青枯菌诱导的马铃薯转录因子类StWRKY8基因的克隆及表达分析

发布时间：2018-05-18 19:05

本文选题：马铃薯 + 转录因子　；参考：《山西师范大学》2016年硕士论文

【摘要】：马铃薯一直以来都是全球性的重要农作物,对于人类具有重要的经济、食用价值,而其产量、质量受青枯病害的严重制约。针对青枯病害,至今尚无化学药剂能有效防控,故可以从植物抗病方面来做研究,以期发现更多的防卫相关基因并了解其功能发挥及机制特点,为预防植物病害提供些许理论依据。本文分别采用伤根灌菌法、5’-RACE(5’末端快速扩增)法对实验材料进行接种和基因序列的克隆。从接种了青枯菌(Ralstonia solanacearum)的马铃薯中薯3号(Solanum tuberosum)中获得了类StWRKY8基因的cDNA序列,该序列长度为563 bp,其中包含一个长度为258bp的完整开放阅读框,可编码85个氨基酸。类StWRKY8基因序列内部具有一个典型的WRKYGQK保守结构域。C2H2为其锌指结构类型,因此隶属于WRKY家族中的第二类。经由邻接法系统进化树的构建,分析其氨基酸序列发现与茄科内的(Solanaceae)其他成员具有较高程度的同源性,甚至与马铃薯转录因子StWRKY8相似性高达98%,故命名为类StWRKY8。而类StWRKY8相关理化性质、等电点等则通过运用相应的生物信息学软件来预测:等电点大约为9.10,半衰期大于5.6 h,属于亲水性蛋白,且亲水性较高。三维结构为非球型,含有3个磷酸化位点,其中两个为Ser磷酸化位点,其中一个为Tyr磷酸化位点。亚细胞定位发现其属于胞内蛋白。经青枯菌诱导后,两种不同材料中类StWRKY8基因的定量分析运用RT-PCR和qRT-PCR来进行,结果显示:类StWRKY8受青枯菌诱导后表达量上调,并且在感、抗病两基因型马铃薯中表达有较明显的差异。类StWRKY8基因在抗病基因型马铃薯接种青枯菌6 h内就能高强度的表达,发挥其抗性,而在感病基因型马铃薯接种青枯菌6 h之内的表达较为缓慢,强度也较低,不能在侵染之初很好地抗病。利用地高辛原位杂交手段对目的基因的表达进行组织定位后显示:类StWRKY8基因的表达主要集中在茎叶组织中,即有维管束、韧皮部的维管束系统中,这说明其表达具有明显的组织特异性。经过分析,总结:类StWRKY8基因具有植物转录因子的典型结构特征,能起到一定的抗病防卫作用,受青枯菌等病原菌的诱导而表达,与此同时,也受寄主植物固有的抗性影响,因此在感、抗病基因型中表达模式区别很大。这也支持所推测的观点,即转录因子类StWRKY8基因作为防卫基因,在植物防御抗病等反应中发挥一定作用并参与机体调控。
[Abstract]:Potato is an important crop all over the world. It has important economic and edible value for human being, but its yield and quality are seriously restricted by bacterial wilt. There is no chemical agent that can effectively control bacterial wilt, so it can be studied from the aspect of plant disease resistance, in order to find more defense-related genes and understand their function and mechanism. To provide some theoretical basis for the prevention of plant diseases. In this paper, the method of rapid amplification of 5'end of 5 'RACE-RACE was used to inoculate the experimental material and clone the gene sequence. The cDNA sequence of StWRKY8 like gene was obtained from potato (Solanum tuberosumum) inoculated with Ralstonia solanacearum. The sequence was 563 BP in length and contained a complete open reading frame with length of 258bp, which could encode 85 amino acids. There is a typical conserved WRKYGQK domain. C2H2 is the zinc-finger structural type within the StWRKY8 gene sequence, so it belongs to the second kind of WRKY family. By constructing the phylogenetic tree, the amino acid sequence was found to have high homology with other members of Solanaceae in Solanaceae, and the similarity with potato transcription factor StWRKY8 was as high as 98%, so it was named StWRKY8. The StWRKY8 related physicochemical properties and isoelectric point were predicted by using the corresponding bioinformatics software: the isoelectric point was about 9.10, the half-life was more than 5.6 h, and it was a hydrophilic protein with high hydrophilicity. The three-dimensional structure is non-spherical and contains three phosphorylation sites, two of which are Ser phosphorylation sites, one of which is Tyr phosphorylation site. Subcellular localization revealed that it belonged to intracellular protein. The quantitative analysis of StWRKY8 gene in two different materials was carried out by RT-PCR and qRT-PCR. The results showed that the expression of StWRKY8 was up-regulated and the expression of StWRKY8 was sensitive. There were significant differences in the expression of resistant potato genotypes. The expression of StWRKY8 like gene was high intensity in resistant potato inoculated with bacterial wilt bacteria within 6 h, but slow and low in susceptible potato inoculated with bacterial wilt bacteria within 6 h. The disease cannot be well diseased at the beginning of an infection. The expression of target gene by digoxin in situ hybridization showed that the expression of StWRKY8 gene was mainly concentrated in stem and leaf tissues, that is, vascular bundle and phloem vascular bundle system. This indicated that its expression had obvious tissue specificity. After analysis, it was concluded that the StWRKY8 like gene had the typical structural characteristics of plant transcription factors, and could play a certain role in disease resistance and defense. It was expressed by pathogens such as bacterial wilt, and was also affected by the inherent resistance of host plants. Therefore, the expression patterns in susceptible and disease-resistant genotypes vary greatly. This also supports the hypothetical view that transcription factor StWRKY8 gene, as a defense gene, plays a certain role in plant defense and disease resistance responses and participates in organism regulation.
【学位授予单位】：山西师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S435.32

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