【作者】 张玮；

【导师】 徐怀亮；

【作者基本信息】 四川农业大学 ， 动物遗传育种与繁殖， 2009， 硕士

【摘要】 本实验采用PCR扩增、DGGE分离基因和基因克隆等分子生物学方法对猕猴DRB等位基因多样性测定与分析,测得四川5个猕猴群体DRB基因2号外显子部分序列。利用测得的基因序列,列出76个猕猴实验样本DRB基因种类,并对新等位基因命名。分析所有等位基因氨基酸序列,在NCBI上下载其它灵长类DRB基因2号外显子序列,采用N-J法构建DRB基因聚类树。并分析5个群体内基因平均遗传距离,群体间遗传平均距离分析。各项分析主要结果如下:1)利用PCR、DGGE和基因克隆测序技术得到69个DRB等位基因,其中57个为本实验测定出的新等位基因。5个群体共检测出DRB1、3、4、5和W座位基因和21个亚座位,DRB1座位检测出8个亚座位,DRB4座位和DRB5座位都只检测出一个亚座位。各亚座位具有群体特殊性:DRB~*W25和DRB~*W37亚座位为小金群体独有;DRB~*W31和DRB~*W40亚座位为黑水群体独有;其它亚座位都为两个或多个群体共有,DRB~*W53亚座位为本实验首次报道的一个新的DRB~*W亚座位2)利用MEGA4.0软件将获得的基因序列翻译成氨基酸序列后,所有氨基酸序列对比分析得出在不同座位和亚座位间氨基酸差异体现在二级结构β区域,同一亚座位中等位基因氨基酸序列差异主要体现在α区域。通过与人HLA-DRB基因2号外显子相应位点基因氨基酸序列对比,实验猕猴DRB基因与相应的人HLA-DRB基因在β区域差异不大,差异主要体现在α区域,证明猕猴与人这两物种间DRB基因氨基酸差异规律一样。3)聚类树分析得出DRB*W07、DRB1*07、DRB*W02、DRB*W31三个亚座位在其它灵长类中无对应亚座位,可能属于猕猴特有亚座位,这些猕猴独有亚座位在人HLA-DRB相关免疫医学研究中,只能采用猕猴作为研究模型。DRB1*03、DRB1*10、DRB*W01亚位点和人HLA-DRB1*13亚座位聚成一个大支,DRB1*04和HLA-DRB1*04聚成一支,DRB3*04和HLA-DRB*02、01聚成一支。这几个亚座位与人HLA-DRB相应亚座位相近,可以作为人类DRB基因免疫医学用。4)各群体内平均遗传距离采用Nei-Gojibori-p和Kimura-2-parameters两种方法得出各群体内平均遗传距离。Kimura一2-parameters计算出各群体内遗传距离由大到小顺序为:汉源群体＞巴中群体＞九龙群体＞小金群体＞黑水群体。表明由总的核苷酸变异造成的平均遗传距离汉源最大,黑水最小,即汉源群体DRB等位基因多样性最丰富,黑水群体基因多样性最弱。Nei-Gojibori-p法计算出各群体平均遗传距离由大到小为:小金群体＞汉源群体＞九龙群体＞黑水群体＞巴中群体,表明由氨基酸差异造成的平均遗传距离小金最大,巴中最小。由组内总的核苷酸差异引起的遗传距离黑水群体最小,汉源群体最大,但是由氨基酸差异引起的组内平均遗传距离,小金群体最大,巴中群体最小。利用软件MEGA4.0软件计算出5个猕猴群体间遗传距离表明各群体间遗传距离实际地理位置不符合。各群体DRB等位基因均受到正向选择压力,九龙群体和巴中群体受到选择压力分别为最大和最小。更多还原

【Abstract】 76 indivalduals from 5 Sichuan Macaca mulatta wild groups have been selected to uesed for DRB gene exon 2 partial sequences diversity analysis by PCR amplitication, DGGE seperation and gene colone.In all 76 Macaca mulatta samples,the gene typles was listed for each sample,meanwhile,new allelic gene was named.After all amimo acid sequences was analysised,applied N-J methouds to constract DRB gene phylogenetic trees that contant the other specis primates genes which were downloaded from NCBI gene bank. Finally,in 5 groups,gene evolution pressurs,inter-group gene average genetic distance, between-group genetic distance,each one was anlysised.The results for each analysis were listed:1) 69 alleles has been detected by PCR amplification,DGGE and gene colone.57 of the 69 alleles are newly.In 5 populations,DRB1,3,4,5 and W,the 5 local alleles has been detected,meanwhile,21 sub-locus have been observed.Each sub-locus show the populational perticularies:Sub-locus DRB*W25 and DRB*W37 the two sub-locus only were detected in population Xiaojin;Sub-locus DRB*W31 and DRB*W40 have been only detected in population Heishui.The other sub-loci,have been observed in more than 2 populations simultaneously.2) After all gene sequences be translated into amimo acid ensued with sequence-blast by solftware MEGA4.0,the difference between locus amimo acids focus on the secondary constractureβregion,althrough,in the same locus,the distinction between two alleles focus onαregion.Compared with human equivalents the difference amimo acids comcentrate onαregions,aboutβregion the difference showed lower thanα.3) Gene phylogenetic tree by solftware MEGA4.0 was used by method N-J.From tree, consenqued that DRB*W07、DRB1*07、DRB*W02、DRB*W31 the 3 sub-locus did not detected in other primers except Macaca mulatta,may they belongs to Macaca mulatta perticulary,other loci can be finded in other primates yet.Locus DRB1*03、DRB1*10、DRB*W01 clusterd with human HLA-DRB1*13,DRB1*04 clusterd with human HLA-DRB1*04,DRB3*04 clusterd with human HLA-DRB*02、01,all the loci adhered with human counterparter may could be used for human immun-medicology.4) The average genetic distance in each group was caculated by solfteware MEGA4.0 with methods Nei-Gojibori-p and Kimular-2-parameters respectly.Results by method Kimular-2-parameters,the genetic distance in each group were arranged from high to low: group Hanyuan＞group Bazhong＞group Jiulong＞group Xiaojin＞group Heishui.This category document that,the average genetic distance influenced by uncletide acid disparity were the lowsted in group HeiShui and the hightest in group HanYuan.But results by method Nei-Gojibori-p,the genetic distance in each group were arranged from high to low were dismarch with results by method Kimura-2-parameter:group Xiaojin＞group Hanyuan＞group Jiulong＞group Heishui＞group Bazhong.This categry recover different with results by method Kimura-2-paramete,by method Nei-Gojibori-p,the average genetic distance influenced by amimo acid disparity were the lowest in group Bazhong and the highest in group Xiaojin.All the above conclude that:genetic distance influenced by nucletide acid disparity in inner-group,group Heishui was the lowest and group Hanyuan were the highest,however,by amimo acid disparity,group Xiaojin and Bazhong show the hightest and lowest respectily.The average genetic distance in 5 groups were caculated by sorlftware MEGA4.0 resoults show that the genetic distance between two groups dis-accordant with the real geographic setting.In DRB alleles evolution,although the dynamic in all populations were positive selections pressure,the pressure in each population show the differences that in population Xiaojin the pressure showed the hightest, but in Bazhong the pressure showed the lowest.更多还原