水貂脾脏cDNA文库构建及MuLu-DRA基因克隆与多态性分析
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摘要
主要组织相容性复合体(major histocompatibility complex,MHC)是脊椎动物中发现的编码组织相容性抗原的高度多态、紧密连锁的基因群,在免疫应答中起重要作用。由于其在动物抗病性能上发挥着重要作用,成为人类及畜牧业发展中的一个重要的研究课题。水貂是我国毛皮动物饲养的主要品种之一,水貂皮是高档裘皮,在我国已有50多年的养殖历史。毛皮动物养殖的关键因素是疾病的预防,而通过免疫来达到这一目的是最有效的手段之一。但目前对水貂免疫机制等方面的研究还比较薄弱。本研究通过构建cDNA全长文库,为水貂的功能基因的克隆、分析奠定基础;通过对水貂MuLu-DRA基因的克隆及多态性分析,为进一步研究水貂的群体进化、抗病相关候选基因的克隆提供理论数据。
(1)本研究通过SMART(Switching Mechanism At 5’end of the RNA Transcript)技术构建了美国短毛黑水貂脾脏全长文库。大肠杆菌XL1-Blue平皿测定和PCR鉴定表明,所构建的文库库容为1.07×109pfu/ml,重组率为91.1%,达到了用于目的基因的分离筛选及克隆表达的建库要求。
(2)首次克隆了水貂主要组织相容性复合体基因MuLu-DRA的全长cDNA序列。得到了全长765bp的cDNA全长序列,共编码254个氨基酸,将得到的序列与其他动物进行比对,结果发现水貂的MuLu-DRA基因与狗、猫、海驴、人、猕猴的核苷酸同源型分别达到97%、93%、93%、89%、88%,并构建了物种同源性发育树;氨基酸同源性分别达到96%、90%、93%、85%、85%。应用分子生物学软件对推导的氨基酸进行分析,结果表明,蛋白质的分子量为29KD,等电点约为4.8,二级结构表明该蛋白质为混合性蛋白质,其中α螺旋占38.6%,β片层占29.5%,Turn(转角)占17.3%,Coil(无规则卷曲)占14.6%。
(3)对6个水貂群体MuLu-DRA*exon2进行了PCR-SSCP分析,共出现6种基因型,分别为AA、BB、CC、AB、BC、AC,说明MuLu-DRA*exon2基因共由三个等位基因A、B、C控制,说明MuLu-DRA*exon2序列进化较为保守,多态性不高。计算了等位基因频率、遗传杂合度、多态信息含量、遗传距离。构建了6个水貂群体的NJ聚类图,丹麦红眼白水貂先是与咖啡水貂聚在一起,然后再与金洲水貂聚在一起,珍珠水貂与银蓝水貂单独聚为一组,最后再与美国短毛黑水貂聚在一起。说明丹麦红眼白水貂、咖啡水貂、金州水貂这三个品种遗传关系较近,银蓝色水貂和珍珠水貂遗传关系较近,能正确反应我国目前饲养水貂种群的遗传关系。进行了MuLu-DRA*exon2多态性与血液指标的相关研究,结果表明基因型与血液指标均不相关;品种是影响WBC数量的重要因素。
The major histocomapatibility complex (MHC) is a group of closely-linked and highly polymorphic gene in vertebrate animals. The gene encode the major histocompatibility antigen and plays a critical role in immune response. Due to its crucial role in animal disease-resistance, MHC has become an important research subject of human and animal husbandry. Mink is one of the major species of animals in the fur industry, and we have more than fifty years histories of farming minks in our country. Successful farming fur animal critically depends on the effective prevention of epidemic diseases, and immunological control is the most powerful way to achieve the aim. However, at present only few studies on mink immunity mechanism have reported. Therefore ,the current situation has seriously hindered the development of effctive means of prevention and treatment of these diseases. This research constructed the cDNA library ,which laid the foundation for the mink function gene clone and analysis; The mink MuLu-DRA gene cDNA sequence was cloned and analyzed for polymorphism, which provides the theoretical data for further study on the population evolution and the disease-resistace related candidate genes.
(1) The full-length cDNA Library of American mink spleen was been constructed by using SMART (Switching Mechanism At 5 , end of RNA Transcript) technology. According to the results of phage plaques bright selection of host bacterial strain XL1-Blue and PCR detection, the percentage of recombinant phages were 91.1% and the titer was 1.07×109 pfu/ml, the library was of high quality for cloning target genes and expressing target proteins.
(2) In order to study function and molecular polymorphism of MuLu-DRA gene of mink. Full-length of the cDNA sequence was 765bp, code 254 amino acid. The result of blasting indicated: the nucleotide had 97%,93%,93%,89%,88% identity with dog, cat, zalophus, homo spiens and macaca respectively; the deduced amimo acid had 96%,90%,93%,85%,85% identity with dog,cat,zalophus, homo spiens and macaca respectively, and the homology tree was constructed. The analysis of deduced amino acid sequence by molecular biology software indicated: the protein molecular weight was 29KD, isoelectric point approximately was at pH 4.8. The analysis of secondary structure of protein indicated: the protein was the mixing property protein, Alapha helix 38.6%, Beta sheets 29.5%, loop (corner) 17.3%, Coil (non-regular curl) 14.6%.
(3) The genetic polymorphism of six mink breeds was investigated for MuLu-DRA gene exon 2 by PCR-SSCP, the results showed that the gene six kinds of genotypes, respectively was AA、BB、CC、AB、AC、BC. It explained that the MuLu-DRA*exon2 gene altogether was controlled by three allele A, B, C, the polymorphism is relatively low. We calculated the allele frequency, the heredity heterozygosity value(H),polymorphism information content (PIC),the heredity distance, constructed NJ cluster chart of six breeds mink, the Regal White mink first clustered with Pastel mink , then gathered JinZhou mink, the Pearl mink and Silverblue mink gathered the same group alone, finally gathered with the American mink. It explained that the Regal White mink and the Pastel mink and JinZhou mink has nearly genetic affinity, the Pastel mink and Silverblue mink had close genetic affinity. The results were consistent with mink population in our country at present, andcan respond the mink population correctly the genetic affinity. We carried on the MuLu-DRA*exon2 polymorphism and the blood target related research. The results explained that the genotype and the blood traits are non-correlated.The variety has the remarkable influence to WBC.
(1)本研究通过SMART(Switching Mechanism At 5’end of the RNA Transcript)技术构建了美国短毛黑水貂脾脏全长文库。大肠杆菌XL1-Blue平皿测定和PCR鉴定表明,所构建的文库库容为1.07×109pfu/ml,重组率为91.1%,达到了用于目的基因的分离筛选及克隆表达的建库要求。
(2)首次克隆了水貂主要组织相容性复合体基因MuLu-DRA的全长cDNA序列。得到了全长765bp的cDNA全长序列,共编码254个氨基酸,将得到的序列与其他动物进行比对,结果发现水貂的MuLu-DRA基因与狗、猫、海驴、人、猕猴的核苷酸同源型分别达到97%、93%、93%、89%、88%,并构建了物种同源性发育树;氨基酸同源性分别达到96%、90%、93%、85%、85%。应用分子生物学软件对推导的氨基酸进行分析,结果表明,蛋白质的分子量为29KD,等电点约为4.8,二级结构表明该蛋白质为混合性蛋白质,其中α螺旋占38.6%,β片层占29.5%,Turn(转角)占17.3%,Coil(无规则卷曲)占14.6%。
(3)对6个水貂群体MuLu-DRA*exon2进行了PCR-SSCP分析,共出现6种基因型,分别为AA、BB、CC、AB、BC、AC,说明MuLu-DRA*exon2基因共由三个等位基因A、B、C控制,说明MuLu-DRA*exon2序列进化较为保守,多态性不高。计算了等位基因频率、遗传杂合度、多态信息含量、遗传距离。构建了6个水貂群体的NJ聚类图,丹麦红眼白水貂先是与咖啡水貂聚在一起,然后再与金洲水貂聚在一起,珍珠水貂与银蓝水貂单独聚为一组,最后再与美国短毛黑水貂聚在一起。说明丹麦红眼白水貂、咖啡水貂、金州水貂这三个品种遗传关系较近,银蓝色水貂和珍珠水貂遗传关系较近,能正确反应我国目前饲养水貂种群的遗传关系。进行了MuLu-DRA*exon2多态性与血液指标的相关研究,结果表明基因型与血液指标均不相关;品种是影响WBC数量的重要因素。
The major histocomapatibility complex (MHC) is a group of closely-linked and highly polymorphic gene in vertebrate animals. The gene encode the major histocompatibility antigen and plays a critical role in immune response. Due to its crucial role in animal disease-resistance, MHC has become an important research subject of human and animal husbandry. Mink is one of the major species of animals in the fur industry, and we have more than fifty years histories of farming minks in our country. Successful farming fur animal critically depends on the effective prevention of epidemic diseases, and immunological control is the most powerful way to achieve the aim. However, at present only few studies on mink immunity mechanism have reported. Therefore ,the current situation has seriously hindered the development of effctive means of prevention and treatment of these diseases. This research constructed the cDNA library ,which laid the foundation for the mink function gene clone and analysis; The mink MuLu-DRA gene cDNA sequence was cloned and analyzed for polymorphism, which provides the theoretical data for further study on the population evolution and the disease-resistace related candidate genes.
(1) The full-length cDNA Library of American mink spleen was been constructed by using SMART (Switching Mechanism At 5 , end of RNA Transcript) technology. According to the results of phage plaques bright selection of host bacterial strain XL1-Blue and PCR detection, the percentage of recombinant phages were 91.1% and the titer was 1.07×109 pfu/ml, the library was of high quality for cloning target genes and expressing target proteins.
(2) In order to study function and molecular polymorphism of MuLu-DRA gene of mink. Full-length of the cDNA sequence was 765bp, code 254 amino acid. The result of blasting indicated: the nucleotide had 97%,93%,93%,89%,88% identity with dog, cat, zalophus, homo spiens and macaca respectively; the deduced amimo acid had 96%,90%,93%,85%,85% identity with dog,cat,zalophus, homo spiens and macaca respectively, and the homology tree was constructed. The analysis of deduced amino acid sequence by molecular biology software indicated: the protein molecular weight was 29KD, isoelectric point approximately was at pH 4.8. The analysis of secondary structure of protein indicated: the protein was the mixing property protein, Alapha helix 38.6%, Beta sheets 29.5%, loop (corner) 17.3%, Coil (non-regular curl) 14.6%.
(3) The genetic polymorphism of six mink breeds was investigated for MuLu-DRA gene exon 2 by PCR-SSCP, the results showed that the gene six kinds of genotypes, respectively was AA、BB、CC、AB、AC、BC. It explained that the MuLu-DRA*exon2 gene altogether was controlled by three allele A, B, C, the polymorphism is relatively low. We calculated the allele frequency, the heredity heterozygosity value(H),polymorphism information content (PIC),the heredity distance, constructed NJ cluster chart of six breeds mink, the Regal White mink first clustered with Pastel mink , then gathered JinZhou mink, the Pearl mink and Silverblue mink gathered the same group alone, finally gathered with the American mink. It explained that the Regal White mink and the Pastel mink and JinZhou mink has nearly genetic affinity, the Pastel mink and Silverblue mink had close genetic affinity. The results were consistent with mink population in our country at present, andcan respond the mink population correctly the genetic affinity. We carried on the MuLu-DRA*exon2 polymorphism and the blood target related research. The results explained that the genotype and the blood traits are non-correlated.The variety has the remarkable influence to WBC.
引文
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