【作者】 欧翔；

【导师】 谭艾娟；

【作者基本信息】 贵州大学 ， 微生物与生化药学， 2008， 硕士

【摘要】 蝉拟青霉既是名贵中药,又有较高的致病性。使用蝉拟青霉做材料研究它在生物防治上的开发价值,具有深远的意义。本文研究蝉拟青霉对蚕豆蚜虫和小菜蛾的杀虫作用。得出8株蝉拟青霉都具有不同程度的杀虫作用,其中GZDXIFR4611的杀虫效果最好。孢子悬液的最高致死率可达到88.7%。影响昆虫病原真菌毒力有酶的因素,本研究主要从酶的方面研究它的杀虫作用。所以对蝉拟青霉蛋白酶、几丁质酶、溶菌酶和酯酶的酶活力研究,在8种菌株中,GZDXIFR4611酯酶的活性明显比其它三种酶的活力高,初步得到蝉拟青霉酯酶与毒力有一定的相关性。为了更好的研究蝉拟青霉酯酶与杀虫作用的关系,优化产酯酶的条件,从而提高其产率。再进行分离纯化,对分离物质进行杀虫实验。筛选蝉拟青霉酯酶的最佳液体培养基:可溶性淀粉10g/L,酵母膏7.5g/L,K₂HPO₄ 0.35g/L,MgSO₄.7H₂O0.35g/L。GZDXIFR4611在48小时产酯酶最多,酶的最适反应温度为40℃,最适pH为6.5,最适反应时间为40min,在25℃条件下,蝉拟青酶酯酶的半衰期约是58h,蝉拟青霉酯酶在40℃时稳定性最高。粗酶液经硫酸铵分级盐析,取40—70%硫酸铵饱和度下的蛋白质沉淀溶解,用Sephadex G-50柱层析脱盐,收集高活性管;使用Sephadex G-100对酶液进行分离纯化,也收集高活性管。我们得出酯酶收率是20.4%,纯化倍数是8.05。把分离纯化的每个阶段得到的酶液进行杀虫实验,得出通过Sephadex G-100过柱收集管的毒力效果最好,致死率可达到86.7%,说明蝉拟青霉酯酶有很大的毒力作用。研究了8株蝉拟青霉胞内几丁质酶、蛋白酶、溶菌酶活力,从中筛选出酶活力高的菌株（GZDXIFR3716）;并对3种酶的产酶条件进行了研究。结果表明,GZDXIFR3716几丁质酶、蛋白酶和溶菌酶含量最高的发酵时间分别为48h、60h和84h;3种酶的最适反应温度分别为50℃、40℃和40℃;3种酶的最适反应pH分别为6.0、7.0和5.0。更多还原

【Abstract】 Paecilomyces cicadaes（PC） is a precious Chinese herbal medicine with high pathogenicity.It is significant to study the virulence of ogenic fungi in the biocontrol field. This thesis is to study the insecticidal effect of PC to aphides and Plutella xylostlla.All of the 8 strains of PC used in this thesis have different insecticidal effect.One of the strains, GZDXIFR4611,has the highest insecticidal function and its insecticidal rate could reach 88.7%especially in spore liquid medium.Study of four different enzymes in PC including chitinase,proteinase,lysozyme and esterase indicates GZDXIFR4611 esterase has a higher enzyme activity then others,hence,shows us the correlation between PC’s esterase and virulence.In order to further research the relationship between PC esterase and insecticidal effect, we optimize the producing conditions of esterase,thereby raising the yield.By purifying the production,we got a relatively pure substances which was used in the insecticidal experimental again.The recipe of the optimal liquid medium used in selecting strains is soluble amylum（10g·L-1_-）,yeast extract paste（7.5g·L-1）,K₂HPO₄（0.35g·L-1）, MgSO₄.7H₂O（0.35 g·L-1）.Indicated by the research,the optimum incubation time is 48h, the optimum reaction factors of PC’s esterase proves at PH=6.5,40℃,40 minutes with the half-life is 58 hours at normal temperature（25℃）,and the enzyme remains stable at 40℃. Crude enzyme was salting out by 40—70%ammonium sulfate and filtrated by sephadex G-50 gel,Collecting the high active tubes and filtrated again by sephadex G-100 gel.In the experiment,the yield rate reaches 20.4%and the purification times is 8.05.The insecticide experiment with enzyme obtained at different stages tells us the enzyme purified with Sephadex G-100 has the highest insecticidal effect,the insecticidal rate could reach 86.7%, and indicates PC esterase is endowed with high virulent property.Based on the enzyme activity experiments of chitinase,proteinase,lysozyme among 8 strains of PC,we select GZDXIFR3716 as the highest activity strain.We also do some studies on enzyme’s yield condition.The incubation time for the highest concentration of hitinase,proteinase,lysozyme in the GZDXIFR3716 liquid medium are 48h,60h and 84 h respectively;and the optimal reaction temperature are 50℃,40℃and 40℃in that order; the optimal reaction pH value are 6.0,7.0 and 5.0 in that order.更多还原