【作者】 李波；

【导师】 马建章；

【作者基本信息】 东北林业大学 ， 野生动植物保护与利用， 2008， 博士

【摘要】 为了梅花鹿(Cervus nippon)的保护管理和养鹿业的可持续发展,本文研究了梅花鹿MHC-DRB基因的遗传特性,分析了它在3个圈养梅花鹿群体中的多态性,探讨了它作为辅助梅花鹿遗传管理的分子标记的可行性。得到以下结果:1.比较了传统的梅花鹿DRB基因方法PCR-SSCP-cloning sequencing与新设计Motif specific-PCR-SSCP-direct sequencing的方法。结果表明传统的方法中出现了28例点突变和7例重组的假阳性等位基因,而新方法能完全消除假阳性序列。2.确定了15个DRB等位基因(Ceni-DRB1～Ceni-DRB15),这些序列中有34.9%核苷酸位点和49.4%氨基酸位点是多态的。由单一个体具有2-5个等位基因推测梅花鹿存在2个以上的DRB基因座。并首次发现梅花鹿与马鹿间共享1个DRB等位基因(Ceni-DRB14同于Ceel-DRB45)。两个物种之间另有2个等位基因(Ceni-DRB5与Ceel-DRB35)只有1个核苷酸(1个氨基酸残基)的差异。3.建立了Allele-specific PCR方法,它可以简便、快速检测梅花鹿个体的DRB基因型。新设计了14个引物(上游引物10个,下游引物4个),引物的有效性检测证实它们的特异性。该方法的建立为梅花鹿的保护遗传学中DRB基因的研究提供了技术支持。4.运用Allele-specific PCR方法,分析了松花湖、西丰和兴凯湖梅花鹿群体中DRB基因的多态性。结果表明3个群体都只含有10个DRB等位基因,其余5个等位基因只在10份肌肉样品中出现。且同一等位基因在不同群体中出现的频率差异显著。在松花湖梅花鹿群体中发现了12种DRB基因单倍型,而西丰和兴凯湖梅花鹿群体中都有14种单倍型。5.梅花鹿DRB基因与产茸性状的相关分析表明,DRB3与二杠茸产量呈显著正相关(R=0.376,P＜0.01),而DRB8和DRB11与二杠茸产量均呈显著负相关(R=-0.403和-0.445,P＜0.01)。在兴凯湖群体中,DRB2与三叉茸产量呈显著负相关(R=-0.27,P＜0.05),具有等位基因世系类型1个体的产茸量显著大于杂合型个体(平均产量高14%,P＜0.05)。而在松花湖和西丰梅花鹿群体中没有等位基因与三叉茸产量间存在相关性,等位基因世系类型问也不存在显著的差异。另外,3个鹿群组合后发现具有111单倍型个体的产茸量显著低于11111单倍型个体(平均产量低15%,P＜0.05)。依据上述结果的分析,得出如下结论:1.新建立的Motif specific-PCR-SSCP-direct sequencing方法可以对梅花鹿DRB基因进行精确分型,该方法检测到15个梅花鹿DRB等位基因,其中13个是新的等位基因。2.新建立的Allele-specific PCR方法可以对梅花鹿DRB基因进行简便而快速的分型。3.首次揭示梅花鹿有2个以上DRB基因座,首次发现梅花鹿与马鹿间共享1个DRB等位基因。4.梅花鹿圈养群体维持了较高的DRB基因的多态性,表明依据鹿茸表型的人工选择不能大幅度降低该基因的多态性。5.DRB基因对梅花鹿的二杠茸重量有明显的影响,但对三叉茸重量则不明显。6.MHC基因在免疫系统和鹿茸生长中均起作用,提示DRB基因可以作为圈养梅花鹿种群遗传管理潜在的分子标记。更多还原

【Abstract】 To conserve sika deer and develop deer breeding continuablely,here investigation of genetic characters of MHC-DRB genes was operated in sika deer.Their polymorphisms were typed in three captive populations,and the feasibility of being genetic marker was discussed for assistant genetic management.The results were revealed as follows:1.A new molecular typing of DRB genes was established in sika deer,i.e.motif specific-PCR-SSCP-direct sequencing.It was compared with PCR-SSCP-cloning sequencing a conventional method.The results indicated artificial alleles from 28 point mutations and 7 recombinations were appeared in the conventional method,and the new method eliminated artificial sequences completely.2.Using this method,15 alleles of DRB genes(named Ceni-DRB1～Ceni-DRB15) were identified in sika deer.34.9%nucleotide sites and 49.4%amino acid sites were variation among these sequences.Because 2-5 alleles were detected for each individual,we can presume the existence of two or more DRB loci in sika deer.Sharing one DRB allele between sika deer and wapiti was discoved firstly,i.e.Ceni-DRB14 is same as Ceel-DRB45.Moreover,two alleles(Ceni-DRB5 and Ceel-DRB35) were differed only one nucleotide or one amino acid between this two species.3.Allele-specific PCR method was found to type DRB genes,which could examine DRB genotype briefly and fleetly in sika deer.14 primers were designed,including 10 forward primers and 4 reverse primers,which were validated by experiment.This method helped to study DRB genes in sika deer conservation genetics.4.Using allele-specific PCR,DRB genes diversities were scaned in Song Huahu,Xi Feng and Xing Kaihu three sika deer populations.The results revealed that only 10 alleles were detected in three populations,and the other 5 alleles were appeared in 10 muscle samples.The frequency of same allele was difference significantly among variational population.12 DRB haplotypes were disclosed in Song Huahu population,and 14 DRB haplotypes were defined in Xi Feng and Xing Kaihu population,respectively.5.Association analysis between DRB genes and antler productivity indicated that DRB3 allele related positively with two bars antler productivity(R=0.376,P＜0.01),but DRB8 and DRB11 two alleles were negative correlation with two bars antler productivity(R=-0.403 and -0.445, respectively,P＜0.01).In Xing Kaihu population,DRB2 allele was also negative correlation with three forks antler productivity(R=-0.27,P＜0.05).Antler productivity of deer with type 1 DRB allelic lineage was 14%greater than heterozygosity(P＜0.05).However,no allele was related with three forks antler productivity in Song Huahu and Xi Feng populations, and there was not difference significantly among DRB allelic lineage in these two populations. Antler productivity of deer with haplotype 111 was 15%lower than haplotype 11111 in three populations(P＜0.05).According to above results,the conclusions were suggested as follows:1.New method i.e.motif specific-PCR-SSCP-direct sequencing could type DRB genes of sika deer exactly.Using this method,15 alleles of DRB genes were identified in captive sika deer population,among which 13 alleles were found firstly.2.Allele-specific PCR method could examine sika deer DRB genotype briefly and fleetly.3.Two or more DRB loci were approved in sika deer,and sharing one DRB allele between sika deer and wapiti was discoved firstly.4.High level polymorphism of DRB genes was maintained in captive sika deer population. It was indicated that artificial selection based on antler could not reduce polymorphism of DRB genes greatly.5.DRB genes worked on productivity of two bars antler obviously,no on productivity of three forks antler.6.MHC genes made important role in immune system and development of antler,which illuminated it could be potential molecular marker for genetic management of captive sika deer population.更多还原