玫烟色棒束孢Pr1酶与毒力的关系及对小菜蛾幼虫血细胞凋亡的影响

发布时间：2018-12-11 10:43

【摘要】：玫烟色棒束孢(Isaria fumosorosea)及球孢白僵菌(Beauveria bassiana)作为两种常见的昆虫病原真菌,通过分泌蛋白酶等多种水解酶来降解昆虫体壁,从而侵染寄主昆虫,在害虫的生物防治中起重要作用。本试验以玫烟色棒束孢Pf9606、Pf7606、Pf904、Pf941、Pf14及球孢白僵菌作为供试菌株,比较不同菌株之间的毒力差异和各菌株Prl蛋白酶活力,采用荧光定量PCR技术检测了不同菌株蛋白酶Prl基因的表达量,此外,通过荧光显微技术检测了被玫烟色棒束孢侵染的小菜蛾幼虫的血细胞凋亡,结果如下：1.玫烟色棒束孢及球孢白僵菌对桃蚜和小菜蛾幼虫的毒力测定表明：供试各菌株毒力存在明显的差异,对桃蚜致病力变化范围为78.43～96.08%,LT50的变化范围为2.39-3.10 d,其中菌株Pf904对桃蚜的致病力最高达96.08%,致死中时最短为2.39d；球孢白僵菌对桃蚜的致病力最低为78.43%,致死中时最长为3.10d。玫烟色棒束孢Pf904对小菜蛾3龄幼虫的致病力为96.67%,LT50为1.37d；对小菜蛾4龄幼虫的致病力为40%,可见3龄幼虫的抗病能力显著低于4龄幼虫。2.采用专一性短肽底物Sue-Ala-Ala-Pro-Phe-pNA法对6株供试菌株进行Prl蛋白酶活性测定,结果发现：不同菌株Prl酶活力不同,Prl蛋白酶酶活力从高到低依次是：玫烟色棒束孢Pf904、Pf7606、Pf14、Pf9606、Pf941、球孢白僵菌。其中菌株Pf904 Pr1酶活力水平最高达0.0857U/mg,球孢白僵菌Prl酶活力水平最低仅0.0052 U/mg。3.实时荧光定量PCR技术检测了不同菌株蛋白酶Prl基因的表达量,结果显示：供试各菌株蛋白酶Prl基因的表达量不同。玫烟色棒束孢904的表达量最高,为对照的5.9倍;白僵菌的表达量最低,为对照的1.1倍。玫烟色棒束孢7606、14、9606、941的表达量依次为对照的5.8、5.2、4.8、3.1倍。4.采用spss软件对玫烟色棒束孢及球孢白僵菌的致病力、Prl蛋白酶活力及基因的表达量三者之间的联系进行了分析：Prl酶活力与菌株的致病力呈正相关,Prl蛋白酶基因的表达量与Prl酶活之间存在正比关系,故Prl蛋白酶活力、Prl蛋白酶基因表达量均对致病力有显著影响,决定着菌株的毒力。5.荧光显微镜技术检测接菌后的小菜蛾幼虫血细胞凋亡,研究表明：正常的小菜蛾3龄与4龄幼虫血细胞的形态、颜色基本一致。在玫烟色棒束孢904作用下,小菜蛾幼虫血细胞发生了不同程度的细胞凋亡,3龄幼虫处理12h后就出现早期凋亡与晚期凋亡细胞,72h后3龄幼虫细胞全部凋亡;而4龄24h后才逐渐出现早期凋亡细胞,72h后4龄幼虫细胞半数凋亡;3龄发生凋亡现象明显比4龄早24h左右,且相同处理时间3龄凋亡细胞数量也明显高于4龄,并随着时间累积凋亡细胞数量不断增多而正常细胞数量逐渐减少。在荧光显微镜下可清晰观察到发生细胞凋亡的细胞核浓缩、染色质凝聚、细胞核碎裂等形态特征.与正常的血细胞均有明显差别。
[Abstract]:As two common entomopathogenic fungi, (Isaria fumosorosea) and (Beauveria bassiana) of Rosa fumigatus, as two common entomopathogenic fungi, degrade the body wall of insects by secreting protease and other hydrolases to infect host insects. It plays an important role in biological control of pests. In this experiment, Pf9606,Pf7606,Pf904,Pf941,Pf14 and Beauveria bassiana were used to compare the virulence of different strains and the activity of Prl protease. Fluorescence quantitative PCR technique was used to detect the expression of protease Prl gene in different strains. In addition, the apoptosis of xylostella larvae infected by rosacea roxburghii was detected by fluorescence microscopy. The results were as follows: 1. The virulence of Beauveria bassiana and Beauveria bassiana to the larvae of Peach aphid and Plutella xylostella showed that the virulence of the tested strains was significantly different, and the pathogenicity of the tested strain was in the range of 78.436.08. The variation range of LT50 was 2.39-3.10 days, and the pathogenicity of strain Pf904 was 96.08 and 2.39 days respectively. The pathogenicity of Beauveria bassiana was 78.43 and the longest was 3.10 days. The pathogenicity of Pf904 to the 3rd instar larvae of Plutella xylostella was 96.6767 and LT50 was 1.37 days, and the pathogenicity to the 4th instar larvae of Plutella xylostella was 40, which showed that the disease resistance of the 3rd instar larvae was significantly lower than that of the 4th instar larvae. 2. The activity of Prl protease was determined by specific short peptide substrate Sue-Ala-Pro-Phe-pNA method. The results showed that the activity of Prl enzyme was different among different strains. The enzyme activity of Prl protease from high to low is: Pf904,Pf7606,Pf14,Pf9606,Pf941, Beauveria bassiana. The highest level of Pf904 Pr1 enzyme activity was 0.0857 U / mg, and the lowest level of Prl activity of Beauveria bassiana was only 0.0052 U / mg 路3. The expression of protease Prl gene in different strains was detected by real-time fluorescence quantitative PCR. The results showed that the expression of protease Prl gene in different strains was different. The highest amount of expression was found in roseosporium roxburghii 904, which was 5.9 times higher than that of the control, and the lowest in Beauveria bassiana was 1.1 times as much as that of the control. The expression level of Rosa fumigatus 7606 was 3.1 times higher than that of the control, 5.85.2and 4.84.80.The expression level of 9606941 was 3.1 times higher than that of the control. The relationship among pathogenicity, Prl protease activity and gene expression of Beauveria bassiana and Bassia bassiana were analyzed by spss software. The results showed that the activity of Prl was positively correlated with the pathogenicity of the strain. There was a direct relationship between the expression of Prl protease gene and the activity of Prl, so the activity of Prl protease and the expression of Prl protease gene had significant influence on the pathogenicity, which determined the virulence of the strain. Fluorescence microscopy was used to detect the blood cell apoptosis of diamondback moth larvae after inoculation. The results showed that the blood cells of the 3rd and 4th instar larvae of normal Plutella xylostella were the same in shape and color. The blood cells of Plutella xylostella larvae had different degrees of apoptosis under the action of rose904. Early and late apoptotic cells appeared in the 3rd instar larvae after 12h treatment, and all the 3rd instar larvae cells were apoptotic after 72 hours. However, early apoptotic cells appeared in the 4th instar after 24h, and half of the apoptotic cells in the 4th instar larvae at 72 h. The number of apoptotic cells at the 3rd instar was significantly higher than that at the 4th instar, and the number of the normal cells decreased gradually with the increasing of the number of the apoptotic cells at the 3rd instar. The morphological features of apoptosis, such as nuclear condensation, chromatin condensation and nuclear fragmentation, can be clearly observed under fluorescence microscope. There were significant differences between the blood cells and normal blood cells.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S476.12

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