蝉拟青霉杀虫作用的研究
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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,K_2HPO_4 0.35g/L,MgSO_4.7H_2O0.35g/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。
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),K_2HPO_4(0.35g·L-1), MgSO_4.7H_2O(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.
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,K_2HPO_4 0.35g/L,MgSO_4.7H_2O0.35g/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。
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),K_2HPO_4(0.35g·L-1), MgSO_4.7H_2O(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.
引文
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[2]王中康,殷幼平,彭国雄,夏玉先.杀虫真菌功能基因克隆和菌株改良研究进展.国虫生真菌研究与应用,2003,3(5):18-28.
[3]王四宝,刘竞男,吴刚等.两种虫生真菌酯酶同工酶酶谱多样性研究.生物学杂志,2002,18(2):23-25.
[4]王春雷,芦柏震,侯桂兰.中国蝉花的研究进展[J].中国药学杂志,2006,41(4):244-246.
[5]孙中涛,闫艳春,姚良同等.R—工程菌表达产物酯酶B1的纯化及其特性.安全与环境学报,2003,3(6):31-34.
[6]刘广玉,胡菽英.天然蝉花和人工培养品镇静镇痛作用比较.现代应用药学,1991,8(2):5-8.
[7]刘莹,孙荣丹,扬翔华等.溶菌酶的应用现状.饲料工业,2006,4(6):27-36
[8]安建梅.粉质拟青霉侵染菜青虫致病过程的研究.植物保护,2002,28(4):14-16.
[9]安建梅.粉质拟青酶代谢产物生理活性的研究.山西农业大学学报,2003,23(2):103-105
[10]李文华,王中康,殷幼平.虫生真菌附着胞研究进展.中国虫生真菌研究与应用,2003,3(5):54-59.
[11]李烨。现代生物技术在环境保护中的应用.淮阴工学学报.2002,11(5):30-33.
[12]李增智,樊美珍.林用真菌杀虫剂生产工艺及应用技术研究.安徽农业大学学报,1996,23(3):233-238.
[13]李敏.溶菌酶及其应用.生物学教学,2006,31-41.
[14]肖林榕.菌类本草[M].北京:中国医药科技出版社,2003,35-36.
[15]陈万群,陈古荣.冬虫夏草代用品研究进展.中草药,1994,25(5):269-271.
[16]陈秀芳,金丽琴,吕建新.蝉拟青霉减轻环磷酰胺所致免疫抑制效应的实验研究.温州医学院学报.2002,32(6):23-29.
[17]陈祝安,黄基荣,许益伟,姚丽娟.蝉拟育霉防治菜粉蝶幼虫试验.生物防治通报 1990,6(3):131-133.
[18]陈驹声.近代工业微生物学.上海科学技术出版社,1982:355-358
[19]宋玉良,宋捷民,严建伟.高压液相色谱法测定蝉花中腺苷的含量.中药新药与临床药理,2000,11(6):367-367.
[20]金丽琴,吕建新,杨介钻.蝉拟青霉总多糖对大鼠非特异性免疫调节作用的研究.中国病理生理杂志,2001,17(12):1232-1235.
[21]张永军,刘爱英,梁宗琦.紫外线对蝉拟青霉原生质体的诱变效果.贵州农业科学,1998,26(6):1-3.
[22]张树政.酶制剂工业[M].北京:科学出版社,1984,655.
[23]周德庆.微生物学教程.北京:高等教育出版社.1997,265-266.
[24]武觐文.应用粉拟青酶防治松毛虫.安徽农业大学学报,1996,23(3):438-445..
[25]姚剑,李增智,樊美珍.虫生真菌入侵过程中酶的研究.安徽农业大学学报,1996,23(3):303-308.
[26]胡学智.酶制剂生产技术[M].北京:化学工业出版社,1994,313-319.
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