大白猪与梅山猪肝脏组织差异表达基因的分离以及基因功能的研究
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摘要
随着猪基因组研究工作的不断深入,与猪生产性状(生长、胴体与肉质性状)相关的很多新基因被证实。但相对猪的整个基因组来说,仍然有大量基因还未被发现。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选育方式等的不同,从而形成了各自的种质特性,使其在生产性状方面存在很大的差异。肝脏是机体最大的腺体,具有重要的生理功能,它在机体糖代谢、蛋白质代谢、脂肪代谢、维生素代谢和激素代谢过程中均具有重要的作用。而中外猪种间的品质差异可能与肝脏中的物质代谢有关。因此,本研究以猪肝脏组织为试验材料,利用mRNA差异显示技术分离大白猪和梅山猪肝脏组织的差异表达基因,对所获得的差异表达基因进行功能分析选出SMNDC1(Survival motor neuron domain containing 1)、HOXA5(Homeobox A5)、TIAF1(TGFB1-induced anti-apoptotic factor 1)、MYO18A(MyosinⅩⅧa)和POLDIP2(Polymerase(DNA-directed),delta interacting protein 2)基因,以及通过候选基因法获得的差异表达基因PHKG2(Phosphorylase kinase,gamma 2),对这些基因作了进一步研究,取得了如下结果:
1.利用mRNA差异显示技术对60日龄大白猪和梅山猪肝脏组织的基因表达情况进行了分析,试验中共检测了200对引物组合(10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1000多条可在重复PCR中出现。
2.共获得70条差异显示EST,通过BLAST比对发现,一些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,结果表明所获得的差异条带中大部分表现有差异,但也有一部分为假阳性。
3.采用Real-time PCR技术与半定量RT-PCR技术相结合的方法对这6个差异表达基因在大白猪和梅山猪肝脏组织的表达情况进行了进一步的鉴定,结果表明HOXA5、TIAF1、MYO18A以及POLDIP2基因在梅山猪肝脏组织中高表达;SMNDC1和PHKG2基因在大白猪肝脏组织中高表达。
4.结合生物信息学克隆、比较基因组学和RACE(Rapid amplification of cDNA end)技术,获得了6个差异表达基因的cDNA序列,除MYO18A基因外均包括完整的开放阅读框(Open reading frame,ORF),MYO18A基因所获得的cDNA序列包括部分编码区序列;(1)SMNDC1,获得cDNA序列1875bp,其中ORF为717bp,编码238个氨基酸,GenBank登录号为EU571478;(2)HOXA5,获得cDNA序列1395bp,其中ORF为813bp,编码270个氨基酸,GenBank登录号为EU_024116;(3)TIAF1,获得cDNA全长序列1633bp,其中ORF为348bp,编码115个氨基酸,GenBank登录号为EU872207;(4)MYO18A,共2个转录本,获得了MYO18A_1 cDNA序列6231bp,MYO18A_2 cDNA序列6186bp,两者
的区别在于MYO18A_2缺失第39外显子;(5)POLDIP2,获得cDNA序列2410bp,其中ORF为1107bp,编码368个氨基酸;(6)PHKG2,获得cDNA序列1469bp,其中ORF为1221bp,编码406个氨基酸,GenBank登录号为EU169240。另外,还获得了6个基因的基因组DNA序列,并分析了其基因组结构,基因的所有内含子和外显子的拼接位点都符合GT-AG规则。
5.利用DNAStar、CLUSTAL W等软件对猪差异表达基因SMNDC1、HOXA5、TIAF1、MYO18A、POLDIP2和PHKG2的基因结构、所编码的蛋白质结构以及功能等特征进行了预测和分析,并构建了分子系统进化树。
6.为了进一步研究这些基因的功能,根据差异表达基因已有的研究结果,采用Real-time PCR技术和半定量RT-PCR技术对候选基因进行了时空表达谱分析。结果显示,SMNDC1基因在大白猪和梅山猪背最长肌的发育过程中呈现了不同的表达模式。在大白猪出生后的背最长肌中的表达量随着日龄的增长而递减;而在梅山猪出生后背最长肌中SMNDC1的表达量随着日龄的增长而递增。HOXA5基因在大白猪出生后的背最长肌中的表达量随着日龄的增长而递减,在3d的表达量最高,之后随着日龄的增长而逐渐减少;TIAF1基因在大白猪出生后各时期的背最长肌中均有表达,表达量差异不显著。另外,对6个差异表达基因在不同组织(心脏、肝脏、脾脏、肺脏、肾脏、胃、小肠、子宫、卵巢、背最长肌以及脂肪)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。
7.SNPs的检测结果证实HOXA5基因有3个潜在的SNPs,其中一个可用于PCR-RFLP的SNP位点,即C1817A-Bpu1102 I-RFLP,位于内含子中;TIAF1基因有5个潜在的SNPs,其中一个可用于PCR-RFLP的缺失突变位点,即PCR-Eco47 I-RFLP位于外显子中;POLDIP2基因有6个潜在的SNPs,其中一个可用于PCR-RFLP的SNPs位点,即C306A-BsuR I-RFLP位于第三外显子中;PHKG2基因有3个可用于PCR-RFLP的SNPs位点,即位于第八外显子中的G785A-Mst I-RFLP和G866A-Tag I-RFLP,以及位于第九外显子中的G875A-Pst I-RFLP。
8.对HOXA5、TIAF1、POLDIP2以及PHKG2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、2003年和2004年大白×梅山猪F_2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HOXA5基因C1817A-Bpu1102 I-RFLP位点所形成的不同基因型在眼肌面积性状存在显著差异(P＜0.05);(2)猪TIAF1基因缺失突变位点(PCR-Eco47 I-RFLP),不同基因型在胴体长性状存在显著差异(P＜0.05),在背最长肌大理石纹评分和肌内脂肪含量这2个性状存在极显著差异(P＜0.01);(3)猪POLDIP2基因C306A-BsuR I-RFLP位点,不同基因型在胴体长性状存在极显著差异(P＜0.01),在肩部背膘厚和平均背膘厚性状存在显著差异(P＜0.05);(4)猪PHKG2基因G785A位点(PCR-Mst I-RFLP)多态性与瘦肉率呈极显著相关(P＜0.01),与内脂率呈显著相关(P＜0.05);G866A位点(PCR-Taq I-RFLP)多态性与瘦肥肉比例呈极显著相关(P＜0.01),与瘦肉率、内脂率、肩部背膘厚和平均背膘厚呈显著相关(P＜0.05);G875A位点(PCR-Pst I-RFLP)多态性与瘦肉率呈显著相关(P＜0.05)。
9.利用基因组PCR步移的方法从猪基因组中扩增了SMNDC1基因1242bp的5'侧翼序列,利用NNPP、MethPrimer、TFSEARCH等生物信息学软件对猪SMNDC1基因的核心启动子区、转录起始位点、CpG岛的分布以及与启动子相结合的转录因子进行了预测和分析。
10.采用5'端缺失策略,结合启动子预测结果,构建了10个SMNDC1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SMNDC1基因5'侧翼序列的10个重组子中,除pGL-SMNDC931和pGL-SMNDC349外,其它重组子的荧光素酶活性与阴性对照均达到显著水平(P＜0.05),表明它们均具有启动子活性。pGL-SMNDC688活性较强,到pGL-SMNDC349活性下降了3倍多,说明这一区域可能存在负的调控位点,从pGL-SMNDC349到pGL-SMNDC109活性升高,达到最大值,表明该区域可能存在正的调控位点。
With the development of porcine genomic studies,many genes associated with pig production traits have been identified,however,additional genes need be identified and further characterized.Due to the difference of origin,genetic background,local environment and artificial selection between Chinese indigenous and foreign commercial pig breeds,they have different intrinsic features.Meishan pigs(Chinese indigenous pigs) have lower lean meat content in their carcasses compared to Large White pigs(Western pigs),but the lean meat of Meishan pigs is of better quality.Liver is the most important organ in the procedure of substance metabolism of organism.Glycometabolism,lipid and protein metabolism were occurred in the liver.Phenotypic variance between the Large White and Meishan pigs may be related with substance metabolism which occurred in the liver.Thus,mRNA differential display technique was used to isolate and identify the differentially expressed genes in livers between Large White and Meishan pigs in the present study,and the gene functions were also primarily analyzed.Porcine differentially expressed genes SMNDC1(Survival motor neuron domain containing 1),HOXA5 (Homeobox A5),TIAF1(TGFB1-induced anti-apoptotic factor 1),MYO18A(MyosinⅩⅧa),POLDIP2(Polymerase(DNA-directed),delta interacting protein 2) and PHKG2 (Phosphorylase kinase,gamma 2) were choosen for further analyses of the potential functions.These main results were as follows:
1.The gene expression was analyzed in liver between 60 days old Large White and Meishan pigs by mRNA differential display.We also observed nearly 3000 bands in differentially displayed PAGE gel,and almost 1000 ones can be repeated in duplicate PCR.
2.There are 70 differentially displayed ESTs in PAGE gel.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.
3.The differential expression of the six genes between Large White and Meishan pigs were further identification by real-time quantitative PCR and semi-quantitative RT-PCR.The result showed that the mRNA expression level of HOXA5,TIAF1, MYO18A and POLDIP2 gene was higher in Meishan pigs than Large White pigs, whereas the mRNA expression level of SMNDC1 and PHKG2 gene was more abundant in Large White pigs than Meishan pigs.
4.We cloned the cDNA sequences of six genes by electronic cloning,comparative genomic technology and rapid amplification of cDNA ends(RACE).(1) SMNDC1, obtained cDNA sequence 1875bp,the ORF is 717bp and encodes 238 amino acids, GenBank accession number EU571478.(2) HOXA5,obtained cDNA sequence 1395bp,the ORF is 813bp and encodes 270 amino acids,GenBank accession number EU_024116.(3) TIAF1,obtained cDNA sequence 1633bp,the ORF is 348bp and encodes 115 amino acids,GenBank accession number EU872207.(4) MYO18A, which has two isoforms,MYO18A_1(6231bp) and MYO18A_2(6186bp),the difference of the two transcripts is the exon 39.(5) POLDIP2,obtained cDNA sequence 2410bp, the ORF is 1107bp and encodes 368 amino acids.(6) PHKG2,obtained cDNA sequence 1469bp,the ORF is 1221bp and encodes 406 amino acids,GenBank accession number EU169240.The DNA sequences of these genes were obtained and the genomic structures were analyzed.All splice sites of exon/intron of these genes conformed to the GT/AG rule.
5.Using DNAStar,CLUSTAL W and some other related software,we analyzed the gene structure,protein structure and conserved motifs of these six genes.In addition, the corresponding phylogenetic trees were constructed.
6.In order to study potential functions of target genes,temporal and spatial expression profiles were analyzed using real-time PCR.It was demonstrated that during seven stages of skeletal muscle development in Large White pigs after birth,the mRNA expression levels of the SMNDC1 gene decreased gradually with age.But in Meishan pigs,the SMNDC1 gene mRNA expression was up-regulated from postnatal 3 days to 150 days.The mRNA expression levels of the HOXA5 gene decreased gradually with age and the TIAF1 gene had no significant differences during the skeletal muscle development.The tissue distribution of the six 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.
7.Single nucleotide polymorphisms(SNPs) in the six genes were detected.It was demonstrated that the HOXA5 gene contained one SNP for PCR-RFLP (C1817A-Bpu1102 I-RFLP),which was located in the intron among three potential polymorphism sites following screening the genome DNA sequence.The TIAF1 gene contained a 6 base-pair deletion mutation for PCR-RFLP(PCR-Eco47 I-RFLP), which was located in the exon among five potential polymorphism sites.The POLDIP2 gene contained one SNP for PCR-RFLP(C306A-BsuR I-RFLP),which was located in the 3~(th) exon among six potential polymorphism sites.The PHKG2 gene included three SNPs for PCR-RFLP(G785A-Mst I-RFLP,G866A-Tag I-RFLP and G875A-Pst I-RFLP),which were respectively located in the 8~(th)(for the first two sites) and 9~(th) exons.
8.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 F_2 offspring, and the results showed:(1) For C1817A-Bpu1102 I-RFLP polymorphism of HOXA5 gene,significant effects were observed on LEA(P＜0.05);(2) There are significant difference between TIAF1-Eco47 I-RFLP polymorphism and CL(P＜0.05),MM1 and IMF(P＜0.01);(3) For C306A-BsuR I-RFLP polymorphism of POLDIP2 gene, significant effects were observed on CL(P＜0.01),SFT and AFT(P＜0.05);(4) For the PHKG2 gene,highly significant associations in the G785A(PCR-Mst I-RFLP) site was detected between genotypes with LMP(P＜0.01) and significant association between genotypes with IFR(P＜0.05).In the G866A(PCR-Taq I-RFLP) site,there were highly significant association between genotype with RLF(P＜0.01) and significant association with LMP,IFR,SFT and AFT(P＜0.05).In the G875A (PCR-Pst I-RFLP) site,there were significant association with LMP(P＜0.05).
9.Genome PCR walking technique was used to clone the proximal promoter region of porcine SMNDC1 gene,and we obtained a fragment of 1242bp.Bioinformatics approaches were adopted to analyze the proximal promoter region of porcine SMNDC1 gene,and the putative binding sites of transcription factors in the regulatory region of the gene were analyzed by online software.
10.To determine the location of the promoter activity of porcine SMNDC1 gene,we constructed 10 recombinants of progressively 5'-deleted DNA fragment linked to the pGL3 reporter,respectively.These recombinants were transiently transfected into PK15 cells.Transcriptional activity of SMNDC1 recombinants normalized by Renilla was significant difference with pGL3-Basic expect for construct 931 and 349 (P＜0.05).Construct 688 contains highest activity and 3 times reduction of transcriptional activity to 349 indicated that negative regulation factors located probably in this region.From the 349 to 109 enhanced the promoter activity by 5.5-fold indicated that positive regulation factors located probably in this region.
1.利用mRNA差异显示技术对60日龄大白猪和梅山猪肝脏组织的基因表达情况进行了分析,试验中共检测了200对引物组合(10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1000多条可在重复PCR中出现。
2.共获得70条差异显示EST,通过BLAST比对发现,一些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,结果表明所获得的差异条带中大部分表现有差异,但也有一部分为假阳性。
3.采用Real-time PCR技术与半定量RT-PCR技术相结合的方法对这6个差异表达基因在大白猪和梅山猪肝脏组织的表达情况进行了进一步的鉴定,结果表明HOXA5、TIAF1、MYO18A以及POLDIP2基因在梅山猪肝脏组织中高表达;SMNDC1和PHKG2基因在大白猪肝脏组织中高表达。
4.结合生物信息学克隆、比较基因组学和RACE(Rapid amplification of cDNA end)技术,获得了6个差异表达基因的cDNA序列,除MYO18A基因外均包括完整的开放阅读框(Open reading frame,ORF),MYO18A基因所获得的cDNA序列包括部分编码区序列;(1)SMNDC1,获得cDNA序列1875bp,其中ORF为717bp,编码238个氨基酸,GenBank登录号为EU571478;(2)HOXA5,获得cDNA序列1395bp,其中ORF为813bp,编码270个氨基酸,GenBank登录号为EU_024116;(3)TIAF1,获得cDNA全长序列1633bp,其中ORF为348bp,编码115个氨基酸,GenBank登录号为EU872207;(4)MYO18A,共2个转录本,获得了MYO18A_1 cDNA序列6231bp,MYO18A_2 cDNA序列6186bp,两者
的区别在于MYO18A_2缺失第39外显子;(5)POLDIP2,获得cDNA序列2410bp,其中ORF为1107bp,编码368个氨基酸;(6)PHKG2,获得cDNA序列1469bp,其中ORF为1221bp,编码406个氨基酸,GenBank登录号为EU169240。另外,还获得了6个基因的基因组DNA序列,并分析了其基因组结构,基因的所有内含子和外显子的拼接位点都符合GT-AG规则。
5.利用DNAStar、CLUSTAL W等软件对猪差异表达基因SMNDC1、HOXA5、TIAF1、MYO18A、POLDIP2和PHKG2的基因结构、所编码的蛋白质结构以及功能等特征进行了预测和分析,并构建了分子系统进化树。
6.为了进一步研究这些基因的功能,根据差异表达基因已有的研究结果,采用Real-time PCR技术和半定量RT-PCR技术对候选基因进行了时空表达谱分析。结果显示,SMNDC1基因在大白猪和梅山猪背最长肌的发育过程中呈现了不同的表达模式。在大白猪出生后的背最长肌中的表达量随着日龄的增长而递减;而在梅山猪出生后背最长肌中SMNDC1的表达量随着日龄的增长而递增。HOXA5基因在大白猪出生后的背最长肌中的表达量随着日龄的增长而递减,在3d的表达量最高,之后随着日龄的增长而逐渐减少;TIAF1基因在大白猪出生后各时期的背最长肌中均有表达,表达量差异不显著。另外,对6个差异表达基因在不同组织(心脏、肝脏、脾脏、肺脏、肾脏、胃、小肠、子宫、卵巢、背最长肌以及脂肪)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。
7.SNPs的检测结果证实HOXA5基因有3个潜在的SNPs,其中一个可用于PCR-RFLP的SNP位点,即C1817A-Bpu1102 I-RFLP,位于内含子中;TIAF1基因有5个潜在的SNPs,其中一个可用于PCR-RFLP的缺失突变位点,即PCR-Eco47 I-RFLP位于外显子中;POLDIP2基因有6个潜在的SNPs,其中一个可用于PCR-RFLP的SNPs位点,即C306A-BsuR I-RFLP位于第三外显子中;PHKG2基因有3个可用于PCR-RFLP的SNPs位点,即位于第八外显子中的G785A-Mst I-RFLP和G866A-Tag I-RFLP,以及位于第九外显子中的G875A-Pst I-RFLP。
8.对HOXA5、TIAF1、POLDIP2以及PHKG2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、2003年和2004年大白×梅山猪F_2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HOXA5基因C1817A-Bpu1102 I-RFLP位点所形成的不同基因型在眼肌面积性状存在显著差异(P＜0.05);(2)猪TIAF1基因缺失突变位点(PCR-Eco47 I-RFLP),不同基因型在胴体长性状存在显著差异(P＜0.05),在背最长肌大理石纹评分和肌内脂肪含量这2个性状存在极显著差异(P＜0.01);(3)猪POLDIP2基因C306A-BsuR I-RFLP位点,不同基因型在胴体长性状存在极显著差异(P＜0.01),在肩部背膘厚和平均背膘厚性状存在显著差异(P＜0.05);(4)猪PHKG2基因G785A位点(PCR-Mst I-RFLP)多态性与瘦肉率呈极显著相关(P＜0.01),与内脂率呈显著相关(P＜0.05);G866A位点(PCR-Taq I-RFLP)多态性与瘦肥肉比例呈极显著相关(P＜0.01),与瘦肉率、内脂率、肩部背膘厚和平均背膘厚呈显著相关(P＜0.05);G875A位点(PCR-Pst I-RFLP)多态性与瘦肉率呈显著相关(P＜0.05)。
9.利用基因组PCR步移的方法从猪基因组中扩增了SMNDC1基因1242bp的5'侧翼序列,利用NNPP、MethPrimer、TFSEARCH等生物信息学软件对猪SMNDC1基因的核心启动子区、转录起始位点、CpG岛的分布以及与启动子相结合的转录因子进行了预测和分析。
10.采用5'端缺失策略,结合启动子预测结果,构建了10个SMNDC1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SMNDC1基因5'侧翼序列的10个重组子中,除pGL-SMNDC931和pGL-SMNDC349外,其它重组子的荧光素酶活性与阴性对照均达到显著水平(P＜0.05),表明它们均具有启动子活性。pGL-SMNDC688活性较强,到pGL-SMNDC349活性下降了3倍多,说明这一区域可能存在负的调控位点,从pGL-SMNDC349到pGL-SMNDC109活性升高,达到最大值,表明该区域可能存在正的调控位点。
With the development of porcine genomic studies,many genes associated with pig production traits have been identified,however,additional genes need be identified and further characterized.Due to the difference of origin,genetic background,local environment and artificial selection between Chinese indigenous and foreign commercial pig breeds,they have different intrinsic features.Meishan pigs(Chinese indigenous pigs) have lower lean meat content in their carcasses compared to Large White pigs(Western pigs),but the lean meat of Meishan pigs is of better quality.Liver is the most important organ in the procedure of substance metabolism of organism.Glycometabolism,lipid and protein metabolism were occurred in the liver.Phenotypic variance between the Large White and Meishan pigs may be related with substance metabolism which occurred in the liver.Thus,mRNA differential display technique was used to isolate and identify the differentially expressed genes in livers between Large White and Meishan pigs in the present study,and the gene functions were also primarily analyzed.Porcine differentially expressed genes SMNDC1(Survival motor neuron domain containing 1),HOXA5 (Homeobox A5),TIAF1(TGFB1-induced anti-apoptotic factor 1),MYO18A(MyosinⅩⅧa),POLDIP2(Polymerase(DNA-directed),delta interacting protein 2) and PHKG2 (Phosphorylase kinase,gamma 2) were choosen for further analyses of the potential functions.These main results were as follows:
1.The gene expression was analyzed in liver between 60 days old Large White and Meishan pigs by mRNA differential display.We also observed nearly 3000 bands in differentially displayed PAGE gel,and almost 1000 ones can be repeated in duplicate PCR.
2.There are 70 differentially displayed ESTs in PAGE gel.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.
3.The differential expression of the six genes between Large White and Meishan pigs were further identification by real-time quantitative PCR and semi-quantitative RT-PCR.The result showed that the mRNA expression level of HOXA5,TIAF1, MYO18A and POLDIP2 gene was higher in Meishan pigs than Large White pigs, whereas the mRNA expression level of SMNDC1 and PHKG2 gene was more abundant in Large White pigs than Meishan pigs.
4.We cloned the cDNA sequences of six genes by electronic cloning,comparative genomic technology and rapid amplification of cDNA ends(RACE).(1) SMNDC1, obtained cDNA sequence 1875bp,the ORF is 717bp and encodes 238 amino acids, GenBank accession number EU571478.(2) HOXA5,obtained cDNA sequence 1395bp,the ORF is 813bp and encodes 270 amino acids,GenBank accession number EU_024116.(3) TIAF1,obtained cDNA sequence 1633bp,the ORF is 348bp and encodes 115 amino acids,GenBank accession number EU872207.(4) MYO18A, which has two isoforms,MYO18A_1(6231bp) and MYO18A_2(6186bp),the difference of the two transcripts is the exon 39.(5) POLDIP2,obtained cDNA sequence 2410bp, the ORF is 1107bp and encodes 368 amino acids.(6) PHKG2,obtained cDNA sequence 1469bp,the ORF is 1221bp and encodes 406 amino acids,GenBank accession number EU169240.The DNA sequences of these genes were obtained and the genomic structures were analyzed.All splice sites of exon/intron of these genes conformed to the GT/AG rule.
5.Using DNAStar,CLUSTAL W and some other related software,we analyzed the gene structure,protein structure and conserved motifs of these six genes.In addition, the corresponding phylogenetic trees were constructed.
6.In order to study potential functions of target genes,temporal and spatial expression profiles were analyzed using real-time PCR.It was demonstrated that during seven stages of skeletal muscle development in Large White pigs after birth,the mRNA expression levels of the SMNDC1 gene decreased gradually with age.But in Meishan pigs,the SMNDC1 gene mRNA expression was up-regulated from postnatal 3 days to 150 days.The mRNA expression levels of the HOXA5 gene decreased gradually with age and the TIAF1 gene had no significant differences during the skeletal muscle development.The tissue distribution of the six 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.
7.Single nucleotide polymorphisms(SNPs) in the six genes were detected.It was demonstrated that the HOXA5 gene contained one SNP for PCR-RFLP (C1817A-Bpu1102 I-RFLP),which was located in the intron among three potential polymorphism sites following screening the genome DNA sequence.The TIAF1 gene contained a 6 base-pair deletion mutation for PCR-RFLP(PCR-Eco47 I-RFLP), which was located in the exon among five potential polymorphism sites.The POLDIP2 gene contained one SNP for PCR-RFLP(C306A-BsuR I-RFLP),which was located in the 3~(th) exon among six potential polymorphism sites.The PHKG2 gene included three SNPs for PCR-RFLP(G785A-Mst I-RFLP,G866A-Tag I-RFLP and G875A-Pst I-RFLP),which were respectively located in the 8~(th)(for the first two sites) and 9~(th) exons.
8.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 F_2 offspring, and the results showed:(1) For C1817A-Bpu1102 I-RFLP polymorphism of HOXA5 gene,significant effects were observed on LEA(P＜0.05);(2) There are significant difference between TIAF1-Eco47 I-RFLP polymorphism and CL(P＜0.05),MM1 and IMF(P＜0.01);(3) For C306A-BsuR I-RFLP polymorphism of POLDIP2 gene, significant effects were observed on CL(P＜0.01),SFT and AFT(P＜0.05);(4) For the PHKG2 gene,highly significant associations in the G785A(PCR-Mst I-RFLP) site was detected between genotypes with LMP(P＜0.01) and significant association between genotypes with IFR(P＜0.05).In the G866A(PCR-Taq I-RFLP) site,there were highly significant association between genotype with RLF(P＜0.01) and significant association with LMP,IFR,SFT and AFT(P＜0.05).In the G875A (PCR-Pst I-RFLP) site,there were significant association with LMP(P＜0.05).
9.Genome PCR walking technique was used to clone the proximal promoter region of porcine SMNDC1 gene,and we obtained a fragment of 1242bp.Bioinformatics approaches were adopted to analyze the proximal promoter region of porcine SMNDC1 gene,and the putative binding sites of transcription factors in the regulatory region of the gene were analyzed by online software.
10.To determine the location of the promoter activity of porcine SMNDC1 gene,we constructed 10 recombinants of progressively 5'-deleted DNA fragment linked to the pGL3 reporter,respectively.These recombinants were transiently transfected into PK15 cells.Transcriptional activity of SMNDC1 recombinants normalized by Renilla was significant difference with pGL3-Basic expect for construct 931 and 349 (P＜0.05).Construct 688 contains highest activity and 3 times reduction of transcriptional activity to 349 indicated that negative regulation factors located probably in this region.From the 349 to 109 enhanced the promoter activity by 5.5-fold indicated that positive regulation factors located probably in this region.
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