粉纹夜蛾中肠氨肽酶N（APN）亚型的基因克隆、组织定位及表达量研究
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摘要
昆虫中肠氨肽酶在昆虫食物蛋白消化方面具有非常重要的功能。对食物中蛋白的消化是昆虫中肠氨肽酶的重要生理功能，中肠氨肽酶活性的抑制会导致幼虫的生长和发育的停止，最终导致其死亡。近二十年来转基因作物的大规模种植，使得害虫长期处于转基因作物Bt蛋白的高压选择下，害虫对Bt作物抗性问题凸显。鳞翅目APNs的基因克隆及其鉴定已经在昆虫与Bt毒素的关系方面证明中肠APNs与Bt毒素的发病机制相关。为进一步明确粉纹夜蛾中肠氨肽酶N基因的种类及其各亚型的分子特征以及粉纹夜蛾氨肽酶N对BT毒素的抗性机制，论文对粉纹夜蛾中肠氨肽酶N进行系统的研究，在成功构建粉纹夜蛾中肠cDNA文库的基础上，运用自身连接反向PCR法分别克隆了纹夜蛾中肠氨肽酶N5(APN5)和中肠氨肽酶N6(APN6)，并结合已经获得的氨肽酶N亚型TnAPN1、 TnAPN2、TnAPN3和TnAPN4,分析了6个粉纹夜蛾的氨肽酶N序列特征，明确了粉纹夜蛾氨肽酶N活性特点、采用荧光定量PCR分析了不同营养饲料对粉纹夜蛾中肠氨肽酶N基因表达的影响、同时探讨了粉纹夜蛾氨肽酶N对Cry1Ac的抗性机制，明确了粉纹夜蛾中肠氨肽酶N对Bt毒素抗性的生化和分子机理在粉纹夜蛾Bt抗性治理中所具意义。本研究主要结果如下：
1.成功构建了高质量的粉纹夜蛾中肠cDNA表达文库；原始文库滴度为4.22×l09pfu/mL，蓝白斑测定重组率为98.8%，平均插入片段约为2.0kb。
2.克隆获得了氨肽酶N APN5和APN6两个基因核苷酸序列全长分别为3012bp和2900bp。推导的APN5和APN6基因氨基酸序列合成的前体蛋白分子量分别为112和118kDa。粉纹夜蛾氨肽酶亚型的序列特征包括N端的信号肽C端蛋白前体区的GPI锚定信号位点，锌指基元HEX2X18E(X代表任意氨基酸)、苏氨酸富集区、GAMEN基元和糖基化位点。
3.用RT-PCR法对分离的6个氨肽酶N基因在粉纹夜蛾不同组织的表达进行了定位，结果显示：TnAPN1、TnAPN3、TnAPN4、TnAPN5基因主要存在于中肠、马氏管、唾腺、脂肪体上；TnAPN6存在于中肠；而TnAPN2在中肠、马氏管、唾腺、脂肪体等组织中检测微弱。不同的组织酶活性测定表明，氨肽酶N的生物活性主要位于中肠；如果不计食物中的蛋白和氨基酸成分，每克中肠组织蛋白质的氨肽酶N活性是恒定的。明确了不同来源营养食物资源对粉纹夜蛾幼虫中肠氨肽酶N活性的影响。
4.采用real time PCR法分析不同食物营养源对粉纹夜蛾幼虫6种中肠氨肽酶N亚型活性表达的影响。qRT-PCR分析表明，不同的组织酶活性测定表明氨肽酶N的生物活性主要位于中肠，6个粉纹夜蛾APN基因在抗性品系幼虫体内转录水平变化不同。APN1基因的转录水平在抗性株明显较低，APN1基因的转录水平抗性株是敏感株的0.026倍；与此相反，APN6基因的转录水平抗性株是敏感株的39倍。APN2、APN3、APN4和APN5基因的转录水平抗性株和敏感株幼虫没有明显的区别。
克隆和鉴定具有消化功能的粉纹夜蛾氨肽酶N基因有助于理解粉纹夜蛾与BT毒素之间的关系，以及氨肽酶N基因在粉纹夜蛾对BT抗性方面所发挥的作用。随研究的进一步深入，新的Cry毒素受体将陆续被发现，新受体基因的功能及其与Bt抗性的关系将成为未来昆虫Bt抗性机制研究的重要内容。这些研究将为害虫的抗性监测和实施预防性的抗性治理策略提供科学依据，将对我国Bt作物的可持续应用具有重要意义。
APN plays an important role in the digestion of insect food. The digestion of protein ininsect food is a major physiological function APN has. The refraining of midgut APN activityresults in damage to larve’s growth and halt of development, finally death. Due to thegrowing of transgenetic plants on large scale in recent years, insects are exposed to theenvironment with high level of Bt and are gradually becoming resistant to Bt. The resistantmechanism of insects to Bt is complex and various. One of the major mechanisms is thechange of binding spot of midgut. Cabbage looper, T.ni is one of the insects which areresistant to Bt. The isolation and identification of Cry1Ac binding protein indicate that APNis one of the receptor of Cry1Ac toxin.
This thesis aimed to clarify the types of midgut APN genes of T.ni, the molecularfeatures of the subtypes, and T.ni resistant mechanism of APN to Cry1Ac. On the basis ofcreating midgut cDNA library, the study cloned APN5and APN6of T.ni by self-ligaton ofinverse PCR. In combination with obtained TnAPN1, TnAPN2, TnAPN3, and TnAPN4, allsubtypes of APN, the sequence features of the six APNs’ genes were analyzed. The activityfeatures of T.ni APN and the effect of different diet on the genetic expression of T.ni APNwere revealed. The study also explored the resistant mechanism of T.ni to Cry1Ac. Thesignificance of physiochemical and molecular mechanism of resistance of T.ni APN toCry1Ac toxin in management and control of Bt resistance of T.ni. The major findings are asfollows:
1. By using cDNA synthesis kits from Stratagene, the midgut cDNA library of T.ni wascreated. The original titer of the library, the combination of blue-white selection and theaverage insertion fragment were4.22×109pfu/Ml,98.8%and2.0kb respectively.
2. After the removal of the signal peptide and the C-terminal prepeptide, the predictedmolecular weights of TnAPN5and TnAPN6wer112and118kDa, respectively. Twosequence features of APNs included the presence of a signal peptide at their N-termini and aprepeptide at the C-termini for the GPI anchor, the zinc binding/gluzincin motifHEX2HX18E, the gluzincin aminopeptidase motif GAMENWG and the presence ofglycosylation sites.
3. By RT-PCR, the expression of genes from the isolated six APNs in different tissueswere localized. The findings showed that the genes of TnAPN1, TnAPN3, TnAPN4, andTnAPN5were mainly in midgut, malpighian tuble，salivary gland and fat body, TnAPN6inmidgut. TnAPN2was weak in malpighian tuble，salivary gland and fat body. Enzymaticactivity assays of various larval tissues showed that aminopeptidase activities were mainlylocalized in the midgut and the specific enzyme activity per mg of midgut tissue proteins wasconstant in T. ni larvae regardless of the composition of dietary proteins and amino acids.
4. By using real-time PCR, the effect of different diet on the activity expression of sixAPN subtypes T.ni larve was analyzed. The enzymatic activity assays indicated the activity ofAPN was mainly in midgut. Six APN subtypes differed in the vivo transcription level ofresistant larve. According to qRT-PCR anaysis, the transcription level of APN1gene was lowin resistant strain, and the level was0.026times as high as that of sensitive strain. Thetranscription level of APN6gene in resistant strain was39times as that of sensitive strain.There was no distinct difference between resistant strain and sensitive strain concerning thetranscription level of APN2, APN4, and TnAPN5genes.
Cloning and identification aminopeptidase N gene with the digestive function helps tounderstand the relationship between the cabbage looper and BT toxin,and the role whichaminopeptidase N genes play in BT resistance.
1.成功构建了高质量的粉纹夜蛾中肠cDNA表达文库；原始文库滴度为4.22×l09pfu/mL，蓝白斑测定重组率为98.8%，平均插入片段约为2.0kb。
2.克隆获得了氨肽酶N APN5和APN6两个基因核苷酸序列全长分别为3012bp和2900bp。推导的APN5和APN6基因氨基酸序列合成的前体蛋白分子量分别为112和118kDa。粉纹夜蛾氨肽酶亚型的序列特征包括N端的信号肽C端蛋白前体区的GPI锚定信号位点，锌指基元HEX2X18E(X代表任意氨基酸)、苏氨酸富集区、GAMEN基元和糖基化位点。
3.用RT-PCR法对分离的6个氨肽酶N基因在粉纹夜蛾不同组织的表达进行了定位，结果显示：TnAPN1、TnAPN3、TnAPN4、TnAPN5基因主要存在于中肠、马氏管、唾腺、脂肪体上；TnAPN6存在于中肠；而TnAPN2在中肠、马氏管、唾腺、脂肪体等组织中检测微弱。不同的组织酶活性测定表明，氨肽酶N的生物活性主要位于中肠；如果不计食物中的蛋白和氨基酸成分，每克中肠组织蛋白质的氨肽酶N活性是恒定的。明确了不同来源营养食物资源对粉纹夜蛾幼虫中肠氨肽酶N活性的影响。
4.采用real time PCR法分析不同食物营养源对粉纹夜蛾幼虫6种中肠氨肽酶N亚型活性表达的影响。qRT-PCR分析表明，不同的组织酶活性测定表明氨肽酶N的生物活性主要位于中肠，6个粉纹夜蛾APN基因在抗性品系幼虫体内转录水平变化不同。APN1基因的转录水平在抗性株明显较低，APN1基因的转录水平抗性株是敏感株的0.026倍；与此相反，APN6基因的转录水平抗性株是敏感株的39倍。APN2、APN3、APN4和APN5基因的转录水平抗性株和敏感株幼虫没有明显的区别。
克隆和鉴定具有消化功能的粉纹夜蛾氨肽酶N基因有助于理解粉纹夜蛾与BT毒素之间的关系，以及氨肽酶N基因在粉纹夜蛾对BT抗性方面所发挥的作用。随研究的进一步深入，新的Cry毒素受体将陆续被发现，新受体基因的功能及其与Bt抗性的关系将成为未来昆虫Bt抗性机制研究的重要内容。这些研究将为害虫的抗性监测和实施预防性的抗性治理策略提供科学依据，将对我国Bt作物的可持续应用具有重要意义。
APN plays an important role in the digestion of insect food. The digestion of protein ininsect food is a major physiological function APN has. The refraining of midgut APN activityresults in damage to larve’s growth and halt of development, finally death. Due to thegrowing of transgenetic plants on large scale in recent years, insects are exposed to theenvironment with high level of Bt and are gradually becoming resistant to Bt. The resistantmechanism of insects to Bt is complex and various. One of the major mechanisms is thechange of binding spot of midgut. Cabbage looper, T.ni is one of the insects which areresistant to Bt. The isolation and identification of Cry1Ac binding protein indicate that APNis one of the receptor of Cry1Ac toxin.
This thesis aimed to clarify the types of midgut APN genes of T.ni, the molecularfeatures of the subtypes, and T.ni resistant mechanism of APN to Cry1Ac. On the basis ofcreating midgut cDNA library, the study cloned APN5and APN6of T.ni by self-ligaton ofinverse PCR. In combination with obtained TnAPN1, TnAPN2, TnAPN3, and TnAPN4, allsubtypes of APN, the sequence features of the six APNs’ genes were analyzed. The activityfeatures of T.ni APN and the effect of different diet on the genetic expression of T.ni APNwere revealed. The study also explored the resistant mechanism of T.ni to Cry1Ac. Thesignificance of physiochemical and molecular mechanism of resistance of T.ni APN toCry1Ac toxin in management and control of Bt resistance of T.ni. The major findings are asfollows:
1. By using cDNA synthesis kits from Stratagene, the midgut cDNA library of T.ni wascreated. The original titer of the library, the combination of blue-white selection and theaverage insertion fragment were4.22×109pfu/Ml,98.8%and2.0kb respectively.
2. After the removal of the signal peptide and the C-terminal prepeptide, the predictedmolecular weights of TnAPN5and TnAPN6wer112and118kDa, respectively. Twosequence features of APNs included the presence of a signal peptide at their N-termini and aprepeptide at the C-termini for the GPI anchor, the zinc binding/gluzincin motifHEX2HX18E, the gluzincin aminopeptidase motif GAMENWG and the presence ofglycosylation sites.
3. By RT-PCR, the expression of genes from the isolated six APNs in different tissueswere localized. The findings showed that the genes of TnAPN1, TnAPN3, TnAPN4, andTnAPN5were mainly in midgut, malpighian tuble，salivary gland and fat body, TnAPN6inmidgut. TnAPN2was weak in malpighian tuble，salivary gland and fat body. Enzymaticactivity assays of various larval tissues showed that aminopeptidase activities were mainlylocalized in the midgut and the specific enzyme activity per mg of midgut tissue proteins wasconstant in T. ni larvae regardless of the composition of dietary proteins and amino acids.
4. By using real-time PCR, the effect of different diet on the activity expression of sixAPN subtypes T.ni larve was analyzed. The enzymatic activity assays indicated the activity ofAPN was mainly in midgut. Six APN subtypes differed in the vivo transcription level ofresistant larve. According to qRT-PCR anaysis, the transcription level of APN1gene was lowin resistant strain, and the level was0.026times as high as that of sensitive strain. Thetranscription level of APN6gene in resistant strain was39times as that of sensitive strain.There was no distinct difference between resistant strain and sensitive strain concerning thetranscription level of APN2, APN4, and TnAPN5genes.
Cloning and identification aminopeptidase N gene with the digestive function helps tounderstand the relationship between the cabbage looper and BT toxin,and the role whichaminopeptidase N genes play in BT resistance.
引文
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Lorence A, Darszon A, Bravo A.1997. Aminopeptidase dependent pore formation of Bacillus thuringiensis Cry1Actoxin on Trichoplusia ni membranes．FEBS Lett,414:303~307.
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Mario Soberón, Claudia Rodriguez-Almazán, Carlos Mu óz-Garay, Liliana Pardo-López, Helena Porta, AlejandraBravo.2012.Bacillus thuringiensis Cry and Cyt mutants useful to counter toxin action in specific environmentsand to overcome insect resistance in the field. Pesticide Biochemistry and Physiology, In Press, AcceptedManuscript, Available online30May.
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Kasorn Tiewsiri,Ping Wang.2011.Differential alteration of two aminopeptidases N associated withresistance to Bacillus thuringiensis toxin Cry1Ac in cabbage looper.PNAS,108(34):14037~14042
Kaur R，Agrawal N,Bhatnagar R.2007. Purification and characterization of aminopeptidase N from Spodoptera lituraexpressed in Sf21insect cells.Protein Expres Purif,54:267~274.
Kikkert JR, et al.2006. Detection of Contarinia nasturtii (Diptera: Cecidomyiidae) in New York, a new pestof cruciferous plants in the United States. J Econ Entomol,99:1310~1315.
Knight P J,Carroll J,Ellar D J.2004. Analysis of glycan structures on the120kDa aminopeptidase N of Manduca sextaand their interactions with Bacillus thuringiensis Cry1Ac toxin.Insect Biochem Mol Biol,34:101~112.
Knight P J，Knowles B H，Ellar D J.1994. The receptor for Bacillus thuringiensis CrylA (c) delta-endotoxin in thebrush border membrane of the lepidopteran Manduca sexta is aminopeptidase N． Mol Microbiol,11(3):429~436.
Knight P J，Knowles B H，Ellar D J.1995. Molecular cloning of an insect aminopeptidase N that serves as a receptorfor Bacillus thuringiensis CryIA (c) toxin．J Biol Chem,270:17765~17770.
Laustsen P G, Vang S, Kristensen T.2001.Mutational analysis of the active site of human insulin-regulatedaminopeptidase． Eur J Biochem,268:98~104.
Lee M K Milen R E, Ge A Z, et al.1992. Location of a Bombyx mori receptor binding region on a Bacillusthuringiensis deltaendotoxin. J Biol Chem,267:3115~3121.
Lee M K，You T H，Young B A.1996. Aminopeptidase N purified from gypsy moth brush border membrane vesiclesis a specific receptor for Bacillus thuringiensis CryIAc toxin． Appl Environ Microbiol,62:2845~2849.
Lorence A, Darszon A, Bravo A.1997. Aminopeptidase dependent pore formation of Bacillus thuringiensis Cry1Actoxin on Trichoplusia ni membranes．FEBS Lett,414:303~307.
Luo K, Lu Y J, Adang M J.1996. A106-kDa form of aminopeptidase is a receptor for Bacillus thuringiensis Cry1C δ-endotoxin in the brush border membrane of Manduca sexta．Insect Biochem Mol Biol,26:783~791.
Luo K,Sangadala S,Masson L,et al.1997.The Heliothis virescens170kDa aminopeptidase functions as " receptor A "bymediating specific Bacillus thuringiensis Cry1A delta-endotoxin binding and pore formation．Insect BiochemMol Biol,1997,27:735~743.
Mario Soberón, Claudia Rodriguez-Almazán, Carlos Mu óz-Garay, Liliana Pardo-López, Helena Porta, AlejandraBravo.2012.Bacillus thuringiensis Cry and Cyt mutants useful to counter toxin action in specific environmentsand to overcome insect resistance in the field. Pesticide Biochemistry and Physiology, In Press, AcceptedManuscript, Available online30May.
Masson L,Lu Y J,Mazza A,et al.1995. The Cry1Ac receptor purified from Manduca sexta displays multiplespecificities.J Biol Chem,270:20309~20315.
Morin S, et al.2003.Three cadherin alleles associated with resistance to Bacillus thuringiensis in pinkbollworm. Proc Natl Acad Sci USA,100:5004~5009.
Nakanishi K,Yaoi K,Nagino Y,et al.2002. Aminopeptidase N isoforms from the midgut of Bombyx mori andPlutella-their classification and the factors that determine their binding specificity of Bacillus thuringiensisCry1Ac toxin．FEBS Lett,519:215~220.
Nakanishi K, Yaoi K, Nagino Y, Hara H., Kitami M, Atsumi S,Muira N, Sato R.2009. Aminopeptidase N isoformsfrom the midgut of Bombyx mori and Plutella xylostella—their classification and the factors that determine theirbinding specificity of Bacillus thuringiensis Cry1Ac toxin. FEBS Lett.,519:215~220.
Oltean Di, Pullikuth AK, Lee HK,et al.1999. Partial purification and characterization of Bacillus thuringiensisCry1A toxin receptor A from Heliothis virescens and cloning of the corresponding cDNA．Appl EnvironMicrobiol，65:4760~4766.
Oltean Di, Lee HK,et al.2010. Three cadherin alleles associated with resistance to Bacillus thuringiensis inpink bollworm. Appl Environ Microbiol，65:4760~4766.
Pacheco S, et al.2009. Domain II loop3of Bacillus thuringiensis Cry1Ab toxin is involved in a“ping-pong” binding mechanism with Manduca sexta aminopeptidase-N and cadherin receptors. J BiolChem,284:32750~32757.
Pigott C R,Ellar D J.2007. Role of receptors in Bacillus thuringiensis Crystal toxin activity.Microbiol Mol Biol R，71(2):255~281.
Pigott CR, Ellar DJ.2007. Role of receptors in Bacillus thuringiensis crystal toxin activity. Microbiol MolBiol Rev,71:255~281.
Ping Wang, Xin Zhang, Jie Zhang.2005. Molecular characterization of four midgut aminopeptidase N isozymes fromthe cabbage looper, Trichoplusia ni. Insect Biochem. Mol.. Biol,35(6):611~620.
Rawlings N D,Barrett A J.1995. Evolutionary families of metallopeptidases.Methods Enzymol,248:183~228.
Rajagopal et al.2003. Role of receptors in Bacillus thuringiensis crystal toxin activity. Microbiol MolBiol Rev,71:255~281.
Redding AM, Mukhopadhyay A, Joyner DC,Hazen TC, Keasling JD.2006. Study of nitrate stress inDesulfovibrio vulgaris Hildenborough using iTRAQ proteomics. Brief Funct Genomics Proteomics,5:133~143.
Reed B.J, Chandler D.S, Sandeman R.M.1999. Aminopeptidases as potential targets for the control of the Australiansheep blowfly,Lucilia cuprina. Int. J. Parasitol,29:839~850.
Sangadala S,Walters F S,English L H,et al.1994. A mixture of Manduca sexta aminopeptidase and phosphataseenhances Bacillus thuringiensis insecticidal CryIA (c) toxin binding and86Rb(+)-K(+) efflux in vitro.J BiolChem,269:10088~10092.
Schnepf E, et al.1998.Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev,62:775~806.
Schnepf E，Crickmore N，Van rie J，et al.1998. Bacillus thuringiensis and its pesticidal crystal proteins.Microbiol MolRev,62:775~806.
Sunita Tajne, Ramadevi Sanam, Rambabu Gundla, Neha S. Gandhi, Ricardo L. Mancera, Dayakar Boddupally,Dashavantha Reddy Vudem, Venkateswara Rao Khareedu.2012.Molecular modeling of Bt Cry1Ac(DI–DII)–ASAL (Allium sativum lectin)–fusion protein and its interaction with (APN) receptor of Manducasexta.Journal of Molecular Graphics and Modelling,33:61~76.
Tabashnik BE, Cushing NL, Finson N, Johnson MW.1990. Field development of resistance to Bacillusthuringiensis in diamondback moth (Lepidoptera: Plutellidae).J Econ Entomol,83:1671~1676.
Tabashnik BE, et al.2003.nsect resistance to transgenic Bt crops: Lessons from the laboratory and field. JEcon Entomol,6:1031~1038.
Tabashnik BE, et al.1998.Insect resistance to Bacillus thuringiensis: Uniform or diverse? Philos Trans RSoc Lond B. Biol Sci,53:1751~1756.
Tabashnik BE,Van Rensburg JB,Carrière Y.2009. Field-evolved insect resistance to Bt crops: Definition,theory, and data. J Econ Entomol,102:2011~2025.
Terra, W.R., Ferreira, C.1994.Insect digestive enzymes:properties, compartmentalization and function. Comp.Biochem. Physiol,109B:1~62.
Vincent Vachon,Raynald Laprade,Jean-Louis Schwartz.2012.Journal of Invertebrate Pathology, In Press, UncorrectedProof, Available online19May.
Wang P,et al.2005.Molecular Chanracrtarion of four aminopeptidase N of the cabbage looper, Trichoplusiani. Appl Environ Microbiol,73:1199~1207.
Wang P,et al.2007.Mechanism of resistance to Bacillus thuringiensis toxi Cry1Ac in a greenhousepopulation of the cabbage looper, Trichoplusia ni. Appl Environ Microbiol,73:1199~1207.
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