褐飞虱可溶性海藻糖酶的分子特性及dsRNA抑制表达分析
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摘要
为探究不同昆虫可溶性海藻糖酶的潜在功能,在获得褐飞虱2个可溶性海藻糖酶NlTRE1-1和NlTRE1-2的cDNA全长序列的基础上,采用生物信息学相关软件分析其序列特征以及二级、三级结构等分子特性及生理功能,同时构建不同昆虫海藻糖酶的系统进化树,进一步采用RNAi技术分析单个海藻糖酶基因抑制效果.结果显示,褐飞虱可溶性海藻糖酶NlTRE1-1和NlTRE1-2蛋白相似性高,其蛋白质二级和三级结构均类似,二级结构中含有相似比例的α螺旋、β-折叠和无规卷曲;系统进化树分析表明褐飞虱海藻糖酶与大豆蚜、豌豆蚜和灰飞虱等昆虫的TRE1具有较近的亲缘关系;且两个褐飞虱特异性TRE1-1和TRE1-2的dsRNA注射后48h,本基因的表达极显著下降.研究结果为以抑制海藻糖酶基因表达来进行的害虫防治技术奠定基础.
In order to explore the potential functions of different insects' soluble trehalase,basing on the full-length sequence of two soluble trehalase NlTRE1-1and NlTRE1-2cDNAs,the sequence characteristics as well as the secondary and tertiary structures of two TRE1 in Nilaparvata lugens were analyzed using the bioinformatics software.Then,the phylogenetic tree of trehalase from different insects was constructed.And the effect of single trehalase gene by RNAi technique was studied.These results showed that the soluble trehalase NlTRE1-1 and NlTRE1-2 proteins had high similarity,their protein secondary and tertiary structures were similar.The secondary structure contained similar ratioα-helix,β-sheet and random coil.The phylogenetic tree analysis showed that two trehalase of N.lugens had a close genetic relationship with the TRE1 of Aphis glycines,Acyrthosiphon pisumand Laodelphax striatella.The expression of this gene was significantly decreased when the two specific TRE1-1and TRE1-2dsRNAs were injected 48 h.This study results provided a basis for the prevention and treatment of pests that inhibited trehalase gene expression.
In order to explore the potential functions of different insects' soluble trehalase,basing on the full-length sequence of two soluble trehalase NlTRE1-1and NlTRE1-2cDNAs,the sequence characteristics as well as the secondary and tertiary structures of two TRE1 in Nilaparvata lugens were analyzed using the bioinformatics software.Then,the phylogenetic tree of trehalase from different insects was constructed.And the effect of single trehalase gene by RNAi technique was studied.These results showed that the soluble trehalase NlTRE1-1 and NlTRE1-2 proteins had high similarity,their protein secondary and tertiary structures were similar.The secondary structure contained similar ratioα-helix,β-sheet and random coil.The phylogenetic tree analysis showed that two trehalase of N.lugens had a close genetic relationship with the TRE1 of Aphis glycines,Acyrthosiphon pisumand Laodelphax striatella.The expression of this gene was significantly decreased when the two specific TRE1-1and TRE1-2dsRNAs were injected 48 h.This study results provided a basis for the prevention and treatment of pests that inhibited trehalase gene expression.
引文
[1]XI Y,PAN P L,YE Y X,et al.Chitin deacetylase family genes in the brown planthopper,Nilaparvata lugens(Hemiptera:Delphacidae)[J].Insect Molecular Biology,2014,23(6):695-705.
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[6]唐斌,魏苹,陈洁,等.昆虫海藻糖酶的基因特性及功能研究进展[J].昆虫学报,2012,55(11):1315-1321.
[7]MERZENDORFER H,ZIMOCH L.Chitin metabolism in insects:structure,function and regulation of chitin synthases and chitinases[J].Journal of Experimental Biology,2003,206(24):4393-4412.
[8]CHEN J,TANG B,CHEN H X,et al.Different functions of the insect soluble and membrane-bound trehalase genes in chitin biosynthesis revealed by RNA interference[J].PLoS One,2010,5(4):e10133.
[9]ZHAO L N,YANG M M,SHEN Q D,et al.Functional characterization of three trehalase genes regulating the chitin metabolism pathway in rice brown planthopper using RNA interference[J].Scientific Reports,2016,6:27841.
[10]WYATT G R.The biochemistry of sugars and polysaccharides in insects[J].Advances in Insect Physiology,1967,4:287-360.
[11]于彩虹,卢丹,林荣华,等.海藻糖:昆虫的血糖[J].昆虫知识,2008,45(5):832-837.
[12]MITSUMASU K,AZUMA M,NIIMI T,et al.Membrane-penetrating trehalase from silkwormBombyx mori.Molecular cloning and localization in larval midgut[J].Insect Molecular Biology,2005,14(5):501-508.
[13]TANG B,CHEN X F,LIU Y,et al.Characterization and expression patterns of a membrane-bound trehalase from Spodoptera exigua[J].BMC Molecular Biology,2008,9(1):51.
[14]SHI Z K,LIU X J,XU Q Y,et al.Two novel soluble trehalase genes cloned from Harmonia axyridis and regulation of the enzyme in a rapid changing temperature[J].Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,2016,198:10-18.
[15]张文庆,陈晓菲,唐斌,等.昆虫几丁质合成及其调控研究前沿[J].应用昆虫学报,2011,48(3):475-479.
[16]ZHU K Y,MERZENDORFER H,ZHANG W Q,et al.Biosynthesis,turnover,and functions of chitin in insects[J].Annual Review of Entomology,2016,61:177-196.
[17]TANG B,YANG M M,SHEN Q D,et al.Suppressing the activity of trehalase with validamycin disrupts the trehalose and chitin biosynthesis pathways in rice brown planthopper,Nilaparvata lugens[J].Pesticide Biochemistry and Physiology,2017,137:81-90.
[18]赵丽娜.海藻糖酶和海藻糖合成酶基因对褐飞虱海藻糖代谢途径关键基因的调控研究[D].杭州:杭州师范大学,2014.
[19]张露,朱世城,郑好,等.褐飞虱海藻糖酶基因在表皮几丁质代谢中的调控作用[J].中国农业科学,2017,50(6):1047-1056.
[20]TAKIGUCHI M,NIIMI T,SU Z H,et al.Trehalase from male accessory gland of an insect,Tenebrio molitor.cDNA sequencing and developmental profile of the gene expression[J].The Biochemical Journal,1992,288(1):19-22.
[21]TANG B,QIN Z,SHI Z K,et al.Trehalase in Harmonia axyridis(Coleoptera:Coccinellidae):effects on beetle locomotory activity and the correlation with trehalose metabolism under starvation conditions[J].Applied Entomology and Zoology,2014,49(2):255-264.
[22]TANG B,WEI P,ZHAO L N,et al.Knockdown of five trehalase genes using RNA interference regulates the gene expression of the chitin biosynthesis pathway in Tribolium castaneum[J].BMC Biotechnology,2016,16(1):67.
[23]刘晓健,张欢欢,李大琪,等.飞蝗可溶型海藻糖酶基因的序列分析及mRNA表达特性[J].昆虫学报,2012,55(11):1264-1271.
[24]刘晓健,孙亚文,崔淼,等.飞蝗海藻糖酶基因的分子特性及功能[J].中国农业科学,2016,49(22):4375-4386.
[25]GU J H,SHAO Y,ZHANG C W,et al.Characterization of putative soluble and membrane-bound trehalases in a hemipteran insect,Nilaparvata lugens[J].Journal of Insect Physiology,2009,55(11):997-1002.
[26]TATUN N,SINGTRIPOP T,SAKURAI S.Dual control of midgut trehalase activity by 20-hydroxyecdysone and an inhibitory factor in the bamboo borer Omphisa fuscidentalis Hampson[J].Journal of Insect Physiology,2008,54(2):351-357.
[27]YAO Q,ZHANG D W,TANG B,et al.Identification of 20-hydroxyecdysone late-response genes in the chitin biosynthesis pathway[J].PLoS One,2010,5(11):e14058.
[2]XI Y,PAN P L,YE Y X,et al.Chitinase-like gene family in the brown planthopper,Nilaparvata lugens[J].Insect Molecular Biology,2015,24(1):29-40.
[3]YANG M M,ZHAO L N,SHEN Q D,et al.Knockdown of two trehalose-6-phosphate synthases severely affects chitin metabolism gene expression in the brown planthopper Nilaparvata lugens[J].Pest Management Science,2017,73(1):206-216.
[4]ZHAO Y,HUANG F K,DONG X L,et al.HPLC analysis of rice variety resistance to differentbiotypes of Nilaparvata lugens[J].Journal of South China Agricultural University,2005,26(2):52-55.
[5]WANG Y C,TANG M,HAO P Y,et al.Penetration into rice tissues by brown planthopper and fine structure of the salivary sheaths[J].Entomologia Experimentalis et Applicata,2008,129(3):295-307.
[6]唐斌,魏苹,陈洁,等.昆虫海藻糖酶的基因特性及功能研究进展[J].昆虫学报,2012,55(11):1315-1321.
[7]MERZENDORFER H,ZIMOCH L.Chitin metabolism in insects:structure,function and regulation of chitin synthases and chitinases[J].Journal of Experimental Biology,2003,206(24):4393-4412.
[8]CHEN J,TANG B,CHEN H X,et al.Different functions of the insect soluble and membrane-bound trehalase genes in chitin biosynthesis revealed by RNA interference[J].PLoS One,2010,5(4):e10133.
[9]ZHAO L N,YANG M M,SHEN Q D,et al.Functional characterization of three trehalase genes regulating the chitin metabolism pathway in rice brown planthopper using RNA interference[J].Scientific Reports,2016,6:27841.
[10]WYATT G R.The biochemistry of sugars and polysaccharides in insects[J].Advances in Insect Physiology,1967,4:287-360.
[11]于彩虹,卢丹,林荣华,等.海藻糖:昆虫的血糖[J].昆虫知识,2008,45(5):832-837.
[12]MITSUMASU K,AZUMA M,NIIMI T,et al.Membrane-penetrating trehalase from silkwormBombyx mori.Molecular cloning and localization in larval midgut[J].Insect Molecular Biology,2005,14(5):501-508.
[13]TANG B,CHEN X F,LIU Y,et al.Characterization and expression patterns of a membrane-bound trehalase from Spodoptera exigua[J].BMC Molecular Biology,2008,9(1):51.
[14]SHI Z K,LIU X J,XU Q Y,et al.Two novel soluble trehalase genes cloned from Harmonia axyridis and regulation of the enzyme in a rapid changing temperature[J].Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,2016,198:10-18.
[15]张文庆,陈晓菲,唐斌,等.昆虫几丁质合成及其调控研究前沿[J].应用昆虫学报,2011,48(3):475-479.
[16]ZHU K Y,MERZENDORFER H,ZHANG W Q,et al.Biosynthesis,turnover,and functions of chitin in insects[J].Annual Review of Entomology,2016,61:177-196.
[17]TANG B,YANG M M,SHEN Q D,et al.Suppressing the activity of trehalase with validamycin disrupts the trehalose and chitin biosynthesis pathways in rice brown planthopper,Nilaparvata lugens[J].Pesticide Biochemistry and Physiology,2017,137:81-90.
[18]赵丽娜.海藻糖酶和海藻糖合成酶基因对褐飞虱海藻糖代谢途径关键基因的调控研究[D].杭州:杭州师范大学,2014.
[19]张露,朱世城,郑好,等.褐飞虱海藻糖酶基因在表皮几丁质代谢中的调控作用[J].中国农业科学,2017,50(6):1047-1056.
[20]TAKIGUCHI M,NIIMI T,SU Z H,et al.Trehalase from male accessory gland of an insect,Tenebrio molitor.cDNA sequencing and developmental profile of the gene expression[J].The Biochemical Journal,1992,288(1):19-22.
[21]TANG B,QIN Z,SHI Z K,et al.Trehalase in Harmonia axyridis(Coleoptera:Coccinellidae):effects on beetle locomotory activity and the correlation with trehalose metabolism under starvation conditions[J].Applied Entomology and Zoology,2014,49(2):255-264.
[22]TANG B,WEI P,ZHAO L N,et al.Knockdown of five trehalase genes using RNA interference regulates the gene expression of the chitin biosynthesis pathway in Tribolium castaneum[J].BMC Biotechnology,2016,16(1):67.
[23]刘晓健,张欢欢,李大琪,等.飞蝗可溶型海藻糖酶基因的序列分析及mRNA表达特性[J].昆虫学报,2012,55(11):1264-1271.
[24]刘晓健,孙亚文,崔淼,等.飞蝗海藻糖酶基因的分子特性及功能[J].中国农业科学,2016,49(22):4375-4386.
[25]GU J H,SHAO Y,ZHANG C W,et al.Characterization of putative soluble and membrane-bound trehalases in a hemipteran insect,Nilaparvata lugens[J].Journal of Insect Physiology,2009,55(11):997-1002.
[26]TATUN N,SINGTRIPOP T,SAKURAI S.Dual control of midgut trehalase activity by 20-hydroxyecdysone and an inhibitory factor in the bamboo borer Omphisa fuscidentalis Hampson[J].Journal of Insect Physiology,2008,54(2):351-357.
[27]YAO Q,ZHANG D W,TANG B,et al.Identification of 20-hydroxyecdysone late-response genes in the chitin biosynthesis pathway[J].PLoS One,2010,5(11):e14058.