【作者】 王军；

【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， 2006， 博士

【摘要】 分子标记辅助选择与传统育种方法的结合加速了猪遗传改良的进程。分子标记辅助选择的基础是寻找与重要经济性状相关的主效基因和分子标记。随着猪分子遗传图谱、QTL定位以及人、鼠等功能基因组研究的深入，通过报道的QTL-定位结果，在与之同源的人鼠等染色体区域内，确定经济性状相关候选基因是切实可行的，基于此，本研究筛选了8个与肌肉代谢相关基因作为猪生产性状候选基因进行了研究，并取得了以下结果：1、HK2（HexokinaseⅡ，己糖激酶2）：（1）获得了大白猪、长白猪、梅山猪三个猪种基因编码区序列和部分基因组序列，并提交到GenBank中，获得四个登录号为DQ432056、DQ432057、AY818385、AY821788；（2）序列比对发现了13处cSNPs（single nucleotide polymorphism in coding sequence）和2处3’非翻译区的SNP；内含子中发现了38个SNPs；（3）建立了第10内含子A／G替换的PCR-MspⅠ-RFLP（MspⅠ酶切片段长度多态性）和第17外显子的T／G替换的PCR-MspⅠ-RFLP分型方法，在所检测的6个不同猪种中，第10内含子的多态在大白和长白猪中只出现了A等位基因；国内猪种可检测到A和B等位基因；第17外显子的多态在所检测的6个不同猪群中A等位基因只存在于杂合子个体中，各猪群中两等位基因频率没有多大差异。（4）在309头大×梅F₂代资源家系中进行标记性状关联分析，第10内含子的多态位点与眼肌高度、平均皮厚和股二头肌大理石纹差异显著，并且加性效应显著；在186头试验猪群（大白、长白、梅山、大白×梅山、长白×大白、大白×长白）中进行标记性状关联分析，第17外显子的多态位点与胴体长、肋骨数、肩部背膘厚和眼肌面积差异极显著，与胸腰椎间背膘厚、三点平均背膘厚、眼肌高度、眼肌宽度、肥肉率、瘦肉率和肌内脂肪差异显著。（5）该基因在猪骨骼肌中特异性表达。2、LMCD1（LIM and cysteine-rich domains 1，LIM富半胱氨酸区域蛋白1）：（1）获得了大白猪、长白猪、梅山猪三个猪种基因编码区序列并提交到GenBank中，登录号为NM₀01008692；（2）序列比对发现了4个cSNPs，其中1处预测可导致氨基酸改变；（3）建立了第三外显子G／A替换的SSCP（polymerase chain reaction-single strand conformational polymorphism）基因分型方法。在所检测的6个猪种中，大白、长白、鄂西黑和皖南花猪种G等位基因的基因频率为1或接近1，而梅山和清平品种的等位基因G基因频率分别为0.2889和0；（4）在178头大×梅F₂代资源家系中进行标记性状关联分析，该位点与胸腰椎间背膘厚和臀部平均背膘厚差异显著，该位点以显性作用方式为主，且方向一致；（5）该基因在所检测的各组织中均有表达，在心脏和肌肉中表达量最高。3、NRAP（Nebulin-related anchoring protein，伴肌动蛋白相关锚定蛋白）：（1）获得了大白猪、杜洛克猪、梅山猪三个猪种基因编码序列、5’RACE（rapid amplificationof cDNA end，快速扩增cDNA末端）及其第39、40内含子序列，序列已提交到GenBank中，登录号为DQ157553和DQ480149；（2）发现了35个SNPs，其中3处预测可导致氨基酸改变；（3）建立了第39内含子A／G替换的PCR-Eco721-RFLP分型方法；在所检测的8个猪种中，大白、长白猪种B等位基因的基因频率为1，中国地方猪群可检测到A和B等位基因；（4）在302头大×梅F₂代资源家系中进行标记性状关联分析，该位点与出生重、内脂率、眼肌高度、肩部背膘厚、背最长肌pH值、失水力、系水力、背最长肌肌肉色值、股二头肌pH值和股二头肌色值显著或极显著相关，该位点在胴体性状以显性作用方式为主，肉质性状则以加性作用为主；（5）该基因特异性的在心脏和骨骼肌中表达。4、ECHI （Enoyl CoA hydramse 1，烯酯酰辅酶A水合酶1）：（1）获得了大白猪、长白猪、梅山猪三个猪种基因编码序列、5’RACE及第3内含子外的所有内含子序列，并提交到序列的GenBank登录号为DQ157552和DQ480146；（2）序列比对发现了17个SNPs，其中1处cSNP；（3）建立了第1内含子A／G替换的PCR-BamHⅠ-RFLP、第5内含子A／G替换的PCR-PstⅠ-RFLP分型方法和第4外显子T／C突变的PCR-SSCP分型方法；第1内含子的酶切多态，在所检测的7个猪种中，大白和长白猪种B等位基因的基因频率接近为1，而中国猪种均可检测到A和B等位基因，除清平猪种外，其余品种均为B等位基因占优势；关于第5内含子多态性分布，在所检测的7个猪种中，监利猪未检测到A等位基因，其余猪种均可检测到A和B等位基因，均为B等位基因占优势；（4）在296头大×梅F₂代资源家系中进行标记性状关联分析，第1内含子多态的不同基因型间内脂率、眼肌高度和肌内水分等生产性状差异显著，以显性效应为主；在306头大×梅F₂代资源家系中进行标记性状关联分析，第5内含子位点与屠宰率、平均皮厚和股二头肌色值差异显著；（5）该基因在所检测的9个组织中均表达。5、CKMT2 （crcatine kinase，mitochondrial 2（sarcomeric），肌节线粒体肌酸激酶2）：（1）获得了大白猪、长白猪、梅山猪三个猪种基因cDNA序列，该基因GenBank登录号为DQ363337，获得的第4内含子序列见附录14；（2）序列比对，发现了9个SNPs，其中3处预测可导致氨基酸改变；（3）建立了第4内含子A／G替换的PCR-MspⅠ-RFLP分型方法，在检测的9个猪种中，梅山和二花脸猪中B等位基因频率为1或接近1，其他7个猪种均为A等位基因频率为1；（4）在302头大×梅F₂代资源家系中进行标记性状关联分析，该位点与出生重、内脂率、肥肉率、胴体长、肩部背膘厚、胸腰椎间背膘厚、臀部背膘厚和肌内水分等性状差异显著，以加性作用方式为主；（5）该基因在心脏和骨骼肌中表达丰度最高，在脾脏、胃、小肠和子宫中不表达，在其余组织不同程度的表达。6、TTID （Titin Immunoglobul in Domain Protein；肌联蛋白免疫球蛋白区域蛋白）：（1）获得了大白猪、长白猪、梅山猪三个猪种基因全长cDNA序列以及除第2、7内含子外的所有内含子序列，提交到GenBank的登录号为DQ157551和DQ480148；（2）序列比对发现了51个SNPs，该基因cDNA中没有发现碱基突变或缺失；（3）建立了第6内含子T／C替换的PCR-HinfⅠ-RFLP分型方法，在检测的10个猪种中，在大白和长白猪种中B等位基因的频率为1或是接近1，国内品种中除鄂西黑猪、清平猪和八眉猪中B等位基因频率略占优势外，其余5个品种均为A等位基因占绝对优势；（4）在279头大×梅F₂代资源家系中进行标记性状关联分析，胴体性状方面，该位点与皮率和眼肌面积差异显著，与平均皮厚差异极显著；并且表现为加性效应显著或极显著，肉质方面，该位点与股二头肌pH值、背最长肌和肌内水分差异显著，表现为显性效应显著；（5）该基因在心脏和骨骼肌中表达量很高，在脂肪和肺中微量表达，其余组织中不表达。7、PFKM （Phosphofructokinase Muscle Type，肌型磷酸果糖激酶）：（1）获得了大白猪、长白猪、梅山猪三个猪种基因全长cDNA序列及第2到21内含子序列，验证猪中第1内含子的大小，序列GenBank的登录号为DQ363336和DQ480147；（2）序列比对发现了111处SNPs，其中4处预测可导致氨基酸改变；（3）建立了第13外显子T／C替换和第17外显子的PCR-TaqⅠ-RFLP分型方法，在所检测的7个猪种中，第13外显子多态性分布频率在检测的大白、长白和鄂西黑猪中B等位基因频率为1，另外四个国内猪种均为B等位基因占优势，在所检测的7个猪种中，第17外显子多态性分布频率基本同第13外显子位点的分布，（4）在214头大×梅F₂代资源家系中进行标记性状关联分析，第13外显子位点基因型不同时，出生重、内脂率、肥肉率、瘦肉率、瘦肥比率、眼肌高度、6—7胸腰椎间背膘厚、胸腰椎间背膘厚、肩部背膘厚、三点平均背膘厚、股二头肌色值、背最长肌大理石纹、肌内水分和肌内脂肪差异显著或极显著；该位点在部分胴体性状加性效应和显性效应均为极显著；在241头大×梅F₂代资源家系中进行标记性状关联分析，第17外显子位点与眼肌高度、臀部背膘厚、三点平均背膘厚、大理石纹、失水率、系水力、肌内脂肪和肌内水分差异显著或极显著；（5）该基因在心脏和骨骼肌中表达量很高，胃和小肠没有表达，其他组织均有不同程度的表达。8、MYLPF （myosin light chain，phosphorylatable，fast skeletal muscle，肌浆球蛋白轻链可磷酸化蛋白）：（1）获得了大白猪、长白猪和梅山猪的基因组序列；序列GenBank的登录号为DQ533994；（2）序列比对，发现了43处SNPs，有5处cSNPs，其中3处预测可导致氨基酸改变；（3）建立了第1内含子G／A替换的PCR-TaqⅠ-RFLP和TIC替换的PCR-MspⅠ-RFLP分型方法；在所检测的7个猪种中，第1内含子MspⅠ-RFLP多态性分布频率在长白猪中A等位基因与B等位基因频率的比例大约为1：2，其余猪种为B等位基因占绝对优势；在所检测的7个猪种中，第1内含子TaqⅠ-RFLP多态性分布频率，在大白和长白猪中A等位基因与B等位基因频率的比例大约为1：2，国内猪种均为B等位基因占绝对优势；（4）在140头试验猪群（长白、梅山、长白、长白×大白、大白×长白）中进行标记性状关联分析，基因型不同时，瘦肉率、皮率、股二头肌大理石纹、背最长肌大理石纹和肌内水分差异显著，眼肌宽度、眼肌面积和肌内脂肪差异极显著，肩部背膘厚和眼肌高度差异接近显著水平；该位点在这些性状上均表现为加性作用方式，但作用方向不同；（5）该基因在各组织中均有表达，尤以心脏和骨骼肌中表达丰度最高。上述实验结果为进一步探讨这些基因对于猪肌肉生产和代谢相关性状影响的分子机制，以及应用标记辅助选择进行分子育种提供了一些有价值的依据，从而推进猪遗传改良工作的进程，为我国功能基因组的研究和源头创新奠定基础。更多还原

【Abstract】 The use of the marker-assisted selection （MAS） combining with traditional breeding methods promoted the progress of genetic improvement in pigs. The basis of MAS was to seek the major gene or marker which contributed genetically much to the economic trait. With the development of pig genetic map, QTL mapping and functional genome research of human or mouse, it is practice that determining candidate gene associated with economic trait in the chromosome region which homologous to human or mouse based on the results of QTL mapping. In this study, eight candidate genes associated with muscle metabolism were selected by comparative positional genomics to research in pigs. The main results are as follows:1、HK2（Hexokinase II）: （1） The full coding sequence and partial genomic sequences of Large White, Landrace, Meishan and Duroc breeds were isolated and deposited to GenBank under accession number DQ432056、DQ432057、AY818385、AY821788. （2） 13 cSNPs （single nucleotide polymorphism in coding sequence）, two SNPs within 3’ untranslated region and 38 SNPs within intron sequences were identified. （3） Two PCR-Msp I -RFLP were developed to detect A/G substitution in intron 10 and T/G substitution in exon 17. Allele frequencies for Msp I PCR-RFLP in intron 10 were studied in unrelated pigs from six breeds and allele A was found only in Large White and Landrace pigs, while allele A and B were both detected in Chinese pigs. For Msp I PCR-RFLP in exon 17 in unrelated pigs from six breeds, allele A was found only in heterozygocytous pig and the frequencies of two alleles were similar in all studied pig breeds. （4） In the 309 F₂ animals in LargeWhite×Meishan resource family were genotyped by Msp I PCR-RFLP in intron 10 for association analysis. For carcass traits, significant effects were observed for this locus on LEH and AST, including their additive effects; For meat quality traits, this locus was significantly associated with MM（BF）. Msp I PCR-RFLP in exon 17 was typed in 186 pigs （Landrace pigs, LargeWhite×Landrace and Landrace×LargeWhite pigs） to study the association between carriers of different genotype and the trait values, statistically significant associations with CL, RNS, BFT1, LEH, LEW, LEA, BFT2, ABF, FMP, LMP and IFR were found. （5） The expression of HK2 gene was specially in skeleton muscle of pigs.2、LMCD1 （LIM and cysteine-rich domains 1） : （1） The full coding sequence of Large White, Landrace and Meishan breeds were isolated and deposited to GenBank under accession number NM₀01008692. （2） 4 cSNPs were identified , of which one was predicted to change amino acid. （3） PCR-SSCP （polymerase chain reaction-Single strand conformational polymorphism） typing was developed to detect G/A substitution in intron 3. The frequencies of allele G were 1 or near to in Large White, Landrace, Erxihei and Wannanhua, while Meishan and Qingping breed were 0.2889 and 0 separately. （4） In the 178 F₂ animals were genotyped for association analysis. For carcass traits, significant effects were observed for this locus on BFT1 and BFT2; For meat quality traits, this locus was near to significantly associated with pH（LD） and pH（SC） and seemed to be dominant in action. （5） LMCD1 gene was highly expressed in heart and muscle while it had a broad expression spectrum across all tissues examined.3、NRAP （Nebulin-related anchoring protein）: （1） The full coding sequence of LargeWhite, Landrace and Meishan breeds were isolated. The nucleotide sequence of 5’RACE （rapid amplification of cDNA end） and intron 39 and 40 have been deposited to GenBank under accession number DQ157553 and DQ480149. （2） 35 SNPs were identified by comparing sequences, three of which resulted in amino acid change. （3） PCR-Eco721-RFLP was developed to detect A/G substitution in intron 39. In unrelated pigs from eight breeds, both allele A and B were found in Chinese pig breeds, while only B was in LargeWhite and Landrace. （4） Association analysis in 302 F₂ animals showed significant associations between Eco721-RFLP genotypes with BWT, CFW, LEH and BFT1 for carcass traits and pH（LD）, pH（BF）, MCV1, MCV2, DLR and WHC for meat quality traits. This locus was predominant in dominance for carcass trait, but in addition for meat quality. （5） NRAP mRNA was transcribed especially in heart and skeleton muscle.4、ECH1 （Enoyl CoA hydratase 1）: （1） The full coding sequence of LargeWhite, Landrace and Meishan breeds, 5’RACE and all introns except intron 3 were isolated and deposited to GenBank under accession number DQ157552 and DQ480146DQ157552. （2） 17 SNPs were identified, of which one was cSNP. （3） Two A/G substitution were detected by PCR-BamHⅠ-RFLP in intron 1 and PCR- PstⅠ-RFLP in intron 5 separately, and T/C mutation in exon 4 was detected by PCR-SSCP. Among seven different pig breeds examined, results of BamHⅠ-RFLP in intron 1 showed that the frequency of allele B was near to 1 in LargeWhite and Landrace; both allele A and B were found in Chinese pig breeds, and allele B was predominant in other breeds except Meishan and Qingping. For PCR- PstⅠ-RFLP in intron 5, in seven commercial pig breeds, both alleles C and D （allele D was dominant） were detected in all other examined breeds except Jianli pig which only had allele D.（4） Association analysis in 296 F₂ animals showed significant associations between BamHⅠ-RFLP genotypes and CFW, LEH and WM. And this locus seemed to be dominant in action. For PCR- PstⅠ-RFLP in intron 5 in 306 F₂ animals, for carcass traits, significant effects were observed for this locus on DP and AST; For meat quality traits, this locus was significantly associated with MCV2. （5） Expression of ECH1 gene can be detected in all tissues examined: heart, liver, lung, kidney, spleen, stomach, small intestine, muscle and fat.5、CKMT2 （creatine kinase, mitochondrial 2）: （1） The full coding sequence of LargeWhite, Landrace and Meishan breeds were isolated and deposited GenBank under accession number DQ363337. In addition, the sequence of intron 4 can be seen in appendix 14. （2） Of nine SNPs identified within these sequences, three of which resulted in amino acid change. （3） An PCR- MspⅠ-RFLP was developed to detect the A/G polymorphism in intron 4. Among nine pig breeds examined, allele B was 1 or near to 1 in Meishan and Erhualian, while in other seven breeds all were 1 of allele A. （4） 302 F₂ animals were typed for association analysis. For carcass traits, significant effects were observed for this locus on BWT, IFR, FMP, CL, BFT1, BFT2 and BFT3, and their additive effects were predominant; For meat quality traits, this locus was significantly associated with WM. （5） Expression of CKMT2 gene was predominant in heart and skeleton muscle and moderate in lung, kidney and fat, although a weak signal was observed for liver, ovary and testis, and no expression in spleen, stomach, small intestine and uterus.6、TTID （Titin Immunoglobulin Domain Protein）: （1） The full coding sequence of LargeWhite, Landrace and Meishan breeds and sequences of all introns except intron 2 and 7 were isolated and deposited to GenBank under accession number DQ157551 and DQ480148. （2） Total 51 SNPs within these intron sequences and no mutation or deletion was found in cDNA sequences. （3） An PCR- HinfⅠ-RFLP was developed to detect T/C substitution in intron 6. Among ten pig breeds which were typed by HinfⅠ-RFLP, results showed that there were significantly different in allele frequencies between Chinese indigenous pig breeds and foreign pig breeds: the frequencies were 1 or near to1 in LargeWhite and Landrace, while allele A was predominant in other five breeds although allele B were only a bit dominant in Erxihei, Qinsping and Bamei. （4） 279 F₂ animals were typed for association analysis. For carcass Waits, significant effects were observed for this locus on SP, LEA and AST, and their additive effects were significant; For meat quality traits, this locus was significantly associated with pH（BF）, MCV1 and WM, and their additive effects were significant. （5） Expression of TTID gene was highest in skeleton muscle and heart, with weak level in fat and lung, and no signal in other tissues.7、PFKM （Phosphofructokinase Muscle Type）: （1） The full cDNA sequence of LargeWhite, Landrace and Meishan breeds and sequences of 2-21 introns were isolated and deposited to GenBank under accession number DQ363336 and DQ480147. And the length of sequence of intron 1 was identified. （2） Of the 111 SNPs identified within all sequences, four of which resulted in amino acid change. （3） PCR-TaqⅠ-RFLP were developed to detect T/C substitution in exon 13 and exon 17. In unrelated individuals from seven different pig breeds, allele frequencies of the TaqⅠ-RFLP in exon 13 was compared: frequencies of allele B in LargeWhite, Landrace, Erxihei were 1 or near to 1, and a little dominant of allele B in other four Chinese pig breeds. For the same polymorphism in exon 17, the trend of allele frequencies distribution was the similar to that of exon 13. （4） For association analysis in the 214 F₂ animals, significant effects were observed for different genetype of exon 13 on BWT, IFR, FMP, LMP, RLF, LEH, BFT1, BFI2, BFT4 and ABF of carcass traits. It is notable that both additive effects and dominant effects in BWT, LMP, RLF and LEH were very significant; For meat quality traits, this locus was significantly associated with MCV2, MM1, WM and IMF. Association analysis in 241 F2 animals showed there were significant effects on BFI3 and ABF of carcass traits and on MM1, MM2, DLR, WHC, IMF and WM of meat quality traits with TaqⅠ-RFLP in exon 17. （5） Expression of PFKM gene had different levels in each examined tissue except stomach and small intestine, with highest in skeleton muscle, then in heart.8、MYLPF （myosin light chain, phosphorylatable, fast skeletal muscle）: （1） The full genomic sequence of of LargeWhite, Landrace and Meishan breeds were isolated and deposited to GenBank under accession number DQ533994. （2）43 SNPs, including 5 cSNPs, were found by comparing sequences of three pig breeds, three of which resulted in amino acid change. （3） PCR-TaqⅠ-RFLP and PCR-MspⅠ-RFLP were developed to detect G/A substitution and T/C substitution in intron 1 separately. For MspⅠ-RFLP among seven different pig breeds examined, the frequency of allele B was two times of that of allele A in Landrace, while allele B were predominant in other breeds. For TaqⅠ-RFLP, the frequency of allele B was two times of that of allele A in LargeWhite and Landrace. （4） The locus of MspⅠ-RFLP was typed in 140 pigs （Landrace, LargeWhite, Meishan, LargeWhite×Landrace and Landrace×Large White pigs） to study the association between carders of different genotype and the trait values. For carcass traits, significant effects were observed for this locus on LMP, SP, LEW and LEA, with approximative significant on BFT1 and LEH（P＜0.06）. This locus appeared to be additive effects with different action direction; For meat quality traits, this locus was significantly associated with MM1, MM2 and WM and IMF, which appeared to be predominant on addictive effects. （5） RT-PCR analysis of MYLPF showed its expression in all tissues examined, with highest levels in heart and skeleton muscle.The results described above provide some valuable basis for the further research of the molecular mechanism of these genes with the production and metabolism of muscle in pig and the application of molecular breeding with MAS. All these works would facilitate the progress of genetic improvement in pig, and establish the basis for the research of functional genome in our country.更多还原