【作者】 张凤伟；

【导师】 邓昌彦； 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】 基因组印记是指二倍体生物的某些组织或细胞中，来源于不同亲本的一对等位基因发生差异性表达，即只有一方亲本的等位基因表达，而另一亲本的等位基因不表达或很少表达的生物学现象，而相应的基因就称为印记基因。对哺乳动物的研究表明，印记基因在胎儿、胎盘的生长发育及胎儿出生后的表现等方面，都发挥着重要的调节作用。目前，对基因组印记的研究主要集中在人和小鼠中，在家畜特别是猪中研究比较少。因此在猪中鉴定印记基因对于基因组印记在物种间的保守性研究具有重要意义。另外，大部分已研究的印记基因都是重要的调控基因，包括转录因子，细胞周期调节基因，生长因子或者是参与复杂的信号通路。基于印记基因在生长和发育中的重要调控作用，预测这些基因可能对猪等家畜动物的生产性状产生较大的影响。因而，在育种中能否将印记基因作为分子标记进行标记辅助选择，是一个值得探讨的新课题。所以本研究主要进行了以下两个方面的工作：（1）利用大白猪×梅山猪和梅山猪×大白猪正反交模型，通过RT-PCR-RFLP分析和PCR产物直接测序法检测“表达的SNP”，鉴定了猪7个候选印记基因的印记状态，以期探讨基因组印记在猪与其他物种间的保守性；（2）在大白猪×梅山猪F₂代群体中，利用PCR-RFLP技术，进行了4个猪候选印记基因的基因型与重要经济性状的关联分析，以期探讨印记基因在育种中作为分子标记的可行性。目前，取得了如下结果：1．根据人和小鼠印记基因的印记状况及生理功能，筛选了PLAGL1、PEG10、PPP1R9A、XIST、MEST、NAP1L5和PEG3基因作为猪候选印记基因。利用电子克隆和比较基因组学方法获得了7个候选印记基因的cDNA序列，其中3个基因包括完整的ORF；（1）PLAGL1，获得cDNA序列2238bp，其中开放阅读框（Open reading frame，ORF）为1392bp，GenBank登录号为DQ28899；（2）PEG10，获得cDNA序列6379bp，其中ORF为1212bp，GenBank登录号为DQ779285；（3）PPP1R9A，获得cDNA序列1946bp，GenBank登录号为EF619476；（4）XIST，获得cDNA序列1039b，GenBank登录号为EF619477；（5）uesr，获得cDNA序列2167bp，其中ORF为981bp，GenBank登录号为EF619473；（6）NAP1L5，获得cDNA序列1158bp，GenBank登录号为EF619474；（7）PEa3，获得cDNA序列2052bp，GenBank登录号为EF619475。比较了所获得的7个候选印记基因cDNA序列与人和鼠同源序列的相似性，表明7个基因cDNA序列的猪-人相似性均大于猪-鼠相似性和人-鼠相似性。2．对所获得的猪7个候选印记基因的cDNA序列，在3头大白猪和3头梅山猪间进行了比对分析：（1）PLAGL1，在CDS和3’UTR各发现1个SNP，全为转换突变；（2）PEa10，在3’UTR发现4个SNP，其中3个转换突变，1个插入／缺失突变；（3）PPP1RgA，在3’UTR发现8个SNP，其中4个转换突变，3个颠换突变，1个插入／缺失突变；（4）XIST，在3’UTR发现4个SNP，其中3个转换突变，1个插入／缺失突变；（5）MEST，在3’UTR发现2个SNP，全为转换突变；（6）NAP1L5，在3’UTR发现2个SNP，其中1个转换突变，1个插入／缺失突变；（7）PE63，在3’UTR发现3个SNP，其中1个转换突变，2个插入／缺失突变。3．利用大白猪×梅山猪和梅山猪×大白猪正反交模型，通过RT-PCR-RFLP分析和PCR产物直接测序法，鉴定了7个候选印记基因在二月龄猪14个组织和器官中的印记状况。（1）PLAGL1基因在肌肉、脂肪、心、肝、脾、肺、肾、胃、小肠、子宫、卵巢和睾丸中母系印记；（2）PEG10基因在肌肉、脂肪、心、肝、脾、肺、肾、胃、小肠、子宫、卵巢和睾丸中母系印记；（3）PPP1R9A基因在肌肉、脂肪、心、肝、脾、肺、肾、胃、小肠、子宫、卵巢和垂体中双等位基因表达；（4）XIST基因在肌肉、脂肪、心、肝、脾、肺、肾、胃、小肠、子宫、卵巢和垂体中双等位基因表达：（5）MEST基因在肝脏、脾和卵巢中双等位基因表达，在肌肉、脂肪、心、肺、肾、胃、子宫和垂体中母系印记；（6）NAP1L5基因在肌肉、脂肪、肝、脾、肺、肾、胃、小肠、子宫、卵巢和垂体中母系印记；（7）PEG3基因在脂肪、心、肺、胃、小肠和卵巢中双等位基因表达，在肌肉、肝、脾、肾和子宫中母系印记。结果表明，虽然7个候选基因的印记状况在不同组织间有些差异，但从总体来看，它们在人、小鼠和猪中是趋于保守的。4．利用PCR-RFLP技术，对4个候选印记基因中存在的SNP位点在不同猪群中进行了基因分型，并在大白×梅山F₂代群体中进行了性状关联分析。结果表明：（1）PLAGL1基因，C1428T位点与肩部背膘厚、内脂率和股二头肌pH值的相关达到显著水平（p＜0.05）；（2）PEG10基因，C5274T位点与眼肌高度和股二头肌pH值的相关达到了显著水平（p＜0.05），与眼肌宽度的相关达到了极显著水平（p＜0.01）；（3）PPP1R9A基因，A1688C位点与肩部背膘厚、臀部膘厚和肌内脂肪的相关达到了显著水平（p＜0.05），和平均背膘厚、胴体长、眼肌面积和骨率的相关达到了极显著水平（p＜0.01）；（4）XIST基因，在公猪群体中，598-TCCATGG位点与臀部膘厚、平均膘厚、肌内脂肪和肌内水分的相关达到了显著水平（p＜0.05），与肥肉率、瘦肉率、6-7胸椎间膘厚、胸腰椎间膘厚达到了极显著水平（p＜0.01），在母猪群体中，598-TCCATGG位点与肥肉率、平均膘厚和眼肌高度的相关达到了显著水平（p＜0.05），与臀部膘厚和6-7胸椎间膘厚达到了极显著水平（p＜0.01）。4个候选印记基因中有3个与脂肪沉积相关，这些结果表明，将印记基因特别是位于性染色体上的XIST基因，作为分子标记应用于育种实践中，具有非常好的前景。更多还原

【Abstract】 Genomic imprinting indicates that alleles from one parent are expressed, but allelesfrom the other parent are not expressed or express little in some tissues or cell types ofdiploid organism. Imprinted genes are genes that preferentially expressed from either thematernally inherited allele or the paternally inherited allele. The researches on genomicimprinting in mammals show that imprinted genes have important roles in the regulationof fetal growth, development, function of the placenta and postnatal behavior. At present,most studies on genomic imprinting are in human and mouse but little in livestock. Inpigs, there is no report on the identification of imprinted genes at the molecular level.Therefore, it is of interest to identify more imprinted genes in pigs for analyzing theconservation of genomic imprinting among different species. Most of the imprinted genesthat have been studied are regulatory: transcription factors, alternative splicer, tumorsuppressors, growth factors, or are involved in complex signal pathways. Because of theimportant regulatory function of imprinted genes on growth and development, we couldpredict that imprinted genes may affect growth and development largely in pigs. Whetherthe imprinted genes may be used as molecular mark in pigs’ breeding is not known. Basedon above, our research includes two main aspects: first, analyzing the imprinted status of7 candidate imprinted genes with RT-PCR-RFLP or sequencing directly in the F₁ hybridsof Large White boars×Meishan sows and Meishan boars×Large White sows; second,PCR-RFLP analysis of 4 genes is used to detect the association of the polymorphism inimprinted genes and the carcass traits in the pigs of Large White×Meishan F₂ hybrids.Our results are as follows:1. We chose PLAGL1、PEG10、PPP1R9A、XIST、MEST、NAP1L5 and PEG3 genesas candidate imprinted genes in pigs according to the imprinted status of the genes inhuman and mouse and their biological functions. We obtained some sequences includesome complete ORF of the genes with electronic cloning and comparative genomictechnology. （1） PLAGL1, obtained cDNA sequence 2238bp, ORF （Open reading frame）1392bp, GenBank accession number DQ28899; （2） PEG10, obtained cDNA sequence6379bp, ORF 1212bp, GenBank accession number DQ779285; （3） PPP1R9A, obtainedcDNA sequence 1946bp, GenBank accession number EF619476; （4） XIST, obtainedcDNA sequence 1039b, GenBank accession number EF619477; （5） MEST, obtainedcDNA sequence 2167bp, ORF 981bp, GenBank accession number DQ EF619473; （6）NAP1L5, obtained cDNA sequence, GenBank accession number EF619474; （7） PEG3,obtained cDNA sequence 2052bp, GenBank accession number EF619475. Comparing thesimilarity between the cDNA sequences of the 7 genes and the corresponding homology in human and mouse show that the sequences similarities between human and pigs are allhigher than the sequences similarities between mouse and pigs.2. Aligning the cDNA sequences of the 7 genes between 3 Large White pigs and 3Meishan pigs revealed that: （1） PLAGL1, one SNP existed in coding region and one SNPexisted in the 3’ UTR region, both of them are transition mutation. （2） PEG10, weidentified 4 SNPs in the 3’ UTR region. Of 3 are transition mutation, 1 isinsertion/deletion mutation. （3） PPP1R9A, we identified 8 SNPs in the 3’ UTR region. Of4 are transition mutation, 3 are transversion mutation, 1 is insertion/deletion mutation. （4）XIST, we identified 4 SNPs in the 3’ UTR region. Of 3 are transition mutation, 1 isinsertion/deletion mutation. （5） MEST, two SNPs existed in the 3’ UTR region, both ofthem are transition mutation. （6） NAP1L5, two SNPs existed in the 3’ UTR region, one istransversion mutation and the other is insertion/deletion mutation. （7） PEG3, weidentified 3 SNPs in the 3’ UTR region. Of 1 is transition mutation, 2 areinsertion/deletion mutation.3. Using the model of F₁ hybrids of Large White boar×Meishan sow and Meishanboar×Large White sow, we identified the imprinted status of the 7 genes in 14 differenttissues of 12 two-month pigs in the model through RT-PCR-RFLP analysis andsequencing directly. （1） PLAGL1gene is paternally expressed in skeletal muscle, fat, heart,liver, spleen, lung, kidney, stomach, small intestine, uterus, ovary and testicle. （2） PEG10gene is paternally expressed in skeletal muscle, fat, heart, liver, spleen, lung, kidney,stomach, small intestine, uterus and ovary. （3） PPP1R9A gene escapes genomicimprinting in skeletal muscle, fat, heart, liver, spleen, lung, kidney, stomach, smallintestine, uterus, ovary and pituitary. （4） Xist gene is biallelically expressed in skeletalmuscle, fat, heart, liver, spleen, lung, kidney, stomach, small intestine, uterus, ovary andpituitary. （5） MEST gene is biallelically expressed in liver, spleen and ovary, butpaternally expressed in skeletal muscle, fat, heart, lung, kidney, stomach, uterus, ovaryand pituitary. （6）NAP1L5 gene is paternally expressed in skeletal muscle, fat, liver,spleen, lung, kidney, stomach, small intestine, uterus, ovary and pituitary. （7） PEG3 geneis biallelically expressed in fat, heart, lung, stomach, small intestine and ovary, butpaternally expressed in skeletal muscle, liver, spleen, kidney and uterus. Although theimprinted status of the 7 genes is different in various tissues, they are relativelyconservative among pigs, human and mouse as a whole.4. Using PCR-RFLP, we detected 4 SNPs of 4 genes in different pig populations andobserved associations with traits in Large White and Meishan F₂ hybrids. The resultsshowed: （1） PLAGL1, C1428T-TaqI-RFLP is significant association with shoulder backfat thickness, internal fat ratio and biceps femoris pH （p＜0.05）. （2） PEG10,C5274T-TaqI-RFLP is significant association with lion eye height and biceps femoris pH（p＜0.05）, higher significant association with lion eye width （p＜0.01）. （3） PPP1R9A,A1688C-Eco47I-RFLP is significant association with shoulder backfat thickness, buttockfat thickness and intramuscular fat （p＜0.05）, higher significant association with averagebackfat thickness, carcass length, lion eye area and bone percentage （p＜0.01）. （4） Xist,598-TCCATGG-Eco47I-RFLP is significant association with buttock fat thickness,average backfat thickness, intramuscular fat and water moisture （p＜0.05）, highersignificant association with fat percentage, lean meat percentage, 6～7 rib fat thickness andthorax-waist fat thickness （p＜0.01） in the population of male pigs. But in the populationof female pigs, 598-TCCATGG-Eco47I-RFLP is significant association with fatpercentage, average backfat thickness and lion eye height （p＜0.05）, higher significantassociation with buttock fat thickness and 6～7 rib fat thickness （p＜0.01）. Three of the 4candidate genes are associated with fat deposition, which showed that imprinted genes inparticular Xist gene located on sexual chromosome are fit for the molecular breeding.更多还原