【作者】 刘薇；

【导师】 李宁；

【作者基本信息】 中国农业大学 ， 生物化学与分子生物学， 2004， 博士

【摘要】 为研究丝羽乌骨鸡体内黑色素合成和沉积的分子机制，本研究构建了丝羽乌骨鸡的基因组细菌人工染色体(BAC)文库。利用构建的BAC文库对Z染色体上黑色素相关基因酪氨酸酶相关蛋白1基因(TYRP1)的基因结构以及该基因的表达与黑色素沉积的关系进行了研究，同时构建了位于Z染色体上缺乏标记区段的表皮黑色素抑制因子基因(ID)的BAC重叠群。 (一) 本文构建了中国特有的丝羽乌骨鸡的BAC文库，结果如下： (1) 该文库共有138，240个BAC克隆(分为72个超级池，每个超级池由20块96孔板组成)。整个文库以两级3维的结构保存，建立了高效快速的PCR筛选系统。 (2) 对随机挑选的452个BAC克隆的插入片段进行鉴定估计文库的平均插入为118kb，文库的基因组覆盖率为13.34倍，预计从文库中筛选到单拷贝基因的机率为99.984％。用40个微卫星标记和8个功能基因对整个文库进行PCR筛选，得到的平均阳性克隆数分别为10.423和11.125。对32个BAC克隆的荧光原位杂交结果表明，文库的最大嵌合率为6％。对筛选得到的6个含LMBR1序列的BAC克隆酶切分析的结果表明BAC克隆之间是相互重叠的。 (二) 本文对Z染色体上与黑色素相关的基因TYRP1和ID的研究结果如下： (1) 获得了10，066bp的TYRP1的全长基因组序列，分析结果表明：丝羽乌骨鸡的TYRP1外显子与人的TYRP1外显子同源性为70.1％。鸡TYRP1基因比人TYRP1基因缺少1个内含子；内含子中有2个微卫星序列。利用内含子1中的微卫星对家系进行基因组扫描，卡方检验结果表明TYRP1与皮肤色和爪色显著相关(0.01＜P＜0.05)，和胫色极显著相关(P＜0.01)。用荧光原位杂交进行TYRP1的染色体定位，首次将TYRP1定位在Z和W染色体长臂近着丝粒的位置。 (2) 在丝羽乌骨鸡、白莱航鸡、绿壳蛋鸡和寿光鸡的TYRP1的7个外显子和内含子6中寻找SNP，比较PCR产物测序结果，只在内含子6中发现两个SNP。经过预测，发现其中的一个SNP可能使丝羽乌骨鸡的内含子6中出现了新的转录因子的结合位点。对丝羽乌骨鸡TYRP1内含子6中相对于其他3种鸡的突变序列进行的凝胶阻滞试验，证实了丝羽乌骨鸡的TYRP1在这个突变位置产生了与转录因子的结合位点。 (3) 用RT-PCR检测丝羽乌骨鸡0.5到16天胚胎各组织中TYRP1的表达，结果表明TYRP1在6天的鸡胚眼中开始表达，10天以后开始在其它组织依次表达。RT-PCR检测白莱航和乌骨鸡的8天、16天胚胎和出生后2天各组织的TYRP1表达，结合对表型的观察发现，乌骨鸡各组织中黑色素的沉积随TYRP1的表达出现，而白莱航(除肾脏外)只在有黑色素沉积的眼睛中表达TYRP1，其他组织既无TYRP1的表达也无黑色素沉积。可见TYRP1的表达与黑色素的沉积和分布是一致的。 (4) 以离ID最近的ACO1(aconitase 1，顺乌头酸酶)基因为起点，设计引物筛选含ACO1的BAC克隆，用染色体步行的方法得到16个BAC克隆。通过BAC指纹分析，构建起包含13个克隆，覆盖了387kb的BAC重叠群。通过荧光原位杂交确认了这个重叠群处于Z染色体末端。中国农业人学博卜学位论文摘要 本研究构建了中国特有鸡种丝羽鸟骨鸡基因组BAC文库并进行了文库质量鉴定，它所具有的13倍高基因组覆盖率、6%的嵌合率和部分重叠的克隆使其成为完善鸡的基因组图谱、研究基因功能和构建BAC重叠群的优质资源。其次，本文对首次测定的TYR尸了的全基因序列和SNP进行分析，发现丝羽鸟骨鸡的TJ坂Pj具有与其它鸡种不同的微卫星标记和转录调控位点，说明了y尺尸了的序列和sNP造成的表达调控的变化可能与乌骨鸡独特的黑色素沉积方式有关。通过对鸟骨鸡与自莱航鸡不同发育期胚胎的T圣’R尸了表达谱和对鸡胚的黑色素沉积时间和分布的观察，进一步证明了Tr尺Pj与黑色素沉积存在密切的关系，为研究丝羽鸟渭·鸡体内黑色素沉积的分子机理提出了新的假设和新的证据。最后，本文在ID基因区域进行染色体步行，构建了覆盖387kb的BAC重叠群，建_、父了构建染色体未知区域物理图谱的技术方法，为ID基因未来的定位和克隆打卜了良女r的基础。更多还原

【Abstract】 To research melanin synthesis and its accumulation mechanism, we constructed a Silky bacterial artificial chromosome (BAC) library. The library was used to study the two genes on chicken Chr. Z which involve in melanin, tyrosinase related protein 1 gene (TYRPI) and dermal melanin inhibitor gene (ID). The genomic structure of TYRP1 was determined and its correlation between melanin accumulation and TYRP1 expression was studied. Moreover, we constructed a BAC contig near ID locus which was on a region short of DNA marker in Chr.Z.I. Results of Silky BAC library construction:(1) The chicken BAC library consists of 138,240 BAC clones in total(72 superpools, each for 20 96-well plates). The stored library was organized in two grades and 3-dimension structure, and a PCR screening system with high efficiency was built.(2) Average insert size of the library is 118 kb evaluated from analysis of 452 BACs randomly picked from library. So the genome coverage of the library is 13.34 and the possibility to find a single-copy gene in the library is 99.984%. Results of screening by 40 microsatellite markers and 8 functional genes showed-the average positive clone numbers of them were 10.423 and 11.125 respectively. And maxi chimerism rate is 6% estimated from FISH results of 32 BACs. PFGE analysis of 6 EcoR] digested BACs clones which were derived from the library and contained LMBR1 sequence proved the overlap among the BACs.II. Results of study on TYRP1 and ID:(1) We first sequenced the 10,066 bp whole genomic sequences of chicken TYRP1. Analysis of the sequences indicates that: compared with human TYRP1, chicken TYRP1 is short of an intron and there is two microsatellites in introns. GeneScanning result in resource family with the microsatellite in intronl indicates that TYRP1 affects the color of skin and claw on a significant level (0.01<P<0.05). And TYRP1 affects leg color on exteme significant level (P<0.01). Chicken TYRP1 exons show 70.1% nucleotide sequence homology with human exons. FISH result shows that TYRP1 locates on the long arm of Z and W near to the centromere.(2) We searched for SNPs in 7 exons and intron 6 of TYRP1 by PCR and sequencing using genomic DNA from 4 breeds. They are Silky, White Leghorn, Blue Shell Layer and Shouguang. There are 2 SNPs in intron 6. One of them was forecasted to produce transcript factor binding site in intron 6 of Silky TYRP1. Gel-shift result of this mutation proved that there is transcript factor binding site around the mutated site.(3) TYRP1expression was detected in chicken embryo from 0.5 to 16 day by RT-PCR. Results showed that TYRP1 started to express in eyes of 6 day embryo, and then expressed in other tissues of 10 day embryos or later. Observation of chicken phenotype and RT-PCR results of TYRP1 expression in 8 day, 16 day embryos and 2 day hatched chicken also showed that melanin accumulation was appeared after detection of TYRP1 in Silky. Meanwhile, TYRPI expression was only detected ineyes of White Leghorn (except for kidney) , the only tissue where melanin exists. All these results show that TYRP1 expression is consistent with the melanin accumulation time.(4) Started with ACO1 (aconitase I), we got positive clone of ACO1 then obtained 16 clones by chromosome walking. After analysis fingerprinting of 16 BAC clones. We obtained a 387kb BAC contig containing 13 BACs. The contig was confirmed to locate on the end of long arm of Chr. Z by FISH.In summary, we constructed and characterized a BAC library of Silky, a unique Chinese native breed of chicken. The library has high genome coverage (13-fold), chimerism (6%) and overlapped BACs, which made it a valuable resource to complete chicken physical map, study functional genes and construct BAC contigs. We analyzed the whole genomic sequence and SNPs of TYRP1 and found that Silky TYRP1 is different with other breeds both in microsatellite and transcription regulation site. This might indicate that the variation of Silky transcription regulation may更多还原