樗蚕孵化酶基因的克隆与表达特征分析
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摘要
樗蚕(Philosamia cynthia cynthia)是重要的野蚕资源。为从分子水平研究樗蚕的孵化机制,以催青第9天的樗蚕卵为材料提取总RNA并反转录合成c DNA,根据鳞翅目昆虫孵化酶基因的保守序列设计简并引物,通过RACE-PCR获得樗蚕孵化酶基因全长c DNA,命名为Pcc HE(Gen Bank登录号:MH119084)。Pcc HE基因c DNA全长为964 bp,由5'-UTR、3'-UTR和885bp的ORF组成,编码294个氨基酸;Pcc HE蛋白序列含有孵化酶特征序列锌指结合基序和Met-转角基序;基于Pcc HE氨基酸序列与16种动物孵化酶同源氨基酸序列构建的系统进化分析树显示,樗蚕与蓖麻蚕之间的亲缘关系最近。半定量RT-PCR检测Pcc HE在樗蚕胚胎发育早期的表达维持在较低水平,催青第3天小幅度增加,第9天时表达水平开始大幅上升,至孵化前达到最高水平;Pcc HE在5龄3 d幼虫中肠中高水平表达,并且在马氏管中也有表达,这是首次在昆虫马氏管中发现孵化酶基因的表达。上述结果表明,Pcc HE与樗蚕的胚胎发育和孵化密切相关,还可能参与幼虫期的食物消化、蛋白质代谢等生物学过程。
Philosamia cynthia cynthia is an important resource for wild silkworm. To investigate the hatching mechanism of Philosamia cynthia cynthia at moleculae level,total RNA was isolated from newly laid eggs on the ninth day of incubation and the c DNA was obtained by reverse transcription method. Degenerate primers were designed according to the conserved sequence of hatching enzyme gene( HE) in Lepidoptera insect. Full length c DNA of Philosamia cynthia cynthia hatching enzyme was amplified by RACE-PCR,and named Pcc HE( Gen Bank accession No: MH119-084). The full length c DNA of Pcc HE was 966 bp,which contained 5'-UTR,3'-UTR and an ORF sequence of 885 bp encoding 294 amino acids. This protein contains the characteristic sequences of hatching enzyme,zinc binding motif and Met-turn motif. Phylogenetic analysis based on amino acid sequence of hatching enzymes indicated that Philosamia cynthia cynthia and Philasamia cynthia ricini had the closest genetic relationship. Semi-quantitative RT-PCR analysis indicated that the expression of Pcc HE transcripts in embryos at early stage remained at a relative low level,increased slightly on the 3 rd day,began to rise sharply on the9 th day and reached to the maximal level before hatching. Moreover,Pcc HE had the highest expression level in midgut on the 3 rd day of 5 th instar larval stage. In addition,its expreesion was also observed in Malpighian tubules,which was the first report on expression of hatching enzyme gene in insect Malpighian tubules. The above results indicate that Pcc HE is involved in embryonic development and hatching process at embryonic stage,and it may also participate in the biological process of digestion and protein metabolism at larval stage.
Philosamia cynthia cynthia is an important resource for wild silkworm. To investigate the hatching mechanism of Philosamia cynthia cynthia at moleculae level,total RNA was isolated from newly laid eggs on the ninth day of incubation and the c DNA was obtained by reverse transcription method. Degenerate primers were designed according to the conserved sequence of hatching enzyme gene( HE) in Lepidoptera insect. Full length c DNA of Philosamia cynthia cynthia hatching enzyme was amplified by RACE-PCR,and named Pcc HE( Gen Bank accession No: MH119-084). The full length c DNA of Pcc HE was 966 bp,which contained 5'-UTR,3'-UTR and an ORF sequence of 885 bp encoding 294 amino acids. This protein contains the characteristic sequences of hatching enzyme,zinc binding motif and Met-turn motif. Phylogenetic analysis based on amino acid sequence of hatching enzymes indicated that Philosamia cynthia cynthia and Philasamia cynthia ricini had the closest genetic relationship. Semi-quantitative RT-PCR analysis indicated that the expression of Pcc HE transcripts in embryos at early stage remained at a relative low level,increased slightly on the 3 rd day,began to rise sharply on the9 th day and reached to the maximal level before hatching. Moreover,Pcc HE had the highest expression level in midgut on the 3 rd day of 5 th instar larval stage. In addition,its expreesion was also observed in Malpighian tubules,which was the first report on expression of hatching enzyme gene in insect Malpighian tubules. The above results indicate that Pcc HE is involved in embryonic development and hatching process at embryonic stage,and it may also participate in the biological process of digestion and protein metabolism at larval stage.
引文
[1]LEPAGE T,GACHE C.Purification and characterization of the sea urchin embryo hatching enzyme[J].J Biol Chem,1989,264(9):4787-4793
[2]FAN T J,KATAGIRI C.Properties of the hatching enzyme from Xenopus laevis[J].FEBS J,2001,268(18):4892-4898
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[4]GEIER G,ZWILLING R.Cloning and characterization of a c DNAcoding for Astacus embryonic astacin,a member of the astacin family of metalloproteases from the crayfish Astacus astacus[J].FEBS J,1998,253(3):796-803
[5]KATAGIRI C,MAEDA R,YAMASHIKA C,et al.Molecular cloning of Xenopus hatching enzyme and its specific expression in hatching gland cells[J].Int J Dev Biol,1997,41(1):19-25
[6]BOND J S,BEYNON R J.The astacin family of metalloendopeptidases[J].J Biol Chem,1991,266(32):21381-21385
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[8]唐顺明,卢福浩,沈兴家,等.家蚕孵化酶样基因Bm HEL的原核表达及蛋白纯化与功能鉴定[J].蚕业科学,2010,36(3):407-412
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[11]唐顺明,丁丽平,沈兴家,等.柞蚕孵化酶样基因的全长c DNA克隆及表达特征分析[J].蚕业科学,2011,37(6):1000-1007
[12]贺华.蓖麻蚕孵化酶基因的克隆及其转基因载体构建[D].镇江:江苏科技大学,2015
[13]唐顺明,赵新慧,吴俊,等.野蚕孵化酶基因的克隆与生物信息学分析[J].江西农业学报,2011,23(10):1-5
[14]王焕英.鳞翅目害虫桑蟥与小菜蛾孵化酶基因的克隆、表达与特性分析[D].镇江:江苏科技大学,2014
[15]朱弘复,王林瑶.中国动物志[M].北京:科学出版社,1996:163-167
[16]万前进.樗蚕的生物学特性与科学防治措施[J].现代农业科技,2011(1):206-207
[17]王秋炎,易红仔.樟树樗蚕生物学特性及其防治要点[J].中国园艺文摘,2016,32(1):103-104
[18]杜占军,王建业,张喜山.樗蚕卵的发育起点温度和有效积温[J].蚕业科学,2008,34(2):351-353
[19]武松,李玉萍,夏润玺,等.樗蚕和蓖麻蚕的DNA条形编码与系统进化分析[J].蚕业科学,2009,35(3):533-538
[20]SIMA Y H,CHEN M,YAO R,et al.The complete mitochondrial genome of the Ailanthus silkmoth,Samia cynthia cynthia(Lepidoptera:Saturniidae)[J].Gene,2013,526(2):309-317
[21]GUEVARA T,YIALLOUROS I,KAPPELHOFF R,et al.Proenzyme structure and activation of astacin metallopeptidase[J].J Biol Chem,2010,285(18):13958-13965
[22]KAWAGUCHI M,NAKAGAWA M,NODA T,et al.Hatching enzyme of the ovoviviparous black rockfish Sebastes schlegelii—environmental adaptation of the hatching enzyme and evolutionary aspects of formation of the pseudogene.[J].FEBS J,2008,275(11):2884-2898
[23]SANO K,KAWAGUCHI M,YOSHIKAWA M,et al.Hatching enzyme of Japanese eel Anguilla japonica and the possible evolution of the egg envelope digestion mechanism[J].FEBS J,2011,278(19):3711-3723
[2]FAN T J,KATAGIRI C.Properties of the hatching enzyme from Xenopus laevis[J].FEBS J,2001,268(18):4892-4898
[3]LEPAGE T,GACHE C.Early expression of a collagenase-like hatching enzyme gene in the sea urchin embryo[J].EMBO J,1990,9(9):3003-3012
[4]GEIER G,ZWILLING R.Cloning and characterization of a c DNAcoding for Astacus embryonic astacin,a member of the astacin family of metalloproteases from the crayfish Astacus astacus[J].FEBS J,1998,253(3):796-803
[5]KATAGIRI C,MAEDA R,YAMASHIKA C,et al.Molecular cloning of Xenopus hatching enzyme and its specific expression in hatching gland cells[J].Int J Dev Biol,1997,41(1):19-25
[6]BOND J S,BEYNON R J.The astacin family of metalloendopeptidases[J].J Biol Chem,1991,266(32):21381-21385
[7]LU F H,TANG S M,SHEN X J,et al.Molecular cloning and characterization of hatching enzyme-like gene in the silkworm,Bombyx mori[J].Mol Biol Rep,2010,37(3):1175-1182
[8]唐顺明,卢福浩,沈兴家,等.家蚕孵化酶样基因Bm HEL的原核表达及蛋白纯化与功能鉴定[J].蚕业科学,2010,36(3):407-412
[9]TANG S M,WU J,ZHAO X H,et al.Molecular cloning and characterization of hatching enzyme-like geneⅡ(Bm HELⅡ)in the silkworm,Bombyx mori[J].Biochem Biophys Res Commun,2012,419(2):194-199
[10]陈加佳,唐顺明,刘龙山,等.家蚕孵化酶基因(Bm HE和Bm HEⅡ)启动子特性与EMSA初步分析[C]//中国生物化学与分子生物学会农业生物化学与分子生物分会.全国农业生物化学与分子生物学第十五届学术研讨会会议论文集.兰州:中国生物化学与分子生物学会农业生物化学与分子生物分会,20016:88-94
[11]唐顺明,丁丽平,沈兴家,等.柞蚕孵化酶样基因的全长c DNA克隆及表达特征分析[J].蚕业科学,2011,37(6):1000-1007
[12]贺华.蓖麻蚕孵化酶基因的克隆及其转基因载体构建[D].镇江:江苏科技大学,2015
[13]唐顺明,赵新慧,吴俊,等.野蚕孵化酶基因的克隆与生物信息学分析[J].江西农业学报,2011,23(10):1-5
[14]王焕英.鳞翅目害虫桑蟥与小菜蛾孵化酶基因的克隆、表达与特性分析[D].镇江:江苏科技大学,2014
[15]朱弘复,王林瑶.中国动物志[M].北京:科学出版社,1996:163-167
[16]万前进.樗蚕的生物学特性与科学防治措施[J].现代农业科技,2011(1):206-207
[17]王秋炎,易红仔.樟树樗蚕生物学特性及其防治要点[J].中国园艺文摘,2016,32(1):103-104
[18]杜占军,王建业,张喜山.樗蚕卵的发育起点温度和有效积温[J].蚕业科学,2008,34(2):351-353
[19]武松,李玉萍,夏润玺,等.樗蚕和蓖麻蚕的DNA条形编码与系统进化分析[J].蚕业科学,2009,35(3):533-538
[20]SIMA Y H,CHEN M,YAO R,et al.The complete mitochondrial genome of the Ailanthus silkmoth,Samia cynthia cynthia(Lepidoptera:Saturniidae)[J].Gene,2013,526(2):309-317
[21]GUEVARA T,YIALLOUROS I,KAPPELHOFF R,et al.Proenzyme structure and activation of astacin metallopeptidase[J].J Biol Chem,2010,285(18):13958-13965
[22]KAWAGUCHI M,NAKAGAWA M,NODA T,et al.Hatching enzyme of the ovoviviparous black rockfish Sebastes schlegelii—environmental adaptation of the hatching enzyme and evolutionary aspects of formation of the pseudogene.[J].FEBS J,2008,275(11):2884-2898
[23]SANO K,KAWAGUCHI M,YOSHIKAWA M,et al.Hatching enzyme of Japanese eel Anguilla japonica and the possible evolution of the egg envelope digestion mechanism[J].FEBS J,2011,278(19):3711-3723