本文選題：棉花 + GhWRKY23　； 參考：《山東農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：棉花在其生長發(fā)育過程中經(jīng)常遭受各種逆境脅迫,包括生物脅迫(黃萎病菌、枯萎病菌、立枯病菌和植食性昆蟲咬食等)和非生物脅迫(干旱、高鹽等),對棉花的品質(zhì)和產(chǎn)量造成一定的不利影響。培育抗病抗逆新品種是解決棉花病害問題的有效途徑,而這依賴于其抗病抗逆分子機制的研究。信號轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄水平的基因表達調(diào)控在植物抵御外界脅迫中起著重要作用。轉(zhuǎn)錄因子,如DREB、NAC、WRKY、MYB等,在響應(yīng)逆境脅迫反應(yīng)中起重要的調(diào)節(jié)作用。分離、鑒定參與逆境脅迫應(yīng)答的轉(zhuǎn)錄因子并開展其功能研究是揭示植物抗病及抗逆分子機制的重要途徑。鑒于此,本研究以陸地棉為材料,利用同源克隆與RACE PCR的方法分離得到一個II類WRKY基因,同時對該基因進行了序列特征分析、表達特性分析及生物學(xué)功能鑒定,主要結(jié)果如下:(1)序列分析表明,GhWRKY23 cDNA全長1194bp,包括121bp的5'UTR,125bp的3'UTR和948bp的開放閱讀框。該ORF編碼一個含有315個氨基酸的多肽,預(yù)測其分子量約為35.6kDa,等電點為5.94�；蚪M序列分析、同源序列比對和蛋白聚類分析表明,GhWRKY23屬于IIc類WRKY蛋白轉(zhuǎn)錄因子。(2)亞細(xì)胞定位分析表明GhWRKY23定位在細(xì)胞核中,推測該基因在細(xì)胞核中發(fā)揮一定的生物學(xué)功能。(3)qRT-PCR分析棉花幼苗中GhWRKY23基因的表達模式,結(jié)果表明干旱、機械損傷和氧化脅迫能上調(diào)GhWRKY23的表達,而高鹽、青枯勞爾氏菌和立枯絲核菌則抑制它的表達。此外,一些病程相關(guān)信號分子如SA、MeJA、ABA、ET在不同程度上誘導(dǎo)了GhWRKY23的表達。以上結(jié)果表明GhWRKY23能夠響應(yīng)多種非生物和生物脅迫,并可能受多種信號分子的調(diào)節(jié),初步推測其可能在防衛(wèi)反應(yīng)中發(fā)揮重要作用。(4)構(gòu)建GhWRKY23正義植物表達載體,通過農(nóng)桿菌侵染的方法獲得穩(wěn)定表達的轉(zhuǎn)基因本生煙植株。對超表達植株的種子進行萌發(fā)實驗和根長統(tǒng)計,結(jié)果表明GhWRKY23對SA和ET敏感,對ABA有一定的抗性。推測該基因能參與到SA、ABA和ET介導(dǎo)的信號轉(zhuǎn)導(dǎo)途徑中。(5)GhWRKY23超表達植株離體葉片接種煙草青枯勞爾氏菌、丁香假單胞菌番茄致病變種和立枯絲核菌后,相對野生型植株表現(xiàn)出明顯的感病癥狀。DAB染色和臺盼藍染色結(jié)果進一步表明超表達植株中積累了更多ROS并產(chǎn)生了更多壞死組織,MDA含量測定結(jié)果說明超表達植株細(xì)胞膜損傷情況更嚴(yán)重。同時用MV模擬氧化脅迫,在轉(zhuǎn)基因的葉片中觀察到更多的活性氧積累。推測該基因可能在抵御病原菌侵染中起到負(fù)調(diào)控作用,并可能參與到氧化脅迫響應(yīng)中。(6)利用qRT-PCR技術(shù),對接種病原菌后前后SA、ABA、JA、ET信號通路中抗病相關(guān)基因及其他病程相關(guān)基因的表達量進行檢測,發(fā)現(xiàn)超表達植株中的有些基因表達量較野生型植株有明顯的下調(diào)趨勢,推測GhWRKY23可能通過抑制SA、JA/ET信號途徑中相關(guān)基因的表達來負(fù)調(diào)控抗病反應(yīng)。(7)利用qRT-PCR分析病原菌侵染前后活性氧產(chǎn)生及清除相關(guān)基因的表達量,結(jié)果顯示超表達GhWRKY23能抑制活性氧產(chǎn)生基因的表達,也能抑制活性氧清除相關(guān)基因的表達,可能在抗氧化系統(tǒng)中起負(fù)調(diào)節(jié)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.
【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】：2016
【分類號】：Q943.2;S562
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本文編號：1952991