本文選題：Alternaria + brassicicola��； 參考：《山東農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：蕓薹生鏈格孢(Alternaria brassicicola)引起的黑斑病是十字花科蔬菜農(nóng)業(yè)生產(chǎn)上的一大難題,早期研究顯示,Fus3-MAPK信號(hào)途徑在A.brassicicola的致病性過程中起到重要的調(diào)控作用,前期在信號(hào)傳導(dǎo)特異性方面對錨定作用已進(jìn)行研究,而未對其支架蛋白作用進(jìn)行詳細(xì)研究。本研究主要以蕓薹生鏈格孢菌(A.brassicicola)為對象,通過構(gòu)建AbSte5基因缺失突變體與野生菌對比研究,進(jìn)而針對MAPK信號(hào)轉(zhuǎn)導(dǎo)途徑中的Ste5基因功能進(jìn)行研究。其主要結(jié)果如下;利用釀酒酵母saccharomyces cerevisiae Ste5基因序列在建立的A.brassicicola基因組數(shù)據(jù)庫中進(jìn)行本地比對,找到A.brassicicola中Ste5同源基因。根據(jù)下載的Ste5基因序列,設(shè)計(jì)特異性引物從A.brassicicola中成功擴(kuò)增Ab Ste5基因以及結(jié)合RT-PCR技術(shù)擴(kuò)增得到cDNA,該基因全長3327bp,cDNA3276bp,含有1個(gè)內(nèi)含子且符合GT-AG法則。通過SMART蛋白質(zhì)功能預(yù)測網(wǎng)站對A.brassicicola Ste5蛋白質(zhì)序列進(jìn)行分析,并與來源于其它真菌中的Ste5同源基因比對發(fā)現(xiàn)在序列上同源性雖低,但氨基酸序列在結(jié)構(gòu)上具有極高的相似性,發(fā)現(xiàn)都具有RING、PH以及vWA功能域,因此推測A.brassicicola中的Ste5基因也是一種支架蛋白。構(gòu)建AbSte5基因缺失突變載體pUCATPH-Ab Ste5以及M13F/M13R引物擴(kuò)增新構(gòu)建質(zhì)粒PCR產(chǎn)物利用PEG介導(dǎo)方法轉(zhuǎn)化A.brassicicola原生質(zhì)體,通過PCR法及RT-PCR技術(shù)進(jìn)一步驗(yàn)證并篩選得到了AbSte5基因缺失突變體,并通過southern雜交技術(shù)得出基因單拷貝插入缺失。在菌落表型、菌絲和分生孢子形態(tài)、黑色素以及致病性檢測上將AbSte5基因缺失突變體與野生菌進(jìn)行比較研究。結(jié)果發(fā)現(xiàn)AbSte5基因缺失突變體在PDA上生長速度下降,而且不能產(chǎn)生分生孢子,當(dāng)采用離體葉片接種法接種未人工刺傷白菜葉片時(shí),Ab Ste5基因缺失突變體不能侵染白菜葉片,當(dāng)接種刺傷白菜葉片時(shí),亦喪失致病力。擴(kuò)增AbSte5全基因以pCB1532質(zhì)粒為載體構(gòu)建互補(bǔ)載體轉(zhuǎn)化AbSte5突變菌株原生質(zhì)體得到互補(bǔ)體后,其生物學(xué)性狀等均恢復(fù)到野生菌水平。由此可以推測,AbSte5基因與菌絲形態(tài)、分生孢子的形成、致病性等有密切的關(guān)系。
[Abstract]:The black spot caused by Alternaria brassicicola (Alternaria brassicicola) is a difficult problem in the agricultural production of cruciferous vegetables. Early studies showed that Fus3-MAPK signaling pathway plays an important role in regulating the pathogenicity of A.brassicicola. The effect of anchoring on signal transduction specificity has been studied, but not on the role of scaffold protein. The purpose of this study was to study the function of Ste5 gene in MAPK signal transduction pathway by constructing AbSte5 gene deletion mutants and wild bacteria. The main results are as follows: using the sequence of saccharomyces cerevisiae Ste5 gene of Saccharomyces cerevisiae to carry out local alignment in the established A.brassicicola genomic database, the homologous gene of Ste5 in A.brassicicola was found. According to the downloadable Ste5 gene sequence, a specific primer was designed to amplify the Ab Ste5 gene from A.brassicicola and to amplify the cDNA by combining RT-PCR technique. The gene was 3327bpcDNA3276bp in length, containing an intron and conforming to the GT-AG rule. The sequence of A.brassicicola Ste5 protein was analyzed by SMART protein function prediction website, and compared with Ste5 homology gene from other fungi. Although the sequence homology was low, the amino acid sequence had very high similarity in structure. Both RINGP PH and vWA functional domains were found, so it is assumed that the Ste5 gene in A.brassicicola is also a scaffold protein. The AbSte5 gene deletion vector pUCATPH-Ab Ste5 and M13F/M13R primers were constructed to amplify the newly constructed plasmid PCR products. The A.brassicicola protoplasts were transformed into A.brassicicola protoplasts by PEG mediated method. The AbSte5 gene deletion mutants were further verified and screened by PCR and RT-PCR techniques. The single copy insertion deletion was obtained by southern hybridization. In colony phenotype, mycelium and conidial morphology, melanin and pathogenicity test, AbSte5 gene deletion mutants were compared with wild bacteria. The results showed that the growth rate of AbSte5 gene deletion mutants on PDA was decreased and conidia could not be produced. The absence of Ste5 gene mutant could not infect Chinese cabbage leaves when it was inoculated in vitro without artificial prick. When inoculated to injure cabbage leaves, it also loses pathogenicity. When the AbSte5 gene was amplified from pCB1532 plasmid and transformed into the protoplast of AbSte5 mutant strain, the biological characters were recovered to the level of wild bacteria. It can be inferred that the AbSte5 gene is closely related to hyphal morphology, conidial formation and pathogenicity.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S432.44

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 王洪凱,林福呈,王政逸;植物病原真菌附著胞的機(jī)械穿透力[J];菌物學(xué)報(bào);2004年01期

2 易龍,肖崇剛,馬冠華,王萬能,龍良鯤;防治煙草赤星病有益內(nèi)生細(xì)菌的篩選及抑菌作用[J];微生物學(xué)報(bào);2004年01期

3 王革,周曉罡,方敦煌,李天飛;木霉拮抗煙草赤星病菌菌株的篩選及其生防機(jī)制[J];云南農(nóng)業(yè)大學(xué)學(xué)報(bào);2000年03期

，

本文編號(hào)：1799442