热激蛋白对蜡蚧轮枝菌抗高温胁迫的功能分析

发布时间：2018-01-20 03:45

本文关键词： 蜡蚧轮枝菌热激蛋白高温胁迫敲除菌株　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：蜡蚁轮枝菌(Verticillium lecanii是一种可用于防治植物病虫害的昆虫病原真菌,生防真菌制剂贮存过程和田间杀虫效果的稳定性是影响其应用推广的重要原因,温度是影响真菌孢子的贮存和田间侵染力的重要因子,热激蛋白对生物体耐热性能的提高发挥着重要作用,因此,研究热激蛋白对昆虫病原真菌的耐热性能的影响,对于解决真菌制剂的开发研制和田间应用推广等问题有重要作用。本试验以蜡蚧轮枝菌VL17、VL18菌株为供试菌株,比较了温度对两菌株的杀蚜活性、菌丝生长以及孢子萌发的差异,进而比较耐热菌株和敏感菌株高温胁迫下热激蛋白基因表达的差异,找出耐热蜡蚁轮枝菌在高温胁迫时发挥主要作用的关键热激蛋白种类,克隆关键热激蛋白基因,并构建关键热激蛋白基因敲除菌株,比较原始菌株和敲除菌株的生物学特性。本试验揭示了热激蛋白在蜡蚧轮枝菌中抗高温胁迫的功能,为蜡蚧轮枝菌制剂的推广和田间应用奠定理论基础。研究结果如下:34! 下VL17、VL18菌株菌落直径差异最显著,VL18菌株比VL17大63.16%,37℃培养条件下,两菌株均停止生长。37℃时对两菌株孢子萌发的抑制作用最大,VL18菌株的孢子萌发率是VL17菌株的2.03倍。温度影响蜡蚧轮枝菌对蚜虫的致病力,28℃时两菌株对蚜虫的致病力最大,VL18菌株的蚜虫校正死亡率是VL17菌株的1.02倍,37℃时两菌株对蚜虫的致病力最小,VL18菌株的蚜虫校正死亡率是VL17菌株的2.67倍。通过温度对蜡蚧轮枝菌的杀蚜活性、菌丝生长以及孢子萌发的研究,结果表明:蜡蚁轮枝菌VL18菌株比VL17更具有耐高温胁迫的能力。蜡蚧轮枝菌 VL17、VL18 菌株经 42℃高温胁迫 15 min、30 min、60 min、120 min后,蜡蚧轮枝菌VL17菌株在胁迫15-60min范围内,HSP70和SHSP基因的相对表达量均随胁迫时间的增多而升高,60 min时SHSP基因表达量是HSP70的7.95倍,当胁迫时间达到120 min时,两基因表达量均比60 min时降低,SHSP表达量是HSP70的4倍;耐热菌株VL18菌株在胁迫30 min以上时,胁迫时间越长,HSP70和SHSP基因的相对表达量越大,胁迫30 min时,SHSP的表达量是HSP70的5.85倍,当胁迫时间达到120 min时,两基因表达量差异最大,SHSP表达量是HSP70的6.41倍,表明SHSP基因对蜡蚧轮枝菌抗高温胁迫具有更关键的作用,是蜡蚧轮枝菌耐热的关键热激蛋白基因。通过克隆SHSP基因,将基因组DNA序列与转录组序列比对,发现SHSP基因不含内含子。通过构建敲除载体pCAMBIA-barl-hsp,利用农杆菌介导的遗传转化法,获得了SHSP基因的敲除菌株,PCR筛选鉴定,最终得到蜡蚧轮枝菌的敲除株。对原始菌株和敲除菌株的生物学特性进行比较,发现在相同碳源培养基上,敲除菌株菌落直径比原始菌株小,在相同氮源培养基上,也是如此。敲除菌株的菌丝生长抑制率大于化学胁迫剂相同时的原始菌株菌丝生长抑制率,敲除菌株的孢子萌发率比化学胁迫剂相同时的原始菌株孢子萌发率小,说明敲除菌株对化学胁迫剂更敏感。在45℃高温胁迫下,随着胁迫时间的增长,蜡蚧轮枝菌原始菌株VL18菌株和敲除菌株的孢子萌发率均逐渐减小,在胁迫时间相同的情况下,敲除菌株的孢子萌发率比原始菌株的孢子萌发率小,当胁迫时间达到120min时,原始菌株的孢子萌发率是敲除菌株的3.52倍。在紫外线辐射下,随着辐射时间的增长,蜡蚧轮枝菌原始菌株VL18菌株和敲除菌株的孢子萌发率均逐渐减小,且辐射时间相同时,敲除菌株的孢子萌发率比原始菌株小,当胁迫时达到10 min时,原始菌株与敲除菌株孢子萌发率差异最大,原始菌株是敲除菌株的2.83倍。在天数相同的情况下,敲除菌株对蚜虫的致病力比原始菌株弱,到第5天时,原始菌株的蚜虫校正死亡率比敲除菌株大7.53%。通过对原始菌株和敲除菌株的生物学特性比较,发现SHSP基因的缺失导致蜡蚧轮枝菌对碳氮源的利用率降低,对化学胁迫剂的抗性降低,对孢子耐高温、耐紫外辐射的能力降低及对蚜虫的致病力降低。
[Abstract]:Verticillium lecanii (Verticillium lecanii is used to control plant diseases and insect pests of entomopathogenic fungi, fungal biocontrol agents during storage and field insecticidal effect stability is an important reason for the effect of its application, the temperature affects the fungal spore storage and field infection force important factor, heat shock protein of organisms improvement of thermal resistance plays an important role, therefore, the research of heat shock proteins of entomopathogenic fungi resistance effect, to solve the fungal agent development and field application problems has an important role. In this experiment, Verticillium lecanii VL17 strain VL18 as tested strains, the temperature of two strains of aphid killing activity, mycelial growth and spore germination of the differences, and then compare the resistant and sensitive strains under high temperature stress, heat shock protein gene expression, find out the heat resisting Verticillium lecanii The key of heat shock protein species play a major role in high temperature stress, heat shock protein gene cloning of key, and the key to construct a heat shock protein gene knockout strains, compared to the original strain and knockout strains. The biological characteristics of this experiment reveals the heat shock protein in Verticillium lecanii in high temperature stress, which the theoretical basis for the preparation of leacanii promotion and field application. The results are as follows: 34! VL17, the most significant strain VL18 colony diameter difference, strain VL18 is 63.16% more than VL17, 37 C conditions, two strains were.37 degrees to stop the growth inhibition of two strains of spore germination, VL18 strains of spore germination rate is 2.03 times higher than the VL17 strain. The pathogenicity of temperature effect of Verticillium lecanii on aphids, the maximum 28 degrees on the pathogenicity of two strains of aphids, VL18 strains of aphids corrected mortality is 1.02 times that of VL17 strains, 37 C two The pathogenicity of aphids was minimal, VL18 strains of aphids corrected mortality is 2.67 times that of VL17 strain. The temperature of Verticillium lecanii activity, mycelial growth and spore germination of the study, the results show that the Verticillium lecanii VL18 strain has more tolerance to heat stress than VL17 wax. Lecanicillium VL17, VL18 strain was 42 degrees high temperature stress of 15 min, 30 min, 60 min, 120 min, Verticillium lecanii VL17 strain stress in the range of 15-60min, the relative expression of HSP70 and SHSP genes were increased with the stress time increasing, the 60 min SHSP gene expression is 7.95 times of HSP70, when the stress time reaches 120 min, two gene expression were higher than 60 min decreased, the expression of SHSP is 4 times that of HSP70 VL18 strain; heat stress in the above 30 min, the stress time was longer, the relative expression of HSP70 and SHSP genes and the larger the amount of stress of 30 min SHSP. The expression is 5.85 times that of HSP70, when the stress time reaches 120 min, the two biggest differences in gene expression, the expression of SHSP is 6.41 times that of HSP70, suggesting that the SHSP gene of Verticillium lecanii against high temperature stress has a key role, is the key of heat shock protein leacanii due to heat through the cloning of SHSP gene, the genomic DNA sequence and transcriptome sequence alignment revealed that SHSP gene contains no intron. Through the construction of knockout vector pCAMBIA-barl-hsp by Agrobacterium mediated transformation, the SHSP gene knockout strains, screening and identification of PCR, finally get the leacanii knockout the original strains and strains. Knockout biological characteristics were compared, found in the same medium carbon source, knockout strains colony diameter than the original strain, under the same nitrogen source medium, too. Knockout strains of mycelial growth inhibition rate was higher than the chemical At the same time, the original stress agent phase mycelial growth inhibition rate, knockout germination rate than the chemical stress agent and original strain spore germination rate of small strain spores that knockout strains of chemical agent is more sensitive. Stress at 45 DEG C under high temperature stress, with the stress time increasing, the original strain of Verticillium lecanii VL18 knockout strains and germination rate decreased in spore strains, stress time under the same conditions, the germination rate of knockout germination rate is smaller than the original strain of spore spore strains, when the stress time reaches 120min, the original strain of spore germination rate is 3.52 times the knockout strain. In the ultraviolet radiation. Along with the radiation time the growth of Verticillium lecanii VL18 strain and original strain knockout strains decreased the germination rate of spores, and radiation at the same time, knockout strains of spore germination than the original strain, when the stress reaches 10 Min, the original strain and knockout strains of spore germination rate difference is 2.83 times of the original strain knockout strains. In the days under the same knockout pathogenicity on aphid strain than the original strain is weak, the fifth day, the mortality rate than the original strain of aphid knockout strains based on 7.53%. the original strain and biological characteristics of knockout strains, found that deletion of the SHSP gene caused by Verticillium lecanii on carbon and nitrogen source rate, reduce the resistance to chemical stress agent, high temperature resistance of spores, ultraviolet radiation and decrease on aphid virulence.

【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S476.12

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