桑椹缩小性菌核病菌（Scleromitrula shiraiana）小柱孢酮脱水酶基因的功能研究

发布时间：2018-02-24 22:00

本文关键词： 桑椹缩小性菌核病黑色素小柱孢酮脱水酶基因敲除　出处：《西南大学》2017年硕士论文　论文类型：学位论文

【摘要】：桑椹菌核病是危害桑椹最为严重的真菌病害之一,其病原菌在桑树开花期专一性侵染桑树花器官。桑椹缩小性菌核病是桑椹菌核病的一种,由核地杖菌(Scleromitrula shiraiana)侵染引起。本研究从桑椹菌核病发病区采集病原菌子囊壳、病果及菌核作为分离材料,最终分离到核地杖菌,鉴定出其在侵染过程中产生的黑色物质为多聚二羟萘类(DHN)黑色素。DHN黑色素不仅对病原菌在逆境中的生存起到不可估量的作用,而且与植物病原菌的致病性密切相关。DHN黑色素的生物合成由多个基因控制,任何一个基因缺失都将阻碍DHN黑色素的生物合成,进而影响病原菌的致病性。小柱孢酮脱水酶(SCD)为DHN黑色素合成途径中的关键酶。在本研究中,根据基因保守结构设计兼并引物和利用RACE技术获得ShSCD基因完整的CDS序列,采用基因敲除技术,获得ShSCD基因敲除菌株,证实了ShSCD基因在黑色素生物合成途径中的重要性。主要实验结果如下:1.ShSCD基因的克隆与信息分析根据其他已知植物病原真菌的小柱孢酮脱水酶基因的保守结构设计兼并引物并利用RACE技术,获得1083bp的全长基因组序列。该基因由2个内含子和3个外显子组成且含有保守的催化及底物结合位点。进化分析表明ShSCD与Botryotinia cinerea T4、Sclerotinia sclerotiorum的SCD基因亲缘关系最近。蛋白质二级结构主要由α螺旋和无规则卷曲组成,三级结构与稻瘟病菌SCD蛋白的晶体结构相似。2.ShSCD敲除突变体的获得利用Genome Walking技术,分别获得ShSCD上游片段I(870bp)和下游片段II(866bp)。以PSKH为敲除载体,以潮霉素基因为筛选基因,构建敲除载体PSKH-ShSCD,经转化和筛选共得到7个转化子。PCR验证表明在转化子中未扩增到ShSCD基因,但扩增到与敲除载体相同的部分序列。半定量PCR及qRT-PCR都未在转化子中检测到ShSCD基因的表达。Southern blotting检测结果表明在野生型菌株中ShSCD为单拷贝基因且在转化子中未检测到ShSCD基因,但检测到潮霉素基因片段。表明筛选得到的转化子为阳性突变体。3.ShSCD基因的功能验证观察菌落形态发现,野生型菌株菌落正面为灰白色,背面为墨绿色,ΔShSCD突变体正面为浅黄色,背面为褐色,其他形态与野生型菌株一致。ΔShSCD突变体比野生型菌株生长较慢。测定菌株黑色素的含量,结果表示ΔShSCD突变体中黑色素的含量显著低于野生型菌株。经高盐处理后ΔShSCD突变体生长比未处理前生长较快,但与相同处理后的野生型菌株生长无显著差异。在浓度为0.5M时野生型菌株生长受到抑制,在0.9M高盐浓度时ΔShSCD突变体生长受到抑制,说明ΔShSCD突变体的渗透调节能力强于野生型菌株,受盐胁迫影响较小。经5mM H2O2处理,野生型菌株与ΔShSCD突变体的抑制率无显著差异。浓度为10mM H2O2时,野生型菌株的抑制率为89%,而ΔShSCD突变体菌株完全不生长。说明野生型菌株的抗氧胁迫能力强于ΔShSCD突变体。以上结果表明,ShSCD基因不仅对黑色素的生物合成,而且在菌丝生长、渗透调节及抗氧化方面具有重要作用。这为进一步研究桑椹缩小性菌核病的致病机理提供了理论依据,为新型杀真菌剂的研制提供了一条新的思路。
[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.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S436.639

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