黑线仓鼠白化突变系白化性状产生机理的研究
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摘要
黑线仓鼠白化突变系（A：CHA）的培育在国内外都属首次，本品系的育成为医学生物学提供了新的动物模型，也是我国为国际实验动物领域增添的一个新型品系，具有较高的学术价值。黑线仓鼠白化突变系（A：CHA）建立后显示出比黑线仓鼠更好的应用前景，在皮肤微循环、辐射防护、体表寄生虫以及流行病学等应用方面，都表现出较黑线仓鼠更多的优越性。目前只知道黑线仓鼠白化突变系的基因遗传属于单一隐性遗传，但并不清楚黑线仓鼠白化性状发生的原因，因此非常有必要揭示黑线仓鼠白化突变群白化性状发生机理，弄清楚该突变种群的遗传背景，进一步完善白化黑线仓鼠突变群的基础生物学数据，为其在生物医学中的推广应用奠定基础。本研究以黑线仓鼠与白化突变系皮肤组织为研究对象，利用组织学、分子生物学和转录组学等方法，对黑线仓鼠与白化突变系的皮肤组织进行比较研究，综合探讨了黑线仓鼠与其白化突变系皮肤组织组织学与分子水平的差异。主要研究内容包括黑线仓鼠及其白化突变系毛囊中黑色素细胞的组织学分析，皮肤组织黑色素细胞超微结构的比较分析，皮肤组织白化相关基因的克隆与生物信息学分析，白化相关基因的表达量比较分析以及基于Illumina测序技术对黑线仓鼠与白化突变系皮肤组织进行转录组测序和分析。现将实验研究报告如下:
1.黑线仓鼠及其白化突变系毛囊中黑色素细胞的组织学分析
本研究采用多巴染色来显示黑色素细胞，结果表明，黑线仓鼠与白化突变系的毛囊组织中均有多巴的阳性着色，多巴阳性着色显示的是成熟的黑色素细胞，说明在黑线仓鼠与白化突变系毛囊组织中都有成熟黑色素细胞分布，但分布数量不同。黑线仓鼠的多巴反应阳性区要明显高于白化突变系。多巴阳性区间接反应酪氨酸酶（TYR）的活性，说明白化突变系TYR表达水平显著低于黑线仓鼠。多巴-甲苯胺蓝复染充分显示多巴阳性黑色素在毛囊组织中的分布，黑线仓鼠与白化突变系毛囊组织中黑色素细胞主要存在于毛囊的毛乳头中。
2.黑线仓鼠与白化突变系皮肤黑色素细胞的超微结构分析
⑴黑线仓鼠的黑色素含量显著高于白化突变系。可见，皮肤组织中黑色素的缺乏是导致黑线仓鼠白化突变系发生的直接原因。
⑵相对于黑线仓鼠，白化突变系皮肤组织的黑色素细胞明显较少,黑色素细胞中黑色素体的发育程度低，大多黑色素体为未有黑色素沉积的黑色素前体（pre-melanosome），黑色素细胞中黑色素体的密度明显较小。可见，成熟黑色素细胞的缺乏以及黑色素体中黑色素沉积的缺乏是黑线仓鼠白化发生的组织学特征。
3.黑线仓鼠与白化突变系白化相关基因的克隆与序列分析
本实验通过对黑线仓鼠及其白化突变系的白化相关基因TYR、TYRP1、TYRP2、P、MITF、Agouti基因进行克隆与序列分析，明确黑线仓鼠白化突变系白化性状产生是否是由于上述基因发生突变所致，结果首次克隆得到了黑线仓鼠及其白化突变系白化相关基因TYR、TYRP1、TYRP2、P、MITF、Agouti基因完整的编码区序列，但对其基因编码区进行生物信息学分析并未发现突变位点，其白化性状产生的机理与已发现的白化机制不同，还有待进一步的研究。
4.黑线仓鼠及其白化突变系白化相关基因表达水平比较分析
本研究结果表明TYR、TYRP1、TYRP2、P、MITF、Agouti mRNA表达量在黑线仓鼠中是白化突变系的2.5、5.3、1.12、1.7、1.8、1.7倍,结论提示TYR、TYRP1、P、MITF、Agouti的基因表达量与黑线仓鼠白化突变性状的产生存在一定的相关性，本研究为阐明白化相关基因在色素沉着过程中的调控机制,以及黑线仓鼠白化突变系白化性状产生机理奠定了一定的理论基础。
5.基于Illumina测序技术对黑线仓鼠及其白化突变系皮肤组织进行转录组测序和分析
本研究以黑线仓鼠与白化突变系皮肤组织为研究对象，应用Illumina/Solexa高通量测序技术对其转录组进行深度测序，使用短序列组装软件SOAP denovo对其转录组测序结果进行从头组装（De novo），建立转录组数据库，并与蛋白数据库Nr、Swiss-Prot、KEGG和COG进行序列比对、功能注释及代谢通路分析；在此基础上，进行差异表达基因分析，筛选与色素合成相关的基因，对白化机制进行探究。
通过本研究共获得7千多万条高质量配对短序列，通过序列组装共获得52,439条Unigenes。通过对黑线仓鼠与白化突变系皮肤转录组进行差异表达基因分析，共筛选出6788条差异表达基因（︱log2Ratio︱≥1且FDR≤0.001），其中表达上调5476条，表达下调1312条，通过GO功能富集分析及KEGG通路富集分析，这些差异表达基因主要参与细胞代谢、蛋白质翻译、细胞外基质受体相互作用等生物学进程。共筛选出1212条参与色素沉着的Unigenes，其中发生表达上调的有130条，表达下调的有13条，Unigenes基因分布于258条经典Kegg代谢通路图，而筛选得到的色素合成基因位于两个代谢通路中，分别为酪氨酸Kegg代谢通路图和黑色素合成代谢通路图。这些实验结果为进一步研究黑线仓鼠白化突变系白化机制奠定基础并为研究黑线仓鼠的基因结构及功能奠定基础。
Cultivation of Cricetulus barabensis albino mutant lines (A: CHA) at home and abroad areconsidered for the first time, The strains of Medical Biology provides a new animal model, alsohas a high academic value for the field of Laboratory Animal Care International that adds a newstrain. Cricetulus barabensis albino mutant lines (A: CHA) after establishment shows betterapplication prospect than the black line hamster in the skin microcirculation, radiation protection,ectoparasites and epidemiological applications, which have more superiority than the black linehamster. Currently only know the black line hamster albino mutant lines genes that belong to asingle genetic recessive inheritance, but do not know the reason of black line hamster in albinotraits, so it is necessary to reveal the mechanism of black line hamsters in albino mutation ofalbino trait, figure out the mutations in the genetic background of the population, to furtherimprove the albino black line hamster mutant group on the basic of biological data, to lay a goodfoundation for the popularization and application in biomedical. This study use the skin tissue ofblack line hamster albino mutation as research material, using the methods of biology, histology,molecular biology to comparative study the skin tissue of black line hamster albino mutant line,comprehensively discussing the machanism of the albino characteristics of the Cricetulusbarabensis albino line. The thesis includes two parts, the first literature of thesis is review, thesecond literature of thesis is research papers, including five chapters, and the last is the summaryand outlook. The main research content includes histological analysis of the melanocytes withinhair folliciles which locate in black line hamster and albino mutant lines, comparing skin tissuemelanoma cell ultrastructure, albino gene cloning and bioinformatics analysis, albino relatedgenes expression and analysis of the albino correlation, based on Illumina sequencing technologyto transcriptome sequencing and analysis of the skin tissue which is in black line hamster andalbino mutant lines. Experiment results will now report as follows:
1. Histological analysis of the hair follicle melanocytes which locate inCricetulus barabensis and its albino mutant lines
Dopa staining results showed that Cricetulus barabensis and its albino mutation of skin tissuewere positive staining of dopamine, dobutamine positive staining shows the dopa positive melanin cells, this results demonstrate that mature melanocytes within black line hamsters and albinomutant lines are distributed in the skin tissue, but the distribution is different. Black line hamsterin dopa-positive area was significantly higher than the albino mutant lines. Dopa is a specificsubstrate of tyrosinase, shows that the TYR expression level of albino mutation was significantlylower than the black line hamster. Dopa-toluidine blue redyeing fully indicates that thedistribution of dopa-positive melanin in the skin tissue, the skin tissue melanocytes which locatein the black line and albino hamster exist in the hair follicle dermal papilla.
2. Cricetulus barabensis and albino mutant skin melanoma cellultrastructure study
(1) Cricetulus barabensis melanin content was significantly higher than the albino mutant.Visible, lack of melanin in the skin tissue is the direct reason that causing Cricetulus barabensisalbino mutation.
(2) Comparing with Cricetulus barabensis, the albino mutation has significantly fewer skinmelanin cells, the development of melanosomes in melanocytes is low, most melanosomes arepre-melanosome that has not deposit of melanin. For the albino mutation, the density ofmelanosomes in melanocytes is obviously smaller. Visible, the lack of melanocytes and lack ofmelanin deposition in melanosomes are the histologic features of Cricetulus barabensis albinooccurrence.
3. Cloning and sequence analysis about an albino genes that is in Cricetulusbarabensis and its albino mutation
In this test, through the Cricetulus barabensis and albino mutation of TYR, TYRP1, TYRP2,P, MITF, Agouti gene cloning and sequence analysis, clear Cricetulus barabensis albino mutantalbino trait is due to the gene mutation, according to the site of gene conservation in mice and ratsdesign a few pairs of primers, using RT-PCR method to clone and sequence. Successfully obtainthe Cricetulus barabensis and albino mutation of TYR, TYRP1, TYRP2, P, MITF, agouti gene,first clone coding region sequence of TYR, TYRP1, TYRP2, P, MITF, Agouti gene which is inCricetulus barabensis and albino mutation, but from coding regions did not mutant, the albino traitmechanism needs further research.
4. Comparative analysis about the level of an albino genes expression which isin Cricetulus barabensis and albino mutation
The results showed that TYR, TYRP1, TYRP2, P, MITF, Agouti mRNA expression quantityin black line hamsters is2.5、5.3、1.12、1.7、1.8、1.7time to albino mutation, the conclusion prompt that the expression level of TYR, TYRP1, TYRP2, P, MITF, Agouti has certain correlationwith Cricetulus barabensis color phenotype, suggesting that several members play a regulatingrole in the pigmentation process. This study clarify an albino genes regulation mechanism ofmelanin traits, as well as laid a theoretical foundation of the mechanism for Cricetulus barabensisalbino mutation albino traits.
5. Based on Illumina sequencing technology to transcriptome sequencingand analysis of the skin tissue which is in Cricetulus barabensis and albinomutant lines
This study take the skin tissue of black line hamsters and albino mutation as the researchobject, using Illumina/Solexa high-throughput sequencing technology to the depth of theirtranscriptome sequencing, using short sequence assembly software SOAP denovo to de novoassembly the results of its transcriptome sequencing, establishing database of the transcriptome,and proceeding sequence alignment, functional annotation and metabolic pathway analysis forprotein database with nr, Swiss-Prot, KEGG and COG; on this basis, proceeding the analysis ofdifferentially expressed genes, screening pigment synthesis genes, and to explore the bleachingmechanism.
Through this study, a total of more than80million high quality matching short sequencesare obtained, through sequence assembly received116,504Unigenes. By black line hamsters andalbino mutation skin transcriptome analysis of differentially expressed genes were screened16,905differentially expressed genes(|log2Ratio︱≥1and FDR≤0.001), through GO functionenrichment analysis and KEGG pathway enrichment analysis, these differentially expressed genesinvolved in cellular metabolism, protein translation, extracellular matrix receptor interaction andbiological processes. These differentially expressed genes were screened out53kinds of geneswhich play important regulating effect in protein synthesis, cell proliferation and cell apoptosisduring the process of growth in black line hamsters,12kinds of differentially expressed geneswhich is related to the growth of black line hamsters, and7kinds of differentially expressed geneswhich is ralated to pigment biosynthesis, the results of these experiments lay the foundation forfurther study about the structure and function of genes which is in black line hamsters.
1.黑线仓鼠及其白化突变系毛囊中黑色素细胞的组织学分析
本研究采用多巴染色来显示黑色素细胞，结果表明，黑线仓鼠与白化突变系的毛囊组织中均有多巴的阳性着色，多巴阳性着色显示的是成熟的黑色素细胞，说明在黑线仓鼠与白化突变系毛囊组织中都有成熟黑色素细胞分布，但分布数量不同。黑线仓鼠的多巴反应阳性区要明显高于白化突变系。多巴阳性区间接反应酪氨酸酶（TYR）的活性，说明白化突变系TYR表达水平显著低于黑线仓鼠。多巴-甲苯胺蓝复染充分显示多巴阳性黑色素在毛囊组织中的分布，黑线仓鼠与白化突变系毛囊组织中黑色素细胞主要存在于毛囊的毛乳头中。
2.黑线仓鼠与白化突变系皮肤黑色素细胞的超微结构分析
⑴黑线仓鼠的黑色素含量显著高于白化突变系。可见，皮肤组织中黑色素的缺乏是导致黑线仓鼠白化突变系发生的直接原因。
⑵相对于黑线仓鼠，白化突变系皮肤组织的黑色素细胞明显较少,黑色素细胞中黑色素体的发育程度低，大多黑色素体为未有黑色素沉积的黑色素前体（pre-melanosome），黑色素细胞中黑色素体的密度明显较小。可见，成熟黑色素细胞的缺乏以及黑色素体中黑色素沉积的缺乏是黑线仓鼠白化发生的组织学特征。
3.黑线仓鼠与白化突变系白化相关基因的克隆与序列分析
本实验通过对黑线仓鼠及其白化突变系的白化相关基因TYR、TYRP1、TYRP2、P、MITF、Agouti基因进行克隆与序列分析，明确黑线仓鼠白化突变系白化性状产生是否是由于上述基因发生突变所致，结果首次克隆得到了黑线仓鼠及其白化突变系白化相关基因TYR、TYRP1、TYRP2、P、MITF、Agouti基因完整的编码区序列，但对其基因编码区进行生物信息学分析并未发现突变位点，其白化性状产生的机理与已发现的白化机制不同，还有待进一步的研究。
4.黑线仓鼠及其白化突变系白化相关基因表达水平比较分析
本研究结果表明TYR、TYRP1、TYRP2、P、MITF、Agouti mRNA表达量在黑线仓鼠中是白化突变系的2.5、5.3、1.12、1.7、1.8、1.7倍,结论提示TYR、TYRP1、P、MITF、Agouti的基因表达量与黑线仓鼠白化突变性状的产生存在一定的相关性，本研究为阐明白化相关基因在色素沉着过程中的调控机制,以及黑线仓鼠白化突变系白化性状产生机理奠定了一定的理论基础。
5.基于Illumina测序技术对黑线仓鼠及其白化突变系皮肤组织进行转录组测序和分析
本研究以黑线仓鼠与白化突变系皮肤组织为研究对象，应用Illumina/Solexa高通量测序技术对其转录组进行深度测序，使用短序列组装软件SOAP denovo对其转录组测序结果进行从头组装（De novo），建立转录组数据库，并与蛋白数据库Nr、Swiss-Prot、KEGG和COG进行序列比对、功能注释及代谢通路分析；在此基础上，进行差异表达基因分析，筛选与色素合成相关的基因，对白化机制进行探究。
通过本研究共获得7千多万条高质量配对短序列，通过序列组装共获得52,439条Unigenes。通过对黑线仓鼠与白化突变系皮肤转录组进行差异表达基因分析，共筛选出6788条差异表达基因（︱log2Ratio︱≥1且FDR≤0.001），其中表达上调5476条，表达下调1312条，通过GO功能富集分析及KEGG通路富集分析，这些差异表达基因主要参与细胞代谢、蛋白质翻译、细胞外基质受体相互作用等生物学进程。共筛选出1212条参与色素沉着的Unigenes，其中发生表达上调的有130条，表达下调的有13条，Unigenes基因分布于258条经典Kegg代谢通路图，而筛选得到的色素合成基因位于两个代谢通路中，分别为酪氨酸Kegg代谢通路图和黑色素合成代谢通路图。这些实验结果为进一步研究黑线仓鼠白化突变系白化机制奠定基础并为研究黑线仓鼠的基因结构及功能奠定基础。
Cultivation of Cricetulus barabensis albino mutant lines (A: CHA) at home and abroad areconsidered for the first time, The strains of Medical Biology provides a new animal model, alsohas a high academic value for the field of Laboratory Animal Care International that adds a newstrain. Cricetulus barabensis albino mutant lines (A: CHA) after establishment shows betterapplication prospect than the black line hamster in the skin microcirculation, radiation protection,ectoparasites and epidemiological applications, which have more superiority than the black linehamster. Currently only know the black line hamster albino mutant lines genes that belong to asingle genetic recessive inheritance, but do not know the reason of black line hamster in albinotraits, so it is necessary to reveal the mechanism of black line hamsters in albino mutation ofalbino trait, figure out the mutations in the genetic background of the population, to furtherimprove the albino black line hamster mutant group on the basic of biological data, to lay a goodfoundation for the popularization and application in biomedical. This study use the skin tissue ofblack line hamster albino mutation as research material, using the methods of biology, histology,molecular biology to comparative study the skin tissue of black line hamster albino mutant line,comprehensively discussing the machanism of the albino characteristics of the Cricetulusbarabensis albino line. The thesis includes two parts, the first literature of thesis is review, thesecond literature of thesis is research papers, including five chapters, and the last is the summaryand outlook. The main research content includes histological analysis of the melanocytes withinhair folliciles which locate in black line hamster and albino mutant lines, comparing skin tissuemelanoma cell ultrastructure, albino gene cloning and bioinformatics analysis, albino relatedgenes expression and analysis of the albino correlation, based on Illumina sequencing technologyto transcriptome sequencing and analysis of the skin tissue which is in black line hamster andalbino mutant lines. Experiment results will now report as follows:
1. Histological analysis of the hair follicle melanocytes which locate inCricetulus barabensis and its albino mutant lines
Dopa staining results showed that Cricetulus barabensis and its albino mutation of skin tissuewere positive staining of dopamine, dobutamine positive staining shows the dopa positive melanin cells, this results demonstrate that mature melanocytes within black line hamsters and albinomutant lines are distributed in the skin tissue, but the distribution is different. Black line hamsterin dopa-positive area was significantly higher than the albino mutant lines. Dopa is a specificsubstrate of tyrosinase, shows that the TYR expression level of albino mutation was significantlylower than the black line hamster. Dopa-toluidine blue redyeing fully indicates that thedistribution of dopa-positive melanin in the skin tissue, the skin tissue melanocytes which locatein the black line and albino hamster exist in the hair follicle dermal papilla.
2. Cricetulus barabensis and albino mutant skin melanoma cellultrastructure study
(1) Cricetulus barabensis melanin content was significantly higher than the albino mutant.Visible, lack of melanin in the skin tissue is the direct reason that causing Cricetulus barabensisalbino mutation.
(2) Comparing with Cricetulus barabensis, the albino mutation has significantly fewer skinmelanin cells, the development of melanosomes in melanocytes is low, most melanosomes arepre-melanosome that has not deposit of melanin. For the albino mutation, the density ofmelanosomes in melanocytes is obviously smaller. Visible, the lack of melanocytes and lack ofmelanin deposition in melanosomes are the histologic features of Cricetulus barabensis albinooccurrence.
3. Cloning and sequence analysis about an albino genes that is in Cricetulusbarabensis and its albino mutation
In this test, through the Cricetulus barabensis and albino mutation of TYR, TYRP1, TYRP2,P, MITF, Agouti gene cloning and sequence analysis, clear Cricetulus barabensis albino mutantalbino trait is due to the gene mutation, according to the site of gene conservation in mice and ratsdesign a few pairs of primers, using RT-PCR method to clone and sequence. Successfully obtainthe Cricetulus barabensis and albino mutation of TYR, TYRP1, TYRP2, P, MITF, agouti gene,first clone coding region sequence of TYR, TYRP1, TYRP2, P, MITF, Agouti gene which is inCricetulus barabensis and albino mutation, but from coding regions did not mutant, the albino traitmechanism needs further research.
4. Comparative analysis about the level of an albino genes expression which isin Cricetulus barabensis and albino mutation
The results showed that TYR, TYRP1, TYRP2, P, MITF, Agouti mRNA expression quantityin black line hamsters is2.5、5.3、1.12、1.7、1.8、1.7time to albino mutation, the conclusion prompt that the expression level of TYR, TYRP1, TYRP2, P, MITF, Agouti has certain correlationwith Cricetulus barabensis color phenotype, suggesting that several members play a regulatingrole in the pigmentation process. This study clarify an albino genes regulation mechanism ofmelanin traits, as well as laid a theoretical foundation of the mechanism for Cricetulus barabensisalbino mutation albino traits.
5. Based on Illumina sequencing technology to transcriptome sequencingand analysis of the skin tissue which is in Cricetulus barabensis and albinomutant lines
This study take the skin tissue of black line hamsters and albino mutation as the researchobject, using Illumina/Solexa high-throughput sequencing technology to the depth of theirtranscriptome sequencing, using short sequence assembly software SOAP denovo to de novoassembly the results of its transcriptome sequencing, establishing database of the transcriptome,and proceeding sequence alignment, functional annotation and metabolic pathway analysis forprotein database with nr, Swiss-Prot, KEGG and COG; on this basis, proceeding the analysis ofdifferentially expressed genes, screening pigment synthesis genes, and to explore the bleachingmechanism.
Through this study, a total of more than80million high quality matching short sequencesare obtained, through sequence assembly received116,504Unigenes. By black line hamsters andalbino mutation skin transcriptome analysis of differentially expressed genes were screened16,905differentially expressed genes(|log2Ratio︱≥1and FDR≤0.001), through GO functionenrichment analysis and KEGG pathway enrichment analysis, these differentially expressed genesinvolved in cellular metabolism, protein translation, extracellular matrix receptor interaction andbiological processes. These differentially expressed genes were screened out53kinds of geneswhich play important regulating effect in protein synthesis, cell proliferation and cell apoptosisduring the process of growth in black line hamsters,12kinds of differentially expressed geneswhich is related to the growth of black line hamsters, and7kinds of differentially expressed geneswhich is ralated to pigment biosynthesis, the results of these experiments lay the foundation forfurther study about the structure and function of genes which is in black line hamsters.
引文
[1] WESTERHOF W.The discovery of the human melanocyte［J］.Pigment Cell Res,2006,19(3):183-193.
[2]潘兴华.黑素细胞及黑素的生成与调节.生理科学进展,1998,29(2):179-181.
[3]姜俊兵，董常生，贺俊平.不同被毛颜色羊驼皮肤组织中成熟黑色素细胞的组织学分析.畜牧兽医学报，2010.41（2）：229-133.
[4] Main, Sharmav, Tambol, et al. interaction of melanin with proteins the importanceof an acidic intramelanosomal PH［J］. Pigment cell Res.2001.14(3):170-179.
[5] Kwon BS, Haq AK, Pomerantz SH, et al. Isolation, chromosomal mapping andexpression of mouse Tyrosinase gene［J］. Invest. Dermatol,1989,93:589-594.
[1] Ito S. and Wakamatsu K. Quantitative analysis of eumelanin and pheomelanin inhumans, mice, and other animals: a comparative review. Pigment Cell Res,2003,16:523–531
[2] Yamaguchi Y., Brenner M. and Hearing V. J. The regulation of skin pigmentation.J BiolChem,2007,282:27557-27561
[1] Sarangarajan R. and Boissy R. E. Tyrp l and oeuloeutaneous albinism type3.pigment Cell Res,2001,14(6):437-444
[2] Jimenez-Cervantes C, Solano F, Kobayashi T, Urabe K,1994. A new enzymaticfunction in the melanogenic pathway. The5,6-dihydroxyindole-2-carboxylic acidoxidase activity of tyrosinase-related protein-1(TRP1).Journal of BiologicalInorganic Chemistry,269(27):17993-18001.
[3] Kim K. S, Mendez E. A, Marklund S, Clutter A. C, Pomp D. and Rothschild M. F.Rapid communication: Linkage mapping of the porcine Agouti gene. Anim. sci,2000,(78):1395-1396
[4] Shibahara S,2000. Induction of melanocyte-specific microphthalmia-associatedtranscription factor by Wnt-3a. The Journal of Biological Chemistry,275:14013-14016.
[5] Prashiela M, Raymond E. B, Sharon P. H, Zhou B. K, Seth J. O,2001.Mislocalization of melanosomal proteins in melanoeytes from mice withoculocutaneous albinism type2. Experimental Eye Research72:695-710.
[1]潘兴华.黑素细胞及黑素的生成与调节[J].生理科学进展,1998,29(2):179-181.
[2] Spritz RA, Chiang PW, Naoki Oiso N, et al. Human and mouse disorders ofpigmentation [J]. Curr Opin Genetics Dev,2003,13:284-289.
[3] Carden SM, Boissy RE, Schoettker PJ, et al. Albinism: moderm moleculardiagnosis [J]. Brit J Ophthalamol,1998,82:189-195.
[4] Shiahara S, Okinaga S, Tomita Y, et al. A point mutation in the tyrosinase gene ofBALB/c albino mouse causing the cysteine-serine substitution at position85[J].Eur J Biochem,1990,189:455-461.
[5] Lefur N, Kelsall SR, Mintz B. Base substitution at different alternative splicedonor sites of the tyrosinase gene in albinism [J]. Genomics,1996,37:245-248.
[6] Schmidt A, Beermann F. Molecular basis of darkened albinism in the mouse. ProcNatl Acad Sci USA,1994,91:4756–4760.
[7] Commos, Gaillardo, Thibauts, et al. Absence of TRP2in melanogenicmelanocytes of human hair [J]. Pigment Cell Res,2004,17(6):488-497.
[1] Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics [J]. NatGenet,2009,10(1):57-63.
[1]徐植岚,王建.白化黑线仓鼠的实验室驯化[J].实验动物科学.1987，3：79-83.
[2]王晶,徐植岚,刘宏伟,等.黑线仓鼠白化突变系的生物学特性与应用[J].北京实验动物科学.1989，2：25-27.
[3]遇秀玲，郑振锋.白化黑线仓鼠解剖学与组织学特性的比较研究.中国实验动物学杂志，1991，3：60－62.
[4] Nordlund J. J, Biossy R. E. and Hearing V. J. The pigment system-physiologicaland pathophysiology. Oxford University Press,1998
[5] Sturm R. A, Teasdele R. D. and Box N. F. Human pigmentation genes:Identification,structure and consequences of polymorphic variation. Gene,2001,277:49-62
[6] Wakamatsu K. and Ito S. Advances chemical methods inmelanin determination.Pigment Cell Res,2002(15):174–183
[7] Slominski A., Tobin D. J, Shibahara S. and Wortsman J. Melanin pigmentation inmammalian skin and its hormonal regulation. Physiol Rev,2004,84:1155-1228
[8] Ito S. and Wakamatsu K. Quantitative analysis of eumelanin and pheomelanin inhumans, mice, and other animals: a comparative review. Pigment Cell Res,2003,16:523–531
[9] Yamaguchi Y., Brenner M. and Hearing V. J. The regulation of skin pigmentation.J BiolChem,2007,282:27557-27561
[10] Sarangarajan R. and Boissy R. E. Tyrp l and oeuloeutaneous albinism type3.pigment Cell Res,2001,14(6):437-444
[11] Hearing V. J. and Tsukamoto K. Enzymatic control of pigmentation in mammals.FASEB J,1991,5:2902-2909
[12] Scherer D. and Kumar R. Genetics of pigmentation in skin cancer-A review.Mutat Res,2010,705:141-153
[13] Oetting W., S.. The tyrosinase gene and oculocutaneous albinism type1(OCA1):a model forunderstanding the molecular biology of melanin formation. PigmentCell Res,2000,13:320–325
[14] Boissy R. E., Moellmann G. E. and Halaban R. Tyrosinase and acid phosphataseactivities inmalanocytes from avian albinos. J Invest Dermatol,1987,88:292-300
[15] Barton D. E, Kwon B. S. and Francke U. Human tyrosinase gene, mapped tochromosome11(q14-q21), defines second region of homology with mousechromosome7. Genomics,1998,3:17-24
[16] Shiahara S,Okinaga S,Tomita Y, et al.A point mutation in the Tyrosinase gene ofBALB/c albino mouse causing the cysteine-serine substitution at position85. EurJ Bioche,1990,189:455-461.
[17] Lefur N,Kelsall SR,Mintz B. Base substitution at different alternative splicedonor sites of the Tyrosinase gene in albinism.Genomics,1996,37:245-248.
[18] Schmidt A and Beermann F. Molecular basis of darkeyed albinism in the mouse.Proc Natl Acad Sci USA1994,91:4756–4760
[19]孙仁山，谭骏,李文维.人类白化并的分子遗传学研究.国外医学遗传学分册，1994，5：250－253.
[20] Rowett M. Albinism in a Suffolk sheep. Mutational analysis of the modulation oftyrosinase by tyrosinase-related proteins1and2in vitro.The journal of Heredity,1999,83:67-69.
[21] Tcbita T.Tyrosinase and acid phosphatase activities in malanocytes from avianalbinos.Invest Dermatol,1987,88:292-300.
[22] Ruppert S, Muller G, Kwon B, et al. Multiple transcripts of the mouse Tyrosinasegene are generated by alternative splicing. EMBO J,1988,7:2715-2722.
[23] Muller G., Ruppert S, Schmid E, et al. Genetic variations in the fine structureand ontogeny of mouse melanin granules. EMBO J,1988,7:2723-2730.
[24] Halaban R, Pomerantz SH, Marshall S. et al. Arch. Himalayan tyrosinase doesnot demonstrate temperature sensitivity. Biochem. Biophys.1984,230:383-387.
[25] Eberle J, Garbe C, Wang NP, et al.Pigm. A distal tyrosinase upstream elementstimulates gene expression in neuralcrest-derived melanocytes of transgenic mice:position-independent and mosaic expression.cell. Res,1995,8:307-313.
[26] Halaban R, Cheng E, Zhang Y, et al. Correction of abnormal retinal pathwaysfound with albinism by introduction of a functional tyrosinase gene in transgenicmice roc. Natl. Acad. Sci. USA,1997,94:6210-6215.
[27] Halaban R, Svedine S, Cheng E, et al. Endoplasmic reticulum retention is acommon defect associated with tyrosinase negative albinism.2000,97:5889-5894.
[28] Sánchez-Ferrer A, Rodríguez-López J. N, García-Cánovas F. and García-Carmona F. Tyrosinase: a comprehensive review of its mechanism. BiochimBiophys Acta.1995,1247(1):1-11
[29] Korner A. and Pawelek J. Mammalian tyrosinase catalyzes three reactions in thebiosynthesis of melanin. Science,1982,217:1163–1165
[30] Cooksey C. J, Garratt P. J., Land E. J, Pavel S., Ramsden C. A, Riley P. A. andSmit N. P. Evidence of the indirect formation of the catecholic intermediatesubstrate responsible forthe autoactivation kinetics of tyrosinase. J Biol Chem,1997,272:26226–26235
[31] Biet E, Alberti C, Faccella P, Sun J. S, Dutreix M. and Larue L. Tyrosinasegenecorrection using fluorescent oligonucleotides. Pigment Cell Res,2003,16:133–138
[32] Jiang Z, Li S, Liu Y, Deng P, Huang J. and He G. Sesamin induces melanogenesisbymicrophthalmia-associated transcription factor and tyrosinase up-regulationvia cAMP signaling pathway. Acta Biochim Biophys Sin (shanghai),2011,43:763-770
[33] Kim K. S, Mendez E. A, Marklund S, Clutter A. C, Pomp D. and Rothschild M. F.Rapid communication: Linkage mapping of the porcine Agouti gene. Anim. sci,2000,(78):1395-1396
[34] Alaluf S, Barret K, Blount M. and Carter N. Ethnic variation in tyrosinase andTYRPl expression in photoexposed and photoproteeted human skin. PigmentCell Res,2003,16(1):35-42
[35] Rieder S, Siricker C. and Joerg H. A comparative genetic approach for theinvestigation of aging grey horse melanoma. J Anim Breed Genet,2000,312(117):73-82
[36]蒋茂森，2002，动物的白化现象，野生动物，23（5）：5
[37] Potterf S. B, Furumura M, Sviderskaya E. V,1998. Normal tyrosinase transportand abnormal tyrosinase routing in pinked-eyed dilution melanocytes.Experimental Cell Research,244:319-326.
[38] Halaban R., Cheng E., Svedine S.,2001. Proper folding and endoplasmicreticulum to Golgitransport of tyrosinase are induced by its substrates, dopa andtyrosine. The Journal of BiologicalChemistry,15:11933-11938.
[39] Halaban R., Svedine S., Cheng E.,2000. Endoplasmice reticulum retention is acommon defectassociated with tyrosinase-negative albinism. Proceedings of theNational Academy of Sciences,9711:5889-5894.
[40] Prashiela M, Raymond E. B, Sharon P. H, Zhou B. K, Seth J. O,2001.Mislocalization of melanosomal proteins in melanoeytes from mice withoculocutaneous albinism type2. Experimental Eye Research72:695-710.
[41] Lamerson C.L, Nordlund J.J,2006. Pigmentary Changes associated withAddison's disease. The pigmentary system: physiology and pathophysiology,Blackwell Publishing,969-972.
[42] Schallreuter K.U, Kothari S, Chavan B,2008. Regulation of melanogenesis-controversies and new concepts. Experimental Dermatology,17(5):395-404.
[43] Cheli Y, Luciani F, Khaled M,2009. Alpha-MSH and cyclic-AMP elevatingagents controlmelanosome pH through a protein kinase A-independentmechanism. Journal of Biological Inorganic Chemistry,284(28):18699-18706.
[44] Busca R, Ballotti R,2000. Cyclic AMP a key messenger in the regulation of skinpigmentation. Pigment Cell Research,13(2):60-69.
[45] Ahn J.H, Jin S.H, Kang H.Y,2008. LPS induces melanogenesis through p38MAPK activation in human melanocytes. Archives of Dermatological Research,300(6):325-329.
[46] Park H.Y, Wu C, Yonemoto L,2004. MITF mediates cAMP-induced proteinkinase C-beta expression in human melanocytes. The Journal of Cell Science,117:3659-3668.
[47] Slominski A, Plonka P.M, Pisarchik A,2005. Preservation of eumelanin hairpigmentation in proopiomelanocortin-deficient mice on a nonagouti (a/a) geneticbackground. Endocrinology,146(3):1245-1253.
[48] Miyamura Y, Coelho S.G, Wolber R,2007. Regulation of human skinpigmentation and responses to ultraviolet radiation. Pigment Cell Research,20(1):2-13.
[49] Ganss R, Schmidt A, Schiitz G., Beermann F,1994. Analysis of the mousetyrosinase promoterin vitro and in vivo. Pigment Cell Research,7(5):275-278.
[50] Zhao H.Q,1996. Retroviral infection with human tyrosinase-related protein-1cDNA upregulate tyrosinase activity and melanin synthesis in a TRP-1deficientmelanoma cell line. The Journal of Investigative Dermatology,106(4):744-752.
[51]舒文,毛华明.黑色素的研究进展[J].国外畜牧学-猪与禽,2003,23(2):31-34.
[52] Shibahara S,2000. Induction of melanocyte-specific microphthalmia-associated
transcription factor by Wnt-3a. The Journal of Biological Chemistry,275:
14013-14016.
[2]潘兴华.黑素细胞及黑素的生成与调节.生理科学进展,1998,29(2):179-181.
[3]姜俊兵，董常生，贺俊平.不同被毛颜色羊驼皮肤组织中成熟黑色素细胞的组织学分析.畜牧兽医学报，2010.41（2）：229-133.
[4] Main, Sharmav, Tambol, et al. interaction of melanin with proteins the importanceof an acidic intramelanosomal PH［J］. Pigment cell Res.2001.14(3):170-179.
[5] Kwon BS, Haq AK, Pomerantz SH, et al. Isolation, chromosomal mapping andexpression of mouse Tyrosinase gene［J］. Invest. Dermatol,1989,93:589-594.
[1] Ito S. and Wakamatsu K. Quantitative analysis of eumelanin and pheomelanin inhumans, mice, and other animals: a comparative review. Pigment Cell Res,2003,16:523–531
[2] Yamaguchi Y., Brenner M. and Hearing V. J. The regulation of skin pigmentation.J BiolChem,2007,282:27557-27561
[1] Sarangarajan R. and Boissy R. E. Tyrp l and oeuloeutaneous albinism type3.pigment Cell Res,2001,14(6):437-444
[2] Jimenez-Cervantes C, Solano F, Kobayashi T, Urabe K,1994. A new enzymaticfunction in the melanogenic pathway. The5,6-dihydroxyindole-2-carboxylic acidoxidase activity of tyrosinase-related protein-1(TRP1).Journal of BiologicalInorganic Chemistry,269(27):17993-18001.
[3] Kim K. S, Mendez E. A, Marklund S, Clutter A. C, Pomp D. and Rothschild M. F.Rapid communication: Linkage mapping of the porcine Agouti gene. Anim. sci,2000,(78):1395-1396
[4] Shibahara S,2000. Induction of melanocyte-specific microphthalmia-associatedtranscription factor by Wnt-3a. The Journal of Biological Chemistry,275:14013-14016.
[5] Prashiela M, Raymond E. B, Sharon P. H, Zhou B. K, Seth J. O,2001.Mislocalization of melanosomal proteins in melanoeytes from mice withoculocutaneous albinism type2. Experimental Eye Research72:695-710.
[1]潘兴华.黑素细胞及黑素的生成与调节[J].生理科学进展,1998,29(2):179-181.
[2] Spritz RA, Chiang PW, Naoki Oiso N, et al. Human and mouse disorders ofpigmentation [J]. Curr Opin Genetics Dev,2003,13:284-289.
[3] Carden SM, Boissy RE, Schoettker PJ, et al. Albinism: moderm moleculardiagnosis [J]. Brit J Ophthalamol,1998,82:189-195.
[4] Shiahara S, Okinaga S, Tomita Y, et al. A point mutation in the tyrosinase gene ofBALB/c albino mouse causing the cysteine-serine substitution at position85[J].Eur J Biochem,1990,189:455-461.
[5] Lefur N, Kelsall SR, Mintz B. Base substitution at different alternative splicedonor sites of the tyrosinase gene in albinism [J]. Genomics,1996,37:245-248.
[6] Schmidt A, Beermann F. Molecular basis of darkened albinism in the mouse. ProcNatl Acad Sci USA,1994,91:4756–4760.
[7] Commos, Gaillardo, Thibauts, et al. Absence of TRP2in melanogenicmelanocytes of human hair [J]. Pigment Cell Res,2004,17(6):488-497.
[1] Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics [J]. NatGenet,2009,10(1):57-63.
[1]徐植岚,王建.白化黑线仓鼠的实验室驯化[J].实验动物科学.1987，3：79-83.
[2]王晶,徐植岚,刘宏伟,等.黑线仓鼠白化突变系的生物学特性与应用[J].北京实验动物科学.1989，2：25-27.
[3]遇秀玲，郑振锋.白化黑线仓鼠解剖学与组织学特性的比较研究.中国实验动物学杂志，1991，3：60－62.
[4] Nordlund J. J, Biossy R. E. and Hearing V. J. The pigment system-physiologicaland pathophysiology. Oxford University Press,1998
[5] Sturm R. A, Teasdele R. D. and Box N. F. Human pigmentation genes:Identification,structure and consequences of polymorphic variation. Gene,2001,277:49-62
[6] Wakamatsu K. and Ito S. Advances chemical methods inmelanin determination.Pigment Cell Res,2002(15):174–183
[7] Slominski A., Tobin D. J, Shibahara S. and Wortsman J. Melanin pigmentation inmammalian skin and its hormonal regulation. Physiol Rev,2004,84:1155-1228
[8] Ito S. and Wakamatsu K. Quantitative analysis of eumelanin and pheomelanin inhumans, mice, and other animals: a comparative review. Pigment Cell Res,2003,16:523–531
[9] Yamaguchi Y., Brenner M. and Hearing V. J. The regulation of skin pigmentation.J BiolChem,2007,282:27557-27561
[10] Sarangarajan R. and Boissy R. E. Tyrp l and oeuloeutaneous albinism type3.pigment Cell Res,2001,14(6):437-444
[11] Hearing V. J. and Tsukamoto K. Enzymatic control of pigmentation in mammals.FASEB J,1991,5:2902-2909
[12] Scherer D. and Kumar R. Genetics of pigmentation in skin cancer-A review.Mutat Res,2010,705:141-153
[13] Oetting W., S.. The tyrosinase gene and oculocutaneous albinism type1(OCA1):a model forunderstanding the molecular biology of melanin formation. PigmentCell Res,2000,13:320–325
[14] Boissy R. E., Moellmann G. E. and Halaban R. Tyrosinase and acid phosphataseactivities inmalanocytes from avian albinos. J Invest Dermatol,1987,88:292-300
[15] Barton D. E, Kwon B. S. and Francke U. Human tyrosinase gene, mapped tochromosome11(q14-q21), defines second region of homology with mousechromosome7. Genomics,1998,3:17-24
[16] Shiahara S,Okinaga S,Tomita Y, et al.A point mutation in the Tyrosinase gene ofBALB/c albino mouse causing the cysteine-serine substitution at position85. EurJ Bioche,1990,189:455-461.
[17] Lefur N,Kelsall SR,Mintz B. Base substitution at different alternative splicedonor sites of the Tyrosinase gene in albinism.Genomics,1996,37:245-248.
[18] Schmidt A and Beermann F. Molecular basis of darkeyed albinism in the mouse.Proc Natl Acad Sci USA1994,91:4756–4760
[19]孙仁山，谭骏,李文维.人类白化并的分子遗传学研究.国外医学遗传学分册，1994，5：250－253.
[20] Rowett M. Albinism in a Suffolk sheep. Mutational analysis of the modulation oftyrosinase by tyrosinase-related proteins1and2in vitro.The journal of Heredity,1999,83:67-69.
[21] Tcbita T.Tyrosinase and acid phosphatase activities in malanocytes from avianalbinos.Invest Dermatol,1987,88:292-300.
[22] Ruppert S, Muller G, Kwon B, et al. Multiple transcripts of the mouse Tyrosinasegene are generated by alternative splicing. EMBO J,1988,7:2715-2722.
[23] Muller G., Ruppert S, Schmid E, et al. Genetic variations in the fine structureand ontogeny of mouse melanin granules. EMBO J,1988,7:2723-2730.
[24] Halaban R, Pomerantz SH, Marshall S. et al. Arch. Himalayan tyrosinase doesnot demonstrate temperature sensitivity. Biochem. Biophys.1984,230:383-387.
[25] Eberle J, Garbe C, Wang NP, et al.Pigm. A distal tyrosinase upstream elementstimulates gene expression in neuralcrest-derived melanocytes of transgenic mice:position-independent and mosaic expression.cell. Res,1995,8:307-313.
[26] Halaban R, Cheng E, Zhang Y, et al. Correction of abnormal retinal pathwaysfound with albinism by introduction of a functional tyrosinase gene in transgenicmice roc. Natl. Acad. Sci. USA,1997,94:6210-6215.
[27] Halaban R, Svedine S, Cheng E, et al. Endoplasmic reticulum retention is acommon defect associated with tyrosinase negative albinism.2000,97:5889-5894.
[28] Sánchez-Ferrer A, Rodríguez-López J. N, García-Cánovas F. and García-Carmona F. Tyrosinase: a comprehensive review of its mechanism. BiochimBiophys Acta.1995,1247(1):1-11
[29] Korner A. and Pawelek J. Mammalian tyrosinase catalyzes three reactions in thebiosynthesis of melanin. Science,1982,217:1163–1165
[30] Cooksey C. J, Garratt P. J., Land E. J, Pavel S., Ramsden C. A, Riley P. A. andSmit N. P. Evidence of the indirect formation of the catecholic intermediatesubstrate responsible forthe autoactivation kinetics of tyrosinase. J Biol Chem,1997,272:26226–26235
[31] Biet E, Alberti C, Faccella P, Sun J. S, Dutreix M. and Larue L. Tyrosinasegenecorrection using fluorescent oligonucleotides. Pigment Cell Res,2003,16:133–138
[32] Jiang Z, Li S, Liu Y, Deng P, Huang J. and He G. Sesamin induces melanogenesisbymicrophthalmia-associated transcription factor and tyrosinase up-regulationvia cAMP signaling pathway. Acta Biochim Biophys Sin (shanghai),2011,43:763-770
[33] Kim K. S, Mendez E. A, Marklund S, Clutter A. C, Pomp D. and Rothschild M. F.Rapid communication: Linkage mapping of the porcine Agouti gene. Anim. sci,2000,(78):1395-1396
[34] Alaluf S, Barret K, Blount M. and Carter N. Ethnic variation in tyrosinase andTYRPl expression in photoexposed and photoproteeted human skin. PigmentCell Res,2003,16(1):35-42
[35] Rieder S, Siricker C. and Joerg H. A comparative genetic approach for theinvestigation of aging grey horse melanoma. J Anim Breed Genet,2000,312(117):73-82
[36]蒋茂森，2002，动物的白化现象，野生动物，23（5）：5
[37] Potterf S. B, Furumura M, Sviderskaya E. V,1998. Normal tyrosinase transportand abnormal tyrosinase routing in pinked-eyed dilution melanocytes.Experimental Cell Research,244:319-326.
[38] Halaban R., Cheng E., Svedine S.,2001. Proper folding and endoplasmicreticulum to Golgitransport of tyrosinase are induced by its substrates, dopa andtyrosine. The Journal of BiologicalChemistry,15:11933-11938.
[39] Halaban R., Svedine S., Cheng E.,2000. Endoplasmice reticulum retention is acommon defectassociated with tyrosinase-negative albinism. Proceedings of theNational Academy of Sciences,9711:5889-5894.
[40] Prashiela M, Raymond E. B, Sharon P. H, Zhou B. K, Seth J. O,2001.Mislocalization of melanosomal proteins in melanoeytes from mice withoculocutaneous albinism type2. Experimental Eye Research72:695-710.
[41] Lamerson C.L, Nordlund J.J,2006. Pigmentary Changes associated withAddison's disease. The pigmentary system: physiology and pathophysiology,Blackwell Publishing,969-972.
[42] Schallreuter K.U, Kothari S, Chavan B,2008. Regulation of melanogenesis-controversies and new concepts. Experimental Dermatology,17(5):395-404.
[43] Cheli Y, Luciani F, Khaled M,2009. Alpha-MSH and cyclic-AMP elevatingagents controlmelanosome pH through a protein kinase A-independentmechanism. Journal of Biological Inorganic Chemistry,284(28):18699-18706.
[44] Busca R, Ballotti R,2000. Cyclic AMP a key messenger in the regulation of skinpigmentation. Pigment Cell Research,13(2):60-69.
[45] Ahn J.H, Jin S.H, Kang H.Y,2008. LPS induces melanogenesis through p38MAPK activation in human melanocytes. Archives of Dermatological Research,300(6):325-329.
[46] Park H.Y, Wu C, Yonemoto L,2004. MITF mediates cAMP-induced proteinkinase C-beta expression in human melanocytes. The Journal of Cell Science,117:3659-3668.
[47] Slominski A, Plonka P.M, Pisarchik A,2005. Preservation of eumelanin hairpigmentation in proopiomelanocortin-deficient mice on a nonagouti (a/a) geneticbackground. Endocrinology,146(3):1245-1253.
[48] Miyamura Y, Coelho S.G, Wolber R,2007. Regulation of human skinpigmentation and responses to ultraviolet radiation. Pigment Cell Research,20(1):2-13.
[49] Ganss R, Schmidt A, Schiitz G., Beermann F,1994. Analysis of the mousetyrosinase promoterin vitro and in vivo. Pigment Cell Research,7(5):275-278.
[50] Zhao H.Q,1996. Retroviral infection with human tyrosinase-related protein-1cDNA upregulate tyrosinase activity and melanin synthesis in a TRP-1deficientmelanoma cell line. The Journal of Investigative Dermatology,106(4):744-752.
[51]舒文,毛华明.黑色素的研究进展[J].国外畜牧学-猪与禽,2003,23(2):31-34.
[52] Shibahara S,2000. Induction of melanocyte-specific microphthalmia-associated
transcription factor by Wnt-3a. The Journal of Biological Chemistry,275:
14013-14016.