發(fā)布時(shí)間：2018-12-11 10:43

【摘要】：玫煙色棒束孢(Isaria fumosorosea)及球孢白僵菌(Beauveria bassiana)作為兩種常見的昆蟲病原真菌,通過分泌蛋白酶等多種水解酶來降解昆蟲體壁,從而侵染寄主昆蟲,在害蟲的生物防治中起重要作用。本試驗(yàn)以玫煙色棒束孢Pf9606、Pf7606、Pf904、Pf941、Pf14及球孢白僵菌作為供試菌株,比較不同菌株之間的毒力差異和各菌株P(guān)rl蛋白酶活力,采用熒光定量PCR技術(shù)檢測了不同菌株蛋白酶Prl基因的表達(dá)量,此外,通過熒光顯微技術(shù)檢測了被玫煙色棒束孢侵染的小菜蛾幼蟲的血細(xì)胞凋亡,結(jié)果如下：1.玫煙色棒束孢及球孢白僵菌對桃蚜和小菜蛾幼蟲的毒力測定表明：供試各菌株毒力存在明顯的差異,對桃蚜致病力變化范圍為78.43～96.08%,LT50的變化范圍為2.39-3.10 d,其中菌株P(guān)f904對桃蚜的致病力最高達(dá)96.08%,致死中時(shí)最短為2.39d；球孢白僵菌對桃蚜的致病力最低為78.43%,致死中時(shí)最長為3.10d。玫煙色棒束孢Pf904對小菜蛾3齡幼蟲的致病力為96.67%,LT50為1.37d；對小菜蛾4齡幼蟲的致病力為40%,可見3齡幼蟲的抗病能力顯著低于4齡幼蟲。2.采用專一性短肽底物Sue-Ala-Ala-Pro-Phe-pNA法對6株供試菌株進(jìn)行Prl蛋白酶活性測定,結(jié)果發(fā)現(xiàn)：不同菌株P(guān)rl酶活力不同,Prl蛋白酶酶活力從高到低依次是：玫煙色棒束孢Pf904、Pf7606、Pf14、Pf9606、Pf941、球孢白僵菌。其中菌株P(guān)f904 Pr1酶活力水平最高達(dá)0.0857U/mg,球孢白僵菌Prl酶活力水平最低僅0.0052 U/mg。3.實(shí)時(shí)熒光定量PCR技術(shù)檢測了不同菌株蛋白酶Prl基因的表達(dá)量,結(jié)果顯示：供試各菌株蛋白酶Prl基因的表達(dá)量不同。玫煙色棒束孢904的表達(dá)量最高,為對照的5.9倍;白僵菌的表達(dá)量最低,為對照的1.1倍。玫煙色棒束孢7606、14、9606、941的表達(dá)量依次為對照的5.8、5.2、4.8、3.1倍。4.采用spss軟件對玫煙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達(dá)量三者之間的聯(lián)系進(jìn)行了分析：Prl酶活力與菌株的致病力呈正相關(guān),Prl蛋白酶基因的表達(dá)量與Prl酶活之間存在正比關(guān)系,故Prl蛋白酶活力、Prl蛋白酶基因表達(dá)量均對致病力有顯著影響,決定著菌株的毒力。5.熒光顯微鏡技術(shù)檢測接菌后的小菜蛾幼蟲血細(xì)胞凋亡,研究表明：正常的小菜蛾3齡與4齡幼蟲血細(xì)胞的形態(tài)、顏色基本一致。在玫煙色棒束孢904作用下,小菜蛾幼蟲血細(xì)胞發(fā)生了不同程度的細(xì)胞凋亡,3齡幼蟲處理12h后就出現(xiàn)早期凋亡與晚期凋亡細(xì)胞,72h后3齡幼蟲細(xì)胞全部凋亡;而4齡24h后才逐漸出現(xiàn)早期凋亡細(xì)胞,72h后4齡幼蟲細(xì)胞半數(shù)凋亡;3齡發(fā)生凋亡現(xiàn)象明顯比4齡早24h左右,且相同處理時(shí)間3齡凋亡細(xì)胞數(shù)量也明顯高于4齡,并隨著時(shí)間累積凋亡細(xì)胞數(shù)量不斷增多而正常細(xì)胞數(shù)量逐漸減少。在熒光顯微鏡下可清晰觀察到發(fā)生細(xì)胞凋亡的細(xì)胞核濃縮、染色質(zhì)凝聚、細(xì)胞核碎裂等形態(tài)特征.與正常的血細(xì)胞均有明顯差別。
[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.
【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S476.12

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