蝗绿僵菌葡聚糖苷转移酶基因的功能研究
发布时间：2018-09-17 16:26
【摘要】：昆虫容易受病原真菌感染而致病,其携带的病原真菌会进一步传播和扩散造成大面积的昆虫群感染并死亡。因此,利用昆虫病原真菌防治农业害虫是一种长效和环保的生防措施。真菌细胞壁是真菌细胞外周的重要结构,为真菌抵御外界不良环境和寄主免疫反应等提供保护,葡聚糖苷转移酶具有水解和转移酶的活性,在葡聚糖分子组装和维持细胞壁完整性方面具有重要作用。但是其在真菌生长发育过程中的作用机制还不清楚。阐明葡聚糖苷转移酶在病原真菌中的功能将为提高真菌农药的毒力及抗真菌药物的筛选提供参考依据。为此,本课题以蝗绿僵菌为试验材料,采用同源重组技术构建了葡聚糖苷转移酶敲除及回复菌株,对其生长、毒力及抗逆特性进行了初步的分析。主要研究内容:1.采用生物信息学分析基因的结构2.构建敲除及回复菌株利用同源重组方法构建了ΔMwg2,ΔMwg3,??Mwg1/Mwg2、??Mwg2/Mwg3敲除及回复菌株。3.基因的功能分析(1)生长特性分析:对野生、敲除及回复菌株的孢子进行接种培养,测定其萌发率和产孢量并观察其菌丝的结构。(2)抗逆特性分析:对野生、敲除和回复菌株的孢子分别进行热激、紫外照射处理,统计萌发率,比较其萌发半抑制时间的差异。(3)毒力分析:用新鲜成熟的野生、敲除型菌株的孢子配制成孢悬液,采用体内注射和体表侵染方法感染东亚飞蝗,统计半致死时间,分析毒力差异。主要研究结果:1.经过生物信息学分析得出,Mwg1:Gen Bank:EFY91318.1,c DNA是1367bp,氨基酸大小是406aa,等电点是5.22p I,分子量是41k Da,其中含有1个内含子;Mwg2:Gen Bank:EFY92943.1,c DNA是998bp,氨基酸大小是277aa,等电点是5.03p I,分子量是26k Da,其中含有1个内含子;Mwg3:Gen Bank:EFY86494.1,c DNA是1669bp,氨基酸大小是511aa,等电点是4.89p I,分子量是52k Da,其中含有2个内含子。2.本实验构建了?Mwg2、?Mwg3和CP1、CP2、CP3菌株,同时构建了??Mwg1/Mwg2、??Mwg2/Mwg3菌株3.葡聚糖苷转移酶基因对蝗绿僵菌生长特性的影响(1)萌发与产孢方面:?Mwg1,?Mwg3与WT和CP相比,产孢量增加,萌发提前;而?Mwg2与WT和CP相比,产孢量减少,萌发延迟;??Mwg1/Mwg2和??Mwg2/Mwg3与WT相比,产孢量增加,萌发提前。(2)菌丝结构方面:?Mwg1与WT和CP相比,菌丝末端更细长,菌丝膈间距离增大;?Mwg2与WT和CP相比,分支数明显增加;?Mwg2与WT和CP相比,菌丝末端更细长,菌丝膈间距离增大;??Mwg1/Mwg2、??Mwg2/Mwg3与WT相比,菌丝末端更细长,菌丝膈间距离增大。4.聚糖苷转移酶基因对蝗绿僵菌抗逆特性的影响(1)紫外耐受性方面:?Mwg1、?Mwg3、??Mwg1/Mwg2、??Mwg2/Mwg3与WT和CP相比,紫外处理后孢子萌发率提前,紫外耐受力增强;?Mwg2与WT和CP相比,紫外处理后孢子萌发率延迟,紫外耐受力减弱。(2)湿热耐受性方面:?Mwg1与WT和CP相比,湿热处理后孢子萌发率提前,湿热耐受力增强;?Mwg2与WT和CP相比,湿热处理后孢子萌发率延迟,湿热耐受力减弱;?Mwg2、??Mwg2/Mwg3与WT和CP相比无显著性差异;??Mwg1/Mwg2与WT相比,湿热处理后孢子萌发率提前。(3)细胞壁敏感剂方面:在加有细胞壁敏感剂刚果红的1/4SADAY平板上,?Mwg1与WT和CP相比,菌落形态明显减小,生长缓慢;??Mwg1/Mwg2与WT相比,也表现为生长缓慢,而其他敲除菌株无显著性影响;各个菌株在添加有Na Cl、Sor的1/4SADAY平板上生长情况与WT相比,无显著性差异,说明葡聚糖苷转移酶不影响绿僵菌对高渗透压的耐受性。5.聚糖苷转移酶基因对蝗绿僵菌毒力的影响主要采用了体表侵染和体内注射,分别制备敲除和WT菌株的孢悬液,感染东亚飞蝗,分析葡聚糖苷转移酶基因对蝗绿僵菌毒力的影响。研究结果表明:体表侵染时,?Mwg1、?Mwg2、?Mwg3与WT相比,毒力无差异;但??Mwg1/Mwg2、??Mwg2/Mwg3与WT相比,毒力明显减弱。体内注射时,?Mwg2与WT相比,毒力无差异;但?Mwg3与WT相比,毒力增强;??Mwg1/Mwg2、??Mwg2/Mwg3与WT相比,毒力增强。
[Abstract]:Insects are susceptible to pathogenic fungi. The pathogenic fungi carried by insects can further spread and cause large-scale insect infections and deaths. Therefore, the use of insect pathogenic fungi to control agricultural pests is a long-term and environmentally friendly biocontrol measure. Fungal cell wall is an important structure in the periphery of fungal cells, which protects fungi from the outside world. Dextransferase has the activities of hydrolysis and transferase. It plays an important role in the assembly of dextran molecules and the maintenance of cell wall integrity. However, the mechanism of its role in fungal growth and development is still unclear. Therefore, we constructed a glucan transferase knockout and recovery strain by homologous recombination technology with Metarhizium anisopliae as experimental material, and preliminarily analyzed its growth, virulence and stress resistance characteristics. Construction of knockout and recovery strains using homologous recombination method to construct Mwg2, _Mwg3,?? Mwg1 / Mwg2,?? Mwg2 / Mwg3 knockout and recovery strains. 3. Functional analysis of genes (1) Growth characteristics: wild, knockout and recovery strains were inoculated and cultured, their germination rate and spore production were measured and observed. Structure of mycelium. (2) Stress resistance analysis: Spores of wild, knockout and recovery strains were treated by heat shock and ultraviolet radiation respectively, and the germination rate was counted and the difference of semi-inhibition time was compared. (3) Toxicity analysis: spore suspension was prepared from spores of fresh and mature wild, knockout strains, and was sensitized by in vivo injection and surface infection. The main results were as follows: 1. Bioinformatics analysis showed that Mwg1: Gen Bank: EFY91318.1, C DNA 1367 bp, amino acid size 406aa, isoelectric point 5.22 pI, molecular weight 41kDa, including 1 intron; Mwg2: Gen Bank: EFY92943.1, C DNA 998 bp, amino acid size 2.22 bp. 77aa, isoelectric point is 5.03pI, molecular weight is 26kDa, which contains one intron; Mwg3: Gen Bank: EFY86494.1, C DNA is 1669bp, amino acid size is 511aa, isoelectric point is 4.89pI, molecular weight is 52kDa, which contains two introns. The effects of glucan transferase gene on the growth characteristics of Metarhizium anisopliae were as follows: (1) In terms of sporulation and sporulation, Mwg1 and Mwg3 produced more spores and germinated earlier than WT and CP, while Mwg2 produced less spores and delayed germination compared with WT and CP, and?? Mwg1/Mwg2 and?? Mwg2/Mwg3 produced more spores and germinated earlier than WT. Compared with CP, Mwg2 had longer mycelial end and larger diaphragm spacing; Mwg2 had more branches than WT and CP; Mwg2 had longer mycelial end and larger diaphragm spacing than WT and CP; Mwg1/Mwg2,?? Mwg2/Mwg3 had longer mycelial end and larger diaphragm spacing than WT. The effects of UV treatment on the ultraviolet tolerance (1) compared with WT and CP, Mwg1, Mwg3, Mwg1 / Mwg2, Mwg2 / Mwg3 had earlier spore germination rate and stronger ultraviolet tolerance; (2) Compared with WT and CP, Mwg2 had longer spore germination rate and weaker ultraviolet tolerance. Compared with WT and CP, the spore germination rate of Mwg2 was delayed and the heat and humidity tolerance was weakened after heat treatment;?Mwg2,?? Mwg2/Mwg3 had no significant difference compared with WT and CP;?? Mwg1/Mwg2 had earlier spore germination rate after heat treatment than WT. (3) Cell wall sensitizer: Congo with cell wall sensitizer Compared with WT and CP, the colony morphology of? Mwg1 decreased significantly and grew slowly on the red 1/4 SADAY plate;?? Mwg1/Mwg2 also grew slowly compared with WT, but other knockout strains had no significant effect; the growth of each strain on the 1/4 SADAY plate with Na Cl and Sor had no significant difference compared with WT, indicating that glucan transferase did not. Effects of glycosyltransferase gene on the virulence of Metarhizium anisopliae were studied. The results showed that the virulence of Metarhizium anisopliae was mainly affected by the infection on the body surface and the injection in vivo. The toxicity of? Mwg1,? Mwg2,? Mwg3 was not different from that of WT, but?? Mwg1 / Mwg2,?? Mwg2 / Mwg3 was significantly weaker than that of WT.
【学位授予单位】：重庆理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S476
本文编号：2246477
[Abstract]:Insects are susceptible to pathogenic fungi. The pathogenic fungi carried by insects can further spread and cause large-scale insect infections and deaths. Therefore, the use of insect pathogenic fungi to control agricultural pests is a long-term and environmentally friendly biocontrol measure. Fungal cell wall is an important structure in the periphery of fungal cells, which protects fungi from the outside world. Dextransferase has the activities of hydrolysis and transferase. It plays an important role in the assembly of dextran molecules and the maintenance of cell wall integrity. However, the mechanism of its role in fungal growth and development is still unclear. Therefore, we constructed a glucan transferase knockout and recovery strain by homologous recombination technology with Metarhizium anisopliae as experimental material, and preliminarily analyzed its growth, virulence and stress resistance characteristics. Construction of knockout and recovery strains using homologous recombination method to construct Mwg2, _Mwg3,?? Mwg1 / Mwg2,?? Mwg2 / Mwg3 knockout and recovery strains. 3. Functional analysis of genes (1) Growth characteristics: wild, knockout and recovery strains were inoculated and cultured, their germination rate and spore production were measured and observed. Structure of mycelium. (2) Stress resistance analysis: Spores of wild, knockout and recovery strains were treated by heat shock and ultraviolet radiation respectively, and the germination rate was counted and the difference of semi-inhibition time was compared. (3) Toxicity analysis: spore suspension was prepared from spores of fresh and mature wild, knockout strains, and was sensitized by in vivo injection and surface infection. The main results were as follows: 1. Bioinformatics analysis showed that Mwg1: Gen Bank: EFY91318.1, C DNA 1367 bp, amino acid size 406aa, isoelectric point 5.22 pI, molecular weight 41kDa, including 1 intron; Mwg2: Gen Bank: EFY92943.1, C DNA 998 bp, amino acid size 2.22 bp. 77aa, isoelectric point is 5.03pI, molecular weight is 26kDa, which contains one intron; Mwg3: Gen Bank: EFY86494.1, C DNA is 1669bp, amino acid size is 511aa, isoelectric point is 4.89pI, molecular weight is 52kDa, which contains two introns. The effects of glucan transferase gene on the growth characteristics of Metarhizium anisopliae were as follows: (1) In terms of sporulation and sporulation, Mwg1 and Mwg3 produced more spores and germinated earlier than WT and CP, while Mwg2 produced less spores and delayed germination compared with WT and CP, and?? Mwg1/Mwg2 and?? Mwg2/Mwg3 produced more spores and germinated earlier than WT. Compared with CP, Mwg2 had longer mycelial end and larger diaphragm spacing; Mwg2 had more branches than WT and CP; Mwg2 had longer mycelial end and larger diaphragm spacing than WT and CP; Mwg1/Mwg2,?? Mwg2/Mwg3 had longer mycelial end and larger diaphragm spacing than WT. The effects of UV treatment on the ultraviolet tolerance (1) compared with WT and CP, Mwg1, Mwg3, Mwg1 / Mwg2, Mwg2 / Mwg3 had earlier spore germination rate and stronger ultraviolet tolerance; (2) Compared with WT and CP, Mwg2 had longer spore germination rate and weaker ultraviolet tolerance. Compared with WT and CP, the spore germination rate of Mwg2 was delayed and the heat and humidity tolerance was weakened after heat treatment;?Mwg2,?? Mwg2/Mwg3 had no significant difference compared with WT and CP;?? Mwg1/Mwg2 had earlier spore germination rate after heat treatment than WT. (3) Cell wall sensitizer: Congo with cell wall sensitizer Compared with WT and CP, the colony morphology of? Mwg1 decreased significantly and grew slowly on the red 1/4 SADAY plate;?? Mwg1/Mwg2 also grew slowly compared with WT, but other knockout strains had no significant effect; the growth of each strain on the 1/4 SADAY plate with Na Cl and Sor had no significant difference compared with WT, indicating that glucan transferase did not. Effects of glycosyltransferase gene on the virulence of Metarhizium anisopliae were studied. The results showed that the virulence of Metarhizium anisopliae was mainly affected by the infection on the body surface and the injection in vivo. The toxicity of? Mwg1,? Mwg2,? Mwg3 was not different from that of WT, but?? Mwg1 / Mwg2,?? Mwg2 / Mwg3 was significantly weaker than that of WT.
【学位授予单位】：重庆理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S476
【参考文献】
相关期刊论文 前1条
1 龚炎杰;王子涵;;酿酒酵母葡聚糖的研究进展[J];现代食品科技;2006年01期
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