【作者】 陈艺燕；

【导师】 方盛国； 万秋红；

【作者基本信息】 浙江大学 ， 动物学， 2009， 博士

【摘要】 主要组织相容性复合体(Major Histocompatibility Complex,MHC)是脊椎动物所特有的多基因家族,能编码种类丰富的细胞表面糖蛋白,具有呈递抗原、启动免疫应答的功能,是动物适应性免疫系统的重要组成部分。哺乳动物Ⅱ类MHC包括DRA、DRB、DQA和DQB等多个经典基因,编码呈递细菌类外源性抗原分子;Ⅱ类MHC基因的exon2编码抗原结合部位,具有高度的多态性,与物种抵抗病原体的能力及对复杂环境的适应性直接相关。本研究以大熊猫指名亚种的“卧龙种群”、“成都种群”和秦岭亚种的“佛坪-楼观台种群”共121只个体为对象,通过对功能性Ⅱ类MHC经典座位的分离和遗传变异分析,揭示了大熊猫Ⅱ类MHC基因多样性的维持机制及其与免疫相关的适应性进化特性。主要的研究结果如下:1)首创了磁珠富集杂交cDNA分离MHC基因的方法。该方法简单、快速、高效。本文运用这一方法分离获得了7个Ⅱ类MHC基因,共10条全长cDNA。该结果不仅重复获得了文献所报道的“大熊猫Ⅱ类MHC基因组计划”已确认的6个表达的经典座位,还新发现和克隆了一个新的座位DRB3,从而表明了所建方法的有效性。2)本文分离获得的10条大熊猫Ⅱ类MHC基因的全长cDNA(DRA 1条,DRB1 2条,DRB3 1条,DQA1 2条,DQA2 1条,DQB1 2条,DQB2 1条)均可以编码完整的蛋白质序列。因此,目前已发现的大熊猫具有功能的经典Ⅱ类MHC基因,包括DRA、DRB1、DRB3、DQA1、DQA2、DQB1和DQB2等7个。3)在以大熊猫“卧龙种群”、“成都种群”和“佛坪-楼观台种群”的121只个体为对象,并利用座位特异性的PCR-SSCP基因分型和测序方法进行6个Ⅱ类MHC功能基因的遗传变异检测中,共克隆了25条等位基因。其结果,发现DRA和DQB2均为单态,而其余4个座位则是中度多态的(6条DRB3等位基因、7条DQA1等位基因、4条DQA2等位基因、6条DQB1等位基因)。与其他濒危的哺乳动物相比,大熊猫的适应性免疫系统(Ⅱ类MHC)具有更丰富的基因多样性。4)在大熊猫的人工圈养种群中,“卧龙种群”和“成都种群”具有相同的等位基因数目,但其分布频率差别明显。进一步的分析发现,“卧龙种群”存在着明显的奠基者效应,其种群中的优势等位基因被主要奠基者携带的等位基因所统治。鉴此,基于维持圈养大熊猫的遗传多样性及提高大熊猫的疾病抵抗能力的必要性,本文建议应加强该种群携带稀有等位基因之大熊猫个体的繁殖成功率。5)大熊猫指名亚种和秦岭亚种在多态的Ⅱ类MHC基因中,具有明显的等位基因丰度差异。其中,指名亚种在DQ区比秦岭亚种具有更多的等位基因(16:10),但秦岭亚种在DR区的等位基因数又多于指名亚种(11:9)。与此同时,在DQ区和DR区,指名亚种和秦岭亚种分别具有9条和5条各自所特有的等位基因。上述结果,充分表明了大熊猫指名亚种和秦岭亚种在其亚种分化之后,各自经历了独立的进化历史,并进化产生了适应不同环境的亚种特异性Ⅱ类MHC基因。6)在大熊猫Ⅱ类MHC基因中,存在着多个方面的平衡选择证据:a.基因的观察杂合度明显高于期望杂合度;b.各基因抗原结合位点的氨基酸杂合度显著地高于非抗原结合位点(P＜0.01);c.抗原结合位点的选择参数ω值(d_N/d_S,即非同义置换率与同义置换率之比)显著地高于非抗原结合位点ω值(P＜0.01);d.5个多态性的座位共存在44个受正选择压力的氨基酸位点,其中95.45%位于抗原结合位点及其附近。这些平衡选择证据,充分表明了Ⅱ类MHC基因在大熊猫的适应性进化过程中,发挥了重要作用。7)大熊猫指名亚种在DRB1和DQA2座位上的选择参数ω值均小于1,属于净化选择模式,表明了净化选择在指名亚种的DRB1和DQA2基因中,是处于统治地位的。相反地,秦岭亚种的选择参数ω值在7个表达的Ⅱ类MHC基因中均大于1,属于正选择模式,表明了正选择广泛地存在于秦岭亚种的Ⅱ类MHC基因中。该结果,明晰地揭示了大熊猫指名亚种和秦岭亚种在Ⅱ类MHC基因的进化机制上,存在着驱动因素的差异。8)无论是大熊猫的指名亚种还是秦岭亚种,在新发现的DRB3座位上,都具有极大的ω值(抗原结合位点的ω值均大于9.27;非抗原结合位点的ω值均大于4.39),表明了DRB3新座位的抗原呈递结构域在其漫长的进化过程中,经历了有别于其它哺乳动物之极强的正选择作用。此外,两亚种的DRA和DQB2基因均为单态,表明了大熊猫的DRA和DQB2基因在其进化过程中,历经的进化选择模式,为极强的净化选择作用。而两亚种在DRB1、DQA1和DQB1三个座位中,出现了基因内的重组现象,表明了重组事件是大熊猫Ⅱ类MHC等位基因多样性的产生机制之一。综上所述,大熊猫Ⅱ类MHC基因的多样性,是由自然选择和基因内重组等动力因素共同驱动来维持的,也就是说“平衡选择、正选择、净化选择和基因内重组,是大熊猫在适应性进化过程中维持其Ⅱ类MHC基因组多态性的主要进化动力”。更多还原

【Abstract】 The major histocompatibility complex(MHC) plays a central role in the adaptive immune system of vertebrates for its function of presenting pathogen peptides to T cells. The exon2 of MHC classⅡclasscical DR and DQ genes encodes antigen binding sites and displays extreme polymorphism,which is believed to be essential for vertebrates to resist pathogen infection.Thus this study aims to:1) isolate all functional classical MHC classⅡloci for the giant panda;2) investigate variations of antigen-binding exon2 of all functional classical MHC classⅡgenes in the giant panda;2) exploring the evolutionary history of immunity-related genes in two subspecies of the giant panda;and 3) reveal the maintaining mechanism of MHC diversity in the giant panda.The results are as follows:1) This study developed a novel magnetic bead hybridization method to isolate cDNA sequences of MHC genes.In the giant panda,a total of 10 full-length MHC cDNA sequences were isolated,nine of which were assigned to 6 known classical MHC classⅡgenes identified from the giant panda MHC genome project.Thus,the othe one DRB cDNA sequence represents a novel locus,which was designated DRB3. The results demonstrated the novel magnetic bead hybridization method is efficient and reliable and thus could be applied to other mammals.2) The complete coding regions in 9 out of the 10 full-length Aime-MHC cDNA sequences revealed no frame-shift mutations,stop codons or indel,which were in good agreement with functional genes.However,the DQA2 was truncated in both the leader peptide and the cytoplasmic tail as compared to Aime-DQA1 and homologs in other mammals,which implies loss of function or an evolving pseudogene.3) Genetic variations and evolutionary patterns of all 7 functional MHC classⅡgenes (1 DRA,2 DRB,2 DQA and 2 DQB) were investigated for 121 giant pandas,using locus-specific single-strand conformation polymorphism genotyping and sequencing techniques.The results revealed the presence of 2 monomorphic loci(DRA and DQB2) and 5 polymorphic loci with different numbers of alleles(7 at DRB1,6 at DRB3,7 at DQA1,4 at DQA2,6 at DQB1).Compared to other endangered animals and species experiencing severe bottlenecks,the giant panda exhibited ample variations in the adaptive immune systems.4) Despite sharing the identical alleles across all MHC loci,the Wolong and the Chengdu captive populations of the Sichuan(i.e.nominal) subspecies exhibited large differences in the distribution of allelic frequencies,reflecting respective founder effects.The main founder’s alleles were distributed widely in the captive population, while rarer alleles not harbored in the founder may have drifted out.For sustainable development of the giant panda,it will be necessary to increase the mating success of giant pandas carrying rare alleles,in order to enhance pathogen resistance in captive populations.5) The two giant panda subspecies,the Sichuan(i.e.nominal) and the Qinling subspecies,exhibited substantial differences in allele distribution and allele richness. The former has more alleles in DQ genes than the latter(16:10),and by contrast,the latter has more alleles in DR genes than the former(11:9).In total,the Sichuan shaped 9 specific alleles,and the Qinling subspecies 5.These differences in allelic distribution and the presence of subspecies-specific alleles implies that the two giant panda subspecies have experienced different evolutionary histories since their division,and have evolved their own functional MHC molecules to resist different habitat-specific pathogens.6) Balancing selection in the giant panda was supported by the following pieces of evidence:(a) the observed heterozygosity was higher than expected.(b) Amino acid heterozygosity was significantly higher at antigen binding sites(ABS) compared to non-ABS sequences(P＜0.01).(c) The selection parameterω(d_N/d_S) was significantly higher at ABS compared to non-ABS sequences(P＜0.01).(d) Approximately 95.45%of the positively selected codons(P＞0.95) were located at or adjacent to an ABS.7) The Sichuan subspecies displayed lowωvalues at DRB1(ω＜0.72) and DQA2(ω＜0.48),suggesting that these sites underwent strong purifying selection.On the contrary,the Qinling subspecies exhibited highωvalues across each locus(all＞1), supporting its extensive positive selection.This contrasting pattern revealed that the Sichuan and the Qinling subspecies developed different evolution-driven mechanisms in the diversity patterns of MHC.8) In both Sichan and Qinling subspecies,the entire exon2 of the DRB3 gene underwent intense positive selection at both ABS(ω＞9.27) and non-ABS(ω＞3.49).For the DRA and DQB2,both subspecies exhibited monomorphism,showing strong purifying selection.On the other hand,intragenic recombination was detected in DRB1,DQA1 and DQB1,providing evidence of recombination serving as one important diversification mechanism.In sum,our present results suggest that balancing,positive and purifying selection accompanied by recombination drive the contrasting diversity patterns of the MHC classⅡgenes between 2 giant panda subspecies.更多还原