摘要
林麝是我国兽类中具有极高经济价值和社会价值的一种资源动物。雄性林麝的麝香囊腺分泌的麝香在医药和香水行业具有非常重要的作用。近几十年来,随着国际市场对麝香的需求量不断增加以及人为的乱捕乱猎和栖息地破坏,林麝的数量不断下降,已到了邻近灭绝的边缘。人工养殖林麝已有50多年,但圈养林麝的种群因疾病等多种限制因素的困扰而发展缓慢。因此,非常有必要对这一物种展开科学研究,进一步加大保护力度。
主要组织相容性复合体(Major Histocompatibility Complex, MHC)是脊椎动物中多态性最高的一个超基因家族,其编码的细胞膜表面蛋白在识别、呈递外来抗原并引发下游免疫应答反应的过程中发挥着非常重要的作用,是脊椎动物适应性免疫系统的重要组成部分。一个有功能的MHCⅡ类分子是由一个α链和一个β链非共价结合形成的二聚体,其分别由MHCⅡ的A基因和B基因编码。MHCⅡ类A基因和B基因exon2编码的抗原结合区具有非常高的多态性,与物种抵抗病原体的能力及适应复杂的环境直接相关。本研究以四川养麝研究所的115只林麝个体为对象,通过对MHCⅡ类经典的DR和DQ座位的分离、遗传变异分析和化脓性疾病相关性的分析,揭示了林麝MHCⅡ基因多态性的维持机制及其与化脓性疾病的密切关系。主要的研究结果如下:
1.利用通用引物获得Mobe-DR基因部分exon2序列的基础上,通过基因组步移法结合长程PCR法?成功地获取了DR基因exon2序列两侧的内含子序列,继而成功地设计了座位特异性引物,并通过群体调查结果证实了其有效性,最终获得一个DRA座位,一个DRB座位(DRB3)。
2.利用通用引物获得Mobe-DQ基因部分exon2序列的基础上,通过长程PCR法,成功地获取了DQ基因完整的intron2序列。通过对长片段序列的比对分析,依据同源序列设计引物,成功地获取到部分intron1序列。所设DQ座位的特异性引物通过群体调查结果证实了其有效性。最终获得了两个DQA座位(DQA1&DQA2)和两个DQB座位(DQB1&DQB2),
3.本研究一共获得6个MHCⅡ经典座位,利用座位特异性的PCR-SSCP基因分型和测序方法,最终分离到34条等位基因(DRA,4条；DRB3,10条；DQAl,4条；DQA2,7条；DQB1,4条；DQB2,5条)。林麝的6个MHCII经典座位均为多态,与其他濒危的哺乳动物相比较,林麝的适应性免疫系统(MHCⅡ类)保留了较丰富的基因多样性。
4.林麝MHCⅡA基因经研究表明均经历了净化选择,B基因则均经历了正选择,尤其是在B基因的抗原结合位点经历了显著的正选择作用,表明不同的座位经历了不同的选择压力。此外,系统发育分析表明跨物种进化是维持林麝各个座位基因多态性的主要机制之一。
5.通过对化脓性疾病易感个体和抗病个体MHCⅡ各个座位上基因型和等位基因数据的统计学分析,与林麝化脓性疾病相关的等位基因一共有9条,其中4条等位基因(Mobe-DQA1*01, Mobe-DQA1*02, Mobe-DQA2*03和Mobe-DQB2*04)与化脓性疾病的抗性显著相关,有5条等位基因(Mobe-DRB3*07, Mobe-DQA1*03(or Mobe-DQAl*04)和Mobe-DQA2*05(or Mobe-DQA2*06)与化脓性疾病的易感性显著相关。与化脓疾病相关的基因型一共有13个,其中与抗性相关的基因型有6个(DRB3*01/03, DQA1*01/01, DQA1*01/02, DQA2*01/03, DQA2*02/04与DQB2*03/04(P<0.05));与易感相关的基因型有7个(DRB3*07/07, DQA1*03/03, DQA2*01/01, DQA2*05/05, DQA2*05/06, DQB1*01/03与DQB2*01/02(P<0.05)).此外,利用各个座位上等位基因的抗原结合区形成的不同单倍型及其基因型数据,通过分析获得与化脓性疾病相关的超级单倍型一共有6条,单倍型H1(DRA*h1/DRB3*h1/DQA1*h1/DQA2*h2/DQB1*h1/DQB2*h2)与H2(DRA*h1/DRB3*h4/DQA1*h1/DQA2*h2/DQB1*h1/DQB2*h1)与化脓性疾病的抗性相关,单倍型H3(DRA*h1/DRB3*h1/DQA1*h3/DQA2*h3/DQB1*h3/DQB2*h2), H4(DRA*h1/DRB3*h5/DQA1*h3/DQA2*h3/DQB1*h1/DQB2*h1), H5(DRA*h1/DRB3*h7/DQA1*h3/DQA2*M/DQB1*h3/DQB2*h2)与H6(DRA*hl/DRB3*h7/DQA1*h3/DQA2*h3/DQB1*hl/DQB2*hl)则与化脓性疾病的易感性相关。推断Mobe-DRA*02及与其相关的基因型在抗病组具有极高的频率,因Mobe-DRA*02与大部分的超级单倍型连锁,尤其是抗病的超级单倍型连锁所致；同时,Mobe-DRA*03釉Mobe-DRA*04的抗原结合区虽然与Mobe-DRA*02完全一致,二者因与易感的超级单倍型连锁,致使其本身和与之相关的基因型在易感组的频率较高。Mobe-DQB2*02和Mobe-DQB2*03的抗原结合区完全一致,二者亦因分别与易感的和抗病的超级单倍型连锁而在易感组和抗病组中的等位基因频率较高。
易感等位基因、基因型与超级单倍型数目均多于抗病的等位基因和基因型数目,暗示圈养林麝易患化脓性疾病确与群体中这些易感等位基因、基因型与超级单倍型有着密切的关系。建议适时地引入新的种源,防止近交引起的易感等位基因的纯合子和杂合子在群体中的数目继续增多,使种源衰退进一步恶化。
Forest musk deer are one of the most valuable game animals in China. The secretion from the preputial gland of the male deer, the musk, plays an important role in the medicine and the perfume industry. Over the past decades, the great demand in the world market, the extensive hunting and poaching and the lost of the habitats push this animal to the edge of extinction. The captive program has been executed for more than fifty years, but the captive population is still small for many reasons, especially the high susceptibility to many diseases. So, it is very necessary to study this animal and protect this species from extinction.
The major histocompatibility complex (MHC) is the most polymorphic super-gene family in the vertebrates. The glycoproteins on the cell surface encoded by MHC genes play an important role in immune responses through recognizing and presenting the foreign peptides to T cells. Functional MHC class II molecules are heterodimers of alpha and beta chains encoded by corresponding A and B genes respectively. Exon2fragments of A and B genes, coding for the first domain of a functional MHC class II molecule and sites associated with the peptide binding are highly polymorphic, science the peptide binding region is considered to be related to the resistance to the parasites and the adaptability to the complex environments. A total of115individuals sampled from2005to2007in Sichuan Musk Deer Breeding Institution were studied here. After the identification of classical DR and DQ genes, the analysis of genetic variation and the correlation analysis with purulent disease, we found the mechanisms that maintain the MHC class II genes polymorphism and the relationship between MHC class II genes and purulent disease of forest musk deer. The main results are listed below:
1. We obtained part of the exon2sequences using the universal primers, and then we used the genome-walking method and the long range PCR to obtain the flanking regions. After that, we designed the locus specific primers based on the obtained sequences. At last, we isolated one DRA and one DKB (DRB3) loci.
2. Regarding the Mobe-DQ genes, we first used the universal primers to amplify the exon2sequences and then adopted the long range PCR to obtain the entire intron2sequences. The intron1fragments were isolated by the primers that were designed based on the homologous sequences. Finally, we obtained two DQA (DQA1&DQA2) and two DOB (DOB1&DQB2) loci.
3. We obtained six classical MHC Ⅱ loci in this study. Thirty-four alleles were confirmed by the locus specific PCR-SSCP genotyping and sequencing methods (DRA,4; DRB3,10; DQA1,4; DOA2,7; DOB1,4; DQB2,5). The six loci of forest musk deer are all polymorphic and showed high numbers of alleles per locus comparing with the other endangered animals, implying that the captive forest musk deer still contain an abundant of genetic diversity.
4. The different loci of forest musk deer experienced different selection pressures. All MHC Ⅱ A genes experienced purifying selection, while all the B genes experienced positive selection, especially the peptide binding region. The phylogenetic analysis showed the trans-species evolution was an important mechanism to maintain the polymorphism of the forest musk deer MHC Ⅱ genes.
5. The relationship between the forest musk deer MHC class Ⅱ genetic variation and the purulent disease was analyzed by the statistical analysis. We found10alleles.13genotypes and6super haplotypes were related to the susceptibility/resistance to the purulent disease. Four alleles (Mobe-DQA1*01, Mobe-DQA2*03, Mobe-DOA2*04and Mobe-DQB2*03) were found to be resistant to the purulent disease significantly, and six alleles (Mobe-DRB3*07, Mobe-DQA1*03(or Mobe-DQA1*04), Mobe-DOA2*05(or Mobe-DOA2*06) and Mobe-DQB2*02) were found to be susceptible to the purulent disease significantly. Six genotypes were related to the resistance to the purulent disease [DRB3*01/DRB3*03, DOA1*01/DQA1*01, DOA1*01/DOA1*02, DQA2*01/DQA2*03, DQA2*02/DOA2*04and DQB2*03/DQB2*04(P<0.05)], and7genotypes related to the susceptibility to the purulent disease [DRB3*07/DRB3*07, DQA1*03/DOA1*03, DQA2*01/DQA2*01, DQA2*05/DOA2*05, DQA2*05/DQA2*06, DOB1*01/DOB1*03and DQB2*01/DOB2*02(P<0.05)]. The super haplotypes related to the resistance to the purulent disease were H1(DRA*hl/DRB3*hl/DOA1*h1/ DQA2*h2/DQB1*h1/DQB2*h2) and H2(DRA*h1/DRB3*h4/DQA1*h1/DQA2*h2/DQB1*h1/DQB2*hl). The haplotypes related to the susceptibility to the purulent disease were H3(DRA*h1/DRB3*h1/DQA1*h3/DQA2*h3/DQB1*h3/DQB2*h2), H4(DRA*h1/DRB3*h5/DQA1*h3/DQA2*h3/DQB1*h1/DQB2*h1), H5(DRA*h1/DRB3*h7/DQA1*h3/DQA2*h1/DQB1*h3/DQB2*h2) and H6(DRA*h1/DRB3*h7/DQA1*h3/DQA2*h3/DQB1*h1/DQB2*h1). The frequency of DRA*02and related genotypes were relatively high in the resistant group for which was linked to the most resistant super haplotyes. Meanwhile, the frequency of DRA*03(or DRA*04) and related genotypes were relatively high in the susceptible group for which was linked to the most susceptible super haplotyes. The peptide binding sites of Mobe-DQB2*02and Mobe-DQB2*03were completely same, but Mobe-DQB2*02had a higher frequency in the susceptible group and Mobe-DQB2*03had a higher frequency in the resistant group for they were linked to different super haplotypes.
The number of the susceptible alleles, genotypes and super haplotypes exceeded that of the resistant ones, implying a correlation with the high susceptibility to the purulent disease of forest musk deer. So, it is necessary to introduce new individuals to timprove current genetic structure of captive populations and enhance ability to resist pathogen infection.
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