本文選題：馬鈴薯 + 轉(zhuǎn)錄因子��； 參考：《山西師范大學(xué)》2016年碩士論文

【摘要】：馬鈴薯一直以來都是全球性的重要農(nóng)作物,對于人類具有重要的經(jīng)濟、食用價值,而其產(chǎn)量、質(zhì)量受青枯病害的嚴重制約。針對青枯病害,至今尚無化學(xué)藥劑能有效防控,故可以從植物抗病方面來做研究,以期發(fā)現(xiàn)更多的防衛(wèi)相關(guān)基因并了解其功能發(fā)揮及機制特點,為預(yù)防植物病害提供些許理論依據(jù)。本文分別采用傷根灌菌法、5’-RACE(5’末端快速擴增)法對實驗材料進行接種和基因序列的克隆。從接種了青枯菌(Ralstonia solanacearum)的馬鈴薯中薯3號(Solanum tuberosum)中獲得了類StWRKY8基因的cDNA序列,該序列長度為563 bp,其中包含一個長度為258bp的完整開放閱讀框,可編碼85個氨基酸。類StWRKY8基因序列內(nèi)部具有一個典型的WRKYGQK保守結(jié)構(gòu)域。C2H2為其鋅指結(jié)構(gòu)類型,因此隸屬于WRKY家族中的第二類。經(jīng)由鄰接法系統(tǒng)進化樹的構(gòu)建,分析其氨基酸序列發(fā)現(xiàn)與茄科內(nèi)的(Solanaceae)其他成員具有較高程度的同源性,甚至與馬鈴薯轉(zhuǎn)錄因子StWRKY8相似性高達98%,故命名為類StWRKY8。而類StWRKY8相關(guān)理化性質(zhì)、等電點等則通過運用相應(yīng)的生物信息學(xué)軟件來預(yù)測:等電點大約為9.10,半衰期大于5.6 h,屬于親水性蛋白,且親水性較高。三維結(jié)構(gòu)為非球型,含有3個磷酸化位點,其中兩個為Ser磷酸化位點,其中一個為Tyr磷酸化位點。亞細胞定位發(fā)現(xiàn)其屬于胞內(nèi)蛋白。經(jīng)青枯菌誘導(dǎo)后,兩種不同材料中類StWRKY8基因的定量分析運用RT-PCR和qRT-PCR來進行,結(jié)果顯示:類StWRKY8受青枯菌誘導(dǎo)后表達量上調(diào),并且在感、抗病兩基因型馬鈴薯中表達有較明顯的差異。類StWRKY8基因在抗病基因型馬鈴薯接種青枯菌6 h內(nèi)就能高強度的表達,發(fā)揮其抗性,而在感病基因型馬鈴薯接種青枯菌6 h之內(nèi)的表達較為緩慢,強度也較低,不能在侵染之初很好地抗病。利用地高辛原位雜交手段對目的基因的表達進行組織定位后顯示:類StWRKY8基因的表達主要集中在莖葉組織中,即有維管束、韌皮部的維管束系統(tǒng)中,這說明其表達具有明顯的組織特異性。經(jīng)過分析,總結(jié):類StWRKY8基因具有植物轉(zhuǎn)錄因子的典型結(jié)構(gòu)特征,能起到一定的抗病防衛(wèi)作用,受青枯菌等病原菌的誘導(dǎo)而表達,與此同時,也受寄主植物固有的抗性影響,因此在感、抗病基因型中表達模式區(qū)別很大。這也支持所推測的觀點,即轉(zhuǎn)錄因子類StWRKY8基因作為防衛(wèi)基因,在植物防御抗病等反應(yīng)中發(fā)揮一定作用并參與機體調(diào)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.
【學(xué)位授予單位】：山西師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S435.32

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1 薛珍;青枯菌誘導(dǎo)的馬鈴薯轉(zhuǎn)錄因子類StWRKY8基因的克隆及表達分析[D];山西師范大學(xué);2016年

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本文編號：1906837