棉花在其生长发育过程中经常遭受各种逆境胁迫,包括生物胁迫(黄萎病菌、枯萎病菌、立枯病菌和植食性昆虫咬食等)和非生物胁迫(干旱、高盐等),对棉花的品质和产量造成一定的不利影响。培育抗病抗逆新品种是解决棉花病害问题的有效途径,而这依赖于其抗病抗逆分子机制的研究。信号转导和转录水平的基因表达调控在植物抵御外界胁迫中起着重要作用。转录因子,如DREB、NAC、WRKY、MYB等,在响应逆境胁迫反应中起重要的调节作用。分离、鉴定参与逆境胁迫应答的转录因子并开展其功能研究是揭示植物抗病及抗逆分子机制的重要途径。鉴于此,本研究以陆地棉为材料,利用同源克隆与RACE PCR的方法分离得到一个II类WRKY基因,同时对该基因进行了序列特征分析、表达特性分析及生物学功能鉴定,主要结果如下:(1)序列分析表明,GhWRKY23 cDNA全长1194bp,包括121bp的5'UTR,125bp的3'UTR和948bp的开放阅读框。该ORF编码一个含有315个氨基酸的多肽,预测其分子量约为35.6kDa,等电点为5.94。基因组序列分析、同源序列比对和蛋白聚类分析表明,GhWRKY23属于IIc类WRKY蛋白转录因子。(2)亚细胞定位分析表明GhWRKY23定位在细胞核中,推测该基因在细胞核中发挥一定的生物学功能。(3)qRT-PCR分析棉花幼苗中GhWRKY23基因的表达模式,结果表明干旱、机械损伤和氧化胁迫能上调GhWRKY23的表达,而高盐、青枯劳尔氏菌和立枯丝核菌则抑制它的表达。此外,一些病程相关信号分子如SA、MeJA、ABA、ET在不同程度上诱导了GhWRKY23的表达。以上结果表明GhWRKY23能够响应多种非生物和生物胁迫,并可能受多种信号分子的调节,初步推测其可能在防卫反应中发挥重要作用。(4)构建GhWRKY23正义植物表达载体,通过农杆菌侵染的方法获得稳定表达的转基因本生烟植株。对超表达植株的种子进行萌发实验和根长统计,结果表明GhWRKY23对SA和ET敏感,对ABA有一定的抗性。推测该基因能参与到SA、ABA和ET介导的信号转导途径中。(5)GhWRKY23超表达植株离体叶片接种烟草青枯劳尔氏菌、丁香假单胞菌番茄致病变种和立枯丝核菌后,相对野生型植株表现出明显的感病症状。DAB染色和台盼蓝染色结果进一步表明超表达植株中积累了更多ROS并产生了更多坏死组织,MDA含量测定结果说明超表达植株细胞膜损伤情况更严重。同时用MV模拟氧化胁迫,在转基因的叶片中观察到更多的活性氧积累。推测该基因可能在抵御病原菌侵染中起到负调控作用,并可能参与到氧化胁迫响应中。(6)利用qRT-PCR技术,对接种病原菌后前后SA、ABA、JA、ET信号通路中抗病相关基因及其他病程相关基因的表达量进行检测,发现超表达植株中的有些基因表达量较野生型植株有明显的下调趋势,推测GhWRKY23可能通过抑制SA、JA/ET信号途径中相关基因的表达来负调控抗病反应。(7)利用qRT-PCR分析病原菌侵染前后活性氧产生及清除相关基因的表达量,结果显示超表达GhWRKY23能抑制活性氧产生基因的表达,也能抑制活性氧清除相关基因的表达,可能在抗氧化系统中起负调节作用。
[Abstract]:Cotton is often subjected to various stresses during its growth and development, including biological stress (Huang Wei, Fusarium wilt, Rhizoctonia and phytophagous insect bite, etc.) and abiotic stress (drought, high salt, etc.), which cause certain adverse effects on the quality and yield of cotton, and the cultivation of new varieties of resistance to disease and inverse is a solution to the problem of cotton disease. An effective way, which depends on the study of its anti disease and anti inverse molecular mechanisms. The regulation of gene expression at signal transduction and transcription levels plays an important role in resisting external stress. Transcription factors, such as DREB, NAC, WRKY, MYB, play an important regulatory role in response to stress responses. In this study, a II class WRKY gene was isolated from the homologous clones and RACE PCR, and the sequence characteristics of the gene, the analysis of the expression characteristics and the identification of the biological functions were analyzed. The results are as follows: (1) sequence analysis shows that GhWRKY23 cDNA is full length 1194bp, including 121bp 5'UTR, 125bp 3'UTR and 948bp open reading frame. The ORF encodes a polypeptide containing 315 amino acids, and predicts its molecular weight is about 35.6kDa, the isoelectric point is the 5.94. genomic sequence analysis, homologous sequence alignment and protein cluster analysis show GhWRKY23 genera IIc WRKY protein transcription factors. (2) subcellular localization analysis showed that GhWRKY23 was located in the nucleus, and that the gene played a certain biological function in the nucleus. (3) the expression pattern of GhWRKY23 gene in cotton seedlings was analyzed by qRT-PCR. The results showed that drought, mechanical damage and oxidative stress could increase the expression of GhWRKY23, and high salt, In addition, some signal molecules such as SA, MeJA, ABA, and ET induced the expression of GhWRKY23 to varying degrees. The above results indicate that GhWRKY23 can respond to a variety of abiotic and biological stresses, and may be regulated by a variety of signal molecules and preliminarily presumed that it may be in defense against a variety of signal molecules. It plays an important role. (4) construct the GhWRKY23 expression vector of just plant and obtain the transgenic transgenic tobacco plants by the method of Agrobacterium infection. The germination experiment and the root length statistics of the super expressed plants show that GhWRKY23 is sensitive to SA and ET and has certain resistance to ABA. It is speculated that the gene can be involved in SA, ABA In the signal transduction pathway mediated by ET. (5) GhWRKY23 overexpressed plant leaves inoculated with lalstonia Solanum, Pseudomonas Syringa and Rhizoctonia Rhizoctonia, the relative wild plants showed obvious symptoms of.DAB staining and trypan blue staining, which further indicated that more ROS was accumulated in the overexpressed plant. More necrotic tissues were produced, and the results of MDA content showed that the cell membrane damage of the overexpressed plant was more serious. At the same time, more active oxygen accumulation was observed in the transgenic leaves with MV simulated oxidative stress. It is presumed that the gene may play a negative regulatory role in resisting the infection of the pathogenic bacteria and may be involved in the response to oxidative stress. (6) using qRT-PCR technology to detect the expression of resistance related genes and other disease related genes in SA, ABA, JA, ET signaling pathways after inoculation of pathogenic bacteria, and found that some genes in the overexpressed plants have a obvious downward trend of downregulation than those of wild type plants. It is presumed that GhWRKY23 may be through the inhibition of SA and JA/ET signal pathways. The expression of related genes negatively regulates the resistance to disease. (7) qRT-PCR is used to analyze the production of reactive oxygen species and the expression of related genes before and after infection. The results show that overexpression of GhWRKY23 can inhibit the expression of reactive oxygen generation genes and inhibit the expression of reactive oxygen scavenging genes, which may be negatively regulated in the antioxidant system. Use.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2;S562
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本文编号：1952991