本文關(guān)鍵詞： 桑椹縮小性菌核病 黑色素 小柱孢酮脫水酶 基因敲除　出處：《西南大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：桑椹菌核病是危害桑椹最為嚴(yán)重的真菌病害之一,其病原菌在桑樹開花期專一性侵染桑樹花器官。桑椹縮小性菌核病是桑椹菌核病的一種,由核地杖菌(Scleromitrula shiraiana)侵染引起。本研究從桑椹菌核病發(fā)病區(qū)采集病原菌子囊殼、病果及菌核作為分離材料,最終分離到核地杖菌,鑒定出其在侵染過(guò)程中產(chǎn)生的黑色物質(zhì)為多聚二羥萘類(DHN)黑色素。DHN黑色素不僅對(duì)病原菌在逆境中的生存起到不可估量的作用,而且與植物病原菌的致病性密切相關(guān)。DHN黑色素的生物合成由多個(gè)基因控制,任何一個(gè)基因缺失都將阻礙DHN黑色素的生物合成,進(jìn)而影響病原菌的致病性。小柱孢酮脫水酶(SCD)為DHN黑色素合成途徑中的關(guān)鍵酶。在本研究中,根據(jù)基因保守結(jié)構(gòu)設(shè)計(jì)兼并引物和利用RACE技術(shù)獲得ShSCD基因完整的CDS序列,采用基因敲除技術(shù),獲得ShSCD基因敲除菌株,證實(shí)了ShSCD基因在黑色素生物合成途徑中的重要性。主要實(shí)驗(yàn)結(jié)果如下:1.ShSCD基因的克隆與信息分析根據(jù)其他已知植物病原真菌的小柱孢酮脫水酶基因的保守結(jié)構(gòu)設(shè)計(jì)兼并引物并利用RACE技術(shù),獲得1083bp的全長(zhǎng)基因組序列。該基因由2個(gè)內(nèi)含子和3個(gè)外顯子組成且含有保守的催化及底物結(jié)合位點(diǎn)。進(jìn)化分析表明ShSCD與Botryotinia cinerea T4、Sclerotinia sclerotiorum的SCD基因親緣關(guān)系最近。蛋白質(zhì)二級(jí)結(jié)構(gòu)主要由α螺旋和無(wú)規(guī)則卷曲組成,三級(jí)結(jié)構(gòu)與稻瘟病菌SCD蛋白的晶體結(jié)構(gòu)相似。2.ShSCD敲除突變體的獲得利用Genome Walking技術(shù),分別獲得ShSCD上游片段I(870bp)和下游片段II(866bp)。以PSKH為敲除載體,以潮霉素基因?yàn)楹Y選基因,構(gòu)建敲除載體PSKH-ShSCD,經(jīng)轉(zhuǎn)化和篩選共得到7個(gè)轉(zhuǎn)化子。PCR驗(yàn)證表明在轉(zhuǎn)化子中未擴(kuò)增到ShSCD基因,但擴(kuò)增到與敲除載體相同的部分序列。半定量PCR及qRT-PCR都未在轉(zhuǎn)化子中檢測(cè)到ShSCD基因的表達(dá)。Southern blotting檢測(cè)結(jié)果表明在野生型菌株中ShSCD為單拷貝基因且在轉(zhuǎn)化子中未檢測(cè)到ShSCD基因,但檢測(cè)到潮霉素基因片段。表明篩選得到的轉(zhuǎn)化子為陽(yáng)性突變體。3.ShSCD基因的功能驗(yàn)證觀察菌落形態(tài)發(fā)現(xiàn),野生型菌株菌落正面為灰白色,背面為墨綠色,ΔShSCD突變體正面為淺黃色,背面為褐色,其他形態(tài)與野生型菌株一致。ΔShSCD突變體比野生型菌株生長(zhǎng)較慢。測(cè)定菌株黑色素的含量,結(jié)果表示ΔShSCD突變體中黑色素的含量顯著低于野生型菌株。經(jīng)高鹽處理后ΔShSCD突變體生長(zhǎng)比未處理前生長(zhǎng)較快,但與相同處理后的野生型菌株生長(zhǎng)無(wú)顯著差異。在濃度為0.5M時(shí)野生型菌株生長(zhǎng)受到抑制,在0.9M高鹽濃度時(shí)ΔShSCD突變體生長(zhǎng)受到抑制,說(shuō)明ΔShSCD突變體的滲透調(diào)節(jié)能力強(qiáng)于野生型菌株,受鹽脅迫影響較小。經(jīng)5mM H2O2處理,野生型菌株與ΔShSCD突變體的抑制率無(wú)顯著差異。濃度為10mM H2O2時(shí),野生型菌株的抑制率為89%,而ΔShSCD突變體菌株完全不生長(zhǎng)。說(shuō)明野生型菌株的抗氧脅迫能力強(qiáng)于ΔShSCD突變體。以上結(jié)果表明,ShSCD基因不僅對(duì)黑色素的生物合成,而且在菌絲生長(zhǎng)、滲透調(diào)節(jié)及抗氧化方面具有重要作用。這為進(jìn)一步研究桑椹縮小性菌核病的致病機(jī)理提供了理論依據(jù),為新型殺真菌劑的研制提供了一條新的思路。
[Abstract]:Mulberry fruit sclerotiniosis is one of the most serious fungal diseases endangering mulberry, the pathogen of mulberry in flowering stage specific infection of mulberry floral organs. Mulberry is a kind of narrow sclerotium disease of Mulberry Sorosis disease, by nuclear rod bacteria (Scleromitrula shiraiana) caused by infection. This study from mulberry sclerotium disease pathogen collection area fruit shell, fruit disease and sclerotia as isolated material, eventually isolated nuclear rod bacteria, identified the black substance in the infection process for poly Dihydroxynaphthalene class (DHN).DHN not only to melanin melanin immeasurable effect on pathogenic bacteria in adversity to survive, the biosynthesis of pathogenicity but with the plant pathogen.DHN is closely related to the melanin biosynthesis is controlled by multiple genes, a gene deletion will prevent DHN melanoma, thereby affecting pathogenic bacteria. Small spore ketone dehydratase (SCD) DHN The key enzyme in melanin biosynthesis. In this study, according to the sequence of CDS gene conserved degenerate primers were designed using RACE technology and get the complete ShSCD gene, using gene knockout, ShSCD gene knockout strains, the ShSCD gene was confirmed in the melanin biosynthetic pathway of the importance of the main results. The following information: cloning and analysis of 1.ShSCD gene conserved primers according to the design of merger column spore ketone dehydration enzyme gene of other known plant pathogenic fungi and the use of RACE technology, the full-length 1083bp genome sequence. The radical cause of 2 introns and 3 exons and contains a conserved binding site and catalytic substrate. Phylogenetic analysis showed that ShSCD and Botryotinia cinerea T4, Sclerotinia sclerotiorum SCD gene had the closest relationship. Two protein structure is mainly composed of alpha helix and random coil, .2.ShSCD knockout mutants obtained by using Genome Walking technology similar crystal structure of three level structure and Magnaporthe grisea SCD protein, respectively ShSCD upstream fragment I (870bp) II (866bp) and downstream fragment using PSKH knockout vector to hygromycin gene for screening the gene knockout vector PSKH-ShSCD, constructed by. Transformation and selection were obtained 7 transformants showed that.PCR transformants were not amplified ShSCD gene amplification, but to knock out partial sequence of the same vector. Semi quantitative PCR and qRT-PCR were not detected in transformants expressing.Southern ShSCD gene blotting test results indicate that in the wild type strain ShSCD is single and copy gene in the transformants was not detected in the ShSCD gene, but detected Hygromycin gene fragment. That transformants screened positive for mutant.3.ShSCD gene functional verification observation of colony morphology, wild type The positive colony was gray, the back is dark green, a ShSCD mutant positive light yellow, brown on the back, the other form with the wild type strain. A ShSCD mutant than the wild type strain growth slow. Determination of strain of melanin, the melanin content in the mutant Delta ShSCD was significantly lower than that of wild type strain the growth of a ShSCD mutant. Than the untreated before growth under high salt treatment, but after treatment with wild type strains of the same growth. No significant difference in the concentration of 0.5M wild type strain inhibited the growth of 0.9M in the high salt concentration ShSCD mutant inhibited the growth that osmotic adjustment ability of mutant ShSCD stronger than the wild type strain, less affected by salt stress. After 5mM H2O2 treatment, the inhibition of wild type strain and a ShSCD mutant was no significant difference between the concentration of 10mM H2O2, inhibition of wild-type strain The rate is 89%, and a ShSCD mutant strain did not grow. That wild type strain of anti oxidative stress ability in ShSCD mutant. These results indicate that ShSCD gene is not only the biosynthesis of melanin, but also in the mycelial growth, osmotic adjustment and antioxidant plays an important role. This provides a theoretical basis for the pathogenic mechanism further study of mulberry sclerotium disease reduced, provides a new way for the development of new antifungal agents.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S436.639

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