【作者】 徐德全；

【导师】 熊远著；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2005， 博士

【摘要】 骨骼肌是动物体内最丰富的组织,占肉用动物体重的45%～60%,其生长发育是肉用家畜最重要的性状之一。不同猪种由于来源、长期所处的地理环境以及所接受的饲养方式和选育性状重点的不同,从而形成了各自的种质特性,使其在肌肉相关性状方面存在较大的差异。两个具有不同性状的亲本杂交产生的杂种在肌肉生长、肌肉品质等方面也往往超过其双亲,产生杂种优势的现象。随着人们对基因结构和功能的研究深入,人们已经认识到这些差异及杂种优势现象应是基因差异表达的结果。所以本研究以猪背最长肌为材料,利用抑制消减杂交技术构建了不同猪种（长白与梅山）以及猪杂交亲代与子代（大白与长大）背最长肌间的正反向消减cDNA文库,进行了差异表达基因的筛选、克隆和鉴定,取得了如下结果: 1.以管家基因G3PDH作为消减指标,对每个消减文库的消减效率进行了检测,结果表明4个消减文库的消减效率都达2⁵倍以上,有的甚至高达2¹⁰倍,这同时也表明了某些特异于两种肌肉组织的差异表达基因的富集效率也达2⁵倍以上,说明所构建的消减cDNA文库质量很好。 2.从以梅山猪为Tester、长白猪为Driver和以杂交母本大白猪为Tester、杂种F1长大猪为Driver的消减cDNA文库中我们分别随机挑取了800多个克隆。利用PCR技术进行鉴定,分别获得了759和773个有效阳性克隆,插入片段长度主要分布于150～750bp之间。 3.利用正向消减cDNA、反向未消减cDNA、反向消减cDNA作探针对两消减cDNA文库中的阳性克隆进行了高通量筛选。在以梅山猪为Tester、长白猪为Driver的消减cDNA文库中,我们选取了27个差异表达克隆进行分析,发现共代表14个ESTs,4个在猪中为已知基因,10个在猪中为未知基因,其中7个与人等其他物种的基因有高同源性,3个未找到明显的同源性。在以杂交母本大白猪为Tester、杂种F1长大猪为Driver的消减cDNA文库中,我们选取了24个差异克隆进行分析,发现共代表12个ESTs,5个在猪中为已知基因,7个在猪中为未知基因,但分别与人等其他物种的基因有高的同源性。并利用含内标化的半定量RT-PCR技术进行了验证,结果表明大部分为差异表达。 4.将电脑克隆和SMART技术相结合,成功克隆了7个差异表达全长基因:（1）CCNG1,GenBank登录号为AY974246,cDNA全长2359bp,ORF为888bp;（2）EEF1A,cDNA全长1777bp,ORF为1389bp;（3） ETFB-like,cDNA全长898bp,ORF为765bp;（4） HUMMLC2B,GenBank登录号为AY754870,cDNA全长681bp,ORF为510 bp;（5） PGK1,GenBank登录号为AY677198,cDNA全长1789bp,ORF为1254bp;（6） TXNIP,cDNA全长1880 bp,ORF为1176 bp;（7） CKM,更多还原

【Abstract】 Skeletal muscle comprises as much as 45%⁶0% of an animal’s body mass. Hence there is much interest in understanding the development, physiology and metabolism of this tissue. Different pig breeds due to the difference of origin, local environment, feeding and selected traits have different intrinsic features, and there is a large difference between their muscle traits. When two breeds that have different traits are crossed, F1 hybrids tend to demonstrate hybrid vigor and have superior muscle traits. Further understanding on gene structure and function has indicated that the trait difference and heterosis are in fact the external exhibition of gene expression and regulation. Thus, we constructed forward and reverse subtracted cDNA libraries between Longissimus muscles from Meishan and Landrace pigs, Fl hybrids Landrace X Yorkshire and their female parents Yorkshire by suppression subtractive hybridization （SSH） technique in order to clone and identify the differentially expressed genes responsible for the trait difference and heterosis. The findings are as follows:1. A housekeeping gene, G3PDH, was used to estimate the subtraction efficiency. In each subtracted cDNA library, G3PDH was subtracted very efficiently at more than 25 folds, and some even near 2¹⁰ folds, indicating that some differentially expressed genes were also enriched at the same folds and the subtracted cDNA libraries were very successful.2. More than 800 clones were randomly selected from each subtracted cDNA library. By PCR analyses, 759 and 773 positive clones were isolated from the subtracted cDNA libraries, by Meishan as Tester, Landrace as Driver and Yorkshire as Tester, Landrace×Yorkshire as Driver, respectively. Most of all plasmids in the clones contain 150⁷50 bp inserts.3. By forward subtractive cDNA, reversal unsubtractive cDNA and reversal subtractive cDNA as probes, high-throughput screening was carried out. In the subtracted cDNAlibrary by Meishan as Tester and Landrace as Driver, 27 differential expressed clones were obtained. Further analyses showed they represent 14 porcine ESTs. Of 4 are known in pig, 7 are unknown in pig but have a high similarity with human and other species genes, and 3 have no significant similarity with known genes. In the subtracted cDNA library by Yorkshire as Tester and LandracexYorshire as Driver, 24 differential expressed clones were obtained. Further analyses showed they represent 12 porcine ESTs. Of 5 are known in pig, 7 are unknown in pig but have a high similarity with human and other species genes. In addition, most of these differential expressed ESTs were subjected to further identification by semi-quantitative RT-PCR analysis with an inner control4. Using in silico cloning and SMART technology, we obtained 7 full-length cDNA: （1） CCNGl, GenBank accession number AY974246, full-length cDNA 2359 bp, ORF 888bp; （2） EEF1A, full-length cDNA 1777 bp, ORF 1389 bp; （3） ETFB-like, full-length cDNA 898 bp, ORF 765bp; （4） HUMMLC2B, GenBank accession number AY754870, full-length cDNA 681bp, ORF 510 bp; （5） PGK1, GenBank accession number AY677198, full-length cDNA 1789 bp, ORF 1254 bp; （6） TXNIP, full-length cDNA 1880 bp, ORF 1176 bp; （7） CKM, GenBank accession number AY754869, full-length cDNA 1572 bp, ORF 1146 bp. CCNGl, EEF1A and ETFB-like demonstrate high-level expression in Meishan pigs. HUMMLC2B, PGK1, TXNIP and CKM demonstrate down-regulated expression in Fl hybrid LandracexYorshire pigs.5. Using CLUSTAL W and some related softwares, we analyzed the gene structure, protein structure, conserved motifs and phylogenetic relation of genes CCNGl, EEFl A, ETFB-like, HUMMLC2B, PGK1, TXNIP and CKM. In addition, the corresponding phylogenetic trees were constructed.6. Using semi-quantitative RT-PCR with an inner control, we carried out the temporal and spatial expression analyses in different pig breeds （Yorkshire; MeishanxYorkshire; YorkshirexMeishan; LandracexYorkshire; Landrace; Meishan; Duroc; Tongcheng）, different development stages （embryo; neonate; two months old; four months old; six months old） and different tissues （heart; liver; spleen; lung; kidney; pancreas; small intestine; stomach; uterus; ovary; testis; adipose tissue; biceps femoris; semispinalls capitis; longissimus dorsi）.7. Using PCR-Walking, we obtained 3 genes’ full genomic sequences containing all introns and 2 genes’ partial genomic sequences, and analyzed their polymorphisms in different pig breeds. （1） CCNGl, 7491 bp, containing 6 introns and 7 exons. In whole genomic sequence, we identified 23 SNPs. Of 4 are transversion mutations, 17 aretransition mutations, 1 is insertion mutation and 1 is deletion mutation. In addition, there is an "AATGAATCTGTGTACTGAATC" insertion in intron 2 of Yangxin pig’s CCNG1. （2） HUMMLC2B, 2936 bp, GenBank accession number AY870651, containing 6 introns and 7 exons. In exon 4 to exon 7 （containing partial exon 4 and exon 7）, we identified 13 SNPs. Of 4 are transversion mutations, 9 are transition mutations. In addition, there is a GA—TC mutation in intron 4. （3） TXNIP, 3331 bp, containing 7 introns and 8 exons. In whole genomic sequence, we identified 11 SNPs. Of 3 are transversion mutations, 8 are transition mutations. （4） PGK1, 1105 bp （containing partial exon 9 and exon 11, complete intron 9, 10 and exon 10）, we identified 8 SNPs. Of 1 is transversion mutations, 7 are transition mutations. （5） KIAA1717, 2032 bp （3’ UTR）, GenBank accession number AY900164, we identified 3 SNPs. all are transition mutations. The locations of splice donor/acceptor sites in all introns follow the "GT/AG" rule.8. Using PCR-RFLP, we detected 4 SNPs in different pig populations and observed associations with traits in YorkshirexMeishan F2 population. The results showed: MS60-2-iVco I -RFLP is significant association with intramuscular fat （p<0.01） and water moisture （p<0.01）. MS60-345-l-IspE I -RFLP is significant association with buttock backfat thickness （p<0.01）, shoulder backfat thickness （p<0.05）, 6-7th thorax backfat thickness （p<0.05）, average backfat thickness （p<0.05） and water moisture （p<0.05）. DB245-2-Msp I -RFLP is significant association with loin eye width （p<0.05）, loin eye area （p<0.05）, longissimus doris pH （p<0.05）, biceps femoris pH （p<0.01）, drip loss rate （p<0.01）, water holding capacity （p<0.01）, longissimus doris color value （p<0.01）, biceps femoris color value （p<0.05） and water moisture （p<0.01）. DB37-Msp I -RFLP is significant association with fat meat percentage （p<Q.Q5）, ratio of lean meat vs. fat meat （p<0.05）, thorax-waist backfat thickness （p<0.05）, buttock backfat thickness （p<0.05）, average backfat thickness （p<0.05）, loin eye height （p<0.05）, loin eye area （p<0.05）, carcass length to 1st spondyle （p<0.01）, carcass length to Is’ rib （p<0.05）, longissimus doris marbling （p<0.05）, biceps femoris marbling （p<0.01）, intramuscular fat （p<0.01）, and water moisture （p<0.01）.更多还原