本文選題：核盤(pán)菌 + 轉(zhuǎn)錄因子��； 參考：《吉林大學(xué)》2015年碩士論文

【摘要】：核盤(pán)菌（Sclerotinia sclerotiorum（Lib.）de Bary）是一種腐生性絲狀病原真菌，它可以造成多種農(nóng)作物的嚴(yán)重病害，從而引起重大經(jīng)濟(jì)損失。核盤(pán)菌的子囊盤(pán)是由菌核在合適的條件下萌發(fā)而成的圓盤(pán)狀子實(shí)體，發(fā)育成熟的子囊盤(pán)將會(huì)釋放子囊孢子，子囊孢子飛散到周?chē)h(huán)境中侵染寄主植物，造成菌核病害的流行。 在核盤(pán)菌基因組中存在4個(gè)Forkhead類轉(zhuǎn)錄因子的同源基因，分別為Ss-FoxE1、Ss-FoxE2、Ss-FoxE3和Ss-FoxE4，我們推斷它們與核盤(pán)菌的有性生殖密切相關(guān)。因此，在本文中我們以Ss-FoxE2為研究對(duì)象，并對(duì)在核盤(pán)菌有性生殖中的作用進(jìn)行了研究。本研究首先根據(jù)核盤(pán)菌基因組序列，克隆得到Ss-FoxE2上游890bp序列以及下游1024bp序列，將上下游序列分別連接到敲除載體pXEH上，成功構(gòu)建了Ss-FoxE2的敲除載體，然后利用裂解酶消化核盤(pán)菌菌絲細(xì)胞壁，從而制備了質(zhì)量較好的原生質(zhì)體細(xì)胞，并用PEG-CaCl2介導(dǎo)的原生質(zhì)體轉(zhuǎn)化法將已構(gòu)建好的敲除載體轉(zhuǎn)化到核盤(pán)菌的原生質(zhì)體中，以潮霉素為篩選抗性進(jìn)行轉(zhuǎn)化子的篩選培養(yǎng)，然后使用快速PCR檢測(cè)方法進(jìn)行初步驗(yàn)證，將驗(yàn)證正確的轉(zhuǎn)化子使用Southern雜交的方法進(jìn)行進(jìn)一步驗(yàn)證。另一方面，克隆Ss-FoxE2編碼基因及其上下游在內(nèi)的3.4kb的片段序列，將克隆得到的序列連接到互補(bǔ)載體上，將構(gòu)建好的互補(bǔ)載體轉(zhuǎn)化至敲除突變體的原生質(zhì)體細(xì)胞中，，通過(guò)G418抗性篩選以及PCR驗(yàn)證得到互補(bǔ)成功的轉(zhuǎn)化子。將Ss-FoxE2敲除突變體和Ss-FoxE2互補(bǔ)突變體對(duì)照野生型菌株，進(jìn)行生物學(xué)特性分析和致病性分析，結(jié)果發(fā)現(xiàn)，Ss-FoxE2敲除突變體與野生型菌株相比，在菌絲生長(zhǎng)速率、菌核數(shù)量、菌核干重以及致病性上幾乎沒(méi)有任何差別，但是，Ss-FoxE2敲除突變體不能誘導(dǎo)發(fā)育形成子囊盤(pán)，這說(shuō)明核盤(pán)菌轉(zhuǎn)錄因子Ss-FoxE2參與調(diào)控核盤(pán)菌子囊盤(pán)的形成，影響核盤(pán)菌的有性生殖，這將為菌核病的防治提供新的理論基礎(chǔ)與技術(shù)支撐。
[Abstract]:Sclerotinia sclerotiorum Lib.de Baryis is a kind of saprophytic filamentous fungi, which can cause serious diseases of many crops and cause great economic losses. Sclerotinia sclerotiorum cotyledon is a disk-shaped fruiting body that germinates from sclerotia under suitable conditions. The mature ascomycetes will release ascospores, and ascospores will fly into the surrounding environment to infect host plants, resulting in the prevalence of sclerotinia disease in Sclerotinia sclerotiorum (Sclerotinia sclerotiorum). There are four Forkhead-like transcription factor homologous genes in the genome of Sclerotinia sclerotiorum, namely Ss-FoxE1, Ss-FoxE2, Ss-FoxE3 and Ss-FoxE4, which are closely related to the sexual reproduction of Sclerotinia sclerotiorum. Therefore, in this paper, we take Ss-FoxE2 as the research object and study the role of Ss-FoxE2 in the sexual reproduction of Sclerotinia sclerotiorum. In this study, the upstream 890bp sequence and downstream 1024bp sequence of Ss-FoxE2 were cloned according to the Sclerotinia sclerotiorum genome sequence. The upstream and downstream sequences were connected to the knockout vector pXEH, respectively, and the knockout vector Ss-FoxE2 was successfully constructed. Then the cell wall of Sclerotinia sclerotiorum was digested by lyase, and the protoplast cells with good quality were prepared. The constructed knockout vector was transformed into the protoplast of Sclerotinia sclerotiorum by PEG-CaCl2 mediated protoplast transformation. Hygromycin was used to screen and culture the transformants, and then the rapid PCR method was used to verify the transformants. The correct transformants were further verified by Southern blotting. On the other hand, the sequence of Ss-FoxE2 coding gene and its upstream and downstream 3.4kb fragments were cloned, the cloned sequence was linked to the complementary vector, and the constructed complementary vector was transformed into the protoplast cells of the knockout mutant. By G418 resistance screening and PCR validation, complementary and successful transformants were obtained. The biological characteristics and pathogenicity of Ss-FoxE2 knockout mutants and Ss-FoxE2 complementary mutants were analyzed. The results showed that Ss-FoxE2 knockout mutants had higher growth rate and sclerotia number than wild-type strains. There was almost no difference in the dry weight and pathogenicity of sclerotia, but Ss-FoxE2 knockout mutant could not induce the development of sclerotia disk, which indicated that Ss-FoxE2 was involved in regulating the formation of sclerotia cotyledon and affecting the sexual reproduction of Sclerotinia sclerotiorum. This will provide a new theoretical basis and technical support for the prevention and treatment of sclerotinia.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S432.44

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相關(guān)期刊論文 前2條

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2 iJ新美;油菜菌核病(Sclerotinia sclerotiorum)在我國(guó)的寄主范圍及生態(tài)特性的娍查研究[J];植物病理學(xué)報(bào);1959年02期

本文編號(hào)：2038965