【作者】 徐学文；

【导师】 刘榜；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2007， 博士

【摘要】 近年来，猪骨骼肌转录谱的研究取得了较大进展，获得了大量差异表达EST，但是，如何建立这些差异表达EST与经济性状表型差异的联系，并利用这些序列信息实现对农业动物经济性状的改良则是今后很长一段时间内需要解决的重大课题。而首先需要解决的一个问题就是通过EST克隆获得相应基因的cDNA信息，并研究基因的功能和相互调控关系。本研究以本实验室前期转录组研究发现的三条猪背最长肌组织中差异表达EST为基础，开展了候选基因的克隆、定位及其功能研究工作，得到如下结果：1．以差异表达EST为基础，结合生物信息学与实验验证克隆获得了猪PPP2R5A、CSRP1和CSRP2基因包含完整CDS的cDNA序列以及CSRP3基因和LDB1基因的全长cDNA序列。2．利用SCHP和RH克隆板对上述5个基因及PP2A相关的另6个基因共11个基因进行了染色体定位，结果如下：PPP2CA定位于SSC 2q21-qter，PPP2CB定位于SSC 15q，PPP2R1A定位于SSC 6q12-q21，PPP2R1B定位于SSC 9p21，PPP2RSA定位于SSC 9q26，PPP2R5B定位于SSC 2p14-p17，PPP2R5D定位于SSC 7q11-q12，CSRP1基因定位于SSC 10，CSRP2基因定位于SSC5，CSRP3基因定位于SSC2p14-p17，LDB1基因定位于SSC14。3．利用QPCR发现猪PPP2R5A基因在心、肝、脾、肺、肾、肌肉、脂肪、小肠组织中广泛表达，但在肌肉组织中表达量明显高于其他组织，并在脂肪组织中高表达；同时发现该基因在通城猪和长白猪胎儿背最长肌中呈上调表达趋势，且在33天和65天，两品种间表达差异显著。4．通过RT-PCR分析发现CSRP1基因和CSRP2基因均呈广泛表达特征，而CSRP3基因仅在横纹肌中表达，定量PCR显示该基因在通城和长白猪肌肉发育过程中上调表达，但是两品种间基因表达差异不显著；推导了猪CSRP家族三个基因的氨基酸序列，构建了该家族基因的系统进化树，并结合氨基酸同源性推测此家族三个基因具有共同的祖先，CSRP1基因与CSRF2基因在结构、功能和进化关系上更为接近，而CSRP3基因则相对独立。5．克隆获得了猪CSRP3基因6293 bp的DNA序列，推测了该基因的基因组结构以及剪接位点信息；并克隆了该基因部分启动子序列，预测发现存在典型的“TATA”Box和E-Box，并包含MEF2、RAR-beta及HNF3-like等转录因子结合位点；发现了猪CSRP3基因的13个潜在SNP位点，并发现可能存在两种较为稳定的单倍型，进一步利用TaqI PCR-RFLP方法对C466T多态位点分析发现，该位点不同基因型频率在国内外品种之间存在显著差异。6．发现猪LDB1基因5’UTR、3’UTR及CDS区中均存在选择性剪接。其中位于CDS区中的一个选择性剪接造成其编码蛋白缺少了46个氨基酸，并进一步发现这种选择性剪接体在肺脏中具有较高表达，其次在肌肉组织中可检测到表达；成功构建了该基因两种转录本的pEGFP-N1融合表达载体，通过转染并利用激光共聚焦观察发现两种转录本均呈现“核质”共定位特征。7．成功克隆了猪LDB1基因上游约2.6 kb的启动子序列，并构建了11个pGL3报告基因载体，转染猪IBRS-2肾细胞，通过双荧光素酶报告基因系统检测发现了对于该基因转录活性起重要调节作用的三个保守区域，通过分析推测SP1、NF-KappaB等转录因子可能参与LDB1基因的转录激活，而ARP-1和TtK等转录因子可能参与对LDB1基因的转录抑制。8．综合分析发现，猪PPP2R5D基因及LDB1基因共有三个位点的选择性剪接不符合“GT-AG”规则，并推测这种剪接现象可能与剪接位点附近存在的短重复序列相关。以上研究工作是本室关于猪骨骼肌基因组学研究的继续和延伸，将为猪功能基因组研究增添新的材料，并为建立差异表达基因与产肉性状表型差异的联系奠定基础。更多还原

【Abstract】 Recent advance in porcine skeletal muscle profile obtained a number of differentially expressed ESTs. In a long period from now, the primary issue will be establishing the relationship between these ESTs and phenotypic difference of economic traits, and translating genomic sequences into enhanced value of the phenotypes. The first challenge is to obtain the corresponding gene based on these ESTs, study their function, and discover the mutual regulatory relationship.Based on three differentially expressed ESTs discovered in our previous transcriptome study of porcine skeletal muscle, the cloning, mapping and functional study of candidate gene had been done, and the results obtained were as follows:1. Combining bioinformatics and experiments validation, the cDNA sequences including complete CDS were obtained for porcine PPP2R5A, CSRP1 and CSRP2 respectively. The full-length cDNA sequence of porcine CSRP3 and LDB1 were also obtained based on corresponding differential expressed ESTs.2. The SCHP and RH panel were used to assign five genes mentioned above and another six PP2A-related genes, and obtained their chromosome location information as follows: PPP2CA to SSC 2q21-qter, PPP2CB to SSC 15q, PPP2R1A to SSC 6q12-q21, PPP2R1B to SSC 9p21, PPP2R5A to SSC 9q26, PPP2R5B to SSC 2pl4-pl7, PPP2R5D to SSC 7q11-q12, CSRP1 to SSC 10, CSRP2 to SSC5, CSRP3 to SSC 2p14-p17, and LDB1 to SSC14.3. QPCR assay indicated that PPP2R5A mRNA expressed widely in porcine heart, liver, spleen, lung, kidney, skeletal muscle, adipose, and small intestine, with the most abundance in skeletal muscle, followed by adipose. Furthermore, PPP2R5A mRNA shows an up-regulated trend during fetal skeletal muscle development of Tongcheng and Landrace pigs. The expression level of porcine PPP2R5A mRNA show significant difference between two pig breeds at both stages of 33-day old and 65-day old embryos.4. RT-PCR assay indicated that CSRP1 and CSRP2 mRNA distributed in all the tissues we detected, however, CSRP3 were only expressed in striated muscle. QPCR assay showed that CSRP3 was up-regulated during skeletal muscle development in Tongcheng and Landrace pigs. We deduced the amino acid sequences and constructed phylogenic tree of the CSRP family members. Combing the similarity information of the amino acid sequences and the cluster results, we deduced that the three members originated from the same ancestor. CSRP1 shows much more similarity with CSRP2 in structure and function, and they have a close evolutionary relationship, but CSRP3 is a separated member.5. We obtained 6293 bp DNA sequence of porcine CSRP3, speculated the genomic structure and analyzed the splicing sites information. We further cloned partial promoter sequence of CSRP3, which contained typical "TATA" box, "E-Box", and transcriptional factor binding sites, including MEF2, RAR-beta and HNF3-like. Thirteen potential SNPs and two possible haplotypes were discovered in CSRP3 DNA, TaqⅠPCR-RFLP assay were further conducted to detect the C466T polymorphic site, and significant difference in genotype frequency between indigenous and exotic pig breeds were discovered.6. Alternative splicing in the 5’UTR, 3’UTR and CDS of porcine LDB1 were detected. One alternative splicing in CDS produced a protein lacking 46 amino acids, which was found to be relatively high expressed in lung, followed by skeletal muscle. We successfully constructed two fused-expression vectors of pEGFP-N1 and two variants of porcine LDBl respectively, and found the dual location pattern in nucleus and cytoplasm of these two variants by observation and analyzing using Laser Scanning Confocal Microscope after transfection.7. The 2.6 kb upstream promoter of porcine LDB1 was obtained and used to construct pG13 reporter vectors. Eleven vectors were obtained and transfected into porcine IBRS-2 cell. Through dual-luciferase reporter assay, we identified three conserved regulatory region in LDB1 promoter, which were used for transcription factor binding sites prediction. SP1 and NF-KappaB were speculated to be involving in transcriptional activation, and ARP-1 and TtK in transcriptional repression.8. These unusual alternative splicing sites detected in porcine PPP2R5D and LDB1 were not accordant with the general "GT-AG" rule, but were speculated to be connected with the short repeat elements around the splicing sites.This research is the extension of our previous study in porcine skeletal muscle development, which will add new materials for porcine functional genomic research, and lay a foundation for establish relationship between these ESTs and phenotypic difference of meat production traits.更多还原