羚牛苦味受体3(Tas2R3)基因的克隆及生物信息学分析
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摘要
Tas2R3是苦味受体基因家族中一个重要的成员,为了进一步了解和研究羚牛(Budorcas taxicolor)苦味受体基因的结构和功能,本研究对羚牛苦味受体3 (Tas2R3)基因进行了克隆和生物信息学分析(GenBank登录号:MG650195)。结果显示,羚牛Tas2R3基因编码区(coding sequence, CDS)序列全长951 bp,共编码316个氨基酸,以亮氨酸含量最高,谷氨酰胺含量最低。其蛋白质等电点为9.68,分子量为51.96 kD。高级结构功能预测显示,二级结构以α-螺旋为主,蛋白质为碱性、稳定的亲水性蛋白,由4个胞外区、7个跨膜区和4个胞内区组成。预测到2种类型共8个糖基化功能位点和4种类型共15个磷酸化功能位点。通过比较Tas2R3基因种间相似性发现,在偶蹄目中具有很高的同源性,羚牛与绵羊(Ovis aries)的相似性最高(0.98),与褐家鼠(Rattus norvegicus)最低(0.52)。用羚牛、绵羊等12个物种的Tas2R3基因CDS序列构建的NJ树与ME树结构一致,表明Tas2R3基因适合用于构建不同物种间的系统进化树。
Tas2 R3 is an important member of the bitter taste receptor(Tas2 Rs) gene families. In order to further study the structure and function of takin(Budorcas taxicolor) bitter taste receptor gene, we performed cloning and bioinformatics analysis of Tas2 R3 gene in takin and submitted the gene sequence to NCBI GenBank(Acc.number: MG650195). The results showed that the full coding DNA sequence(CDS) of takin Tas2 R3 was 951 bp,encoding 316 amino acids, among which leucine occupied the highest content and glutamine with the lowest content, and the calculated molecular weight of the protein is 51.96 kD with an isoelectric point of 9.68. High structure and function prediction suggested that the secondary structures of the protein were dominated of alpha helix and this Tas2 R3 protein is an alkaline, and stable hydrophilic protein, which comprises 7 transmembrane regions, 4 extracellular regions and 4 intracellular regions. Additionally, it turned out that the Tas2 R3 protein were predicted to contain 2 types of 8 glycosylation sites and 4 types of 15 phosphorylation sites. Homology analysis indicated that the Tas2 R3 gene of takin had a high homology with Artiodactyla species, and the closest relationship with Ovis aries(0.98), the lowest relationship with Rattus norvegicus(0.52). Furthermore, the Neighbour-Joining tree constructed by Tas2 R3 gene CDS of 12 species is consistent with their species tree, indicating that the Tas2 R3 gene should be suitable for constructing molecular phylogenetic tree among different species likewise.
Tas2 R3 is an important member of the bitter taste receptor(Tas2 Rs) gene families. In order to further study the structure and function of takin(Budorcas taxicolor) bitter taste receptor gene, we performed cloning and bioinformatics analysis of Tas2 R3 gene in takin and submitted the gene sequence to NCBI GenBank(Acc.number: MG650195). The results showed that the full coding DNA sequence(CDS) of takin Tas2 R3 was 951 bp,encoding 316 amino acids, among which leucine occupied the highest content and glutamine with the lowest content, and the calculated molecular weight of the protein is 51.96 kD with an isoelectric point of 9.68. High structure and function prediction suggested that the secondary structures of the protein were dominated of alpha helix and this Tas2 R3 protein is an alkaline, and stable hydrophilic protein, which comprises 7 transmembrane regions, 4 extracellular regions and 4 intracellular regions. Additionally, it turned out that the Tas2 R3 protein were predicted to contain 2 types of 8 glycosylation sites and 4 types of 15 phosphorylation sites. Homology analysis indicated that the Tas2 R3 gene of takin had a high homology with Artiodactyla species, and the closest relationship with Ovis aries(0.98), the lowest relationship with Rattus norvegicus(0.52). Furthermore, the Neighbour-Joining tree constructed by Tas2 R3 gene CDS of 12 species is consistent with their species tree, indicating that the Tas2 R3 gene should be suitable for constructing molecular phylogenetic tree among different species likewise.
引文
Adler E.,Hoon M.A.,Mueller K.L.,Chandrashekar J.,Ryba N.J.,and Zuker C.S.,2000,A novel family of mammalian taste receptors,Cell,100(6):693-702
Ai H.S.,1999,Habitat and food of takin in the gaoligong mountains of Yunnan,Yesheng Dongwu Xuebao(Chinese Joural of Wildlife),(4):36-37(艾怀森,1999,羚牛在高黎贡山的栖息地及食性,野生动物学报,(4):36-37)
Ai H.S.,2000,Preliminary observation on habitat and feeding habit of budorcas taxicolor in south central section of gaoligongshan,Yunnan Linye Keji(Yunnan Forestry Science and Technology),(3):61-64(艾怀森,2000,高黎贡山中南段羚牛栖息地和食性初步观察,云南林业科技,(3):61-64)
Bufe B.,Breslin P.A.,Kuhn C.,Reed D.K.,Tharp C.D.,Slack J.P.,Kim U.,Drayna D.,and Meyerhof W.,2005,The molecular basis of individual differences in phenylthiocarbamide and propylthiouracil bitterness perception,Current Biology Cb,15(4):322-327
Chandrashekar J.,Mueller K.L.,Hoon M.A.,Adler E.,Feng L.X.,Guo W.,Zuker C.S.,and Ryba N.J.,2000,T2Rs function as bitter taste receptors,Cell,100(6):703-711
Chandrashekar J.,Hoon M.A.,Ryba N.J.,and Zuker C.S.,2006,The receptors and cells for mammalian tast,Nature,444(7117):288-294
Chen J.,Zhang H.X.,Wu X.Y.,Shang S.,Yan J.K.,Chen Y.,Zhang H.H.,and Tang X.X.,2017,Characterization of the gut microbiota in the golden takin(Budorcas taxicolor bedfordi),AMB Express,7(1):81-90
Feng H.,Feng C.L.,Huang Y.,and Wang L.,2016,Analysis on complete mitochondrial genome sequence of golden takin(Budorcas taxicolor),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(9):2299-2305(冯慧,冯成利,黄原,王璐,2016,羚牛线粒体基因组全序列测定与分析,基因组学与应用生物学,35(9):2299-2305)
Feng H.,Feng C.L.,Wang L.,and Huang Y.,2017,Genetic diversity of golden takin(Budorcas taxicolor bedfordi)population from Qinling mountains in China revealed by sequence analysis of mitochondrial DNA control region,Biochemical Systematics and Ecology,70:1-6
Feng H.,Huang Y.,Ren Y.,Feng C.L and Liu X.L.,2014,Structure of mitochondrial DNA control region and genetic diversity of Budorcas taxicolor in Shaanxi province,Xibei Nongye Xuebao(Acta Agriculturae Boreali-occidentalis Sinica),23(1):1-6(冯慧,黄原,任轶,冯成利,刘晓农,2014,陕西省羚牛mt DNA控制区序列结构和种群遗传多样性分析,西北农业学报,23(1):1-6)
Gilbertson T.A.,Damak S.,and Margolskee R.F.,2000,The molecular physiology of taste transduction,Curr.Opin.Neurobiol.,10(4):519-527
Guan T.P.,Ge B.M.,William M.,Hu J.,Song Y.L.,Shen L.M.and Ma W.H.,2012,Analysis of terrain induced impact on Takin(Budorcas taxicolor)home range estimation:an example from fixed kernel estimator and minimum convex polygon,Shoulei Xuebao(Acta Theriologica Sinica),32(2):110-117(官天培,葛宝明,William Mcshea,胡婧,宋延龄谌利民,马文虎,2012,地形因素对羚牛家域估算的影响分析-以固定核域法与最小凸边形法为例,兽类学报,32(2):110-117)
Guan T.P.,Ge B.M.,Shen L.M.,You Z.Q.,Tang Z.H.,Liu H.,and Song Y.L.,2015,Home range and fidelity of Sichuan takin,Shengtai Xuebao(Acta Oecologica),35(6):1862-1868(官天培,葛宝明,谌利民,游章强,唐中海,刘昊宋延龄,2015,四川羚牛的家域与忠诚度,生态学报,35(6):1862-1868)
Hu L.L.,and Shi P.,2009,Latest advances on the studies of function and evolution of bitter taste receptor gene(T2R)family Kexue Tongbao(Chinese Science Bulletin),54(17):2472-2482(胡玲玲,施鹏,2009,苦味受体基因家族功能和演化研究的最新进展,科学通报,54(17):2472-2482)
Jiang Z.,Lian E.,Chen R.,and Zhang Y.,2015,Selection of Budorcas taxicolor habitat in mins han area of gansu,Gansu Linye Keji(Journal of Gansu Forestry Science and Technology),(2):26-29(蒋震,连娥,陈蓉,张英,2015,甘肃岷山地区羚牛的栖息地选择研究,甘肃林业科技,(2):26-29)
Li S.Z.,Chen Y.M.,Yao Y.F.,and Xu H.L.,2013,Cloning and sequence analysis of bitter taste receptor Tas2R2 gene in the masked palm civet(Paguma larvata),Guangdong Nongye Kexue(Guangdong Agricultural Sciences),40(6):143-147(李世宗,陈应梅,姚永芳,徐怀亮,2013,果子狸苦味受体Tas2R2基因的克隆与序列分析,广东农业科学,40(6)143-147)
Matsunami H.,Montmayeur J.P.,and Buck L.B.,2000,A family of candidate taste receptors in human and mouse,Nature404(6778):601-603
Powell D.,Speeg B.,Li S.,Blumer E.,and Mcshea W.,2013,An ethogram and activity budget of captive Sichuan takin(Budorcas taxicolor tibetana)with comparisons to other bovidae,Mammalia,77(4):391-401
Pronin A.N.,Xu H.,Tang H.X.,Zhang L.,Li Q.,and Li X.D.,2007,Specific alleles of bitter receptor genes influence human sensitivity to the bitterness of aloin and saccharin,Current Biology Cb,17(16):1403-1408
Reed D.R.,Nanthakumar E.,North M.,Bell C.,Bartoshuk L.M.,and Price A.,1999,Localization of a gene for bitter-taste perception to human chromosome 5p15,American Journal of Human Genetics,64(5):1478-1480
Ren Y.,Feng H.,Feng C.L.,and Meng S.J.,2012,Phylogenic analysis of takin by mtDNA,Jiying Zuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),31(5):481-484(任轶,冯慧,冯成利,蒙世杰,2012,应用线粒体DNA序列探讨羚牛分类地位,基因组学与应用生物学,31(5):481-484)
Sangay T.,Rajaratnam R.,and Karl V.,2016,Current distribution and conservation status of Bhutan takin budorcas whitei lydekker,Journal of Threatened Taxa,8(14):6930-6947
Shi P.,Zhang J.Z.,Yang H.,and Zhang Y.P.,2003,Adaptive diversification of bitter taste receptor genes in mammalian evolution,Molecular Biology snd Evolution,20(5):805-814
Sun M.H.,Yue C.W.,Liu T.T.,Li L.,and Liu H.Y.,2017,The complete mitochondrial genome of Budorcas taxicolor bedfordi,(Artiodactyla:Bovidae)and phylogenetic studies of caprinae,Conservation Genetics Resources,(9):397-400
Wang S.,and Xie Y.,2009,China species red list(Vo I.Ⅰ),Higher Education Press,Beijing,China,pp.297(汪松,解焱,2009,中国物种红色名录(第一卷),高等教育出版社,中国,北京,pp.297)
Wangchuk T.R.,Wegge P.,and Sangay T.,2015,Habitat and diet of bhutan takin Budorcas taxicolor whitei during summer in Jigme Dorji National Park,Bhutan,Journal of Natural History,50:11-12
Wu J.Y.,1986,Study of system and distribution of chinese takin(Budorcas taxicolor Hodgson),Dongwuxue Yanjiu(Zoological Resecrch),7(2):167-175(吴家炎,1986,中国羚牛分类分布的研究,动物学研究,7(2):167-175)
Yao G.,Li Y.H.,Li D.Y.,Williams P.,and Hu J.,2016,Phylogenetic analysis of the endangered takin in the confluent zone of the Qinling and Minshan Mountains using mt DNA control region,Mitochondrial Dna Part A Dna Mapping Sequencing and Analysis,27(4):2594-2605
Zeng Z.G.,and Song Y.L.,2001,Daily activity rhythm and time budget of golden takin in spring and summer,Shoulei Xuebao(Acta Theriologica Sinica),21(1):7-13(曾治高,宋延龄,2001,秦岭羚牛春夏季昼夜活动节律与时间分配,兽类学报,21(1):7-13)
Zeng Z.G.,Zhong W.Q.,Song Y.L.,Gong H.S.,Wang X.J.,and Wang K.W.,2001,Feeding behavior of golden takin,Dongwuxue Zazhi(Chinese Journal of Zoology),36(6):25-28(曾治高,钟文勤,宋延龄,巩会生,王学杰,王宽武,2001,秦岭羚牛的采食行为,动物学杂志,36(6):25-28)
Zeng Z.G.,Zhong W.Q.,Song Y.L.,Li J.S.,Zhao L.G.,and Gong H.S.,2003,Present status of studies on eco-biology of takin,Shoulei Xuebao(Acta Theriologica Sinica),23(2):161-167(曾治高,钟文勤,宋延龄,李俊生,赵雷刚,巩会生,2003,羚牛生态生物学研究现状,兽类学报,23(2):161-167)
Zhang L.X.,Bai H.B.,Feng Z.Q.,Ma C.,Sun W.M.,Cui L.W.,and Xiao W.,2014,Distribution,population and conservation status of Budorcas taxicolor taxicolor in Dulongjiang township,Sichuan Dongwu(Sichuan Journal of Zoology),33(4):493-497(张利祥,白洪斌,丰志全,马驰,孙文莫崔亮伟,肖文,2014,独龙江流域羚牛的种群,分布和保护,四川动物,33(4):493-497)
Zhong Y.,Pan Y.,Hou W.H.,Yao Y.F.,Xu H.L.,and Zhang S.L.,2014,Molecular cloning and sequence analysis of black bear(Ursus thibetanus)bitter taste receptor Tas2R2 gene,Yesheng Dongwu Xuebao(Chinese Journal of Wildlife),35(4):407-413(钟源,潘阳,侯文海,姚永芳,徐怀亮,张栓玲,2014,黑熊苦味受体Tas2R2基因的分子克隆与序列分析,野生动物学报,35(4):407-413)
Ai H.S.,1999,Habitat and food of takin in the gaoligong mountains of Yunnan,Yesheng Dongwu Xuebao(Chinese Joural of Wildlife),(4):36-37(艾怀森,1999,羚牛在高黎贡山的栖息地及食性,野生动物学报,(4):36-37)
Ai H.S.,2000,Preliminary observation on habitat and feeding habit of budorcas taxicolor in south central section of gaoligongshan,Yunnan Linye Keji(Yunnan Forestry Science and Technology),(3):61-64(艾怀森,2000,高黎贡山中南段羚牛栖息地和食性初步观察,云南林业科技,(3):61-64)
Bufe B.,Breslin P.A.,Kuhn C.,Reed D.K.,Tharp C.D.,Slack J.P.,Kim U.,Drayna D.,and Meyerhof W.,2005,The molecular basis of individual differences in phenylthiocarbamide and propylthiouracil bitterness perception,Current Biology Cb,15(4):322-327
Chandrashekar J.,Mueller K.L.,Hoon M.A.,Adler E.,Feng L.X.,Guo W.,Zuker C.S.,and Ryba N.J.,2000,T2Rs function as bitter taste receptors,Cell,100(6):703-711
Chandrashekar J.,Hoon M.A.,Ryba N.J.,and Zuker C.S.,2006,The receptors and cells for mammalian tast,Nature,444(7117):288-294
Chen J.,Zhang H.X.,Wu X.Y.,Shang S.,Yan J.K.,Chen Y.,Zhang H.H.,and Tang X.X.,2017,Characterization of the gut microbiota in the golden takin(Budorcas taxicolor bedfordi),AMB Express,7(1):81-90
Feng H.,Feng C.L.,Huang Y.,and Wang L.,2016,Analysis on complete mitochondrial genome sequence of golden takin(Budorcas taxicolor),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(9):2299-2305(冯慧,冯成利,黄原,王璐,2016,羚牛线粒体基因组全序列测定与分析,基因组学与应用生物学,35(9):2299-2305)
Feng H.,Feng C.L.,Wang L.,and Huang Y.,2017,Genetic diversity of golden takin(Budorcas taxicolor bedfordi)population from Qinling mountains in China revealed by sequence analysis of mitochondrial DNA control region,Biochemical Systematics and Ecology,70:1-6
Feng H.,Huang Y.,Ren Y.,Feng C.L and Liu X.L.,2014,Structure of mitochondrial DNA control region and genetic diversity of Budorcas taxicolor in Shaanxi province,Xibei Nongye Xuebao(Acta Agriculturae Boreali-occidentalis Sinica),23(1):1-6(冯慧,黄原,任轶,冯成利,刘晓农,2014,陕西省羚牛mt DNA控制区序列结构和种群遗传多样性分析,西北农业学报,23(1):1-6)
Gilbertson T.A.,Damak S.,and Margolskee R.F.,2000,The molecular physiology of taste transduction,Curr.Opin.Neurobiol.,10(4):519-527
Guan T.P.,Ge B.M.,William M.,Hu J.,Song Y.L.,Shen L.M.and Ma W.H.,2012,Analysis of terrain induced impact on Takin(Budorcas taxicolor)home range estimation:an example from fixed kernel estimator and minimum convex polygon,Shoulei Xuebao(Acta Theriologica Sinica),32(2):110-117(官天培,葛宝明,William Mcshea,胡婧,宋延龄谌利民,马文虎,2012,地形因素对羚牛家域估算的影响分析-以固定核域法与最小凸边形法为例,兽类学报,32(2):110-117)
Guan T.P.,Ge B.M.,Shen L.M.,You Z.Q.,Tang Z.H.,Liu H.,and Song Y.L.,2015,Home range and fidelity of Sichuan takin,Shengtai Xuebao(Acta Oecologica),35(6):1862-1868(官天培,葛宝明,谌利民,游章强,唐中海,刘昊宋延龄,2015,四川羚牛的家域与忠诚度,生态学报,35(6):1862-1868)
Hu L.L.,and Shi P.,2009,Latest advances on the studies of function and evolution of bitter taste receptor gene(T2R)family Kexue Tongbao(Chinese Science Bulletin),54(17):2472-2482(胡玲玲,施鹏,2009,苦味受体基因家族功能和演化研究的最新进展,科学通报,54(17):2472-2482)
Jiang Z.,Lian E.,Chen R.,and Zhang Y.,2015,Selection of Budorcas taxicolor habitat in mins han area of gansu,Gansu Linye Keji(Journal of Gansu Forestry Science and Technology),(2):26-29(蒋震,连娥,陈蓉,张英,2015,甘肃岷山地区羚牛的栖息地选择研究,甘肃林业科技,(2):26-29)
Li S.Z.,Chen Y.M.,Yao Y.F.,and Xu H.L.,2013,Cloning and sequence analysis of bitter taste receptor Tas2R2 gene in the masked palm civet(Paguma larvata),Guangdong Nongye Kexue(Guangdong Agricultural Sciences),40(6):143-147(李世宗,陈应梅,姚永芳,徐怀亮,2013,果子狸苦味受体Tas2R2基因的克隆与序列分析,广东农业科学,40(6)143-147)
Matsunami H.,Montmayeur J.P.,and Buck L.B.,2000,A family of candidate taste receptors in human and mouse,Nature404(6778):601-603
Powell D.,Speeg B.,Li S.,Blumer E.,and Mcshea W.,2013,An ethogram and activity budget of captive Sichuan takin(Budorcas taxicolor tibetana)with comparisons to other bovidae,Mammalia,77(4):391-401
Pronin A.N.,Xu H.,Tang H.X.,Zhang L.,Li Q.,and Li X.D.,2007,Specific alleles of bitter receptor genes influence human sensitivity to the bitterness of aloin and saccharin,Current Biology Cb,17(16):1403-1408
Reed D.R.,Nanthakumar E.,North M.,Bell C.,Bartoshuk L.M.,and Price A.,1999,Localization of a gene for bitter-taste perception to human chromosome 5p15,American Journal of Human Genetics,64(5):1478-1480
Ren Y.,Feng H.,Feng C.L.,and Meng S.J.,2012,Phylogenic analysis of takin by mtDNA,Jiying Zuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),31(5):481-484(任轶,冯慧,冯成利,蒙世杰,2012,应用线粒体DNA序列探讨羚牛分类地位,基因组学与应用生物学,31(5):481-484)
Sangay T.,Rajaratnam R.,and Karl V.,2016,Current distribution and conservation status of Bhutan takin budorcas whitei lydekker,Journal of Threatened Taxa,8(14):6930-6947
Shi P.,Zhang J.Z.,Yang H.,and Zhang Y.P.,2003,Adaptive diversification of bitter taste receptor genes in mammalian evolution,Molecular Biology snd Evolution,20(5):805-814
Sun M.H.,Yue C.W.,Liu T.T.,Li L.,and Liu H.Y.,2017,The complete mitochondrial genome of Budorcas taxicolor bedfordi,(Artiodactyla:Bovidae)and phylogenetic studies of caprinae,Conservation Genetics Resources,(9):397-400
Wang S.,and Xie Y.,2009,China species red list(Vo I.Ⅰ),Higher Education Press,Beijing,China,pp.297(汪松,解焱,2009,中国物种红色名录(第一卷),高等教育出版社,中国,北京,pp.297)
Wangchuk T.R.,Wegge P.,and Sangay T.,2015,Habitat and diet of bhutan takin Budorcas taxicolor whitei during summer in Jigme Dorji National Park,Bhutan,Journal of Natural History,50:11-12
Wu J.Y.,1986,Study of system and distribution of chinese takin(Budorcas taxicolor Hodgson),Dongwuxue Yanjiu(Zoological Resecrch),7(2):167-175(吴家炎,1986,中国羚牛分类分布的研究,动物学研究,7(2):167-175)
Yao G.,Li Y.H.,Li D.Y.,Williams P.,and Hu J.,2016,Phylogenetic analysis of the endangered takin in the confluent zone of the Qinling and Minshan Mountains using mt DNA control region,Mitochondrial Dna Part A Dna Mapping Sequencing and Analysis,27(4):2594-2605
Zeng Z.G.,and Song Y.L.,2001,Daily activity rhythm and time budget of golden takin in spring and summer,Shoulei Xuebao(Acta Theriologica Sinica),21(1):7-13(曾治高,宋延龄,2001,秦岭羚牛春夏季昼夜活动节律与时间分配,兽类学报,21(1):7-13)
Zeng Z.G.,Zhong W.Q.,Song Y.L.,Gong H.S.,Wang X.J.,and Wang K.W.,2001,Feeding behavior of golden takin,Dongwuxue Zazhi(Chinese Journal of Zoology),36(6):25-28(曾治高,钟文勤,宋延龄,巩会生,王学杰,王宽武,2001,秦岭羚牛的采食行为,动物学杂志,36(6):25-28)
Zeng Z.G.,Zhong W.Q.,Song Y.L.,Li J.S.,Zhao L.G.,and Gong H.S.,2003,Present status of studies on eco-biology of takin,Shoulei Xuebao(Acta Theriologica Sinica),23(2):161-167(曾治高,钟文勤,宋延龄,李俊生,赵雷刚,巩会生,2003,羚牛生态生物学研究现状,兽类学报,23(2):161-167)
Zhang L.X.,Bai H.B.,Feng Z.Q.,Ma C.,Sun W.M.,Cui L.W.,and Xiao W.,2014,Distribution,population and conservation status of Budorcas taxicolor taxicolor in Dulongjiang township,Sichuan Dongwu(Sichuan Journal of Zoology),33(4):493-497(张利祥,白洪斌,丰志全,马驰,孙文莫崔亮伟,肖文,2014,独龙江流域羚牛的种群,分布和保护,四川动物,33(4):493-497)
Zhong Y.,Pan Y.,Hou W.H.,Yao Y.F.,Xu H.L.,and Zhang S.L.,2014,Molecular cloning and sequence analysis of black bear(Ursus thibetanus)bitter taste receptor Tas2R2 gene,Yesheng Dongwu Xuebao(Chinese Journal of Wildlife),35(4):407-413(钟源,潘阳,侯文海,姚永芳,徐怀亮,张栓玲,2014,黑熊苦味受体Tas2R2基因的分子克隆与序列分析,野生动物学报,35(4):407-413)