cDNA Cloning, Tissue Expression Analysis of MITF Gene in Liancheng White Ducks
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摘要: 对连城白鸭刺参小眼畸形相关转录因子（microphthalmia-associtated transcription factor，MITF）的序列进行克隆及序列分析，预测MITF蛋白的二、三级结构，并利用RT-PCR技术检测MITF基因在连城白鸭不同组织中的表达差异，为深入研究连城白鸭体内黑色素沉积的内在调控机制奠定理论基础。结果显示，连城白鸭MITF基因长度为1 323 bp（登录号：MG516570），包括一个完整的CDS区（34~1 323 bp），编码429个氨基酸；与绿头鸭氨基酸同源性100%，与原鸡等鸟类的同源性达95%以上，而与绵羊等哺乳动物的亲缘关系较远。对MITF蛋白的二级结构预测显示，无规则卷曲及α螺旋是形成其二级结构的主要结构元件；连城白鸭MITF蛋白三级结构为一个环状结构间隔开两个螺旋，从而形成MITF蛋白的基础螺旋-环-螺旋-拉链结构。荧光定量PCR分析显示，连城白鸭皮肤组织中的MITF基因mRNA表达量最高，极显著（P < 0.01）高于其他各组织；各组织表达量依次为：皮肤>肾脏>肌胃>肝脏>肌肉。综上所述，MITF基因在连城白鸭组织中的表达具有组织特异性，为深入探究MITF基因能否作为调控连城白鸭黑色素沉积的主效候选基因及连城白鸭黑色素性状的辅助选择提供理论依据。
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关键词:
- 连城白鸭 /
- 小眼畸形相关转录因子 /
- 序列分析 /
- 基因表达
Abstract: The cloning, tissue expression and structural analysis of Microphthalmia-associated transcription factor (MITF) gene were conducted in Liancheng white duck, which would provide a theoretical basis for the further research of melanin deposition.The MITF gene from the skin of female ducks was cloned through nested-PCR, and the mRNA expression detected in tissues by qRT-PCR. It was shown that the MITF cDNA was 1 323 bp in length (GenBank No. MG516570) with a complete CDS region (34-1 323 bp) encoding 429 amino acids. The amino acid sequence of MITF was 100% homologous to that of mallard and more than 95% to that of Gallus gallus, but farther from sheep and other mammals. The predicted secondary structure of the protein was mainly random coil and α-helix, while the circular tertiary structure contained two separate helices to form the helix-loop-helix-chain structure. The qRT-PCR results showed that the mRNA expression of MITF was significantly higher in the skin than the other organs (P < 0.01) with the relative expressions in the order of skin > kidney > gizzard > liver > muscle. It appeared that the expression of MITF gene was tissue-specific in Liancheng white ducks. The information would aid in the utilization of MITF gene for studies on the melanin deposition and trait selection of the duck. -
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图 1 连城白鸭MIIF基因cDNA扩增片段电泳
注：M为DNA分子量标记DL5000。
Figure 1. Electrophoresis of amplified cDNA fragment of MITF gene from Liancheng white duck
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图 3 MITF系统进化树分析
Figure 3. Phylogenetic analysis of amino acid sequences coded by MITF gene
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图 4 连城白鸭MITF蛋白质二级结构预测
注：h为α螺旋；e为β折叠；t为β转角；c为无规则卷曲。数字表示氨基酸数量。
Figure 4. Predicted secondary structure of Liancheng white duck MITF protein
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图 5 连城白鸭MITF蛋白亲疏水性分布
注：横坐标为序列的位置，纵坐标为氨基酸的标度值(＞0表示疏水性，＜0表示亲水性)。
Figure 5. Hydrophobicity distribution of MITF protein in Liancheng white duck
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图 6 连城白鸭MITF蛋白的三级结构
Figure 6. Tertiary structure of MITF protein of Liancheng white duck
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图 7 MITF基因在连城白鸭各组织中的相对表达量
注：不同大写字母表示差异极显著(P < 0.01)；内参基因：GAPDH；n=10。
Figure 7. Relative mRNA expressions of MITF gene in tissues of Liancheng white duck
表 1 基础饲粮组成及营养水平(饲喂基础)
Table 1 Composition and nutrients in basal diet for ducks (as-fed basis)
饲粮组成 1~21 d含量 22~130 d含量 营养水平 1~21 d含量 22~130 d含量 玉米/% 54.00 50.00 粗蛋白质/% 18.50 16.00 次粉/% 12.00 11.80 代谢能/(MJ·kg-2) 12.12 11.72 去皮豆粕/% 21.00 12.00 赖氨酸/% 0.95 0.70 菜粕/% 2.00 2.00 蛋氨酸+胱氨酸/% 0.78 0.65 米糠粕/% 0.00 12.00 钙/% 0.85 0.80 干酒糟及其可溶物/% 6.00 7.00 有效磷/% 0.40 0.35 豆油/% 1.00 1.20 预混料/% 4.00 4.00 合计/% 100.00 100.00 注：(1)预混料可为每千克饲粮提供：1~21 d: VA 7 000 IU，VD3 2 000 IU，VE 30 mg，VK3 4.0 mg，VB1 3.0 mg，VB2 7.0 mg，VB6 9.0 mg，VB12 0.03 mg，胆碱450 mg，烟酸35 mg，泛酸20 mg，叶酸1.0 mg，生物素0.2 mg，Fe 60 mg，Cu 8 mg，Mn 85 mg，Zn 80 mg，I 0.35 mg，Se 0.3 mg。22~130 d：VA 6 000 IU，VD 2 000 IU，VE 10 mg，VK3 1.0 mg，VB1 2.0 mg，VB2 4.0 mg，VB6 3.0 mg，VB12 0.02 mg，胆碱400 mg，烟酸20 mg，泛酸10 mg，叶酸0.6 mg，生物素0.1 mg，Fe 55 mg，Cu 5 mg，Mn 80 mg，Zn 75 mg，I 0.3 mg, Se 0.2 mg。(2)营养水平为计算值。 
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表 2 RT-PCR扩增引物
Table 2 RT-PCR Primers
引物 引物序列(5′-3′) 扩增长度/bp 熔解温度/℃ L1 Forward CTCTTCCCACAGCAATTCCGAGCA 1431 60 Reverse GGGCTACTGATGAAGCACAAGA L2 Forward CCCTCTAGATGCATGCTCGAGGCTCC TCCAGCATGACCTCAC 1323 60 Reverse CTTGGTACCGAGCTCGGATCCGTATT TGCTAACAAGCATGGTCG
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表 3 荧光定量PCR引物信息
Table 3 Information on qRT-PCR primers
基因名称 基因序列号 引物序列(5′-3′) 扩增长度/bp 熔解温度/℃ 小眼畸形相关转录因子 NM_205029.1 F: GGTCAACAGGGTCATCAAACAAGAA 135 64 R: CCGAGGTTGTCACTGAAGGTGAT 3-磷酸甘油醛脱氢酶 NM_204305.1 F: GGGTAGTGAAGGCTGCTGCTGAT 114 64 R: CCGCATCAAAGGTGGAGGAAT
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表 4 MITF基因编码氨基酸序列(14个物种)的遗传距离和同源性
Table 4 Homology and genetic distance of amino acid sequence encoded by MITF gene in 14 species
1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 0.0000 0.0163 0.0287 0.0164 0.0205 0.0205 0.0164 0.1397 0.1397 0.1936 0.1889 0.1486 0.1974 2 100.0 0.0150 0.0264 0.0150 0.0188 0.0188 0.0188 0.1441 0.1441 0.1994 0.1950 0.1524 0.1969 3 99.1 99.1 0.0416 0.0302 0.0339 0.0339 0.0340 0.1601 0.1601 0.2183 0.2139 0.1685 0.2133 4 98.4 98.5 97.6 0.0188 0.0226 0.0226 0.0226 0.1482 0.1482 0.2141 0.2048 0.1482 0.2015 5 99.1 99.1 98.3 98.9 0.0038 0.0038 0.0038 0.1400 0.1400 0.1995 0.1992 0.1483 0.1930 6 98.8 98.9 98.1 98.7 99.8 0.0000 0.0075 0.1439 0.1439 0.2041 0.1949 0.1522 0.1970 7 98.8 98.9 98.1 98.7 99.8 100.0 0.0075 0.1439 0.1439 0.2041 0.1949 0.1522 0.1970 8 99.1 98.9 98.1 98.7 99.8 99.6 99.6 0.1442 0.1442 0.2014 0.1952 0.1505 0.1947 9 92.1 91.9 91.0 86.3 92.1 91.9 91.9 91.9 0.0000 0.0700 0.0884 0.0380 0.0966 10 92.1 91.9 91.0 91.7 92.1 91.9 91.9 91.9 100.0 0.0700 0.0884 0.0380 0.0966 11 89.6 89.3 88.4 88.6 89.3 89.1 89.1 89.3 96.1 96.1 0.0435 0.0916 0.1462 12 89.6 89.3 88.4 88.9 89.6 89.3 89.3 89.3 95.2 95.2 97.6 0.0837 0.1674 13 91.4 91.2 90.4 91.5 91.5 91.2 91.2 91.4 97.9 97.9 95.0 95.4 0.1035 14 88.8 88.9 88.0 85.1 89.1 88.9 88.9 89.0 90.9 94.7 91.9 91.0 94.3 注：上三角为遗传距离，下三角为同源性，%。1为连城白鸭，2为绿头鸭，3为鸿雁，4为鹦鹉，5为火鸡，6为原鸡，7为白绒乌鸡，8为鹌鹑，9为人，10为黑猩猩，11为猪，12为绵羊，13为兔，14为家鼠。
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