【作者】 方威；

【导师】 徐银学；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2009， 硕士

【摘要】 Lhx8是最新鉴定的LIM家族成员之一,最新研究表明,Lhx8在卵子发生的早期阶段对于维持卵母细胞的分化和存活有着极为重要的作用。对Lhx8基因的克隆测序和初步分析,可以为研究猪的Lhx8基因的生物学功能和品种改良提供基础的基因信息.本文采用电子克隆的方法,结合RT-PCR和RACE技术,获得了猪Lhx8基因的cDNA全序列。利用生物信息学方法预测了该蛋白的结构和功能,并进行了同源序列多重比对和分子系统发生分析。利用实时荧光定量(Real-time) PCR技术分析了猪Lhx8基因的mRNA在不同组织和胚胎发育不同时期的相对表达水平。1 Lhx8基因的克隆测序用人的Lhx8基因的cDNA全序列作为电子探针,分别从NCBI的dbEST和UniGene数据库钓取了共3条猪的EST序列,将其输入DNAstar-SeqMan程序,最后拼接出2个重叠群(Contig)序列。在此基础上,利用Oligo6.0软件手动设计引物,借助RT-PCR和RACE技术一方面验证了电子克隆的正确性；另一方面扩增出cDNA全序列,长度为1681bp,GenBank登录号为FJ 587986。2 Lhx8蛋白的生物信息学分析借助生物信息学网络资源和软件,预测出猪Lhx8基因开放阅读框的长度为885bp,编码295个氨基酸残基,且两侧分别是243bp的5’UTR和553bp的3’UTR。通过对蛋白序列相似性检索,预测出猪Lhx8蛋白不含有信号肽,不含有跨膜区,定位于细胞核中,而且确定其含有LIM结构域。推测猪Lhx8蛋白具有DNA结合活性,参与染色质修饰和基因转录调控。3 Real-time PCR分析Lhx8基因在组织中的相对表达采用RT-PCR技术,分析了猪Lhx8基因在不同组织中的相对表达水平,结果表明：Lhx8基因在性腺、脑组织、免疫组织和骨骼肌中有表达,而在心脏、肝、脾、肾、脂肪和肺组织中没有表达,推测Lhx8基因在猪性腺、免疫器官和脑的发育和功能维持过程具有重要作用.4 qRT-PCR分析Lhx8基因在卵母细胞和附植前胚胎中的表达采用qRT-PCR技术,分析了猪Lhx8基因在卵母细胞和附植前胚胎各个时期的表达水平差异,结果表明：Lhx8基因在GV期到4-细胞期的表达水平显著升高,并且在4-细胞期达到最高水平,从4-细胞期开始到囊胚阶段,该基因的分泌水平逐渐降低。更多还原

【Abstract】 Lhx8 is a member of the LIM-homeobox transcription factor family which encodes a LIM homeodomain transcriptional regulator preferentially expressed in germ cells and is a critical regulator of mammalian oogenesis. The cDNA cloning and preliminary analysis of Lhx8 gene not only provides primary information for better understanding the biological functions of Lhx8 in pig, but also for the establishment of animal model to investigate human diseases in ovary.In this study, using in silico approach combined with reverse transcription polymerase chain reaction (RT-PCR) and 5’rapid amplification of cDNA ends (5’RACE), we cloned the full-length cDNA of Lhx8. Bioinformatics methods are adopted to predict the structure and function of Lhx8 protein, and also to construct a phylogenetic tree to reveal the evolutionary relationship of various species. To check relative expression levels of Lhx8 mRNA in various porcine tissues and preimplantation embryos, the RT-PCR and qRT-PCR was performed.1 cDNA cloning and sequencing of porcine Lhx8 geneThe full-length cDNA sequence of human Lhx8 gene served as an electronic probe, which was submitted to generate BLAST reciprocal best hits from dbEST and UniGene database, and about 3 EST sequences were retrieved. Using DNAstar-SeqMan program, these ESTs were assembled into two contigs. Based on the two contigs, the gene specific primers were designed by Oligo 6.0 for RT-PCR and RACE-PCR, which were carried out not only to check the validity of in silico approach, but also to obtain the full-length sequence of porcine Lhx8 cDNA (1681bp). The sequence data were submitted to the GenBank databases under accession No. FJ587986.2 Bioinformatics analysis of Lhx8 proteinUsing bioinformatics network resources and relevant softwares, we predict that Lhx8 cDNA has a 885 bp open reading frame (ORF) flanked by a 243 bp 5UTR and a 550 bp 3UTR. The deduced amino acid sequence has 295 residues, which contains no signal peptide, typical hydrophobic regions and transmembrane region, but is a nucleoprotein residing in nucleolus. A Conserved Domain Database alignment reveals that Lhx8 protein contains two tandemly repeated LIM domains, which are characterized by cysteine-rich, double-zinc finger motifs and a diatinct homeodomain.3 RT-PCR analysis of mRNA tissue-specific expression profilesMultitissue RT-PCR with RNA derived from adult tissues shows that Lhx8 is expressed in brain, skeletal muscle, ganod and immunity tissues. Other tissues, such as liver, heart, lung, spleen, kidney and lymph are not detected.4 qRT-PCR analysis of the Lhx8 mRNA level in oocytes and preimplantation embryosIn preimplantation embryos, Lhx8 mRNA was detected in all of granulocytes, oocytes, 4-cell embryos,8-cell embryos, morula and blastula. Its expression profiling revealed that its mRNA levels were firstly increased from GV stage oocytes to 4-cell stage embryos and highest in 4-cell stage embryos, and then gradually decreased until the blastocyst stage.更多还原