黑线仓鼠MTNR1A的克隆及种群间的遗传多态性分析
摘要
黑线仓鼠(Cricetulus barabensis)在我国分布广泛,在维护生态系统平衡中起着重要的作用,为农田和草原主要害鼠之一。本研究从分子水平上研究黑线仓鼠的遗传多态性,为综合治理措施的制定提供科学依据。
生物的遗传多样性是引起其存活力、繁殖力差异的主要原因之一,生物遗传多样性的本质是生物体在遗传物质上的变异,即编码遗传信息的核苷酸的改变。一个物种或种群的遗传变异越丰富,对外界环境的适应能力就越强；反之其适应能力就越弱。单核苷酸多态性作为第三代多态性遗传标记,在遗传学中亲缘关系鉴定、遗传图谱构建、遗传多样性分析等研究中应用广泛,发挥着越来越重要的作用,已经成为国内外的研究热点。
褪黑素在多个物种的多个组织中广泛存在,参与生物的昼夜节律、视网膜信号的调节以及季节性繁殖哺乳动物的生殖调控等多种生理功能的调节,其生物学功能的发挥必须与其高亲和性G蛋白耦联受体的结合来实现。过去在黑线仓鼠中研究褪黑素受体国内外未见报道,通过分析比较种群间的单核苷酸变异,可以了解种群内的亲缘关系及进化的有关生物学信息,从而为研究种群遗传多态性及遗传结构提供有力的理论基础。
本实验对山东省三个不同地区黑线仓鼠种群和曲阜两个季节黑线仓鼠种群的遗传多态性进行研究,首先根据GenBank中已注册金仓鼠(Mesocricetus auratus)、黑线毛足鼠(Phodopus sungorus)等近缘物种的MTNR1A序列,利用Primer5.0软件和Oligo6.0软件根据引物设计原则设计2对引物,其次分别对黑线仓鼠MTNR1A基因5’端调控区段和外显子1,外显子2部分区段进行PCR扩增,经连接、转化和测序,得到232条序列,最后利用Chromos、ClustalX、DNAman、DnaSP5.0和MEGA3.1等主要的序列分析软件对济宁曲阜、临沂沂南、潍坊临胸三个黑线仓鼠种群之间及曲阜春秋季种群之间的单核苷酸多态性(SNPs)进行了比较分析。
研究结果表明：(1)首次利用PCR法在山东黑线仓鼠种群中(共116个样本)克隆得到MTNR1A部分序列(约1708 bp),其中包含5’端调控序列(约725 bp)、外显子1(185 bp)和外显子2部分区段(798 bp)。5’端和外显子1区段有多态位点数19个,有55种单倍型,在GenBank注册号为HM099922-HM099976。外显子2区段有多态位点数13个,有18个单倍型,在GenBank注册号为HM099977-HM099994。
(2)分析5’端调控序列发现四种调控序列：CAC、GAGA、GC_SP1和GC；发现少量可能是转录起始位点的AT、TA序列；但未发现典型TATA序列和CAAT序列。
(3)本实验对曲阜两个不同季节种群研究发现,MTNR1A第290、292、325、716、940位碱基变化分别导致第77、78、109、239和314位氨基酸发生改变,依次是天冬酰胺→丝氨酸、亮氨酸→甲硫氨酸、亮氨酸→缬氨酸、天冬酰胺→丝氨酸、缬氨酸→异亮氨酸。第N77S、F239S、V314I位氨基酸变化只发生在秋季,而第L78M、L109V位氨基酸变化只发生在春季,初步推断与季节变化有关。第251～253位三个位点发生颠换、插入、转换三种碱基连带变化的多态位点,不但可导致氨基酸发生改变,而且可以使编码顺序发生改变,与季节变化无关。
(4)不同种群间遗传多样性存在差异,遗传多样性临朐种群大于曲阜种群,曲阜种群大于沂南种群。
(5)不同种群间黑线仓鼠MTNRIA基因均具有一定遗传分化,但分化不明显,利用NJ系统进化树分析也支持分化不明显的结论。
Cricetulus barabensis is widely distributed in China, and its plays an important role in keeping ecosystem balance. Cricetulus barabensis is one of the deleterious rodents for agriculture and grassland. In this experiment we have studied it at molecular level.
Genetic diversity is one of important factors affecting species vitality and reproduction, and is the variation of genetic material which is nucleic acid. Genetic diversity plays a very important role in survival and adaptability of a species. The richer genetic variation of a species (population) is, the stronger its adaptation to the external environment is. On the contrary, the adaptation is weaker. Single nucleotide polymorphism (SNP) which has been considered as the third genetic marker for molecular research is widely used in the genetic relationship, genetic map construction, analysis of genetic diversity. The use of molecular markers, revealing polymorphism at the DNA level, has been playing an increasing part in genetics studies. The new marker type (SNP) is now on the scene and has gained high popularity.
Melatonin elicits its biological effects through high affinity G protein-coupled receptors that are responsible for melatonin to effect on mammalian circadian rhythm, regulation of retinal signals and reproductive seasonality affected by day length and son on. In the past, the study about melatonin receptor 1a in Cricetulus barabensis has never been reported at home and abroad. In order to study biological information about genetic diversity of different populations, genetic relationship within species and so on, the author has analyzed the single nucleotide variation of different populations which could provide the theoretical basis for genetic diversity and genetic structure.
First of all, the author designed two primers with the software:Primer5.0, Oligo6.0 and MTNR1A gene sequences of Cricetulus barabensis related species:Mesocricetus auratus and Phodopus sungorus which have been registered in GenBank. Second, the gene fragments were amplified from three populations in Yinan, Linqu and Qufu areas, Shandong province, China, then cloned and sequenced. The 232 gene fragments are the 5'UTR and exon 1 sequence and partial exon 2 sequence. This experiment mainly analyzed the single nucleotide polymorphisms (SNPs) in three different populations of Cricetulus barabensis, and the comparison of polymorphism among Cricetulus barabensis by use of Chromas, ClustalX, DNAman, DnaSP5.0, MEGA3.1 and other key sequence analysis software in order to explore the reason for the variation in different geography populations of Cricetulus barabensis and the variation level among different subspecies.
The major results were as follows:(1)The 232 partial sequences (1708 bp) were amplified with PCR and the fragments contained the 5'UTR (725 bp) sequence and exon 1 sequence (185 bp) and partial exon 2 sequence (798 bp). There are 19 polymorphic loci and 55 haplotypes (HM099922～HM099976) of the 5'UTR and exon 1 sequence and 13 polymorphic loci and 18 haplotypes (HM099977～HM099994) of partial exon 2 sequence.
(2) There were 4 kind of regulatory sequence in the 5'UTR of MTNR1A:CAC, GAGA, GC_SP1, GC and little transcriptional start site:AT and TA. However, the author did not find the typical TATA box and CAAT box in 5'UTR.
(3) The bases mutation (290,292,325,716,940) lead to amino acid change (N77S, L78M, L109V, F239S and V314I) by comparison between Spring and Autumn population. The importance thing is that the amino acid change (N77S, F239S, V314I) only appear in Autumn population, and the amino acid change (L78M, L109V) only appear in Spring population, so the author concluded that it has relation to season. Transversion, insertion and conversion from 251 to 253 not only lead to amino acid change, but also change the coding order. The three bases change had no relation to season.
(4) There was divergence of genetic diversity in three populations and the Linqu population genetic diversity is greatest, the Qufu population genetic diversity is greater than the Yinan population genetic diversity.
(5) This result demonstrated that the differentiation of three populations was unconspicuous. The NJ phylogenetic tree also indicated the same result.
生物的遗传多样性是引起其存活力、繁殖力差异的主要原因之一,生物遗传多样性的本质是生物体在遗传物质上的变异,即编码遗传信息的核苷酸的改变。一个物种或种群的遗传变异越丰富,对外界环境的适应能力就越强；反之其适应能力就越弱。单核苷酸多态性作为第三代多态性遗传标记,在遗传学中亲缘关系鉴定、遗传图谱构建、遗传多样性分析等研究中应用广泛,发挥着越来越重要的作用,已经成为国内外的研究热点。
褪黑素在多个物种的多个组织中广泛存在,参与生物的昼夜节律、视网膜信号的调节以及季节性繁殖哺乳动物的生殖调控等多种生理功能的调节,其生物学功能的发挥必须与其高亲和性G蛋白耦联受体的结合来实现。过去在黑线仓鼠中研究褪黑素受体国内外未见报道,通过分析比较种群间的单核苷酸变异,可以了解种群内的亲缘关系及进化的有关生物学信息,从而为研究种群遗传多态性及遗传结构提供有力的理论基础。
本实验对山东省三个不同地区黑线仓鼠种群和曲阜两个季节黑线仓鼠种群的遗传多态性进行研究,首先根据GenBank中已注册金仓鼠(Mesocricetus auratus)、黑线毛足鼠(Phodopus sungorus)等近缘物种的MTNR1A序列,利用Primer5.0软件和Oligo6.0软件根据引物设计原则设计2对引物,其次分别对黑线仓鼠MTNR1A基因5’端调控区段和外显子1,外显子2部分区段进行PCR扩增,经连接、转化和测序,得到232条序列,最后利用Chromos、ClustalX、DNAman、DnaSP5.0和MEGA3.1等主要的序列分析软件对济宁曲阜、临沂沂南、潍坊临胸三个黑线仓鼠种群之间及曲阜春秋季种群之间的单核苷酸多态性(SNPs)进行了比较分析。
研究结果表明：(1)首次利用PCR法在山东黑线仓鼠种群中(共116个样本)克隆得到MTNR1A部分序列(约1708 bp),其中包含5’端调控序列(约725 bp)、外显子1(185 bp)和外显子2部分区段(798 bp)。5’端和外显子1区段有多态位点数19个,有55种单倍型,在GenBank注册号为HM099922-HM099976。外显子2区段有多态位点数13个,有18个单倍型,在GenBank注册号为HM099977-HM099994。
(2)分析5’端调控序列发现四种调控序列：CAC、GAGA、GC_SP1和GC；发现少量可能是转录起始位点的AT、TA序列；但未发现典型TATA序列和CAAT序列。
(3)本实验对曲阜两个不同季节种群研究发现,MTNR1A第290、292、325、716、940位碱基变化分别导致第77、78、109、239和314位氨基酸发生改变,依次是天冬酰胺→丝氨酸、亮氨酸→甲硫氨酸、亮氨酸→缬氨酸、天冬酰胺→丝氨酸、缬氨酸→异亮氨酸。第N77S、F239S、V314I位氨基酸变化只发生在秋季,而第L78M、L109V位氨基酸变化只发生在春季,初步推断与季节变化有关。第251～253位三个位点发生颠换、插入、转换三种碱基连带变化的多态位点,不但可导致氨基酸发生改变,而且可以使编码顺序发生改变,与季节变化无关。
(4)不同种群间遗传多样性存在差异,遗传多样性临朐种群大于曲阜种群,曲阜种群大于沂南种群。
(5)不同种群间黑线仓鼠MTNRIA基因均具有一定遗传分化,但分化不明显,利用NJ系统进化树分析也支持分化不明显的结论。
Cricetulus barabensis is widely distributed in China, and its plays an important role in keeping ecosystem balance. Cricetulus barabensis is one of the deleterious rodents for agriculture and grassland. In this experiment we have studied it at molecular level.
Genetic diversity is one of important factors affecting species vitality and reproduction, and is the variation of genetic material which is nucleic acid. Genetic diversity plays a very important role in survival and adaptability of a species. The richer genetic variation of a species (population) is, the stronger its adaptation to the external environment is. On the contrary, the adaptation is weaker. Single nucleotide polymorphism (SNP) which has been considered as the third genetic marker for molecular research is widely used in the genetic relationship, genetic map construction, analysis of genetic diversity. The use of molecular markers, revealing polymorphism at the DNA level, has been playing an increasing part in genetics studies. The new marker type (SNP) is now on the scene and has gained high popularity.
Melatonin elicits its biological effects through high affinity G protein-coupled receptors that are responsible for melatonin to effect on mammalian circadian rhythm, regulation of retinal signals and reproductive seasonality affected by day length and son on. In the past, the study about melatonin receptor 1a in Cricetulus barabensis has never been reported at home and abroad. In order to study biological information about genetic diversity of different populations, genetic relationship within species and so on, the author has analyzed the single nucleotide variation of different populations which could provide the theoretical basis for genetic diversity and genetic structure.
First of all, the author designed two primers with the software:Primer5.0, Oligo6.0 and MTNR1A gene sequences of Cricetulus barabensis related species:Mesocricetus auratus and Phodopus sungorus which have been registered in GenBank. Second, the gene fragments were amplified from three populations in Yinan, Linqu and Qufu areas, Shandong province, China, then cloned and sequenced. The 232 gene fragments are the 5'UTR and exon 1 sequence and partial exon 2 sequence. This experiment mainly analyzed the single nucleotide polymorphisms (SNPs) in three different populations of Cricetulus barabensis, and the comparison of polymorphism among Cricetulus barabensis by use of Chromas, ClustalX, DNAman, DnaSP5.0, MEGA3.1 and other key sequence analysis software in order to explore the reason for the variation in different geography populations of Cricetulus barabensis and the variation level among different subspecies.
The major results were as follows:(1)The 232 partial sequences (1708 bp) were amplified with PCR and the fragments contained the 5'UTR (725 bp) sequence and exon 1 sequence (185 bp) and partial exon 2 sequence (798 bp). There are 19 polymorphic loci and 55 haplotypes (HM099922～HM099976) of the 5'UTR and exon 1 sequence and 13 polymorphic loci and 18 haplotypes (HM099977～HM099994) of partial exon 2 sequence.
(2) There were 4 kind of regulatory sequence in the 5'UTR of MTNR1A:CAC, GAGA, GC_SP1, GC and little transcriptional start site:AT and TA. However, the author did not find the typical TATA box and CAAT box in 5'UTR.
(3) The bases mutation (290,292,325,716,940) lead to amino acid change (N77S, L78M, L109V, F239S and V314I) by comparison between Spring and Autumn population. The importance thing is that the amino acid change (N77S, F239S, V314I) only appear in Autumn population, and the amino acid change (L78M, L109V) only appear in Spring population, so the author concluded that it has relation to season. Transversion, insertion and conversion from 251 to 253 not only lead to amino acid change, but also change the coding order. The three bases change had no relation to season.
(4) There was divergence of genetic diversity in three populations and the Linqu population genetic diversity is greatest, the Qufu population genetic diversity is greater than the Yinan population genetic diversity.
(5) This result demonstrated that the differentiation of three populations was unconspicuous. The NJ phylogenetic tree also indicated the same result.
引文
[1]张知彬,王祖望.农业重要害鼠的生态学及控制对策[M].北京：海洋出版社,1998,128.
[2]董京平,张知彬.鼠害的防治及鼠类对森林更新的研究进展[J].生物学通报,2008,43(1)：10～12.
[3]张知彬.我国草原鼠害的严重性及防治对策[J].中国科学院院刊,2003(5)：343～347.
[4]汪诚信.我国鼠害及其防治对策[J].中国媒介生物学及控制杂志,1996,7,(1)：62～65.
[5]Clark K J, Harcum S W, Griffiths J, Bailey K M. Development of a method to quantify gene expression levels for glycosylation pathway genes in Chinese hamster ovary cell cultures [J]. Applied Biochemistry and Biotechnology,2005,125(3):159～173.
[6]Muller N, Derouazi M, Tilborgh F W, Wulhfard S, Wulhfardl S, Hackerl D L, Jordanl M, Wurm1 F M. Scalable transient gene expression in Chinese hamster ovary cells in instrumented and non-instrumented cultivation systems [J]. Biotechnology Letters,2007, 29(5):703～711.
[7]Yoon S K, Ahn Y H, Jeong M H. Effect of culture temperature on follicle-stimulating hormone production by Chinese hamster ovary cells in a perfusion bioreactor [J]. Applied Microbiology and Biotechnology,2007,76(1):83～89.
[8]罗泽珣,陈卫,高武.中国动物志兽纲第6卷啮齿目下册仓鼠科[M].北京： 科学出版社,2000,28～38.
[9]纪树立.鼠疫[M].北京：人民卫生出版社,1988,156.
[10]郑智民,姜志宽,陈安国.啮齿动物学[M].上海：上海交通大学出版社,2008,168～169.
[11]寿振黄.中国经济动物志(兽类)[M].北京：科学出版社,1962,156～159.
[12]张洁.北京大兴地区黑线仓鼠种群繁殖生态研究[J].兽类学报,1986,6(1)：45～46.
[13]侯希贤,董维惠,张鹏利,杨玉平.呼和浩特栽培牧草地黑线仓鼠生态学的调查[J].中国草地,1989,(5)：53～57.
[14]李焕仁,陈荣.肃南县的主要害鼠[J].甘肃农业科技,2006,(12)：45～46.
[15]穆成旺,任维新.黑线仓鼠的生物学特性及防治研究[J].甘肃农业科技,1999,(1)：39.
[16]李玉春,卢浩泉,张学栋,徐文生.黑线仓鼠的生长指标分析与年龄指标确定[J].兽类学报,1989,9(1)：49～55.
[17]杨玉平,周延林,张朋利,侯希贤,董维惠.黑线仓鼠生长发育研究[J].兽类学报,1996,16(4)：309～311.
[18]柳劲松,王德华,孙儒泳.东北地区黑线仓鼠的代谢产热特征及其体温调节[J].动物学报,2003,49(4)：451～457.
[19]徐承水,杨幼凤,卢浩泉.黑线仓鼠不同季节睾丸组织学研究[J].曲阜师范大学学报,1992,18(2)：60～65.
[20]徐承水,党本元,张祥沛,李亚,张向峰.曲阜孔林黑线仓鼠种群调节机理的研究[J].中国媒介生物学及控制杂志,1996,79(5)：339～344.
[21]李传海.黑线仓鼠微卫星引物的筛选及应用[D].曲阜师范大学硕士学位论文,2006.
[22]高倩.黑线仓鼠SSR标记及地理种群遗传多态性研究[D].曲阜师范大学硕士学位论文,2008.
[23]解学辉, 董晓波, 秦桢, 孔繁华, 徐来祥.大仓鼠SSR座位的筛选及黑线仓鼠MHC基因的克隆与序列分析.曲阜师范大学学报：自然科学版,2009,35(2)：98～103.
[24]王玉志,卢浩泉,陈安,陈宁,苏传东.灰色系统在华北平原旱作区黑线仓鼠种群数量预测预报中的应用[J].动物学报(增刊),1997,43：107～112.
[25]董维惠,候希贤,张耀星,杨玉平,郎炳聚,薛小平.内蒙古中西部地区黑线仓鼠种群生态特征的比较研究[J].中国媒介生物学及控制杂志,2004,15(3)：205～208.
[26]武文华,付和平,武晓东,杨玉平,董维惠,徐胜利.应用马尔可夫链模型预测长爪沙鼠和黑线仓鼠种群数量[J].动物学杂志,2007,42(6)：69～78.
[27]Arendt J. Melatonin and the Mammalian Pineal Gland[M]. London:Chapman and Hall, 1995,295-310.
[28]Gisela M, Aaron B L, John R H. The mechanism of frog skin lightening by acetylcholine[J]. General and Comparative Endocrinology,1974,23(1):45～51.
[29]季从亮,储明星,陈国宏.褪黑激素受体基因的研究进展[J].遗传,2003,25(2)：221～224.
[30]茆达干,杨利国,吴结革.褪黑激素对动物生殖的作用及其调控[J].草食家畜,2001,3： 5～8.
[31]Pozo D, Delgado M, Fernandez-Santos J M, Calvo JR, Gomariz RP, Martin-Lacave I, Ortiz GG, Guerrero J M. Expression of the Mella-melatonin receptor mRNA in T and B subsets of lymphocytes from thymus and spleen[J]. The FASEB Journal,1997,11(6):466～473.
[32]Reppert S M, Weaver D R, Rivkees S A, Stopa E G. Putative melatonin receptors in a human biological clock[J]. Science,1988,242:78～81.
[33]Vanecek J. Melatonin binding sites[J]. Journal of Neurochemistry,1988,51(5):1436～1440.
[34]Reppert S M, Weaver D R. Melatonin madness[J]. Cell,1995,83(7):1059～1062.
[35]Dubocovich M L, Takahashi J. Use of 2-[125I] iodomelatonin to characterize melatonin binding sites in chicken retina[J]. Proc Natl Acad Sci USA,1987,84:3916～3920.
[36]Dubocovich M L. Pharmacology and function of melatonin receptors [J]. The FASEB Journal,1988,2:2765～2773.
[37]Dubocovich M L. Melatonin receptors:are there multiple subtypes?[J]. Trends in Pharmacological Sciences,1995,16(2):50～56.
[38]Dubocovich M L, Bermudez MA, Gerdin M J, Masan M I. Molecular pharmacology, regulation and function of mammalian melatonin receptors[J]. Frontiers in Bioscience, 2003,8,1093～1108.
[39]Reppert, S M. Melatonin receptors:molecular biology of a new family of G protein-coupled receptors[J]. Journal of biological rhythms,1997,12(6):528～531.
[40]Ebisawa T, Karne S, Lerner M R, Reppert S M. Expression cloning of a high-affinity melatonin receptor from Xenopus dermal melanophores[J]. Proc Natl Acad Sci USA,1994, 91:6133～6137.
[41]Reppert S M, Weaver, D R, Ebisawa T. Cloning and characterization of a mammalian melatonin receptor that mediates reproductive and circadian responses[J]. Neuron,1994, 13(5):1177～1185.
[42]Reppert S M, Weaver D R, Cassone V M, Godson C, Kolakowski L F. Melatonin receptor for birds:molecular analysis of two receptor subtypes differentially expressed in chick brain[J]. Neuron,1995,15(5):1003～1015.
[43]Roca A L, Godson C, Weaver D R, Reppert S M. Structure, characterization, and expression of the gene encoding the mouse Mella melatonin receptor[J]. Endocrinology,1996,137(8): 3469～3477.
[44]Weaver D R, Liu C, Reppert S M. Nature's knock-out:the Mellb receptor is not necessary for reproductive and circadian responses to melatonin in Siberian hamster[J]. Mol. Endocrinology,1996,10:1478～1487.
[45]Barrett P, Conway S, Jockers R, Strosberg A D, Lemaitre G B, Delagrange P, Morgan P J. Cloning and functional analysis of a polymorphic variant of the ovine Mel la melatonin receptor[J]. Biochimica et Biophysica Acta,1997,1356(3):299～307.
[46]Carcangiu V, Mura M C, Vacca G M, Pazzola M, Dettori M L, Luridiana S, Bini P P. Polymorphism of the melatonin receptor MT1 gene and its relationship with seasonal reproductive activity in the Sarda sheep breed[J]. Animal Reproduction Science,2009,116: 65～72.
[47]Kokkola T, Watson M A, White J, Dowellb S, Foordb S M, Laitinena J T. Mutagenesis of Human MellaMelatonin Receptor Expressed in Yeast Reveals Domains Important for Receptor Function [J]. Biochemical and Biophysical Research Communications,1998, 249(2):531～536.
[48]Messer L A, Wang L, Tuggle C K, Yerle M, Chardon P, Pomp D, Womack J E, Barendse W, Crawford A M, Notter D R, Rothschild M F. Mapping of the melatonin receptor la (MTNR1A) gene in pigs, sheep, and cattle[J]. Mammalian Genome,1997, (8):368～370.
[49]Archibald A L, Haley C S, Brown J F, et al. The PiGMaP consortium linkage map of the pig (Sus scrofa) [J]. Mammalian Genome,1995,6:157～175.
[50]Pelletier J, Bodin L, Hanocq E, Malpaux B, Teyssier J, Thimonier J, Chemineau P. Association between expression of reproductive seasonality and alleles of the gene for Mella receptor in the ewe[J]. Biology of Reproduction,2000,62:1096～1101.
[51]季从亮, 储明星, 陈国宏,周国利,朱颜.4个绵羊品种褪黑激素受体1a基因第二外显子PCR-RFLP分析[J].华中农业大学学报,2003,22(2)：105～109.
[52]葛颂,洪德元.遗传多样性及其检测方法[A].见：钱迎倩,马克平主编.生物多样性研究的原理与方法[C].北京：中国科学技术出版社,1994,122～140.
[53]孙儒泳.动物生态学原理(第三版)[M].北京：北京师范大学出版社,2001.
[54]季维智,宿兵.遗传多样性研究的原理与方法[C].杭州：浙江科学出版社,1999.
[55]陈灵芝.中国的生物多样性[M].北京：上海科技出版社,99～113.
[56]陈灵芝,马克平.生物多样性科学[M].上海：上海科技出版社,2001,1-6,93～125.
[57]Alain V, Denis M, Magali S, Andre E. A review on SNP and other types of molecular markers and their use in animal genetics[J]. Genet Sel Evol,2002,34:275～305.
[58]Authony B. SNP attack on complex traits[J]. Nature Genet,1998,20(3):217～218.
[59]Linlin MA, YaLi XUE, Yan LIU, Li P, Fu S. Polymorphism study of seven SNPs at ADH genes in 15 Chinese populations [J]. Hereditas,2005,142:103～111.
[60]赵辉,李启寨,李俊,曾长青,胡松年,于军.相邻碱基组分与产生SNP的转换或颠换在植物基因组中的研究[J].中国科学C辑(生命科学),2006,36(1)：128.
[61]Bakker E G, Stahl E A, Toomajian C, Kreitman K. Distribution of genetic variation within and among local populations of Arabidopsis thaliana over it s species range [J]. Molecular ecology,2006,15:1405～1408.
[62]萨姆布鲁克J,弗里奇E F,曼尼阿蒂斯T.分子克隆实验指南(第二版)[M].金冬雁,黎孟枫等译.北京：科学出版社,1992.
[63]Migaud M, Gavet S, Pelletier J. Partial cloning and polymorphism of the melatoninla (Mella) receptor gene in two breeds of goat with different reproductive seasonality [J]. Reproduction,2002,124:59～64.
[64]程笃学.绵-山羊MTNR1A基因的克隆测序及其与繁殖季节性的关联研究[D].山东农业大学硕士学位论文,2005.
[65]何远清,储明星,王金玉,方丽,叶素成.济宁青山羊MTNR1A基因外显子2的克隆与序列分析[J].安徽农业大学学报,2006,33(4)：502～505.
[66]Parker P G, Whiteman H H. Genetic diversity in fragmented populations of Clemmys guttata and Chrysemys picta marginata as shown by DNA fingerprinting[J]. Copeia,1993, 3:841～846.
[67]Tamate H B, Shibata K, Tsuchiya T, Ohtaishi N. Assessment of genetic variations within populations of Sika Deer in Japan by analysis of randomly amplified polymorphic DNA (RAPD) [J]. Zoological Science,1995,12(5):669～673.
[68]Frankham R. Relationship of genetic variation to population size in wildlife[J]. Conservation Biology,1996,10:1500～1508.
[69]汪永庆,张知彬,徐来祥.大仓鼠年龄组及性别种群的遗传多样性[J].兽类学报,2002,(3)：187～200.
[70]张知彬,王祖望.害鼠综合治理的理论和实践[M].北京：科学出版社,1996,145～160.
[71]Saccheri I, Kuussaari M, Kankare M, Vikman P, Fortelius W, Hanski I. Inbreeding and extinction in a butterfly meta population[J]. Nature,1998,392:491～494.
[72]Westemeier R L, Brawn J D, Simpson S A, Esker T L. Tracking the long term decline and recovery of an isolated population [J]. Science,1998,282:1695～1698.
[73]陶峰勇,王小明,郑合勋,方盛国.中国大鲵四种群的遗传结构和地理分化[J].动物学研究,2005,26(2)：162～167.
[74]Wright S. Evolution in Mendelian population[J]. Genetics,1931,16:91～96.
[75]张娟,尹林克,张道远.刚毛柽柳天然居群遗传多样性初探[J].云南植物研究所,2003,25：557～562.
[76]顾万春.统计遗传学[M].北京：科学出版社,2004,150.
[2]董京平,张知彬.鼠害的防治及鼠类对森林更新的研究进展[J].生物学通报,2008,43(1)：10～12.
[3]张知彬.我国草原鼠害的严重性及防治对策[J].中国科学院院刊,2003(5)：343～347.
[4]汪诚信.我国鼠害及其防治对策[J].中国媒介生物学及控制杂志,1996,7,(1)：62～65.
[5]Clark K J, Harcum S W, Griffiths J, Bailey K M. Development of a method to quantify gene expression levels for glycosylation pathway genes in Chinese hamster ovary cell cultures [J]. Applied Biochemistry and Biotechnology,2005,125(3):159～173.
[6]Muller N, Derouazi M, Tilborgh F W, Wulhfard S, Wulhfardl S, Hackerl D L, Jordanl M, Wurm1 F M. Scalable transient gene expression in Chinese hamster ovary cells in instrumented and non-instrumented cultivation systems [J]. Biotechnology Letters,2007, 29(5):703～711.
[7]Yoon S K, Ahn Y H, Jeong M H. Effect of culture temperature on follicle-stimulating hormone production by Chinese hamster ovary cells in a perfusion bioreactor [J]. Applied Microbiology and Biotechnology,2007,76(1):83～89.
[8]罗泽珣,陈卫,高武.中国动物志兽纲第6卷啮齿目下册仓鼠科[M].北京： 科学出版社,2000,28～38.
[9]纪树立.鼠疫[M].北京：人民卫生出版社,1988,156.
[10]郑智民,姜志宽,陈安国.啮齿动物学[M].上海：上海交通大学出版社,2008,168～169.
[11]寿振黄.中国经济动物志(兽类)[M].北京：科学出版社,1962,156～159.
[12]张洁.北京大兴地区黑线仓鼠种群繁殖生态研究[J].兽类学报,1986,6(1)：45～46.
[13]侯希贤,董维惠,张鹏利,杨玉平.呼和浩特栽培牧草地黑线仓鼠生态学的调查[J].中国草地,1989,(5)：53～57.
[14]李焕仁,陈荣.肃南县的主要害鼠[J].甘肃农业科技,2006,(12)：45～46.
[15]穆成旺,任维新.黑线仓鼠的生物学特性及防治研究[J].甘肃农业科技,1999,(1)：39.
[16]李玉春,卢浩泉,张学栋,徐文生.黑线仓鼠的生长指标分析与年龄指标确定[J].兽类学报,1989,9(1)：49～55.
[17]杨玉平,周延林,张朋利,侯希贤,董维惠.黑线仓鼠生长发育研究[J].兽类学报,1996,16(4)：309～311.
[18]柳劲松,王德华,孙儒泳.东北地区黑线仓鼠的代谢产热特征及其体温调节[J].动物学报,2003,49(4)：451～457.
[19]徐承水,杨幼凤,卢浩泉.黑线仓鼠不同季节睾丸组织学研究[J].曲阜师范大学学报,1992,18(2)：60～65.
[20]徐承水,党本元,张祥沛,李亚,张向峰.曲阜孔林黑线仓鼠种群调节机理的研究[J].中国媒介生物学及控制杂志,1996,79(5)：339～344.
[21]李传海.黑线仓鼠微卫星引物的筛选及应用[D].曲阜师范大学硕士学位论文,2006.
[22]高倩.黑线仓鼠SSR标记及地理种群遗传多态性研究[D].曲阜师范大学硕士学位论文,2008.
[23]解学辉, 董晓波, 秦桢, 孔繁华, 徐来祥.大仓鼠SSR座位的筛选及黑线仓鼠MHC基因的克隆与序列分析.曲阜师范大学学报：自然科学版,2009,35(2)：98～103.
[24]王玉志,卢浩泉,陈安,陈宁,苏传东.灰色系统在华北平原旱作区黑线仓鼠种群数量预测预报中的应用[J].动物学报(增刊),1997,43：107～112.
[25]董维惠,候希贤,张耀星,杨玉平,郎炳聚,薛小平.内蒙古中西部地区黑线仓鼠种群生态特征的比较研究[J].中国媒介生物学及控制杂志,2004,15(3)：205～208.
[26]武文华,付和平,武晓东,杨玉平,董维惠,徐胜利.应用马尔可夫链模型预测长爪沙鼠和黑线仓鼠种群数量[J].动物学杂志,2007,42(6)：69～78.
[27]Arendt J. Melatonin and the Mammalian Pineal Gland[M]. London:Chapman and Hall, 1995,295-310.
[28]Gisela M, Aaron B L, John R H. The mechanism of frog skin lightening by acetylcholine[J]. General and Comparative Endocrinology,1974,23(1):45～51.
[29]季从亮,储明星,陈国宏.褪黑激素受体基因的研究进展[J].遗传,2003,25(2)：221～224.
[30]茆达干,杨利国,吴结革.褪黑激素对动物生殖的作用及其调控[J].草食家畜,2001,3： 5～8.
[31]Pozo D, Delgado M, Fernandez-Santos J M, Calvo JR, Gomariz RP, Martin-Lacave I, Ortiz GG, Guerrero J M. Expression of the Mella-melatonin receptor mRNA in T and B subsets of lymphocytes from thymus and spleen[J]. The FASEB Journal,1997,11(6):466～473.
[32]Reppert S M, Weaver D R, Rivkees S A, Stopa E G. Putative melatonin receptors in a human biological clock[J]. Science,1988,242:78～81.
[33]Vanecek J. Melatonin binding sites[J]. Journal of Neurochemistry,1988,51(5):1436～1440.
[34]Reppert S M, Weaver D R. Melatonin madness[J]. Cell,1995,83(7):1059～1062.
[35]Dubocovich M L, Takahashi J. Use of 2-[125I] iodomelatonin to characterize melatonin binding sites in chicken retina[J]. Proc Natl Acad Sci USA,1987,84:3916～3920.
[36]Dubocovich M L. Pharmacology and function of melatonin receptors [J]. The FASEB Journal,1988,2:2765～2773.
[37]Dubocovich M L. Melatonin receptors:are there multiple subtypes?[J]. Trends in Pharmacological Sciences,1995,16(2):50～56.
[38]Dubocovich M L, Bermudez MA, Gerdin M J, Masan M I. Molecular pharmacology, regulation and function of mammalian melatonin receptors[J]. Frontiers in Bioscience, 2003,8,1093～1108.
[39]Reppert, S M. Melatonin receptors:molecular biology of a new family of G protein-coupled receptors[J]. Journal of biological rhythms,1997,12(6):528～531.
[40]Ebisawa T, Karne S, Lerner M R, Reppert S M. Expression cloning of a high-affinity melatonin receptor from Xenopus dermal melanophores[J]. Proc Natl Acad Sci USA,1994, 91:6133～6137.
[41]Reppert S M, Weaver, D R, Ebisawa T. Cloning and characterization of a mammalian melatonin receptor that mediates reproductive and circadian responses[J]. Neuron,1994, 13(5):1177～1185.
[42]Reppert S M, Weaver D R, Cassone V M, Godson C, Kolakowski L F. Melatonin receptor for birds:molecular analysis of two receptor subtypes differentially expressed in chick brain[J]. Neuron,1995,15(5):1003～1015.
[43]Roca A L, Godson C, Weaver D R, Reppert S M. Structure, characterization, and expression of the gene encoding the mouse Mella melatonin receptor[J]. Endocrinology,1996,137(8): 3469～3477.
[44]Weaver D R, Liu C, Reppert S M. Nature's knock-out:the Mellb receptor is not necessary for reproductive and circadian responses to melatonin in Siberian hamster[J]. Mol. Endocrinology,1996,10:1478～1487.
[45]Barrett P, Conway S, Jockers R, Strosberg A D, Lemaitre G B, Delagrange P, Morgan P J. Cloning and functional analysis of a polymorphic variant of the ovine Mel la melatonin receptor[J]. Biochimica et Biophysica Acta,1997,1356(3):299～307.
[46]Carcangiu V, Mura M C, Vacca G M, Pazzola M, Dettori M L, Luridiana S, Bini P P. Polymorphism of the melatonin receptor MT1 gene and its relationship with seasonal reproductive activity in the Sarda sheep breed[J]. Animal Reproduction Science,2009,116: 65～72.
[47]Kokkola T, Watson M A, White J, Dowellb S, Foordb S M, Laitinena J T. Mutagenesis of Human MellaMelatonin Receptor Expressed in Yeast Reveals Domains Important for Receptor Function [J]. Biochemical and Biophysical Research Communications,1998, 249(2):531～536.
[48]Messer L A, Wang L, Tuggle C K, Yerle M, Chardon P, Pomp D, Womack J E, Barendse W, Crawford A M, Notter D R, Rothschild M F. Mapping of the melatonin receptor la (MTNR1A) gene in pigs, sheep, and cattle[J]. Mammalian Genome,1997, (8):368～370.
[49]Archibald A L, Haley C S, Brown J F, et al. The PiGMaP consortium linkage map of the pig (Sus scrofa) [J]. Mammalian Genome,1995,6:157～175.
[50]Pelletier J, Bodin L, Hanocq E, Malpaux B, Teyssier J, Thimonier J, Chemineau P. Association between expression of reproductive seasonality and alleles of the gene for Mella receptor in the ewe[J]. Biology of Reproduction,2000,62:1096～1101.
[51]季从亮, 储明星, 陈国宏,周国利,朱颜.4个绵羊品种褪黑激素受体1a基因第二外显子PCR-RFLP分析[J].华中农业大学学报,2003,22(2)：105～109.
[52]葛颂,洪德元.遗传多样性及其检测方法[A].见：钱迎倩,马克平主编.生物多样性研究的原理与方法[C].北京：中国科学技术出版社,1994,122～140.
[53]孙儒泳.动物生态学原理(第三版)[M].北京：北京师范大学出版社,2001.
[54]季维智,宿兵.遗传多样性研究的原理与方法[C].杭州：浙江科学出版社,1999.
[55]陈灵芝.中国的生物多样性[M].北京：上海科技出版社,99～113.
[56]陈灵芝,马克平.生物多样性科学[M].上海：上海科技出版社,2001,1-6,93～125.
[57]Alain V, Denis M, Magali S, Andre E. A review on SNP and other types of molecular markers and their use in animal genetics[J]. Genet Sel Evol,2002,34:275～305.
[58]Authony B. SNP attack on complex traits[J]. Nature Genet,1998,20(3):217～218.
[59]Linlin MA, YaLi XUE, Yan LIU, Li P, Fu S. Polymorphism study of seven SNPs at ADH genes in 15 Chinese populations [J]. Hereditas,2005,142:103～111.
[60]赵辉,李启寨,李俊,曾长青,胡松年,于军.相邻碱基组分与产生SNP的转换或颠换在植物基因组中的研究[J].中国科学C辑(生命科学),2006,36(1)：128.
[61]Bakker E G, Stahl E A, Toomajian C, Kreitman K. Distribution of genetic variation within and among local populations of Arabidopsis thaliana over it s species range [J]. Molecular ecology,2006,15:1405～1408.
[62]萨姆布鲁克J,弗里奇E F,曼尼阿蒂斯T.分子克隆实验指南(第二版)[M].金冬雁,黎孟枫等译.北京：科学出版社,1992.
[63]Migaud M, Gavet S, Pelletier J. Partial cloning and polymorphism of the melatoninla (Mella) receptor gene in two breeds of goat with different reproductive seasonality [J]. Reproduction,2002,124:59～64.
[64]程笃学.绵-山羊MTNR1A基因的克隆测序及其与繁殖季节性的关联研究[D].山东农业大学硕士学位论文,2005.
[65]何远清,储明星,王金玉,方丽,叶素成.济宁青山羊MTNR1A基因外显子2的克隆与序列分析[J].安徽农业大学学报,2006,33(4)：502～505.
[66]Parker P G, Whiteman H H. Genetic diversity in fragmented populations of Clemmys guttata and Chrysemys picta marginata as shown by DNA fingerprinting[J]. Copeia,1993, 3:841～846.
[67]Tamate H B, Shibata K, Tsuchiya T, Ohtaishi N. Assessment of genetic variations within populations of Sika Deer in Japan by analysis of randomly amplified polymorphic DNA (RAPD) [J]. Zoological Science,1995,12(5):669～673.
[68]Frankham R. Relationship of genetic variation to population size in wildlife[J]. Conservation Biology,1996,10:1500～1508.
[69]汪永庆,张知彬,徐来祥.大仓鼠年龄组及性别种群的遗传多样性[J].兽类学报,2002,(3)：187～200.
[70]张知彬,王祖望.害鼠综合治理的理论和实践[M].北京：科学出版社,1996,145～160.
[71]Saccheri I, Kuussaari M, Kankare M, Vikman P, Fortelius W, Hanski I. Inbreeding and extinction in a butterfly meta population[J]. Nature,1998,392:491～494.
[72]Westemeier R L, Brawn J D, Simpson S A, Esker T L. Tracking the long term decline and recovery of an isolated population [J]. Science,1998,282:1695～1698.
[73]陶峰勇,王小明,郑合勋,方盛国.中国大鲵四种群的遗传结构和地理分化[J].动物学研究,2005,26(2)：162～167.
[74]Wright S. Evolution in Mendelian population[J]. Genetics,1931,16:91～96.
[75]张娟,尹林克,张道远.刚毛柽柳天然居群遗传多样性初探[J].云南植物研究所,2003,25：557～562.
[76]顾万春.统计遗传学[M].北京：科学出版社,2004,150.