【作者】 李洪涛；

【导师】 顾为望；

【作者基本信息】 南方医科大学 ， 人体解剖学与组织胚胎学， 2008， 博士

【摘要】 研究背景藏猪(Tibet hog,Sus scrofa)是世界上体型较小的小型猪之一,原产于青藏高原、海拔2500～4300m的农区和半农半牧区,分布于中国的西藏自治区、云南省、四川省和甘肃省等地。藏猪保存了较为纯正的品种资源,是唯一能够适应高海拔气候和以放牧为主的猪种。由于长期的地理隔离和环境的差异,不同地区的藏猪出现了一定程度的分化,如四川藏猪与云南藏猪相比,四川藏猪偏离Hardy-Weiberg定律的基因座数多于云南藏猪;四川藏猪体型较小,而云南减猪体型较大。目前虽然对四川藏猪或云南藏猪开展了一些研究,但对于来自西藏自治区的藏猪研究报导较少。2004年南方医科大学从西藏自治区工布江达县将50头藏猪(雌、雄各半)引种到广州,首次开展对藏猪进行实验动物化的培育,并将其命名为西藏小型猪(Tibet mini-pig)。2006底获得广东省实验用西藏小型猪生产质量合格证,目前存栏数已达600余头。目的测定西藏小型猪的mtDNA控制区序列和Cyt b基因序列,研究西藏小型猪mtDNA控制区和Cyt b基因序列的遗传分化,及其对血液生理生化指标的影响,并和其他猪的序列比对分析,研究其亲缘关系;探讨西藏小型猪氟烷基因座位的群体结构特征,检测氟烷基因型隐性纯合子个体,寻找西藏小型猪独特的分子遗传标记,从理论上指导西藏小型猪实验动物化的培育。方法1随机抽取120头西藏小型猪血液,利用试剂盒提取血液基因组DNA;设计引物扩增西藏小型猪以及7头巴马小型猪、23头贵州香猪和17头五指山猪的mtDNA控制区,测序并结合Clustalw软件和MEGA3.0软件进行所测序列多重比对,确定变异位点、单倍型,并建立西藏小型猪和国内其他猪的亲缘关系树。对其中58头8月龄的西藏小型猪分别测定血液生理生化指标。血液生理指标14项:白细胞、红细胞、血红蛋白、血小板、淋巴细胞、单核细胞、粒细胞、嗜酸性粒细胞、嗜碱性粒细胞、血细胞比容、平均红细胞体积、平均红细胞血红蛋白含量、平均红细胞血红蛋白浓度、红细胞分布宽度。血液生化指标11项:谷丙转氨酶、谷草转氨酶、谷丙转氨酶与谷草转氨酶比值、总蛋白、碱性磷酸酶、葡萄糖、尿素氮、肌酐、总胆固醇、甘油三酯、白蛋白。分类后进行指标的比较。统计学分析采用SPSS13.0版统计学软件进行两个样本的t检验,所有数据以均数±标准差((?)±SD)表示。2以代表mtDNA控制区5′端序列单倍型的西藏小型猪和部分巴马小型猪、贵州香猪、五指山猪的全基因组DNA为研究对象,设计引物扩增四种小型猪的Cyt b基因序列,测序后利用MEGA3.0软件进行碱基序列和氨基酸序列比对,建立亲缘关系树,分析西藏小型猪的进化地位。3应用PCR-RFLP技术,研究西藏小型猪的氟烷基因多态性。以杜洛克猪和长白猪基因组样本为对照,用特异性引物扩增3个猪种的氟烷基因片段,然后进行限制性内切酶(HhaⅠ)酶切以及酶切结果的鉴定,通过基因型辨读,获得群体的基因型频率特征数据,确定隐性纯合子基因型个体。对酶切电泳结果进行辨读时,HAL~NHAL~N基因型表现出493bp和166bp两条电泳条带,HAL~NHAL~n基因型表现出659bp、493bp和166bp三条电泳条带,HAL~nHAL~n基因型只出现659bp一条电泳条带。结果1前人的研究表明,猪的mtDNA控制区的串联重复区存在长度异质性(15—29个重复片段),重复片段全部是:GTACACGTGC,称之为完全重复。但是本研究显示,西藏小型猪不仅有完全重复(A型),而且有不完全的重复(B型),即一部分西藏小型猪10bp的重复片段不仅有GTACACGTGC,而且还有GTACACATGC和GTACACGTAC这两个片段及其交替排列现象。系谱分析显示,这种排列类型同样按母系遗传方式由母本传给后代,与父本没有关系。西藏小型猪mtDNA控制区3′端侧翼区为340bp,变异位点少,与国内其他家猪的序列一样比较保守;5′端侧翼区704bp,有20个变异位点,由此归纳出26个单倍型。西藏小型猪5′端侧翼区三个转换位点(305,500,691)的变化几乎与串联重复序列所分的A、B两组类型相对应:B型中三个变异位点的碱基分别为t,a,a(100%);A型中三个变异位点的碱基分别是c,g,g的占87%,其他13%。与西藏小型猪相比,巴马小型猪、贵州香猪和五指山猪mtDNA控制区5′端变异位点较少,分别只有4、4、3种单倍型,串联重复区也只有一种类型,A型。对西藏小型猪A型和B型群体与血液生理生化特性的进行相关分析,结果表明A型和B型群体血液红细胞数量有显著性差异(A型＜B型,P＜0.05)。2利用控制区5′端侧翼区序列归纳出的单倍型建立亲缘关系树的分析表明,西藏小型猪与中国地方家猪有较近的亲缘关系,特别与中国西南地区几种家猪的亲缘关系最近。3西藏小型猪与国内家猪的Cyt b基因序列几乎相同;与欧洲猪相比差异较大,共有16个主要变异位点,其中有两个特殊转换位点:420位点T—C转换和883位点的G—A转换,几乎各占一半。利用Mege3.0软件将碱基序列转换为氨基酸序列后的分析表明,西藏小型猪与国内其他猪有一个氨基酸位点(295位点)、与欧洲猪有三个氨基酸位点(89,295,314位点)存在着差异。在295位点,大部分A型西藏小型猪与欧洲猪同为缬氨酸(Ⅴ),而B型和少部分A型为异亮氨酸(Ⅰ)。巴马小型猪、五指山猪和贵州香猪的氨基酸序列在295位点也为异亮氨酸(Ⅰ)。结果进一步证实,西藏小型猪群体内存在分化,mtDNA控制区的序列变异与功能基因的结构变化存在一定相关性。4 120头西藏小型猪个体氟烷基因型全部为HAL~NHAL~N,没有发现应激敏感基因,氟烷基因显性纯合子基因型:HAL~NHAL~N的频率为100%;HAL~NHAL~n型和HAL~nHAL~n型均为0。而在杜洛克猪样本中检测到4份杂合子样本,长白猪样本中检出1份杂合子个体,但都没有发现隐性纯合子个体。西藏小型猪群体内未发现应激敏感性的隐性基因,整个群体抗应激能力强。结论1根据西藏小型猪mtDNA控制区串联重复区的长度和重复片段存在异质性,可将西藏小型猪群体分为A型和B型。A、B类型和5′端的三个碱基转换位点:305,500,691,可以联合组建西藏小型猪的遗传标记。建议通过人工选择,A型B型分为两个群体,淘汰A型中少量变异个体(13%),强化西藏小型猪的标记,培育成两个封闭群体:B型三个转换位点分别为t,a,a;A型封闭群的三个转换位点纯化为c,g,g。2由于西藏小型猪串联重复区的排列类型A型或B型都可以由母本传给后代,而目前所报导的国内家猪全部为A型,没有B型的报导,因此提出A型西藏小型猪和其他中国家猪有共同起源,B型可能是基因突变或者是两种母系起源的结果。3西藏小型猪Cyt b基因的全序列有两个转换位点,与A型和B型西藏小型猪有一定对应关系。利用Mege3.0软件将碱基序列转换为氨基酸序列后的分析表明,西藏小型猪Cyt b第295位氨基酸大部分为缬氨酸(Ⅴ),而少部分为异亮氨酸(Ⅰ)。其他国内家猪的氨基酸序列全部为异亮氨酸(Ⅰ)。结果进一步证实,在西藏小型猪群体内存在分化。4西藏小型猪群体氟烷基因,未发现应激敏感性的隐性基因,说明西藏小型猪的应激敏感性比较低,适合于开展外科手术或器官移植等实验研究。更多还原

【Abstract】 BackgroundTibet hog is one kind of smaller mini-pig in the world,which mainly live in semi-agricultural and semi-pastoral areas(average elevation:2500～4300m) in Qinghai-Tibet plateau located in southwest China.Tibet hogs are distributed mainly in Tibet autonomous region and Yunnan,Sichuan,and Gansu.It is the only pig breed that can adapt to the high altitude climate.Because of long-term geographical isolation and environmental difference,the differentiation of Tibet hog in different regions(such as Sichuan Tibet hog and Yunnan Tibet hog) appear.Sichuan Tibet hog diverses more from the law of Hardy-Weiberg than Yunnan Tibet hog do.Sichuan Tibet hog is smaller than Yunnan Tibet hog.Although some research work has been done on Tibet hog(mostly on Sichuan Tibet hog and Yunnan Tibet hog),little work has been done on Tibet hog from Tibet autonomous region.To develop a laboratory pig strain,50 indigenous Tibet pigs(25 males and 25 females) were moved from Tibet autonomous region to Guangzhou city in 2004,which were raised in the base of Tibet mini-pig breeding in Zengcheng,Guanzhou city.These pigs were bred for the purpose of laboratory animalization and were named as Tibet mini-pig.The number of Tibet mini-pigs are about 600 now.Objectives1.To analyze the sequence of mitochondrial DNA D-loop region and Cyt b gene of Tibet mini-pigs,to investigate genetic differentiation and the impact on partial blood parameters and to explore their cytoplasmic DNA markers used to identify them.To find the phylogenetic relationship between Tibet mini-pigs and several other breeds of Chinese domesticated pigs.2.To investigate the population structure feature of HAL locus,and to detect allozygote individuals of HAL genotype in different Tibet mini-pig,and to guide the breeding of laboratory animal Tibet mini-pigs in abstracto.Methods1.Collecting blood from 120 Tibet mini-pigs randomly and genome DNA was extracted from the blood with Genome DNA Extraction Kit.Primer were designed. The fragment of Mitochondrial DNA D-loop region in 120 Tibet mini-pigs,7 Bama miniature pigs and 23 Guizhou xiang pigs and 17 WZS pigs were amplified and sequenced,followed by being multiple alignment compared with each other using ClustalW software and MEGA3.0 software,then variation sites and haplotype were made.Established genetic relationship tree between Tibet mini-pig and other China domestic pigs with CLUSTALW software.Additionally,blood physiological and biochemical parameters of 58 Tibet mini-pigs(8 months old) were measured which included WBC,RBC,HGB,PLT,LYM,MONO,NEU,EOS,BASO,HCT, MCV,MCH,MCHC,RDW,ALT,AST,AST/ALT,TP,AKP,GLU,BUN, CREA,CHOL,TG and ALB.After classified,these index were compared.The software SPSS13.0 and Independent samples t-test were used to make statistics analysis,and all the data was denoted as mean±standard deviation((?)±SD).2.Making research on genome DNA from Tibet mini-pig(representing haplotype of D-loop sequence),some Bama miniature pigs,Guizhou xiang pigs and WZS pigs.Then designing Primer and amplifing the sequence of Cyt b gene from Tibet mini-pig,Bama miniature pigs,Guizhou xiang pigs and WZS pigs.After sequencing, the base sequence and amino acid sequence were compared and analysed using MEGA3.0 software,then phylogenetic tree and evolution position of Tibet mini-pig were established. 3.PCR-RFLP methods were used in the research of HAL polymorphism. Genome samples from 36 Duroc and 20 Changbai as control group,HAL fragment were amplified using specific primer,then the fragment was digested with Hha I and the result was verified.Through reading genotype,genotypic frequency data of the group were obtained.The genotype can be classified to 3 kinds by electrophoresis analysis,of which HAL~NHAL~N represented two electrophoretic bands of 493bp and 166bp,HAL~NHAL~n represented three electrophoretic bands of 659bp,493bp and 166bp respectively,HAL~nHAL~n represented one electrophoretic band of 659bp.Results1.Previous research showed the length heterogeneity(15～29 10 bp repeat motif) in the tandem repeat sequence of mtDNA D-loop in pigs and all of the motifs is GTACACGTGC,a perfect repeat,but the data from this study demonstrated that there are not only perfect repeats(i.e,type A) but also some imperfect repeats(type B),that is to say,moiety Chinese Tibet mini-pigs contain an alternate array in three motifs:GTACACGTGC,GTACACATGC and GTACACGTAC.By performing pedigree analysis,array types were inherited from mother to offspring and had no relation with father’s types.340 bp sequence at 3’ end of the Tibet mini-pigs D-loop which is very conservative particularly exhibited high homology as in the same region in domestic pigs.704 bp sequence at 5’ end of the Tibet mini-pigs D-loop had 20 polymorphic sites which deduced 26 haplotypes.The three transform sites(305,500 and 691) nearly corresponded to type A and B of tandem repeat sequence.All type B are t,a,a(100%);the bases in three mutation site of type A are all c,g,g(87%);the rest mutation account 13%.The mutation sites in two haplotype of type A is similar to type B.Compared with Tibet mini-pigs,Bama miniature pigs,Guizhou xiang pigs and WZS pigs had 4,4 and 3 haplotypes,respectively,with only one type of tandem repeat sequence,that is type A.Although many studies were performed to investigate the repeat arrays of D-loop previously,little is known about the function of the repeat sequence.In this study,among these blood physiological and biochemical parameters described above,only RBC of Tibet mini-pigs(8 months old) was found to have significant differences between type A and B(P＜0.01),which demostrated tandem repeat sequence has influence on the fuction of blood cells.2.Tibet mini-pigs have close relation with domestic pigs in southwest China,by using phylogenetic tree building by haplotypes deduced from polymorphic sites of 5’ terminal flanking region.3.There are 16 mutation between domestic pig in China and pig from Europe. Besides there was a significant difference in two nucleotide site:a T-C switch in in site 420 and a G-A switch in in site 883,with a proportion about 50%respectively. Compare with pigs from Europe,Tibet mini-pig is different in three amino acids site. In site 295,most of type A Tibet mini-pig and pig from Europe are all Valine,while type B Tibet mini-pig is Isoleucine in this site.Bama miniature pigs,Guizhou xiang pigs and WZS pigs is coincide with type B and a small fraction of type A Tibet mini-pig in site 295 which is isoleucine.Our research showed that Bama miniature pigs,Guizhou xiang pigs and WZS pigs had very close blood relationship with some of Tibet mini-pigs.The research further proved there is differentiation among Tibet mini-pig group.4.The HAL gene type of Tibet mini-pig are all HAL~NHAL~N,no stress sensitive gene is discovered.The HAL genotype frequency of Tibet mini-pig colony is HAL~NHAL~N 100%,HAL~NHAL~n and HAL~nHAL~n 0.4 heterozygote in samples from Duroc pig and 1 heterozygote in samples from Long-white pigs were detected.There is no allozygote in all these samples.As a result,a conclusion is drawed that there is no stress sensitive recessive gene and Tibet mini-pig population have higher ability to defense the press in Tibet mini-pig groups.Conclusions1.Based on the variety of tandem repeat motif and the variety sites of the entire sequence of the hypervariable mtDNA D-loop region,Tibetan mini-pig can be divided into two types:A and B.Types A and B Tibet mini-pig had significant differences in RBC count(type A＜type B).There is a closely genetic relationships between a piece of Tibet mini-pig(type) A and Chinese domestic pigs.Type A,type B and the three base pair transition site(site 305,site 500 and site 691) can be uesd as the united genetic marker of Tibet mini-pigs together.Through artificial selection, classifying type A and type B as two groups,eliminating some of the mutative individuals in type A(13%),reinforcing the genetic marker,we can breed two closed colony of Tibet mini-pigs.All type A is c,g,g and all type B is t,a,a.2.By performing pedigree analysis,array types(A and B) were inherited from mother to offspring and had no relationships with father’s types.Because all of the reported domestic pigs in China are type A,we infer that some of Tibet mini-pigs(A) may be the common origin of Chinese domestic pigs.3.Two variable sites in Cyt b gene of Tibet mini-pigs were discovered,including a T-C transform in site 420 and a G-A transform in site 883,which corresponded with type A and type B Tibet mini-pig.Compare with pigs from Europe,Tibet mini-pig is different in three amino acids site.In site 295,type A Tibet mini-pig and pig from Europe are all Valine,while type B Tibet mini-pig is Isoleucine in this site.Bama miniature pigs,Guizhou xiang pigs and WZS pigs is coincide with type B Tibet mini-pig in site 295.It was confirmed again that there is differentiation in the Tibet mini-pig.4.The HAL genotype frequency of Tibet mini-pig is very low,which demostrate the Tibet mini-pig have a low stress sensibility.So Tibet mini-pig is fit to breed as medical laboratory animal.更多还原