【摘要】： 研究背景 藏豬(Tibet hog,Sus scrofa)是世界上體型較小的小型豬之一,原產(chǎn)于青藏高原、海拔2500～4300m的農(nóng)區(qū)和半農(nóng)半牧區(qū),分布于中國(guó)的西藏自治區(qū)、云南省、四川省和甘肅省等地。藏豬保存了較為純正的品種資源,是唯一能夠適應(yīng)高海拔氣候和以放牧為主的豬種。由于長(zhǎng)期的地理隔離和環(huán)境的差異,不同地區(qū)的藏豬出現(xiàn)了一定程度的分化,如四川藏豬與云南藏豬相比,四川藏豬偏離Hardy-Weiberg定律的基因座數(shù)多于云南藏豬;四川藏豬體型較小,而云南減豬體型較大。目前雖然對(duì)四川藏豬或云南藏豬開展了一些研究,但對(duì)于來(lái)自西藏自治區(qū)的藏豬研究報(bào)導(dǎo)較少。2004年南方醫(yī)科大學(xué)從西藏自治區(qū)工布江達(dá)縣將50頭藏豬(雌、雄各半)引種到廣州,首次開展對(duì)藏豬進(jìn)行實(shí)驗(yàn)動(dòng)物化的培育,并將其命名為西藏小型豬(Tibet mini-pig)。2006底獲得廣東省實(shí)驗(yàn)用西藏小型豬生產(chǎn)質(zhì)量合格證,目前存欄數(shù)已達(dá)600余頭。 目的 測(cè)定西藏小型豬的mtDNA控制區(qū)序列和Cyt b基因序列,研究西藏小型豬mtDNA控制區(qū)和Cyt b基因序列的遺傳分化,及其對(duì)血液生理生化指標(biāo)的影響,并和其他豬的序列比對(duì)分析,研究其親緣關(guān)系;探討西藏小型豬氟烷基因座位的群體結(jié)構(gòu)特征,檢測(cè)氟烷基因型隱性純合子個(gè)體,尋找西藏小型豬獨(dú)特的分子遺傳標(biāo)記,從理論上指導(dǎo)西藏小型豬實(shí)驗(yàn)動(dòng)物化的培育。 方法 1隨機(jī)抽取120頭西藏小型豬血液,利用試劑盒提取血液基因組DNA;設(shè)計(jì)引物擴(kuò)增西藏小型豬以及7頭巴馬小型豬、23頭貴州香豬和17頭五指山豬的mtDNA控制區(qū),測(cè)序并結(jié)合Clustalw軟件和MEGA3.0軟件進(jìn)行所測(cè)序列多重比對(duì),確定變異位點(diǎn)、單倍型,并建立西藏小型豬和國(guó)內(nèi)其他豬的親緣關(guān)系樹。對(duì)其中58頭8月齡的西藏小型豬分別測(cè)定血液生理生化指標(biāo)。血液生理指標(biāo)14項(xiàng):白細(xì)胞、紅細(xì)胞、血紅蛋白、血小板、淋巴細(xì)胞、單核細(xì)胞、粒細(xì)胞、嗜酸性粒細(xì)胞、嗜堿性粒細(xì)胞、血細(xì)胞比容、平均紅細(xì)胞體積、平均紅細(xì)胞血紅蛋白含量、平均紅細(xì)胞血紅蛋白濃度、紅細(xì)胞分布寬度。血液生化指標(biāo)11項(xiàng):谷丙轉(zhuǎn)氨酶、谷草轉(zhuǎn)氨酶、谷丙轉(zhuǎn)氨酶與谷草轉(zhuǎn)氨酶比值、總蛋白、堿性磷酸酶、葡萄糖、尿素氮、肌酐、總膽固醇、甘油三酯、白蛋白。分類后進(jìn)行指標(biāo)的比較。統(tǒng)計(jì)學(xué)分析采用SPSS13.0版統(tǒng)計(jì)學(xué)軟件進(jìn)行兩個(gè)樣本的t檢驗(yàn),所有數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差((?)±SD)表示。 2以代表mtDNA控制區(qū)5′端序列單倍型的西藏小型豬和部分巴馬小型豬、貴州香豬、五指山豬的全基因組DNA為研究對(duì)象,設(shè)計(jì)引物擴(kuò)增四種小型豬的Cyt b基因序列,測(cè)序后利用MEGA3.0軟件進(jìn)行堿基序列和氨基酸序列比對(duì),建立親緣關(guān)系樹,分析西藏小型豬的進(jìn)化地位。 3應(yīng)用PCR-RFLP技術(shù),研究西藏小型豬的氟烷基因多態(tài)性。以杜洛克豬和長(zhǎng)白豬基因組樣本為對(duì)照,用特異性引物擴(kuò)增3個(gè)豬種的氟烷基因片段,然后進(jìn)行限制性內(nèi)切酶(HhaⅠ)酶切以及酶切結(jié)果的鑒定,通過(guò)基因型辨讀,獲得群體的基因型頻率特征數(shù)據(jù),確定隱性純合子基因型個(gè)體。對(duì)酶切電泳結(jié)果進(jìn)行辨讀時(shí),HAL~NHAL~N基因型表現(xiàn)出493bp和166bp兩條電泳條帶,HAL~NHAL~n基因型表現(xiàn)出659bp、493bp和166bp三條電泳條帶,HAL~nHAL~n基因型只出現(xiàn)659bp一條電泳條帶。 結(jié)果 1前人的研究表明,豬的mtDNA控制區(qū)的串聯(lián)重復(fù)區(qū)存在長(zhǎng)度異質(zhì)性(15—29個(gè)重復(fù)片段),重復(fù)片段全部是:GTACACGTGC,稱之為完全重復(fù)。但是本研究顯示,西藏小型豬不僅有完全重復(fù)(A型),而且有不完全的重復(fù)(B型),即一部分西藏小型豬10bp的重復(fù)片段不僅有GTACACGTGC,而且還有GTACACATGC和GTACACGTAC這兩個(gè)片段及其交替排列現(xiàn)象。系譜分析顯示,這種排列類型同樣按母系遺傳方式由母本傳給后代,與父本沒(méi)有關(guān)系。 西藏小型豬mtDNA控制區(qū)3′端側(cè)翼區(qū)為340bp,變異位點(diǎn)少,與國(guó)內(nèi)其他家豬的序列一樣比較保守;5′端側(cè)翼區(qū)704bp,有20個(gè)變異位點(diǎn),由此歸納出26個(gè)單倍型。西藏小型豬5′端側(cè)翼區(qū)三個(gè)轉(zhuǎn)換位點(diǎn)(305,500,691)的變化幾乎與串聯(lián)重復(fù)序列所分的A、B兩組類型相對(duì)應(yīng):B型中三個(gè)變異位點(diǎn)的堿基分別為t,a,a(100%);A型中三個(gè)變異位點(diǎn)的堿基分別是c,g,g的占87%,其他13%。 與西藏小型豬相比,巴馬小型豬、貴州香豬和五指山豬mtDNA控制區(qū)5′端變異位點(diǎn)較少,分別只有4、4、3種單倍型,串聯(lián)重復(fù)區(qū)也只有一種類型,A型。對(duì)西藏小型豬A型和B型群體與血液生理生化特性的進(jìn)行相關(guān)分析,結(jié)果表明A型和B型群體血液紅細(xì)胞數(shù)量有顯著性差異(A型＜B型,P＜0.05)。 2利用控制區(qū)5′端側(cè)翼區(qū)序列歸納出的單倍型建立親緣關(guān)系樹的分析表明,西藏小型豬與中國(guó)地方家豬有較近的親緣關(guān)系,特別與中國(guó)西南地區(qū)幾種家豬的親緣關(guān)系最近。 3西藏小型豬與國(guó)內(nèi)家豬的Cyt b基因序列幾乎相同;與歐洲豬相比差異較大,共有16個(gè)主要變異位點(diǎn),其中有兩個(gè)特殊轉(zhuǎn)換位點(diǎn):420位點(diǎn)T—C轉(zhuǎn)換和883位點(diǎn)的G—A轉(zhuǎn)換,幾乎各占一半。利用Mege3.0軟件將堿基序列轉(zhuǎn)換為氨基酸序列后的分析表明,西藏小型豬與國(guó)內(nèi)其他豬有一個(gè)氨基酸位點(diǎn)(295位點(diǎn))、與歐洲豬有三個(gè)氨基酸位點(diǎn)(89,295,314位點(diǎn))存在著差異。在295位點(diǎn),大部分A型西藏小型豬與歐洲豬同為纈氨酸(Ⅴ),而B型和少部分A型為異亮氨酸(Ⅰ)。巴馬小型豬、五指山豬和貴州香豬的氨基酸序列在295位點(diǎn)也為異亮氨酸(Ⅰ)。結(jié)果進(jìn)一步證實(shí),西藏小型豬群體內(nèi)存在分化,mtDNA控制區(qū)的序列變異與功能基因的結(jié)構(gòu)變化存在一定相關(guān)性。 4 120頭西藏小型豬個(gè)體氟烷基因型全部為HAL~NHAL~N,沒(méi)有發(fā)現(xiàn)應(yīng)激敏感基因,氟烷基因顯性純合子基因型:HAL~NHAL~N的頻率為100%;HAL~NHAL~n型和HAL~nHAL~n型均為0。而在杜洛克豬樣本中檢測(cè)到4份雜合子樣本,長(zhǎng)白豬樣本中檢出1份雜合子個(gè)體,但都沒(méi)有發(fā)現(xiàn)隱性純合子個(gè)體。西藏小型豬群體內(nèi)未發(fā)現(xiàn)應(yīng)激敏感性的隱性基因,整個(gè)群體抗應(yīng)激能力強(qiáng)。 結(jié)論 1根據(jù)西藏小型豬mtDNA控制區(qū)串聯(lián)重復(fù)區(qū)的長(zhǎng)度和重復(fù)片段存在異質(zhì)性,可將西藏小型豬群體分為A型和B型。A、B類型和5′端的三個(gè)堿基轉(zhuǎn)換位點(diǎn):305,500,691,可以聯(lián)合組建西藏小型豬的遺傳標(biāo)記。建議通過(guò)人工選擇,A型B型分為兩個(gè)群體,淘汰A型中少量變異個(gè)體(13%),強(qiáng)化西藏小型豬的標(biāo)記,培育成兩個(gè)封閉群體:B型三個(gè)轉(zhuǎn)換位點(diǎn)分別為t,a,a;A型封閉群的三個(gè)轉(zhuǎn)換位點(diǎn)純化為c,g,g。 2由于西藏小型豬串聯(lián)重復(fù)區(qū)的排列類型A型或B型都可以由母本傳給后代,而目前所報(bào)導(dǎo)的國(guó)內(nèi)家豬全部為A型,沒(méi)有B型的報(bào)導(dǎo),因此提出A型西藏小型豬和其他中國(guó)家豬有共同起源,B型可能是基因突變或者是兩種母系起源的結(jié)果。 3西藏小型豬Cyt b基因的全序列有兩個(gè)轉(zhuǎn)換位點(diǎn),與A型和B型西藏小型豬有一定對(duì)應(yīng)關(guān)系。利用Mege3.0軟件將堿基序列轉(zhuǎn)換為氨基酸序列后的分析表明,西藏小型豬Cyt b第295位氨基酸大部分為纈氨酸(Ⅴ),而少部分為異亮氨酸(Ⅰ)。其他國(guó)內(nèi)家豬的氨基酸序列全部為異亮氨酸(Ⅰ)。結(jié)果進(jìn)一步證實(shí),在西藏小型豬群體內(nèi)存在分化。 4西藏小型豬群體氟烷基因,未發(fā)現(xiàn)應(yīng)激敏感性的隱性基因,說(shuō)明西藏小型豬的應(yīng)激敏感性比較低,適合于開展外科手術(shù)或器官移植等實(shí)驗(yàn)研究。
[Abstract]:Research background
Tibet hog (Sus scrofa) is one of the * * smaller pigs in the world. It is located in the Qinghai Tibet Plateau, with an elevation of 2500 to 4300m in the agricultural area and semi agricultural and semi pastoral areas. It is distributed in Tibet autonomous region of China, Yunnan, Sichuan and Gansu provinces. * Tibetan pigs preserve relatively pure variety resources and are the only ones that can adapt to the high altitude climate. * due to long-term geographical isolation and environmental differences, Tibetan pigs in different areas have been differentiated to some extent. * * compared with Sichuan Tibetan pigs, the number of locus of Tibetan pigs deviating from Hardy-Weiberg's law is higher than that of Yunnan Tibetan pigs, while that of Sichuan Tibetan pigs is smaller than that of Yunnan Tibetan pigs. Tibetan Pig * * or Yunnan Tibetan Pig carried out some researches, but less research reports on Tibetan pigs from Tibet autonomous region *.2004 introduced 50 Tibetan pigs (female, male and half) from Tibet autonomous region to Tibet for the first time. It was the first time to carry out laboratory animal cultivation of Tibetan pigs and named it "Tibet miniature pig" (Tibet min). I-pig) at the bottom of the.2006 *, we have obtained the quality certificate for the production of Tibet miniature pigs in Guangdong province. At present, the number of livestock has reached 600.
objective
The sequence of mtDNA control region and Cyt B gene sequence of Tibet miniature pig were determined. The genetic differentiation of mtDNA control region and Cyt B gene sequence of Tibet minipigs and their effects on blood physiological and biochemical indexes were studied. The genetic relationship between them was compared with other pig sequences, and the genetic structure of halothane gene loci in Tibet miniature pigs was studied. To detect halothane genotype recessive homozygous individuals, search for unique molecular genetic markers of Tibet miniature pigs, and guide the cultivation of Tibet miniature pigs experimentally.
Method
1 randomly selected 120 Tibet miniature pig blood, extracted genomic DNA from blood by kit, designed primers to amplify the mtDNA control area of Tibet miniature pig and 7 Bama miniature pigs, 23 Guizhou Xiang Pigs and 17 Five Fingers Group pigs, sequenced and compared multiple sequences of the tested sequences with Clustalw software and MEGA3.0 software to determine the mutation sites and haplotypes. * * and establish the phylogenetic tree of Tibet miniature pig and other domestic pigs. 58 * 8 month old Tibet miniature pigs were used to measure blood physiological and biochemical indexes. Blood physiological indexes were 14 items: white blood cells, red blood cells, hemoglobin, platelets, lymphocytes, monocytes, granulocytes, eosinophils, basophils, hematocrit, and flat. BLOOD BIOCHEMICAL INDEXES 11 items: ALT, ALT, ALT/AST, ALT/AST ratio, total protein, alkaline phosphatase, glucose, urea nitrogen, creatinine, total cholesterol, triglyceride, albumin. Statistical analysis was performed with SPSS13.0 statistical software for t-test of two samples. All data were expressed as mean (?) + standard deviation ((?) + SD).
2 the genomic DNA of Tibet miniature pigs and haplotype pigs representing Guizhou * * Xiang Pigs and Five Fingers Group pigs, which represent haplotypes of the 5 * end sequence of mtDNA control area, were designed. Primers were used to amplify the Cyt B gene sequences of four miniature pigs. After sequencing, the nucleotide sequences and amino acid sequences were compared with MEGA3.0 software to establish the phylogenetic tree. * the evolution status of Tibet miniature pigs.
3 using PCR-RFLP technology, we studied the polymorphism of halothane gene in Tibet miniature pig * * *, using the genome samples of Duroc and Landrace pigs as controls, we amplified the halothane gene fragments of 3 pig breeds with specific primers, then performed restriction enzyme (Hha I) digestion and enzyme digestion results, and obtained genotype frequencies by genotype analysis. The HAL~NHAL~N genotype showed two electrophoretic bands of 493 BP and 166 bp, the HAL~NHAL~n genotype showed three electrophoretic bands of 659 bp, 493 BP and 166 bp, and the HAL~nHAL~n genotype showed only one electrophoretic band of 659 bp.
Result
1 previous studies have shown that there are length heterogeneity (15 * 29 repeats) in the tandem repeat region of mtDNA control area in pigs. All repeat fragments are GTACACGTGC, which is called complete duplication. However, this study shows that * Tibet miniature pigs not only have complete duplication (type A) but also have incomplete duplication (B *), that is, the weight of part of Tibet miniature pig 10bp. The complex fragments not only contain GTACACGTGC, but also GTACACATGC and GTACACGTAC fragments and their alternate arrangement. The pedigree analysis showed that this arrangement type was also passed from mother to offspring according to maternal inheritance, and had no relationship with father.
The 3 * end flanking area of mtDNA control area in Tibet miniature pig is 340bp, with less variation sites, and * conservative compared with other domestic pig sequences. There are 20 mutation sites in the 5 'flanking region 704bp, and 26 haplotypes are induced. * the change of three transformation sites (305500691) in 5' flanking region of Tibet miniature pigs is almost identical to that of tandem repeats. The bases of three mutation sites in type B were t, a, a (100%) and the bases of three mutation sites in type A were c, g, g (87%) and the others (13%).
Compared with Tibet * * * pigs, Bama miniature pigs, Guizhou Xiang Pigs and Five Fingers Group pigs mtDNA control area had 5 fewer variants, only 4,4,3 haplotypes. There was only one type of tandem repeat type, A type. Correlation analysis between A and B groups and blood physiological and biochemical characteristics of Tibet miniature pigs showed that A and B group blood There was a significant difference in the number of red blood cells (type A < B, P < 0.05).
2 the analysis of the phylogenetic tree based on haplotypes derived from the 5 * flank region of the control area showed that the Tibet miniature pig had a close relationship with the local pig in China, especially with the * * relationship of several pigs in Southwest China.
3 * Tibet miniature pig and domestic pig * * Cyt B gene sequence is almost the same. Compared with the European pig, there are 16 major mutation sites, including two special transformation sites: 420 locus T - C transformation and 883 - site G - A transformation, almost half. There is an amino acid locus (295 loci) in Tibet miniature pig and other pigs in China. There are three amino acid loci (89295314 loci) in European pig breeds. At the 295 locus, most A Tibet miniature pigs are valine (V) compared with European pigs, while B and A are isoleucine (I.) Bama miniature pigs, Five Fingers Group pigs and Guizhou. The amino acid sequence of Xiang pig is also isoleucine at the 295 locus. (1) it is further confirmed that there is differentiation in the Tibet miniature pig population. The sequence variation in the mtDNA control region is related to the structural change of the functional gene.
The individual halothane genotype of 4120 Tibet miniature pigs was HAL~NHAL~N. No stress sensitive genes were found. The dominant homozygote genotype of halothane gene was HAL~NHAL~N: the frequency of HAL~NHAL~N was 100%, HAL~NHAL~n and HAL~nHAL~n were 0., while 4 heterozygote samples were detected in Duroc pig samples, and 1 heterozygous individuals were detected in Landrace samples. No recessive homozygous individuals were found. * the stress sensitive recessive gene was not found in the Tibet miniature pig population, and the whole population had strong anti stress ability.
conclusion
1 * according to the heterogeneity of the tandem repeats and repetitive fragments in the mtDNA control area of Tibet mini pig, the * Tibet miniature pig population can be divided into A base and B type.A, B base type and 5 'end * three base conversion sites: 305500691, which can be combined to form the genetic markers of Tibet miniature pig. Through the artificial selection, the A type B can be divided into two groups. A small number of variant individuals in A * (13%) were strengthened, and the markers of Tibet miniature pigs were strengthened to form two closed populations. The three transformation sites of B type were t, a and a, and three transformation sites of A closed group were purified to C, G, g..
2 * the type of tandem repeats in Tibet minipigs can be passed from maternal to offspring. The reported domestic pigs are all A type. There is no report on B * *. Therefore, it is proposed that A Tibet miniature pig and other Chinese pigs have a common origin. B type may be the result of A mutation or the origin of two maternal origins.
3 * the complete sequence of Cyt B gene in Tibet miniature pig has two conversion sites, which is corresponding to A type and B * Tibet miniature pig. The analysis of the conversion of nucleotide sequence to amino acid sequence using Mege3.0 software shows that most of the 295th amino acids of Cyt * B in Tibet miniature pig are valine (V), while a few are isoleucine (I). * all the amino acid sequences of pigs were isoleucine (*). The results further confirmed that there were differentiation in the Tibet miniature pig population.
4 * the halothane gene in Tibet miniature pig population did not detect stress sensitive recessive genes. * it indicates that the sensitivity of Tibet miniature pigs is relatively low, and is suitable for experimental studies such as surgery or organ transplantation.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R-332

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