【作者】 崔奎青；

【导师】 石德顺；

【作者基本信息】 广西大学 ， 动物遗传育种与繁殖， 2007， 博士

【摘要】 首先分离培养水牛胎儿成纤维细胞，并检测细胞传代培养过程中的核型稳定性和支原体污染情况。结果发现，组织块法和胰酶消化法均可从水牛胎儿皮肤组织获得大量高活力细胞，15代以前的水牛胎儿成纤维细胞生长良好。分别对体外培养5代、15代、25代和30代的胎儿成纤维细胞染色体分析结果显示，核型的正常率随体外传代增加呈下降趋势，但各代细胞之间差异不显著（P＞0.05），核型正常比率均在75％以上。设计两对PCR引物对第5、15、25和30代细胞的支原体检测污染情况进行了检测，结果检测细胞样本和阴性对照均为阴性，说明培养到30代以内的水牛胎儿成纤维细胞没有发生支原体污染。上述结果表明，在现有实验条件下水牛胎儿成纤维细胞能进行稳定传代培养，可满足体细胞核移植与转基因操作的需要。在此基础上，对影响水牛胎儿成纤维细胞在极低细胞接种密度条件下生长增殖和细胞集落形成的各种因素进行了系统的研究。结果发现，低密度接种的水牛胎儿成纤维细胞总体细胞集落形成率低于20％；在培养液中添加适宜浓度的胰岛素对水牛胎儿成纤维细胞的增殖具有显著的促进作用；培养液中添加不同浓度的细胞条件培养液，能明显改善细胞的增长；进口血清比自制血清更有利于提高细胞集落形成率；使用多聚赖氨酸做生长介质可以让细胞集落形成率增加，而琼脂和明胶的作用相反。这些研究结果表明，低密度接种的水牛胎儿成纤维细胞集落形成率较低；在培养液中添加胰岛素、条件培养液、进口血清及以多聚赖氨酸为细胞生长介质，可以提高水牛胎儿成纤维细胞集落形成率。对电穿孔法和阳离子脂质体法转染水牛胎儿成纤维细胞的条件进行系统摸索，获得了较好的DNA转染参数。电穿孔法转染时脉冲时间为15ms，电场强度为40～50V／mM时，细胞的死亡率处于20％～50％之间，比较适合外源DNA转入水牛胎儿成纤维细胞。将脂质体与DNA的比例控制在8μL：2μg，可获得较高的转染率。相同培养条件下，脂质体法比电穿孔能获得更多的转基因抗性细胞集落，转染DNA的纯度和细胞的状态也明显影响脂质体法的转染效率。用含有GFP基因的线性化载体pCE-EGFP-IRES-Neo-dNdB通过lipofectamine^TM2000转染水牛胎儿成纤维细胞，G418筛选2周后挑取抗性细胞集落扩大培养，对转染外源基因的抗性细胞系的生物学特性进行了分析。结果发现，经过G418筛选后细胞增殖活力下降，细胞爬片HE染色后可看到细胞形态发生变化，凋亡分析发现部分抗性细胞表现出早期凋亡征状，但染色体核型正常率与正常组相比差异不明显。这些结果表明，转染本身或随后的筛选过程会对细胞造成一定的损害。从绵羊基因组扩增得到β-casein基因启动子区到第二内含子区4.1kb的5调控序列，从人基因组克隆得到人血小板生成素（TPO）基因长5.5kb的基因组序列，然后将启动子和TPO定向插入到ploxp骨架上。包含CMV超级启动子的GFP片段从质粒pCE-EGFP-IRES-Neo-dNdB中获得，并定向连接到ploxp-CN-TPO中。经酶切、PCR鉴定，成功获得了包含人TPO基因、绵羊β-casein启动子、GFP标记基因和新霉素抗性基因等长达20kb的转基因载体ploxp-EGFP-CN-TPO。通过脂质体介导将构建好的载体线性化后导入水牛胎儿成纤维细胞中，G418筛选后得到35个抗性细胞集落，扩大培养后其中4个表达GFP绿色荧光。经PCR鉴定目的基因整合的完整性，发现1个为完整转EGFP-CN-TPO基因阳性细胞集落。通过显微操作、电融合的方法将阳性细胞移入体外成熟去核的水牛卵母细胞内构建重组胚，囊胚率显著低于对照组（P＜0.05），说明供体细胞经过基因转染及长期体外培养筛选对克隆胚胎的早期发育有明显影响；对转基因体细胞构建核移植重组胚外源基因表达进行检测发现，绿色荧光蛋白开始表达于4细胞以后的重组胚，说明转染的外源基因能够在胚胎早期发育过程中表达。为研究广西沼泽水牛核基因组的多态性，采用23对荧光标记微卫星引物，对随机抽取的60个广西本地水牛样本进行了PCR检测，然后联合应用具有多态性的位点进行培养细胞的遗传鉴定。结果发现，23对引物中，有19对可以获得特异性产物且存在多态，平均有效等位基因数为4.0189，基因座ILSTS086含有13个等位基因；各微卫星基因座的平均杂合度变化范围为0.1852～0.4722，ILSTS019基因座平均杂合度最高（0.4722），而ILSTS017平均杂合度最低（0.1852）；群体基因平均多态信息含量（PIC）为0.6639，19个标记中有18个PIC大干0.5，属高度多态位点。联合应用19个位点对培养细胞的基因组来源进行同一分析，证明培养细胞与供体水牛基因型完全相同，培养细胞基因组来源于供体水牛。研究结果表明，18个微卫星标记可进一步用于水牛亲缘关系的分析研究。为研究广西沼泽型水牛线粒体D-loop区DNA的多态性，比较分析沼泽型水牛与河流型水牛之间的差异，本研究对26头广西本地水牛、8头贵州贵阳水牛、11头广西水牛与河流型水牛杂交后代、9头尼里拉菲和12头摩拉水牛（共4个品种66头水牛个体）的线粒体全长D-loop序列进行了扩增测序。66头水牛912bp的D-loop多重序列比较结果发现了147个多态位点，共形成58个单体型，其中中国沼泽型水牛与河流型水牛后裔单体型明显分成两簇；以牛为外群通过NJ、ML和贝叶斯方法构建的单体型进化树显示，沼泽型与河流型水牛分成明显两大支，河流型水牛聚在一起，而在沼泽型水牛内部又分成两个侧支；观察单体型之间的遗传关系与距离的median joining network网络分析产生三个区，与进化树分析结果一致；66头水牛D-loop区序列的碱基错配分布分析表明，水牛群体在历史上发生两次独立的群体扩增事件。为进一步验证实验结果，我们从genebank上下载了211条水牛D-loop序列与我们得到的序列整合在一起进行分析，在共有序列878bp区域中，包含了158个多态位点，共获得129个单体型；以牛为外群的进化树分析与单体型网络分析表明，沼泽型水牛分成两簇，所有的河流型水牛聚集成一紧密的簇，与进化树结果一致；碱基错配分布分析结果呈现两个明显的峰，进一步推断水牛群体在历史上发生过两次大的群体扩增。根据上述结果推断：沼泽型水牛与河流型水牛来自不同的家养化事件，沼泽型水牛起源于中国，其祖先可能存在两个基因池。更多还原

【Abstract】 Firstly, methods for isolation and in vitro culture of buffalo fetal fibroblasts (BFF) were establishd, and karyotype stability and mycoplasma contamination of BFF were detected during passages. Large number of high vitality cells could be obtained by tissue explant culture and trypsin digestion. Karyotype analysis of 5th, 15th, 25th, 30th passage BFF showed that more than 75% cells had normal karyotype, and there were no significant difference among different passages (P>0.05). Two pair of nested-PCR primers were designed to detect mycoplsma contamination of BFF, and no contamination was found in 5th, 15th, 25th, 30th passages cells. Thus, the present culture system is stable enough to serve BFF cells for somatic cell cloning and transgenic studies.Factors affecting the proliferation and colony formation of BFF cells cultured in low density were studied. The rate of colony formation was less than 20% when BFF cells were cultured in low density. Insulin could stimulate proliferation of BFF cells and more colonies were formed. Addition of cell conditioned medium also could improve the growing of BFF cells. Qualified serum could increase the colony formation in vitro than that of homemade serum. Poly-lysine was also beneficial to the form of the colony when used as growing media, but agar and glutin were harmful to the growing of BFF cells.The transfection efficiency of electroporation and lipofectin mediated methods was compared. The efficiency of electroporation was line up with the electrical field intensity in which 20%～50% of cells died when the field intensity was set at 40 to 50V/mm. Employment of 8μL lipofectin and 2μg DNA resulted in more cells transfected. Quality of DNA and state of cell were associated with transfection efficiency of lipofectin. At the same culture condition, lipofectin reagent method can obtain more transgenic colonies.The growing status of transgenic cells selected by G418 was investigated in this study. Buffalo fetal fibroblasts were transfected with lined plasmid pCE-EGFP-IRES-Neo-dNdB by Lipofectin reagent. Then anti-G418 cell colonies were collected after two weeks of selecting culture and continued culturing to enlarge the cell amount for detecting cytobiology characterization. The results showed that the proliferation vitality of anti-G418 cell colony was decreased, morphology changing can be seen with normal cells as control which grown on coverslips after hematoxylin and eosin staining. Apoptosis symptom also can be detected, though chromosome karyotype had no distinct difference with nomal cells. These results stated clearly that the long time screening in G418 could do harm to the transgenic cells to a certain degree.4.1kb 5’ promoter region of sheepβ-casein gene from the far upstream sequence to the part of exon2 was amplified by the long distance-PCR technique, the human 5.5kb thrombiotin gene was obtained with the same technique. We insert these two segments into ploxp vector after cutting with restriction enzymes. Green fluorescent protein (GFP) gene sequence came from plasmid pCE-EGFP-IRES-Neo-dNdB and then inserted into ploxp-CN-TPO. Enzyme cutting analysis and PCR testing results showed that the transgenic vector ploxp-EGFP-CN-TPO was constructed as designed advance. BFF cells were transfected with the lined ploxp-EGFP-CN-TPO, and 35 anti-G418 cell colonies were harvested after G418 selection. GFP signals was detected in 4 colonies and got 1 positive transgenic cell colony after PCR proving. The positive transgenic cells were used as donors to reconstruct embryos by micromanipulate and fusion method. The development rate of embryos derived from transgenic cells were significant lower than normal cells (P<0.05). The GFP signal could be detected after 4-cell stage of reconstructed transgenic embryos, which means that foreign gene can express normally in the transgenic embryos.Twenty three microsatellite loci were selected to observe the genetic variation of Guangxi swamp buffalo population. Nineteen primer pairs amplified discrete products and gave polymorphic band patterns on a panel of 60 buffaloes. The mean effective number of alleles per polymorphic marker was 4.0189, mean heterozygosity per polymorphic marker ranged from 0.1852 to 0.4722, mean polymorphism information contents (PIC) for Guangxi buffalo was 0.6639, eighteen microsatellite loci displayed high polymorphism. Then genome source of cultured cell was analyzed using nineteen microsatellite loci, results indicated that cultured cell and donor buffalo had same genome pattern. Thus we conclude that eighteen microsatellite loci can be used in identify the genetic relationship of buffaloes.To investigate the polymorphism of mitochondrial DNA in Guangxi swamp buffalo, the entire mitochondrial D-loop region of 66 water buffaloes of four different breeds were analyzed, 58 mitochondrial haplotypes with 147 polymorphic sites were detected. The haplotypes of swamp buffaloes and river buffaloes formed two different clusters. Using bovine D-loop sequences as outgroup, three phylogenetic trees were constructed with NJ, ML and Bayes method respectively. These trees all showed that swamp buffaloes and river buffaloes belong to two different clusters distinctly, and two small clusters were observed in the swamp cluster of the three. By median joining network analysis, three different clusters also be found, the high diverged clusters between swamp and river buffalo, and two little clusters appear in the whole swamp cluster. The mismatch distribution analysis showed that there may be two different indepent events in the evolution history of buffaloes. Pooled with 211 released D-loop sequences of Genbank, we detected 129 haplotypes with 158 polymorphic sites in 878bp multi-aligned region. The phylogenetic tree and median joining network analysis also showed that there were three clusters in total buffaloes, two swamp buffalo’s clusters and one high diverged river swamps. Two obviously wave crests appeared in the mismatch distribution analysis, which means that there maybe two different population expanding event in water buffaloes. We conclude based on the above results, that river and swamp buffalos decented from two different domestication events, and there were at least two ancestor gene pools in the domestication of swamp buffaloes.更多还原