【作者】 任竹青；

【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， 2007， 博士

【摘要】 近年来的研究表明，脂肪组织除对组织器官起保护作用、保持体温、保护内脏器官以及缓冲机械压力等作用外，它还具有重要的内分泌功能，主动参与能量代谢的平衡。长期以来，中外猪种由于遗传背景、长期所处的地理环境以及所接受的饲养方式和选育方式等的不同，从而形成了各自的种质特性，使其在脂肪相关性状方面存在很大的差异。而中外猪种杂交产生的杂种往往在脂肪相关性状等方面表现优于双亲的特点，产生杂种优势。这些差异以及杂种优势现象的产生应该与基因差异表达有关。因此，本研究以猪背膘组织为试验材料，利用mRNA差异显示技术分离大白×梅山猪正反向杂交组合亲子代间差异表达基因，并对这些基因的功能做了研究，取得了如下结果：1．利用mRNA差异显示技术分别对120日龄和180日龄大白猪×梅山猪杂交组合亲子代间基因表达情况进行了分析，发现杂种与亲本之间存在8类基因表达模式：P1，表达一致型；P2，单亲一致型；P3，正交或反交单亲一致型；P4，杂种特异型；P5，正交或反交特异型；P6，正交或反交沉默型；P7，双亲特异表达型；P8，单亲沉默型。试验中共检测了140对引物组合（7条锚定引物和20条随机引物）的差异显示结果，观察了近3000条EST条带，其中近2000条可在重复PCR中出现。此外，对120日龄和180日龄杂种与亲本间基因表达模式进行了比较，结果表明杂种与亲本间基因表达模式在不同的时期是变化的。2．共获得60条差异显示EST，其中120日龄组39条，180日龄组21条。通过BLAST比对发现，大部分EST与GenBank数据库中已知序列无同源性，采用半定量RT-PCR方法对这些EST进行验证，结果表明大部分表现差异，也有相当一部分为假阳性，将部分EST提交GenBank，获得登陆号CV507051-CV507087。3．结合电脑克隆和RACE技术，成功克隆了7个差异表达基因全长：（1）ACL，cDNA全长4378bp，编码1076个氨基酸；（2）SMPX，cDNA全长863bp，编码86个氨基酸；（3）ANGPTL4，cDNA全长1847bp，编码412个氨基酸；（4）IDH1，cDNA全长2264bp，编码402氨基酸；（5）IDH3β，共3个转录本，IDH3β₁cDNA全长1247bp，编码383个氨基酸；IDH3β₂ cDNA全长1540bp，编码385个氨基酸；IDH3β₃ cDNA全长1445bp，编码385个氨基酸；（6）IDH3γ，cDNA全长1346bp，编码392个氨基酸；（7）RPL28，cDNA全长513bp，编码137个氨基酸。4．利用DNAStar、CLUSTALW等软件对猪ACL、SMPX、ANGPTL4、IDH1、IDH3β、IDH3γ和RPL28基因结构、所编码蛋白质结构和功能等特征进行了预测和分析，并构建了分子系统进化树。5．采用Real-time PCR技术对这7个差异表达基因在大白猪×梅山猪杂交组合亲子代间基因表达情况进行了进一步的鉴定，结果表明与半定量RT-PCR的检测结果相比，差异表达的趋势是一致的，其中ACL、IDH3β以及RPL28基因在杂种大梅和梅大猪中增强表达；SMPX、ANGPTL4、IDH1以及IDH3γ基因普遍在大白猪中高表达，其中SMPX基因在杂种梅大猪中几乎不表达，IDH1基因在杂种大梅猪中表达量最低。并对这些基因在不同组织（心、肝、脾、肺、肾、胃、小肠、子宫、卵巢、背膘以及眼肌）的表达情况做了分析，结果显示它们在大多数组织中表达，且表现不同的表达特征。6．克隆了ANGPTL4、IDH1、IDH3β以及IDH3γ基因的全部内含子和其它基因的部分内含子序列，并分析了这4个基因的基因组结构以及序列在不同猪群中的多态性：（1）ANGPTL4基因包含7个外显子和6个内含子，仅发现2处发生突变，一处为第三内含子2737位G→A的转换突变，另一处为第六外显子203位C→T的转换突变；（2）IDH1基因包含10个外显子和9个内含子，共发现9处发生突变，分别为第二外显子1处（T1112C），第六内含子5处（G13194T、C13228T、C13336T、C13422T和A13477G），第十外显子3处（T20531A、G20543A和第20534bp处C插入突变）；（3）利用猪IDH3β₁或IDH3β₂ cDNA序列对比发现，该基因的内含子和外显子组成与预测的一致，即12个外显子和11个内含子；利用猪IDH3β₃ cDNA序列与之对比，发现该基因包含13个外显子和12个内含子，前11个外显子和前10个内含子的组成和大小完全一致，共发现8处发生突变，分别为第一内含子1处（G63A），第二外显子1处（G187C），第二内含子2处（G287C和C336T），第六外显子1处（G2732A），第十一内含子2处，分别为A4277G和第4413bp处25个碱基的小卫星序列；（4）IDH3γ基因包含10个外显子和9个内含子，发现第二内含子存在一段微卫星序列（重复单位为“GT”）。7．对ACL、ANGPTL4、IDH3β以及IDH3γ基因共6个多态性位点在不同猪群中进行了基因分型，结果表明这些位点在这些猪群中具有丰富的多态性，并在2000年和2003年大白×梅山猪F2代资源家系中与重要生产性状进行了关联分析，结果表明：（1）猪ACL基因第24内含子插入突变所形成的不同基因型在瘦肉率、肥肉率、花油重、6-7胸椎间背膘厚、背最长肌pH、骨二头肌pH以及失水率性状存在显著（P＜0.05）或极显著差异（P＜0.01）；（2）猪ACL XhoI-RFLP基因型不同时，皮率、骨率、屠宰率、板油重、花油重、背最长肌pH、骨二头肌pH、头半棘肌pH以及系水率性状存在显著（P＜0.05）或极显著差异（P＜0.01）；（3）猪ANGPTL4基因第3内含子PCR-SSCP不同基因型在眼肌面积、肌内水分、肌内脂肪以及背最长肌大理石纹评分性状存在显著差异（P＜0.05）；（4）猪IDH3β基因小卫星不同基因型在骨率、眼肌面积、肌内水分、肌内脂肪以及头半棘肌pH值性状存在显著（P＜0.05）或极显著（P＜0.01）差异；（5）猪IDH3β基因5’侧翼区插入突变所形成的不同基因型在板油重、眼肌面积、骨二头肌pH值、肌内脂肪和肌内水分性状存在显著差异（P＜0.05）；（6）猪IDH3γ基因微卫星的不同基因型在屠宰率、肋骨数、平均皮厚以及肌内水分性状存在显著（P＜0.05）或极显著差异（P＜0.01）。8．采用TAIL-PCR技术分别扩增了ACL基因936bp和IDH3β基因2447bp的5’侧翼序列，序列分析发现，猪ACL基因的5’侧翼序列富含G+C，高达61.75％，未发现“TATA”盒；猪IDH3β基因的5’侧翼序列同样缺失“TATA”盒，也未发现“CAAT”盒，不同的是其A+T含量相对较高，达55.8％。9．采用5’端缺失策略，结合启动子预测结果，分别构建了8个ACL基因和19个IDH3β基因启动子区重组子，将其转染猪PK15细胞系，并利用荧光素酶双报告基因系统检测了它们的活性，结果表明：在所构建的猪ACL基因5侧翼序列的8个重组子中，除pGL-ACL27和pGL-ACL15外，其它重组子的荧光素酶活性与阴性对照均达到极显著水平（P＜0.01），表明它们均具有启动子活性，pGL-ACL919活性最强，到pGL-ACL679活性下降了将近3倍，说明从-919bp到-679bp区域可能存在负的调控位点，从pGL-ACL621到pGL-ACL73活性有小幅度下降，到pGL-ACL27活性降到与阴性对照水平无显著差异，表明维持该启动子的基本活性区域位于-73bp到+77bp之间；IDH3β基因启动子活性开始于pGL-IB82，然后虽然有些波动，但是活性一直下降直到-164bp处，之后活性又开始升高，直到-279bp处活性达到最高，这些表明维持该启动子的基本活性区域位于-82bp到+16bp之间，从-82到-279之间的179bp区域具有最高的启动子活性。此外，我们还检测了IDH3β基因5’侧翼区插入突变对启动子活性的影响，结果显示携带该插入突变的启动子具有更高的启动子活性。更多还原

【Abstract】 Fat tissue is not only the main location saving up fat, which can store energy, maintain animal heat, protect vital organisms, lighten physical pressure but also an endocrine organ.Due to the difference of origin, genetic background, local environment, feed manner and artificial selection between Chinese indigenous and foreign commercial pig breeds, they have different intrinsic features, and there is a large difference between their fattness traits.When these two breeds are hybridized, F1 hybrids tend to demonstrate hybrid vigor and produce heterosis. Further understanding on gene function and expression regulation has indicated that the difference traits performance and heterosis are in fact the external exhibition of differential genes expression and regulation. Thus, mRNA differential display technique was used to isolate and identify the differentially expressed genes between Large White and Meishan, and their F1 hybrids, Large White×Meishan and Meishan×Large White pigs in the present study, and the gene functions were also primarily analyzed. The results are as follows:1. The gene expression were analyzed between F1 hybrids, White×Meishan and Meishan×Large White, and their parents, Large White and Meishan pigs by mRNA differential display. Eight patterns of gene expression were observed at both four and six months old hybrids and their parents, which included: P1, bands detected in both hybrids and parents; P2, bands occurring in hybrids and one parent; P3, bands occurring in one hybrid and one parent; P4, bands visualized in only hybrids; P5, bands occurring in only one hybrid; P6, bands observed in parents and one hybrid; P7, bands occurring in only parents; and P8, bands occurring in only one parent. We also observed nearly 3000 bands in differentially displayed PAGE gel, and almost 2000 ones can be repreated in duplicate PCR. In addition, the comparison of gene expression patterns between 120 days old and 180 days old hybrids and their parents showed that the gene expression were varied.2. 60 differentially displayed ESTs in PAGE gel were cloned, including 39 ESTs at 120 days old and 21 at 180 days old, between hybrids and their parents. Most ESTs were not homologous to the sequences in GenBank database. The result of validation showed that a majority of ESTs were really differentially expressed, while a few of them weren’t by semi-quantitative RT-PCR. Some ESTs were subsequently deposited in GenBank and the accession numbers were CV507051-CV507087.3. We cloned the full-length cDNA of seven genes by rapid amplification of cDNA ends （RACE） in combination with in silico cloning, which included: （1）ACL, which has 4378bp and encodes 1076 amino acids; （2） SMPX, which has 863bp and encodes 86 amino acids; （3）ANGPTL4, which has 1847bp and encodes 412 amino acids; （4） IDH1, which has 2264bp and encodes 402 amino acids; （5） IDH3β, which has three isoforms, IDH3β₁（1247bp）, IDH3β₂（1540bp）, and IDH3β₃（1445bp）, and encode 383, 385 and 385 amino acids, respectively; （6） IDH3γ, which has 1346bp and encodes 392 amino acids; and （7） RPL28, which has 513bp and encodes 137 amino acids. 4. Using DNAStar, CLUSTAL W and some other related software, we analyzed the gene structure, protein structure and conserved motifs of these seven genes. In addition, the corresponding phylogenetic trees were constructed.5. The differential expression of these seven genes between F1 hybrids and their parents were further identification by real-time quantitative PCR. The result showed that the trend of differential gene expression was consonant with the result by semi-quantitative RT-PCR. The mRNA expression level of ACL, IDH3βand RPL28 was higher in F1 hybrids than in both Meishan and Large White pigs, whereas the mRNA of SMPX, ANPTL4, IDH1 and IDH3γwas more abundant in Large White than both two hybrids and Meishan pigs. The tissue distribution of these seven genes in heart, liver, spleen, lung, kidney, stomach, small intestine, uterus, ovary, backfat and longissmus drosi indicated that they were expressed in most tissues and displayed different expression patterns.6. We cloned all introns of four genes （ANGPTL4、IDH1、IDH3βand IDH3γ） and partial introns of other three genes among these seven genes, and analyzed the genomic structure and polymorphism of these 4 genes. The results are as follows: （1） ANGPTL4 gene contains seven exons and six introns. Only two mutations were found. One is G→A transversion in third intron and the other is C→T transversion in sixth intron; （2） IDH1 gene contains ten exons and nine introns. Nine mutations were found in total, including one in second exon （T1112C）, five in sixth intron （G13194T, C13228T, C13336T, C13422T and A13477G）, three in ten exon （T20531A, G20543A and C inserted mutation at 20534th bp）; （3） The genomic structure of IDH3β₁ and IDH3β₂ is organized in twelve exons, while that of IDH3β₃ contains thirteen exons separated by twelve introns. Eight mutations were found in total, including one in first intron （G63A）, one in second exon （G187C）, two in second intron （G287C and C336T）, one in sixth exon （G2732A） and two in eleventh exon （A4277G and 25bp mini-satellite）; and （4） IDH3γgene contains ten exons and nine introns. Micro-satellite （GT dinucleotide repeats） was found in second intron.7. Genotyping of a total of six polymorphic locus showed that there are abundant polymorphisms in various pig breeds. Association analysis was performed between polymorphisms and important product traits in Large White×Meishan F2 offspring, and the results showed: （1） For insertion-mutated polymorphism in 24th intron of ACL gene, significant effects were observed on LMP, FMP, CFW, RFT, pH（LD）, pH（BF） and WLR; （2） There are significant difference between ACL XhoI-RFLP genotypes and SP, BP, DP, LFW, CFW, pH（LD）, pH（BF）, pH（SC） and WHC; （3） Statistically significant association were found between ANGPTL4 PCR-SSCP in third intron and LEA, WM, IMF and MM1; （4） For minisatellite polymorphism of IDH3βgene, significant effects were observed on BP, LEA, WM, IMF and pH（SC）; （5） For insertion-mutated polymorphism in 5’flanking region of IDH3βgene, significant effects were observed on LFW, LEA, pH（LD）, IMF and WM; and （6） For microsatellite polymorphisms of IDH3γgene, significant effects were observed on DP, RNS, AST and WM.8. 936bp 5’flanking sequence ofACL gene and 2447bp 5’flanking sequence of IDH3βgene were obtained by using TAIL-PCR. The former lacks of TATA box and comprise high G+C content （61.75%）, whereas the latter lacks both TATA box and CAAT box with a percentage of 55.8 % A+T content.9. To determine the location of the promoter activity of pig ACL and IDH3β, we constructed 8 and 19 recombinants of progressively 5’-deleted DNA fragment linked to the pGL3 reporter, respectively. These recombinants were transiently transfected into PK15 cells. Transcriptional activity of ACL recombinants normalized by Renilla was significant difference with pGL3-Basic expect for construct -27 and -15 （P＜0.01）. Construct -919 contains highest activity. 3 times reduction of transcriptional activity from-919 to -679bp indicated that negative regulation factors located probably in this region. The activity started on construct -73 suggested the basal promoter activity was located within the -73 to +77bp region; Transcriptional activity of IDHβrecombinants was not significantly different between the recombinant -58 and pGL3-Basic. The activity started on construct -82, decreasing with the length of the fragment up to -164 in despite of a bit of fluctuation, and kept increasing from construct -164 up to -279. Thus, the basal promoter activity was located within the -82 to +16bp region, whereas the upstream 197bp conferred maximal transcriptional activity. The extension of the 5’flanking sequence up to -2435 diminished the promoter activity. In addition, the IDH3βpromoter with the inserted-mutation possessed higher activity compared with the wild one.更多还原