生物多样性 ›› 2012, Vol. 20 ›› Issue (2): 184-192. DOI: 10.3724/SP.J.1003.2012.09211
收稿日期:
2011-11-22
接受日期:
2012-02-28
出版日期:
2012-03-20
发布日期:
2012-04-09
通讯作者:
吴海龙
作者简介:
* E-mail: whlong@mail.ahnu.edu.cn基金资助:
Fang Li, Yilin Shu, Hailong Wu*()
Received:
2011-11-22
Accepted:
2012-02-28
Online:
2012-03-20
Published:
2012-04-09
Contact:
Hailong Wu
摘要:
两栖类正经历全球范围内的种群衰退, 很多两栖动物集群灭绝事件与环境病原体(如壶菌(Batrachochytrium dendrobatidis)的侵扰有关。MHC基因的表达产物在有颌脊椎动物免疫应答过程中起关键作用, 其多态性通常与动物对疾病的抗性或易感性密切相关, 因而被认为是研究动物适应性进化的最佳候选基因之一。本文对中国特有的无尾两栖动物凹耳蛙(Odorrana tormota)MHC II类B基因多态性进行初步研究。首先, 利用1对通用引物扩增出凹耳蛙MHC II类B基因exon2长约180 bp的DNA片段。在此基础上, 利用ligation-mediated PCR进一步获取侧翼未知序列, 序列拼接后长2,030 bp, 包含exon2以及intron1和intron2的部分序列。基于上述序列设计出凹耳蛙B基因exon2特异性引物(IIQ1BU/IIQ1BD), 对该物种黄山种群32个样品进行PCR扩增和克隆测序, 共获得34个不同的等位基因, 等位基因序列核苷酸和氨基酸变异位点的比例分别为16.17%(33/204)和26.87%(18/67), 大多数氨基酸变异位点位于推测的抗原结合位点(antigen binding sites, ABS)。每个样品包含2-5个等位基因, 结合等位基因序列特征以及cDNA表达分析结果, 推测凹耳蛙至少拥有3个可表达的B基因座位。与文献报道的蛙科其他物种比较后发现, 尽管凹耳蛙目前的分布区非常狭窄, 但其MHC II类 B基因多态性明显高于蛙科其他动物。等位基因碱基替换模式提示凹耳蛙MHC II类B基因曾经历过强烈的正选择作用, ABS区的dN值显著大于dS (P<0.05), PAML软件包CODEML程序中不同模型的似然比检测(likelihood rate test)结果同样支持上述推论, 贝叶斯经验贝叶斯路径(Bayesian Empirical Bayes)共检测出5个显著受正选择作用的氨基酸位点。贝叶斯系统树的拓扑结构显示, 无尾两栖类不同科的等位基因分别形成单系群, 但蛙科不同属的等位基因未能形成单系群, 蛙属绿池蛙(Rana clamitans)的1个等位基因与臭蛙属凹耳蛙的部分等位基因享有共同的谱系关系, 提示蛙科不同属间的B基因存在跨种多态性。
李方, 疏义林, 吴海龙 (2012) 凹耳蛙MHC II类B基因第二外显子多态性分析. 生物多样性, 20, 184-192. DOI: 10.3724/SP.J.1003.2012.09211.
Fang Li, Yilin Shu, Hailong Wu (2012) Polymorphism of exon 2 of MHC Class II B gene in the Chinese concave- eared torrent frog (Odorrana tormota). Biodiversity Science, 20, 184-192. DOI: 10.3724/SP.J.1003.2012.09211.
引物 Primer | 序列 Sequence (5′-3′) |
---|---|
X1 | CGTTGGATATACGATCTGGAGG |
X2 | GTACTTCGACAGTGATAAGGGATT |
S1 | GGGTTTCTGTTCCAGGAGTCAG |
S2 | AGCACTTGGTCTCCCCAGCTCGGT |
IIQ1BU | GTACGGAGGATATCAGGTTTC |
IIQ1BD | CAGGTGCAGTATATTCCTTCAT |
表1 本文用于分析凹耳蛙MHC II类B基因变异的引物序列
Table 1 Sequences of the primers used in the study of MHC II B genes variation in the Chinese concave-eared torrent frog
引物 Primer | 序列 Sequence (5′-3′) |
---|---|
X1 | CGTTGGATATACGATCTGGAGG |
X2 | GTACTTCGACAGTGATAAGGGATT |
S1 | GGGTTTCTGTTCCAGGAGTCAG |
S2 | AGCACTTGGTCTCCCCAGCTCGGT |
IIQ1BU | GTACGGAGGATATCAGGTTTC |
IIQ1BD | CAGGTGCAGTATATTCCTTCAT |
图1 凹耳蛙MHC II类等位基因的氨基酸序列对位排列。* 根据Brown等(1993)的方法界定的抗原结合位点(ABS), # 参照Tong等(2006)的方法界定的ABS位点。
Fig. 1 Amino acid alignment of MHC II alleles of the Chinese concave-eared torrent frog. Sites involved in putative antigen binding sites are indicated by * (according to Brown et al., 1993) or by # (according to Tong et al., 2006), respectively.
位点 Sites | 同义替换率dN | 非同义替换率dS | dN/dS | Z检验P值 P(Z-test) |
---|---|---|---|---|
All | 0.047(0.014) | 0.050(0.018) | 0.710 | 0.909 |
ABS ( | 0.116(0.037) | 0.046(0.029) | 1.081 | 0.028 |
Non-ABS ( | 0.020(0.011) | 0.051(0.025) | 0.510 | 0.075 |
ABS ( | 0.167(0.056) | 0.081(0.066) | 1.480 | 0.032 |
Non-ABS ( | 0.025(0.011) | 0.044(0.018) | 0.491 | 0.176 |
表2 凹耳蛙MHC II类B基因exon2序列、ABS(putative antigen binding sites)区、非ABS区平均非同义替换率(rates of nonsynonymous substitutions per nonsynonymous site, dN)、同义替换率(rates of synonymous substitutions per synonymous site, dS)以及dN/dS比值。
Table 2 The average rates of nonsynonymous substitutions per nonsynonymous site (dN) and synonymous substitutions per synonymous site (dS) as well as the resulting ratio dN/dS for the sequenced section, the ABS (putative antigen binding sites) and non-ABS of MHC II B genes exon 2 of the Chinese concave-eared torrent frog.
位点 Sites | 同义替换率dN | 非同义替换率dS | dN/dS | Z检验P值 P(Z-test) |
---|---|---|---|---|
All | 0.047(0.014) | 0.050(0.018) | 0.710 | 0.909 |
ABS ( | 0.116(0.037) | 0.046(0.029) | 1.081 | 0.028 |
Non-ABS ( | 0.020(0.011) | 0.051(0.025) | 0.510 | 0.075 |
ABS ( | 0.167(0.056) | 0.081(0.066) | 1.480 | 0.032 |
Non-ABS ( | 0.025(0.011) | 0.044(0.018) | 0.491 | 0.176 |
模型 Model | 似然值 Likelihood | 估计参数 Estimate parameters | (LRT) 2△L | 受正选择位点 Positively selected sites |
---|---|---|---|---|
M1 | -655.027 | p0= 0.69482 p1=0.30518 ω0=0.02484 ω1=1.00000 | 52.322 (P<0.001) | None |
M2 | -628.866 | p0=0.48425 p1=0.43958 p2=0.07617 ω0=0.00000 ω1=1.00000 ω2=15.77201 | 1T**, 10Y**, 34W**, 43Q*, 59Y** | |
M7 | -655.106 | p= 0.01273 q= 0.02500 | 52.454 (P<0.001) | None |
M8 | -628.879 | p0=0.92380 p=0.00503 q=0.00516 (p1= 0.07620) ω= 16.20861 | 1T**, 10Y**, 33S, 34W**, 43Q*, 59Y** |
表3 不同密码子进化模型对凹耳蛙MHC II类 B基因外显子2吻合度检验的参数与似然值
Table 3 Summary of parameter estimates and likelihood values of different models of codon evolution for exon 2 of MHC II B gene in the Chinese concave-eared torrent frog
模型 Model | 似然值 Likelihood | 估计参数 Estimate parameters | (LRT) 2△L | 受正选择位点 Positively selected sites |
---|---|---|---|---|
M1 | -655.027 | p0= 0.69482 p1=0.30518 ω0=0.02484 ω1=1.00000 | 52.322 (P<0.001) | None |
M2 | -628.866 | p0=0.48425 p1=0.43958 p2=0.07617 ω0=0.00000 ω1=1.00000 ω2=15.77201 | 1T**, 10Y**, 34W**, 43Q*, 59Y** | |
M7 | -655.106 | p= 0.01273 q= 0.02500 | 52.454 (P<0.001) | None |
M8 | -628.879 | p0=0.92380 p=0.00503 q=0.00516 (p1= 0.07620) ω= 16.20861 | 1T**, 10Y**, 33S, 34W**, 43Q*, 59Y** |
图2 利用贝叶斯方法基于MHC II类B基因外显子2核苷酸序列构建部分无尾两栖类的系统树, 以有尾类的虎纹顿口螈和墨西哥蝾螈的同源序列作为外类群。仅大于0.95的后验概率标在系统树的分支上。
Fig. 2 Phylogenetic relationship of anuran MHC class II B exon 2 nucleotide sequences constructed with Bayesian inference with two outgroup sequences from the caudate amphibian, Ambystoma tigrinum and Ambystoma mexicanum. Posterior probabilities above 0.95 are indicated on branches.
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