【作者】 莫德林；

【导师】 李奎； 刘榜； 冯书堂；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2006， 博士

【摘要】 贯穿于机体整个生命周期的细胞凋亡对机体的发育、存活及维持正常生理功能都有着重要意义，尤其是在个体发育、细胞分化、组织器官形成过程中都发挥着重要作用。而骨骼肌在不同的生长发育过程中涉及大批基因的差异表达及网络调控，而这些差异表达的基因很可能在生长发育过程中调控着某些细胞的增殖与凋亡。根据该推理假设，本研究从构建的猪胎儿骨胳肌cDNA文库中挑选出一个在组织及不同发育时期都有差异表达的未知基因作为研究对象，并通过分子生物学、细胞生物学以及生物信息学等方法从核酸及蛋白质两个水平上进行了结构与功能研究，并取得了如下研究结果：1．猪的PNAS-4基因cDNA全长序列的获得根据已获得的该基因部分信息，采用电脑克隆策略结合RACE技术，获得了猪的PNAS-4基因的5’非翻译区序列，然后通过PCR方法填补了序列中一个82bp的Gap，从而得到该基因的cDNA全长4133bp，并提交GenBank数据库，GenBank收录号DQ435075；2．cDNA序列上的功能元件分析对猪的PNAS-4 cDNA序列进行分析，发现该基因的在5’UTR区有一内源性核糖体进入位点(IRES)，而在3’UTR区除了有两个多(聚)腺苷酸加尾信号(AATAAA)外，还存在着两个功能域框Brd-Box和GY-Box，其中Brd-Box通过影响转录的稳定性和翻译的效率来介导转录后负调控；3．人的同源基因clorf121的启动子区域调控因子的分析利用人的同源基因clorf121的启动子区域进行网络调控分析时发现，有一个CpG岛附近有着较多的转录因子(Sp1、AP-1)的结合点，说明该基因具有较强的转录效率，另外还发现在启动子区域有较多的抑凋亡因子结合位点。根据其CpG岛的GC含量高及无TATA box的特征推测该基因是一个无处不表达的持家基因；4．推导的该编码的基因蛋白质特点由该基因CDS序列推导出猪的PNAS-4编码着是一个含有194个氨基酸残基，是一个既无信号肽又无跨膜结构的非分泌性蛋白质；并对其保守的功能域DUF862进行了三维结构的模拟，发现其有三个可能的蛋白质结合域以及三个裸露在外的氨基酸残基；同源比对后，发现该基因在哺乳动物之间同源性极高达96-98％，与家禽及非洲蟾蜍分别有着94％和74％的相似性，甚至拟南芥的该蛋白质与猪也有着45％的相似性；5．多态检测及连锁不平衡分析利用比较基因组学通过PCR方法获得猪的该基因第二内含子全长，并提交GenBank数据库，GenBank收录号DQ406743。通过PCR-RFLP及DHPLC技术对第二内含子及3’UTR区共计四个SNPs位点进行多态分析，发现这四个多态位点在不同品种中表现出等位基因频率的差异。这四个位点虽相隔20Kb，但却不同程度地相互处于连锁不平衡状态。通过性状关联分析，发现该基因不同基因型个体在内脂率、板油率、腿臀肉骨率、胴体直长、眼肌面积以及血红蛋白浓度等性状存在显著性差异(P＜0.05)；6．应用定量PCR进行时空表达谱分析通过real-time PCR方法研究了猪的PNAS-4基因在成年猪15个组织中的表达情况，发现该基因在所研究的组织中均有表达，但存在表达量的差异，其中，骨胳肌和淋巴组织中表达量最高，肾和大脑中表达量最低。另外还发现该基因在癌化的猪肾上皮细胞中的表达量要比正常肾组织要高。而在胚胎发育时期，该基因在骨胳肌中的表达呈现下调表达模式，但出生后呈现出上调表达模式。将该基因在胚胎时期的表达模式进行比较，发现该基因在中外猪种骨骼肌的发育过程中并不存在差异表达；7．该基因的原核表达按照常规的实验方法对该基因进行原核表达，并没有发现特异性条带，推测可能是由于大肠杆菌密码子的偏爱造成了该基因在原核生物中的表达障碍；8．亚细胞定位利用绿色荧光表达载体pEGFP-C1与该基因编码框进行融合表达，转染并染色后通过共聚焦显微镜进行观察，发现猪的PNAS-4基因的蛋白质CGI-146明显分布于高尔基体，说明该蛋白质存在于高尔基体内并发挥其生物学功能；9．通过真核表达载体pcDNA3.1进行超表达将该基因编码框插入到真核超表达载体peDNA3.1中，进行细胞转染后，利用倒置显微镜在普通视野下观察发现，随着转染后时间的延长，越来越多的细胞出现萎缩，浓缩等凋亡现象；通过细胞凋亡检测试剂盒染色后，在倒置荧光显微镜FITC和若丹明双重滤镜下观察到凋亡早期、中期及晚期细胞，从而初步确定了该基因具有促凋亡的特性。器官移植作为20世纪医学领域发展起来的新兴技术已经成为治疗器官功能衰竭终末期的首要治疗手段。全世界每年有数万名器官功能衰竭患者通过器官移植而获新生。然而供体器官的严重不足，使得许多病人在等待中死去，而物种间异种移植则成为了解决该医学问题的最好方法。进行猪-人异种器官移植，首先要解决的问题是克服超急性排斥反应(HAR)。它是由猪的血管内皮细胞表面α-Gal抗原与人体内的天然抗体结合，从而激活补体级联反应而发生的。经研究证实，该抗原是由一组具有Galα(1，3)Gal双糖末端的糖蛋白或糖脂组成的，它的形成依赖于α1，3-半乳糖基转移酶(α1，3-GT)的催化。因此利用生物工程技术敲除该基因将能在很大程度上有效地克服HAR。基因打靶是80年代发展起来的一项重要的分子生物学技术。它是通过外源DNA与受体细胞基因组中的同源序列之间发生重组，从而定向改变细胞或生物个体遗传信息的实验手段。目前，该技术已先后在小鼠、绵羊、山羊、牛和猪的体细胞中获得成功。本研究利用现有的α1，3-GT基因序列信息，成功构建了正负双筛选基因打靶载体，期望在后续实验中通过体细胞基因打靶技术获得敲除α1，3-GT基因的高近交五指山猪品系，从而为我国的异种器官移植提供更为理想的实验动物。利用长距离PCR(LR-PCR)方法，从高近交系五指山猪的基因组中成功得到了5.4Kb、1.6Kb的扩增产物。其中5.4Kb的DNA片段包括了α1，3-GT完整的第8内含子；1.6Kb的DNA片段覆盖了大部分第9外显子。考虑到5.4Kb的DNA片段中存在限制性内切酶SalⅠ位点，于是通过PCR方法选取其中的5.1Kb DNA片段作为打靶载体的5’同源臂，并将1.6Kb的DNA片段作为3’同源臂，以neo为正选择标记基因，以HVS-tk为负选择标记基因，构建敲除猪α1，3-GT基因部分序列打靶载体PLS-pPNT，全长约15.2Kb，并经测序验证，从而为后续实验打下了良好基础。更多还原

【Abstract】 Part I Analysis of structure and function of porcine PNAS-4 Being involved in the whole lifecycle of organism, apoptosis, which is ubiquitous and also normal of cell death, is known to play an important role during organism development, survival and maintain normal physiological function especially in embryonic development, cell proliferation, differentiation, formation of apparatus. In different developmental stages, the skeletal muscle involves a great number of differential expression genes which are regulating proliferation and apoptosis for certain cells in the developmental process.In our previous work, a novel gene with differential expression in different tissues and different developmental stages was isolated by screening from a full-length cDNA library constructed with the 55-day-old pig fetus skeletal muscle. In order to study the structure and function of this gene from nucleic acid and protein level, several methods including molecular biology, cytobiology as well as bioinformatics was employed in experiment. And the main results are as follows:1. Obtaining the Full-length cDNA of porcine PNAS-4The full-length 4133bp cDNA sequence of porcine PNAS-4 (GenBank accession number: DQ435075) was obtained by PCR and 5’RACE.2. Functional elements located in the cDNAA functional element called Internal Ribosome Entry Site (IRES) was found in the 5’ UTR. Two boxes named Brd-Box and GY-Box located in the 3’ UTR of the porcine gene containing two consensus polyadenylation signals (AATAAA) located upstream of the poly (A) stretch. And the Brd-Box plays an important role in negative regulation for the post- transcription via its influence on the stability and translation efficiency.3. Regulation factors in the promoter of human homologous geneA lot of binding site of transcription factors such as Sp1 and AP-1 were discovered nearby the CpG island after analysis of network regulation for the promoter of human clorf121 which is homologous to porcine PNAS-4, indicating the transcription efficiency will be higher than others. In addition, there are many binding sites of apoptosis repressor in the human promoter. This promoter is GC rich and lacks a TATA box as is typical for promoters of ubiquitously expressed "housekeeping genes".4. Characterization of predicted protein sequences The conceptual translation product of the porcine PNAS-4 transcript encodes 194 amino acids residues, which is a non-secretary protein without signal peptide, or transmembrane regions. Three-dimensional simulation structure was carried out according to the Putative conserved domain related to Eukaryotie protein of unknown function (DUF862), which comprises three possible protein binding domains and four amino acids residues exposing outside. Comparison to seven amino acids sequences of different species reported in GenBank, the porcine putative protein has a high level identity (96-98%) in mammals, and furthermore, it shares 74% and 45% similarity with Xenopus laevis and Arabidopsis thaliana, respectively.5. Detection of Polymorphisms and analysis of linkage disequilibriaThe complete second intron comprising of 3.2kb in length (GenBank accession no. DQ406743) was obtained according to the human clorf121 genome structure. After analysis of four mutation site located on the second intron and 3’UTR respectively using PCR-RFLP and DHPLC methods, the allele frequency of the four SNPs displays variable in different pig breeds. And the four mutation sites appear linkage disequilibria in single population though they are approximately 21Kb apart from each other. There are significant associations between the genotype of PNAS-4 with phenotypes of Percentage of Leaf Fat, Internal Fat Rate, Carcass Length, Rib Numbers, Loin-muscle Area, Ratio of M vs S in Ham and Hemoglobin Concentration (P＜0.05).6. Temporal and spatial express patternReal-time Q-PCR was performed to analyze tissue-specific expression of porcine PNAS-4 transcript using the housekeeping geneβ-actin as endogenous control. Relative mRNA expression of PNAS-4 was high in the lymph as well as in skeletal muscle, and with weakest signal observed in kidney and brain. In addition, the higher mRNA expression was found in cancerization cell than the normal kidney tissue. For temporal expression analysis, the porcine PNAS-4 appears down-regulated pattern during embryonic development while as up-regulated pattern after birth. And in the process of skeletal muscle development, there is no differential expression between indigenous and exotic pig breeds for this novel gone.7. Expression P4-p28c in E. coliA prokaryotic expression plasmids P4-pET28c was Constructed, and transferred into E. coli BL21 (DE3) expression strain. As a result from codon preference, no special signal peptide was found via conventional methods including SDS-PAGE.8. The porcine CGI-146 intracellular distributionThe intracellular distribution of porcine CGI-146 (the product of PNAS-4) in PK-15 cells was analyzed by fluorescence and confocal analysis of transiently transfected with P4-pEGFP-C1. After stained with MitoTracker Red and Hoechst33342, CGI-146-pEGFP fusion proteins were predominantly detected in Golgi apparatus, which was identified using the homo-functional plasma P4-pEGFP-N3 as the complementation.9. Overexpression of the porcine CGI-146Transiently transfection of PK-15 cells with the overexressed plasmid P4-pcDNA3.1 which express 21.4kDa non-tagged protein, the cell shrinkage and the less well defined cell surface was observed in the normal view with normal optical microscope. After the transfected cells were stained with Annexin V and Propidium Iodide, the apoptosis (annexin V-positive/propidium iodide-negative) were observed by the fluorescence microscope with barely vector alone as controls. As a new medical technology born in 20th century, clinical transplantation has become one of the important and powerful treatment methods for end-stage organ failure. There are tens of thousands patients coming to a new life by this medical technology every year in all over the world. However many patients die in the course of waiting because the donor organs are serious insufficiency. Therefore the Xenotransplantation has become to the most appropriate method to solve the urgently problem of medicine.The major obstacle to success is the hyperacute rejection (HAR) in pig-to-human discordant xenotransplantation, which attribute to the presence of the Galactose-α1,3-galactose (αGal) epitopes on the surface of pig cells and tissues. And the Synthesis of theα1,3-Gal epitope is catalyzed by the enzymeα1,3-galactosyltransferase (α1, 3-GT, GGAT1), but humans, apes, and Old World monkeys have lost the corresponding galactosyltransferase activity because only with a non-functional copy of theα1,3-GT gene in the course of evolution and therefore produce a large quantity of natural antibodies to the epitope, which are responsible for hyperacute rejection of porcine organs. Consequently, the genetic knockout of the a1,3-galactosyltransferase locus in pigs would provide permanent and complete protection against the HAR at extant range using gene engineering technology.Gene targeting is an important genetic manipulation technology, which defined as the introduction of site-specific modifications into the genome by homologous recombination, has revolutionarized the field of mouse genetics and allowed the analysis of diverse aspects of gene function in vivo. In present, the cloning of sheep, goat, cattle, and pigs by somatic cell nuclear transfer provides a feasible means of disrupting or deleting theα1,3-GT in mammals other than mice.According to the porcineα1,3-GT fragment obtained from the NCBI, a gene targeting vector, which comprise of positive and negative selection, was used to establish the highly inbred Wuzhishan miniature pig (WZSP) line knocked out theα1,3-GT gene by the method of gene targeting in somatic cells. At the same time, to offer the possible experimental animals used for Xenotransplantation in our country. Using the long-range (LR) PCR method, we have cloned two DNA fragments which are 5.4Kb and 1.6Kb respectively from the highly inbred WZSP genome. The 5.4Kb DNA fragment includes the whole eighth intron of porcineα1,3-GT gene, and the other spans the most part of exon 9, which are cloned to vector for sequencing. And sequencing analysis showed that the two fragment are genomic DNA of the porcineα1,3-GT gene.Because there is a restriction digestion enzyme SalⅠlocated in the former of 5.4Kb DNA fragment, 5.1Kb was amplified for 5’arm of targeting vector using conventional PCR method while the 1.6Kb DNA fragment as the 3’arm. And then, the construction taregeting vector containing 15.2 Kb used for knock-out porcineα1,3-GT gene was completed and conformed by sequencing, in which the neomycin phosphotransferase (neo’) and herpes simplex virus-thymidine kinase (HSV-tk) genes were used as positive and negative selection markers.更多还原