【作者】 王建涛；

【导师】 李祥龙；

【作者基本信息】 河北农业大学 ， 动物遗传育种与繁殖， 2008， 硕士

【摘要】 本试验根据GenBank上已发表的牛（NW₀01502197）和狗（NC₀06612）RAB27A基因序列设计引物,扩增山羊RAB27A基因从内含子1至外显子6部分序列片断,测序总长度为11483bp,已提交到GenBank,序列号为EU595665。为比较不同物种RAB27A基因分化程度,也同时测定了绵羊RAB27A基因部分序列,测序总长度为4728bp,已提交到GenBank,序列号为:EU599576。根据测序结果发现了山羊RAB27A基因序列上的33个突变,其中处于外显子的有2个,UTR的5个,内含子的26个。依据序列号EU595665上的位置来命名这些突变位点,处于外显子上的2个突变分别为183C＞T和9669C＞A,其中引起蛋白质的氨基酸序列的改变的点突变只有1个,是9669C＞A（Leu180Met）。处于UTR和内含子的31个点突变分别为147G＞A、9967T＞C、10101G＞A、10459G＞C、10483C＞A、523T＞C、571T＞C、699T＞C、2306G＞A、2426A＞T、2428T＞G、2719C＞T、3338A＞T、3636T＞C、3660G＞A、3817A＞C、3907G＞C、4165A＞G、4440G＞A、4492C＞T、4634A＞T、4708C＞T、5198C＞A、5300C＞T、5686G＞A、6472T＞C、6754T＞C、8127C＞G、8316G＞A、8778T＞C和8930C＞T。同时发现山羊RAB27A基因序列存在缺失、插入和替代的现象,缺失现象为第2772位缺失一个腺嘌呤（A）、第5109位缺失一个胸腺吡啶（T）和第4238-4252位缺失一个15bp的核苷酸片段（TTTTGTAACTATTTT）;发生的插入现象是第4440位插入两个碱基（AG）;发生的替代现象是第10325-10330位一个6bp核苷酸片段（AAAACA）被一个3bp核苷酸片段（CAA）替代。147G＞A位点上检测到G和A两个等位基因。我们研究的8个山羊品种(承德无角山羊、雷州黑山羊、成都麻羊、南江黄羊快长系和黑色系、济宁青山羊、辽宁绒山羊和唐山奶山羊1333个个体中G为优势等位基因（90.39%）,突变率在不同品种中存在差异。8个群体的GG基因型频率都比较高（63.83-97.37%）,AG基因型频率在各群体间差异较大（2.63-31.91%）,AA基因型仅在雷州山羊、南江黄羊（快长系和黑色系）等三个品种中出现。对8个地方山羊群体RAB27A基因147G＞A位点进行的Hardy—Weinberg平衡检验结果表明,雷州山羊和南江黄羊黑色系未处于Hardy-Weinberg平衡状态（P＜0.05）,其余6个山羊群体均处于平衡状态,认为这6个山羊群体RAB27A基因147G＞A位点上均未进行人工选择,遵循随机交配模式。同时这两个山羊群体的皮毛颜色都是黑色,这就说明山羊RAB27A基因147G＞A位点可能与山羊的黑色毛色有关。山羊RAB27A基因9669C＞A位点检测到C和A两个等位基因,通过对多态位点9669C＞A群体分析,证实9669C＞A是一个稀有突变。总体来说,不同物种RAB27A基因CDS序列变异度不大,以山羊的RAB27A基因CDS序列为参考,于其他物种的核苷酸一致性分别为绵羊99.40%、牛98.04%、马92.94%、猪92.79%、狗91.44%、大猩猩90.69%、人90.39%、恒河猴89.79%、小鼠86.94%、挪威鼠85.44%、短尾负鼠86.49%、鸡81.38%和非洲爪蛙80.63%。各物种间RAB27A基因CDS的聚类接过与动物分类学分法基本一致。更多还原

【Abstract】 On the basis of cattle（NW₀01502197）and dog（NC₀06612）RAB27A sequences from GenBank,part of goat RAB27A gene from intron 1 to exon 6 was amplified.The total length of determined sequence was 11483bp,which had been submitted to GenBank with the accession number EU595665.For the purpose of analyse the degree of differentiation among different species,sheep RAB27A gene fragments was also amplified.The total length of determined sequence of sheep was 4728bp,with the accession number was EU599576.A total of 33 point mutations were found in the determinged goat RAB27A gene sequence,of which 2 were in exons,5 in UTR and 26 in introns.These mutations in exons were named as follows:183C＞T and 9669C＞A.There was only one missense mutation, 9669C＞A（Leu180Met）.The 31 mutations in UTR and introns were 147G＞A,9967T＞C、10101G＞A、10459G＞C、10483C＞A、523T＞C、571T＞C、699T＞C、2306G＞A、2426A＞T、2428T＞G、2719C＞T、3338A＞T、3636T＞C、3660G＞A、3817A＞C、3907G＞C、4165A＞G、4440G＞A、4492C＞T、4634A＞T、4708C＞T、5198C＞A、5300C＞T、5686G＞A、6472T＞C、6754T＞C、8127C＞G、8316G＞A、8778T＞Cand 8930C＞T.There are deletions of Adenine,Thymine and a short sequence（TTTTGTAACTATTTT） at the locus of 2772,5109 and 4238-4252 respectively.An insertion of two bases（AG）was detected at the 4440 site,and a 6bp nucleotide（AAAACA）was repleaced by 3bp nucleotide（CAA）from 10325 to 10330.Two alleles of G and A was detected at the locus of 147G＞A.Then 333 individials from 8 breeds（Chende Polled goat、Leizhou goat、Chengdu Ma goat、Nanjiang Brown goat（B）、Nanjiang Brown goat（F）、Jining Gray goat、Liaoning Cashmere goat and Tangshan Dairy goat）were studied.We found that G is the preponderant allele（90.39%）.The mutation rate is different among different varieties.GG genotype frequency is 63.83-97.37%,GG genotype frequency is different among different varieties（2.63-31.91%）,and the AA genotype were observed in Leizhou goat,Nanjiang Brown goat（B）and Nanjiang Brown goat（F）.Based on the Hardy—Weinberg test for 8 native goat breeds,we found that Leizhou goat and Nanjiang Brown goat（B）were deviation from the Hardy-Weinberg equilibrium,the others showed a good fit to Hardy-Weinberg equilibrium.So,we suggest that these 6 native goat breeds have not been selected by artificial selection at corresponding mutation sites.At the same time,we discovered that Leizhou goat and Nanjiang Brown goat（B） both are black.It could be inferred that the 147G＞A might be related to the black coat color of goat.Two alleles,C and A at 9669C＞A were deteced.Based on population analysis,we confirmed that the 9669C＞A was a rare mutation.Compared with the CDS of goat RAB27A gene,the nucleotide similarity is 99.40%for sheep,98.04%for bovine,92.94%for horse,92.79%for pig,91.44%for dog 90.69% for gorilla,90.39%for human,89.79%for rhesus,86.94%for mice,85.44%for norway rat, 86.49%for short-tailed opossum,81.38%for chicken,and 80.63%for xenopus lesvis. There was lowwer divergency of CDS among different species,and the cluster result of species is basically accord with the traditional taxonomy.更多还原