玫烟色棒束孢Pr1酶活力、Pr1酶基因表达量与其毒力的相关性
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摘要
丝氨酸弹性凝乳蛋白酶Pr1是一类能高效降解昆虫体壁蛋白的重要酶,其活力与虫生真菌的毒力有很大的关系。探索玫烟色棒束孢不同菌株Pr1酶活力、Pr1蛋白酶基因表达量与毒力的相关性对该菌的应用具有重要的意义。文中采用专一性短肽底物Suc-Ala-Ala-Pro-Phe-pNA和荧光定量PCR分别测定了玫烟色棒束孢不同菌株的Pr1酶活和Pr1基因的表达量,并采用坡塔喷雾法测定了供试菌株对桃蚜的毒力。结果表明:不同供试菌株Pr1蛋白酶活力与其毒力的线性回归方程为y=3.64x+0.62,R~2=0.432,两者呈正相关;供试菌株Pr1酶活力、Pr1基因表达量与毒力的回归方程为y=0.236+10.833x_1–0.039x_2 (x_1=Pr1酶活力,x_2=Pr1基因表达量),R~2=0.568,说明线性拟合方程能很好地反映原始数据;序列相关系数D-W为2.444,在0.05水平上相关显著,表明Pr1酶活力、Pr1基因表达量对毒力有显著影响;VIF=12.705表明Pr1酶活力、Pr1基因表达量存在中度多重共线性。因此建议将Pr1蛋白酶的酶活力和Pr1基因表达量作为菌株毒力筛选时的重要指标。
Serine elastic chymotrypsin Pr1 is an enzyme that efficiently degrades insect body wall protein through itsconnection with the virulence of entomogenous fungi.Therefore,it is important to explore the relationship between the Pr1 protease activity,the Pr1 gene expression and the virulence of different strains of entomogenous fungi.Specific peptide substrate Suc-Ala-Ala-Pro-Phe-pNA and fluorogenic quantitative PCR were used for detecting Pr1 protease activity and Pr1 gene expression,and the slope spray method was used for evaluating the virulence of the fungi on the Myzus persicae.The results indicated that the linear regression equation of the Pr1 protease activity and the virulence of different strains were:y=3.64x+0.62,R2=0.432.It was shown that there is a positive correlation between the Pr1 protease activity and virulence of different strains.Moreover,the result of the multiple linear regression analysis between Pr1 protease activity,Pr1 gene expression and the virulence of different strains was:y=0.236+10.833x1–0.039x2(x1 represents Pr1 protease activity while x2 represents Pr1 gene expression),R2=0.568,which suggested that the raw data could be represented by a linear fitting equation.The serial correlation coefficient was high(D-W was 2.444),indicating that Pr1 protease activity and Pr1 gene expression have great effect on the virulence of the fungi.Additionally,VIF=12.705,which shows that moderate multiple collinear exists between Pr1 protease activity and Pr1 gene expression.Therefore,Pr1 protease activity and Pr1 gene expression could be recommended as important indicators for strain virulence selection.
Serine elastic chymotrypsin Pr1 is an enzyme that efficiently degrades insect body wall protein through itsconnection with the virulence of entomogenous fungi.Therefore,it is important to explore the relationship between the Pr1 protease activity,the Pr1 gene expression and the virulence of different strains of entomogenous fungi.Specific peptide substrate Suc-Ala-Ala-Pro-Phe-pNA and fluorogenic quantitative PCR were used for detecting Pr1 protease activity and Pr1 gene expression,and the slope spray method was used for evaluating the virulence of the fungi on the Myzus persicae.The results indicated that the linear regression equation of the Pr1 protease activity and the virulence of different strains were:y=3.64x+0.62,R2=0.432.It was shown that there is a positive correlation between the Pr1 protease activity and virulence of different strains.Moreover,the result of the multiple linear regression analysis between Pr1 protease activity,Pr1 gene expression and the virulence of different strains was:y=0.236+10.833x1–0.039x2(x1 represents Pr1 protease activity while x2 represents Pr1 gene expression),R2=0.568,which suggested that the raw data could be represented by a linear fitting equation.The serial correlation coefficient was high(D-W was 2.444),indicating that Pr1 protease activity and Pr1 gene expression have great effect on the virulence of the fungi.Additionally,VIF=12.705,which shows that moderate multiple collinear exists between Pr1 protease activity and Pr1 gene expression.Therefore,Pr1 protease activity and Pr1 gene expression could be recommended as important indicators for strain virulence selection.
引文
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[2]Charnley AK,St Leger RJ.The role of cuticle-degrading enzymes in fungal pathogenesis in insects//Cole GT,Hoch HC,eds.The Fungal Spore and Disease Initiation in Plants and Animals.Boston,MA:Springer,1991:267-287.
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[5]Gao TN.Infection and influence of reproduction of Bemisia tabaci by Isaria fumosorosea[D].Guangzhou:South China Agricultural University,2016:1-62(in Chinese).高天妮.玫烟色棒束孢对烟粉虱的侵染及对生殖的影响[D].广州:华南农业大学,2016:1-62.
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[10]Wang HM,Zhang H,Hao C,et al.Effects of Isaria fumosorosea infection on different enzyme activities in the larvae of Plutella xylostella.Mycosystema,2013,32(2):269-276(in Chinese).王宏民,张奂,郝赤,等.玫烟色棒束孢侵染对小菜蛾幼虫体内不同酶活的影响.菌物学报,2013,32(2):269-276.
[11]Lei YY,He YR,Xie MQ,et al.Transcriptome analysis of immune responses of Plutella xylostella(Lepidoptera:Plutellidae)infected by the entomopathogenic fungus Isaria fumosorosea.Acta Entomol Sin,2016,59(9):956-964(in Chinese).雷妍圆,何余容,谢梅琼,等.玫烟色棒束孢诱导的小菜蛾免疫响应表达谱分析.昆虫学报,2016,59(9):956-964.
[12]Tang Q.Cloning and expression of the virulence genes from Isaria fumosorosea and generation of transgenic Beauveria bassiana strains with these genes[D].Hefei:Anhui Agricultural University,2009:1-120(in Chinese).汤强.玫烟色棒束孢毒力基因的克隆与表达及球孢白僵菌高毒力重组菌株的获得[D].合肥:安徽农业大学,2009:1-120.
[13]Gu JR.Research on the different expression of pathogenicity-related genes of Isaria fumosorosea[D].Guangzhou:South China Agricultural University,2016:1-51(in Chinese).顾家睿.玫烟色棒束孢致病相关基因的差异表达研究[D].广州:华南农业大学,2016:1-51.
[14]Tang Q,Zhang YP,Xie L,et al.Generation of transgenic Beauveria bassiana strains with chitinase gene from Isaria fumosorosea and its increased virulence against Dendrolimus punctatus(Lepidoptera:Lasiocampidae).Acta Entomol Sin,2009,52(7):755-762(in Chinese).汤强,章玉萍,谢翎,等.玫烟色棒束孢几丁质酶的转基因球孢白僵菌菌株的获得及其对马尾松毛虫的毒力增效作用.昆虫学报,2009,52(7):755-762.
[15]Gillespie RJ,Bateman R,Charnley AK.Role of cuticle-degrading proteases in the virulence of Metarhizium spp.for the desert locust,Schistocerca gregaria.J Invertebr Pathol,1998,71(2):128-137.
[16]Leger RJ,Cooper RM,Charnley AK.Production of cuticle-degrading enzymes by the entomopathogen Metarhizium anisopliae during infection of cuticles from Calliphora vomitoria and Manduca sexta.JGeneral Microbiol,1987,133(5):1371-1382.
[17]Zhang LW,Liu YJ,Yao J,et al.Evaluation of Beauveria bassiana(Hyphomycetes)isolates as potential agents for control of Dendroctonus valens.Insect Sci,2011,18(2):209-216.
[18]Li Z,Jin DC,Liu AY.Correlation between extracellular protease of Paecilomyces cicadae and its toxicity to Lipaphis erysimi.Guizhou Agric Sci,2010,38(12):135-137(in Chinese).李忠,金道超,刘爱英.蝉拟青霉胞外蛋白酶与菌株毒力的关系.贵州农业科学,2010,38(12):135-137.
[19]Tian ZL,Zhu XM,Li QY,et al.Study on correlation between Pr1 protease activity and strain virulence of Beauveria bassiana.J Jilin Agric Univ,2012,34(6):607-611(in Chinese).田志来,朱晓敏,李启云,等.白僵菌菌株毒力与Pr1蛋白酶活性相关性研究.吉林农业大学学报,2012,34(6):607-611.
[20]Lei YY,Lv LH,He YR,et al.Correlation between biological characteristics of Beauveria bassiana and its virulence to Plutella xylostella.Chin J Biol Cont,2010,26(2):143-148(in Chinese).雷妍圆,吕利华,何余容,等.球孢白僵菌生物学特性与其对小菜蛾致病力相关性分析.中国生物防治,2010,26(2):143-148.
[2]Charnley AK,St Leger RJ.The role of cuticle-degrading enzymes in fungal pathogenesis in insects//Cole GT,Hoch HC,eds.The Fungal Spore and Disease Initiation in Plants and Animals.Boston,MA:Springer,1991:267-287.
[3]Fang WG,Feng J,Fan YH,et al.Expressing a fusion protein with protease and chitinase activities increases the virulence of the insect pathogen Beauveria bassiana.J Inverteb Pathol,2009,102(2):155-159.
[4]St Leger RJ,Joshi L,Bidochka MJ,et al.Construction of an improved mycoinsecticide overexpressing a toxic protease.Proc Natl Acad Sci USA,1996,93(13):6349-6354.
[5]Gao TN.Infection and influence of reproduction of Bemisia tabaci by Isaria fumosorosea[D].Guangzhou:South China Agricultural University,2016:1-62(in Chinese).高天妮.玫烟色棒束孢对烟粉虱的侵染及对生殖的影响[D].广州:华南农业大学,2016:1-62.
[6]Pu ZL,Li ZZ.Insect Mycology.Hefei:Anhui Science and Technology Publishing House,1996:1-715(in Chinese).蒲蛰龙,李增智.昆虫真菌学.合肥:安徽科学技术出版社,1996:1-715.
[7]Fang QX,Yang SF,Hu YM,et al.Control of whitefly,Trialeurodes vaporariorum,by Paecilomyces fumosorseus var.beijingensis Fang et.Q.T.Chen in greenhouses.Chin J Biol Control,1986,2(8):129-131(in Chinese).方祺霞,杨淑芳,胡亚梅,等.玫烟色拟青霉北京变种防治温室白粉虱的研究.生物防治通报,1986,2(8):129-131.
[8]Lei YY,He YR,LüLH.Physiological defense responses of Plutella xylostella(Lepidoptera:Plutellidae)larvae infected by entomopathogenic fungus Isaria fumosorosea.Acta Entomol Sin,2011,54(8):887-893(in Chinese).雷妍圆,何余容,吕利华.小菜蛾血淋巴对玫烟色棒束孢入侵的生理防御反应.昆虫学报,2011,54(8):887-893.
[9]Lei YY,LüLH,He YR,et al.The symptoms and histopathological changes of Plutella xylostella larvae infected with Isaria fumosorosea.Acta Phytophyl Sin,2011,38(2):147-152(in Chinese).雷妍圆,吕利华,何余容,等.小菜蛾感染玫烟色棒束孢后的病征及组织病理变化.植物保护学报,2011,38(2):147-152.
[10]Wang HM,Zhang H,Hao C,et al.Effects of Isaria fumosorosea infection on different enzyme activities in the larvae of Plutella xylostella.Mycosystema,2013,32(2):269-276(in Chinese).王宏民,张奂,郝赤,等.玫烟色棒束孢侵染对小菜蛾幼虫体内不同酶活的影响.菌物学报,2013,32(2):269-276.
[11]Lei YY,He YR,Xie MQ,et al.Transcriptome analysis of immune responses of Plutella xylostella(Lepidoptera:Plutellidae)infected by the entomopathogenic fungus Isaria fumosorosea.Acta Entomol Sin,2016,59(9):956-964(in Chinese).雷妍圆,何余容,谢梅琼,等.玫烟色棒束孢诱导的小菜蛾免疫响应表达谱分析.昆虫学报,2016,59(9):956-964.
[12]Tang Q.Cloning and expression of the virulence genes from Isaria fumosorosea and generation of transgenic Beauveria bassiana strains with these genes[D].Hefei:Anhui Agricultural University,2009:1-120(in Chinese).汤强.玫烟色棒束孢毒力基因的克隆与表达及球孢白僵菌高毒力重组菌株的获得[D].合肥:安徽农业大学,2009:1-120.
[13]Gu JR.Research on the different expression of pathogenicity-related genes of Isaria fumosorosea[D].Guangzhou:South China Agricultural University,2016:1-51(in Chinese).顾家睿.玫烟色棒束孢致病相关基因的差异表达研究[D].广州:华南农业大学,2016:1-51.
[14]Tang Q,Zhang YP,Xie L,et al.Generation of transgenic Beauveria bassiana strains with chitinase gene from Isaria fumosorosea and its increased virulence against Dendrolimus punctatus(Lepidoptera:Lasiocampidae).Acta Entomol Sin,2009,52(7):755-762(in Chinese).汤强,章玉萍,谢翎,等.玫烟色棒束孢几丁质酶的转基因球孢白僵菌菌株的获得及其对马尾松毛虫的毒力增效作用.昆虫学报,2009,52(7):755-762.
[15]Gillespie RJ,Bateman R,Charnley AK.Role of cuticle-degrading proteases in the virulence of Metarhizium spp.for the desert locust,Schistocerca gregaria.J Invertebr Pathol,1998,71(2):128-137.
[16]Leger RJ,Cooper RM,Charnley AK.Production of cuticle-degrading enzymes by the entomopathogen Metarhizium anisopliae during infection of cuticles from Calliphora vomitoria and Manduca sexta.JGeneral Microbiol,1987,133(5):1371-1382.
[17]Zhang LW,Liu YJ,Yao J,et al.Evaluation of Beauveria bassiana(Hyphomycetes)isolates as potential agents for control of Dendroctonus valens.Insect Sci,2011,18(2):209-216.
[18]Li Z,Jin DC,Liu AY.Correlation between extracellular protease of Paecilomyces cicadae and its toxicity to Lipaphis erysimi.Guizhou Agric Sci,2010,38(12):135-137(in Chinese).李忠,金道超,刘爱英.蝉拟青霉胞外蛋白酶与菌株毒力的关系.贵州农业科学,2010,38(12):135-137.
[19]Tian ZL,Zhu XM,Li QY,et al.Study on correlation between Pr1 protease activity and strain virulence of Beauveria bassiana.J Jilin Agric Univ,2012,34(6):607-611(in Chinese).田志来,朱晓敏,李启云,等.白僵菌菌株毒力与Pr1蛋白酶活性相关性研究.吉林农业大学学报,2012,34(6):607-611.
[20]Lei YY,Lv LH,He YR,et al.Correlation between biological characteristics of Beauveria bassiana and its virulence to Plutella xylostella.Chin J Biol Cont,2010,26(2):143-148(in Chinese).雷妍圆,吕利华,何余容,等.球孢白僵菌生物学特性与其对小菜蛾致病力相关性分析.中国生物防治,2010,26(2):143-148.