【作者】 黄艳群；

【导师】 邱祥聘； 李宁；

【作者基本信息】 四川农业大学 ， 动物遗传育种与繁殖， 2003， 博士

【摘要】 多趾是一些鸡的品种特征，是脊椎动物常见的肢异常表型。通过比较基因组学研究，发现有相似的基因和机理影响着多趾的发生。Lmbr1是人和鼠轴前多趾(PPD)的候选基因，也可能是鸡多趾的候选基因。 本研究首次克隆了鸡Lmbr1基因的编码序列，检测到鸡Lmbr1基因的一种短剪接方式如人ACHP患者(Acheiropodia，无手无脚)一样正好精确地缺失了c7orf2的外显子4。另外，从鸡Lmbr1基因检测到6个cSNPs(single nucleotide polymorphism in coding sequence，编码序列的单核苷酸多态性)，其中两个突变预测蛋白质变异，4个为沉默突变。在丝羽乌骨鸡和白洛克肉鸡中，A797G和T1254C变异在RNA水平呈现品种特异性变化的趋势。 本研究采用PCR扩增方法克隆了鸡Lmbr1的10-16内含子，发现鸡Lmbr1基因的几个内含子的剪接方式均符合常见的GT-AG的法则。包含鸡Lmbr1 10-17外显子的基因组为相应区域的人C7orf2的1／5，鼠Lmbr1的1／3，预测鸡Lmbr1的基因组基因在50-60kb。通过直接测序，从丝羽乌骨鸡和白洛克肉鸡的内含子区域检测到了63个变异位点，转换和颠换的比例为1.6：1。 在由多趾的丝羽乌骨鸡和四趾的白洛克肉鸡杂交建立的CAU资源群体中，多趾呈不完全显性。本研究首次报道鸡Lmbr1基因编码序列的T1254C位点是鸡多趾表型的特异性突变位点。T1254C位点为纯合的TT对应多趾个体，T1254C位点为纯合的CC对应四趾个体，T1254C位点为杂合对应五趾或四趾，F2代杂合基因型的多趾外显率为84％。T1254C突变能解释资源群体中的趾数表型变异，显示了T1254C突变对多趾的发生起着关键的作用。本研究曾先后采用三对引物对资源群进行PCR-SSCP基因型分析，在P代(parents，亲代)都观察到了基因型和品种(趾型)的紧密相关。在内含子13和外显子16都检测到丝羽乌骨鸡的特异性单倍型。 本研究首次发现鸡Lmbr1基因多态也与一些屠体性状有显著的关联。T1254C相关的PCR-SSCP基因型和屠宰性状的关联分析表明，AA基因型的全净膛率为62.93％，显著地低于BB基因型(63.83％)(P＜0.05)：AA基因型的肌胃率为2.21％，显著地高于BB基因型(2.00％)(P＜0.05)：AA基因型的胫围为4.31cm，显著地高于AB基因型(4.17cm)、极显著地高于BB基因型(4.11cm)(P＜0.01)；AA基因型的胫爪率为6.43％，极显著地高于BB基因型(5.97％)(P＜0.01)。将T1254C和G1255A两个突变联合分析，基因型对胫爪率和胫围的影响仍达到了极显著水平，显示了Lmbr1基因具有一因多效的作用或与这些屠体性状的主效QTL紧密连锁。Lmbr1基因与趾型和胫爪率的极显著关联，表明Lmbr1基因应是调控鸡多趾表型的关键基因。Lmbr1基因的突变对胫爪率和胫围的影响都达到极显著水平，也进一步显示了Lmbr1基因对鸡多趾的形成乃至对骨骼发育都有重要的作用。 采用PCR-SSCP基因型进行连锁分析和采用放射杂交板定位两种方法，把Lmbr1基因定位在鸡2号染色体短臂靠近ADL0270标记(cR=30.6，Lod=8.23)，该段染色体与包含C7orf21Lmbr1基因的人7q36和鼠5B1为同源臂，人、鼠和鸡的PPD也都被定位在这个区间。定位结果和功能分析都证明Lmbr1基因应是调控鸡多趾表型的关键基因。更多还原

【Abstract】 Polydactyly(Po) is a kind of breed identity in chicken, Which is a common limb defect in vertebrates. Through comparative genomics research, it was found there is a homologous gene and similar mechanism underlying polydactyly among different species. Lmbrl is the candidate gene for PPD in human and mouse, It should be the candidate gene for chicken polydactyly.The coding sequence (CDS) of chicken Lmbrl was cloned and an short splicing form of chicken Lmbrl transcripts was detected which is the result of the complete deletion of exon 4 as C7orf2 in human Acheiropodia (ACHP). In addition, six cSNPs were detected in the CDS of chicken Lmbrl by RT-PCR from five-toed Silkies and four-toed White Plymouth Rock broilers. Two of them were missense mutation, and four for silence mutation. A797G and T1254C polymorphisms show the tendency of special change between silikies and White Plymouth Rock broilers on RNA level.The 10-16 introns of chicken Lmbrl were cloned and found their splicing sites follow the consensus (5’AG/N, 3’N/GT). The size of chicken part Lmbrl genomic gene including 10-17 exons is about one fifth to the corresponding region of human C7orf2 and one third to the corresponding region of mouse Lmbrl. We predict the whole genomic size of chicken Lmbrl is about 50-60kb. Through sequencing, 63 SNPs in the introns of chicken Lmbrl were detected. The ratio of transition and transversion is 1.6:1.Po also behaves as an incomplete dominant in CAU resource reference population established by silkies and White Plymouth Rock broilers. We first reported that T1254C in the coding sequence of chicken Lmbrl is the special mutation site for polydactyly in CAU resource population.Homologous TT for T1254C corresponds to polydactyly, homoogous CC for T1254C corresponds to normal, heterozygote for T1254C corresponds to polydactyly or normal, the penetrance of the Polydactyly in heterozygote for T1254C is 84%. T1254C poymorphism could expain the phenotype variation in digit number,which shows that T1254C has a critical role for polydactyly.In our research, we observed clear association between PCR-SSCP genotypes and breeds (or digit phenotype) in Parents population by three pairs of primers. Silkies have special haplotype in intron 13 and exon 16.Our research showed that the mutations of Lmbrl also have a significant association with some carcass characters .The association analysis on PCR-SSCP genotype related with T1254C polymorphism and carcass characters shows ,the eviscerated yield rate for AA genotype was 62.93%,which was significant lower than that of BB genotype (63.83%) (P<0.05),while the gizzard rate for/L4 genotype was 2.21% ,which was significant higher than that of BB genotype (2.00%) (P<0.05); the shank girth for AA genotype was 4.31cm ,which was significant higher than that of AB genotype(4.17cm) and extremely significant higher than that of BB genotype (4.11cm) (PO.OJ );The shank and claw rate for AA genotype was 6.43% ,which was extremely significant higher than that of BB genotype (5.97%) .Combining with T1254C and G1255A polymorphisms, we also observed an extremely significant association between genotype and the shank and claw rate and shank girth(P<0.01).The close association of chicken Lmbrlpolymorphism with digit phenotype and the shank and claw rate demonstrates that Lmbrl gene has multi-effect or is extremely linked with main QTL for these carcass traiteJLmbr] is the key gene for controling polydactyly in chicken. The close association of chicken Lmbrl gene polymorphism with the shank and claw rate and shank girth also gives us a hint that Lmbrl gene may have an important role not only on polydactyly but also on skeleton development.The chicken Lmbrl gene was mapped to chromosome 2p region near ADL0270 by linkage analysis and RH mapping method(cR=30.6,Lod=8.23), this region is the homology with human 7q36 and mouse 5B1 chromosome including C7orf2/Lmbrl where localized the preaxial polydactyly(PPD) mutant in mouse and human. The mapping results and functional analysis demonstrate that Lmbrl更多还原