白鹭MHC-II-DAB-I第二外显子基因的多态性与进化

白鹭MHC II DAB I第二外显子基因的多态性与进化李 力1，罗斯特1，林清贤1，2*，陈小麟1，2 收稿日期：2015-04-16基金项目：国家自然科学基金(，)；福建省自然科学基金(2010Y2007).*通信作者：(林清贤)；(陈小麟)*(1.厦门大学生命科学学院，2.厦门大学环境与生态学院，滨海湿地生态系统教育部重点实验室，福建 厦门)摘要：克隆测序白鹭(Egretta garzetta)5个种群138份个体组织样本的主要组织相容性复合体(MHC)II类B基因(DAB I)第2外显子(exon2)序列，分析探讨第2外显子基因的多态性、进化选择、系统关系和种群遗传结构. 主要结果如下：白鹭MHC II DAB I 第2外显子基因序列长度为270 bp，共计定义了139个等位基因；序列分析显示第2外显子基因有101个核苷酸变异位点(37.4%)和31个氨基酸变异位点(34.4%)；基于贝叶斯法构建的系统树显示白鹭MHC II DAB I 第2外显子基因有5个高支持率的谱系；肽结合位点(PBR)、非肽结合位点(non-PBR)的非同义替换率(dN) 和同义替换率(dS)比值计算显示，PBR的dN/dS为1.99 (p0.05)，而non-PBR的dN/dS则小于1，表明白鹭MHC II DAB I第2外显子基因受到正选择作用；根据等位基因在群体中的分布频率作分子方差分析(AMOVA)，得到FST为0.1941(p1时该位点可认为受到了正选择作用. 分别计算PBR、non-PBR的dS和dN，结果如表1所示， PBR区的dN/dS 显著大于1，而non-PBR的dN/dS 则小于1，这表明白鹭MHC II DAB I exon2的PBR区在进化过程中受到强烈的正选择作用(positive selection)(表1). 分别利用PAML软件包CODEML程序中的模型M1与M2以及M7与M8 检测exon2所受的选择作用，比较分析结果显示，模型M2相对于M1(p0. 01)、M8相对于M7(p0. 01)对本文数据有更好的拟合度. 在模型M2当中共检测到10个氨基酸位点受到正选择作用，其后验概率均大于95%；而在M8检测到11个受正选择作用的氨基酸位点，其中有10个位点与M2的位点完全相同(表2). 表1 白鹭MHC II DAB I exon2 的肽结合位点(PBR)、非肽结合位点(non-PBR)的dN和dS Tab.1 dN and dS of PBR and non-PBR of MHC II DAB I exon2 in little egret区域氨基酸数dN(S. E.)dS(S. E.)dN/dSpPBR240. 22160. 03440. 11530. 04961. 920. 0074Non-PBR660. 03590. 01120. 04050. 01530. 881.000Total900. 08690. 01540. 06570. 01761. 320. 2257表2 不同密码子进化模型对白鹭MHC II DAB I exon2吻合度检验的参数与似然值Tab.2 Parameter estimates and likelihood values of different codon evolution models for MHC II DAB I exon2 in little egret模型似然值估算参数(LRT)2L受正选择位点M12153. 152p0 =0. 52803p1=0. 46082154. 63(p0. 001)Not allowed0 =0. 042231 =1. 000006S* 23E* 32Y* 33A* 42Y* 55Y* 66A* 80V* 81A* 84S*M22073. 833p0 =0. 04938p1=0. 03889p2=0. 120810=0. 090221 =1. 000002=14. 16431M72152. 096p =0. 10623q=0. 86031Not allowed6S* 23E* 32Y* 33A* 42Y* 55Y* 65N* 66A* 80V* 81A* 84S*M82073. 652p0 =0. 04938p=0. 03889155. 52q=0. 11054 =15. 20752(p1=0. 18863)(p0. 001)注：*表示位点的后验概率大于0. 95，*表示后验概率大于0. 99. 2.4 MHC II DAB I exon 2等位基因的系统关系基于白鹭139个等位基因序列与其他3种外群鹭科鸟类(黄嘴白鹭、岩鹭、夜鹭)的MHC II DAB I exon2序列，通过贝叶斯方法所构建的系统发育树显示，白鹭的MHC II DAB I exon2可分为5个谱系(RY、JY、ZS、NB、XY)，这些谱系均有较高的支持率，并且每个谱系内只包括与之相对应的地理种群序列(图2). 在这5个谱系中，谱系XY与NB关系较亲近，并与RY谱系聚为一支，而谱系JY则与ZS关系更亲近. 图中仅大于0. 50的后验概率显示在系统树的分支上.图2 白鹭及3种鹭科鸟类MHC II DAB 1 exon2序列的系统关系树Fig.2 Phylogenetic relationship of MHC II DAB I exon2 sequences among Egretta garzetta，Egretta eulophotes， Egretta sacra and Nycticorax nycticorax2.5 MHC II DAB I exon 2的种群遗传结构在白鹭MHC II DAB I exon 2等位基因分布中，浙江舟山种群拥有49个，是等位基因数量最多的种群，然后依次是福建宁德种群(35)、贵州遵义种群(20)、福建厦门种群(19)，而河南信阳种群最少(16). 用Arlequin v3.1软件分析群体间分化指数(Fst)(表3)，结果显示5个白鹭群体间的Fst均较大，并且这5个群体两两之间Fst值差异显著性检验值p0.05，表明白鹭这5群体间的遗传分化显著，群体间缺乏基因交流.对白鹭5个群体的AMOVA分析显示群体内个体间的变异占总变异来源的80.59%，同时群体间的变异则占19.41%( p0.0001)，该结果进一步说明5个群体间的遗传差异显著(表4).表3 白鹭5个不同种群群体之间的Fst值Tab.3 Fst value between 5 different populations of Egretta garzetta群体鸡屿(JY)南白(NB)日屿(RY)信阳(XY)舟山(ZS)鸡屿(JY)南白(NB)0.10155日屿(RY)0.127820.19392信阳(XY)0.331930.247480.28177舟山(ZS)0.048320.192250.160260.36019表4 白鹭种群MHC II DAB I 第2外显子基因遗传差异的分子方差分析(AMOVA)Tab. 4 Analysis of molecular variance of MHC II DAB I exon2 among 5 little egret populations变异来源自由度平方和方差分量变异百分比Fstp群体间4211. 6811. 7321819. 410.19410.0001群体内个体间134963. 7147. 191980. 59总计1381175. 3968. 924081003 讨 论聚合酶链式反应-单链构象多态性技术(PCR-SSCP)能够检测DNA片段上不同位点的多态性或单个碱基的突变、缺失、插入或置换等变化37-39.本文利用PCR-SSCP技术研究白鹭5个地理种群的MHC II DAB I 基因第2外显子的遗传多态性. 结果显示第2外显子拥有数量众多的等位基因，表现为较高多态性特点. 所有等位基因序列均未发现插入或缺失突变，经翻译成氨基酸序列后，也没有发现终止密码子，这间接表明所分离到的等位基因可能来源于表达的基因座，能够行使正常的功能表达.为了探究白鹭MHC II DAB I基因多态性的维持机制，本研究对第2外显子编码抗原结合区肽结合位点(PBR)以及非抗原结合区肽结合位点(Non-PBR)的dN与dS值进行了统计分析. 结果显示，PBR区dN显著大于dS(p0. 01)，提示白鹭MHC II DAB I第2外显子基因曾经历过强烈的正选择作用(表2). 在CODEML程序选择压力检验模型的比较分析结果中，模型M2和M8分别较M1和M7具有更好的拟合度(p 0.001)(表2)，同样也支持上述的正选择作用. 由此我们推测正选择作用在维持白鹭MHC II DAB I 第2外显子基因多态性起着重要作用，该结论与其他鸟类MHC研究报道一致. Alcaide等在研究黄爪隼(Falco naumanni)时发现其MHC II B第2外显子具有较高的遗传多态性，他们从21个黄爪隼个体样品中共分离获得26种等位基因，其选择压力分析结果显示了黄爪隼的MHC II B 第2外显子基因受到了强烈的正选择作用40. Bollmer等在比较分布于加拉帕戈斯群岛的特有种加岛鵟(Buteo galapagoensis)和其近缘广布种斯温氏鵟(Buteo swainsoni)在MHC II B 基因第2外显子的遗传多样性差异时发现，广布种斯温氏鵟相对于特有种加岛鵟在MHC II B 基因上有更高的遗传多样性，他们在20个斯温氏鵟个体上共发现了20种等位基因，而在32个加岛鵟个体上则只有3种等位基因，同时强烈的正选择作用也在斯温氏鵟的MHC II B基因第2外显子基因上被检测到41. Dearborn等基于巢式PCR和高通量测序技术研究白腰叉尾海燕(Oceanodroma leucorhoa)的MHC II B 基因第2外显子，共获得21种等位基因(48个个体)，研究结果揭示白腰叉尾海燕MHC II B 基因第2外显子存在较高遗传多态性的重要原因是由于曾经长期受到病原体侵扰所引起正选择作用的结果19.基于系统树、群体间分化指数(Fst)和AMOVA分析结果显示，5个白鹭种群的MHC II DAB I第2外显子等位基因存在显著的种群分化.有研究表明当动物在不同环境下的微生物和病原体侵扰时，由于受到选择压力的长期作用，会导致与机体免疫反应重要相关的MHC基因会出现不同种群在等位基因数量上出现较大差异42, 43. 因此本研究推测导致白鹭5个种群间的MHC出现种群分化的原因可能是这5个地理种群的白鹭在应对不同生境时受到了不同病原侵扰，诱发了不同的免疫反应，这种等位基因数量的差异反映了不同的环境选择压力的影响.由于MHC多样性、等位基数量受不同生境的病原种类和数量所影响，因此MHC多样性能够反映不同种群对各自环境的适应能力12, 44, 45.今后的研究有必要针对不同种群生境中的寄生虫和微生物种类进行调查，以深入探讨白鹭种群MHC等位基因数量差异的成因.参考文献:1 Klein, J, Satta Y, Ohuigin C, et al. 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KeyLaboratoryof Ministry of Education for Coast and Wetland Ecosystems, College of the Environment & Ecology, Xiamen University, Xiamen , China)Abstract: The DNA fragments of major histocompatibility complex (MHC) class II DAB I exon2 genes from 138 individual