Ants Gene in Common Carp（Cyprinus carpio,Cyprinidae）and Impact on Scale Development

Abstract

Abstract: ants（adenine nucleotide translocator）, also called ATP/ADP carrier, mediates the exchange of ADP and ATP across the inner mitochondrial membrane, thus playing an essential role in energy metabolism in eukaryotic cells. Genefishing differental display Technique was used to screen different expression products in the transcript level in common carp and germany mirror carp, and obtained a partial sequence of ants gene. To fish for all the expressing ants products in the skin of common carp, RT-PCR was performed, and we obtained 6 different CDS sequences. CLUSTALW was applied to align the translated amino acid sequences and discovered that these 6 CDS can code 4 different amino acid sequences, indicated that ants genes have at least 6 different transcripts, but have 4 different protein products in protein level. Aligned these 2013年第3期121 程安达等：鲤鱼（Cyprinus carpio,Cyprinidae）ants 基因及其对鳞片发育的影响 ants（adenine nucleotide translocators）, 又称为 ATP/ADP 载体,可以调节ATP 和ADP 在线粒体内膜上的转运,所以在真核细胞的能量代谢中扮演着重要的角色
［1-3］。ants 作为线粒体内膜的组成部分, 是线粒体中最丰富的一类蛋白。在呼吸条件下,线粒体内产生的ATP 被转运到胞质来维持细胞活动, 作为交换,线粒体外部的ADP 被转运进入线粒体内, 为在ATP 合成酶的作用下ADP 转变为ATP 提供底物。ants 属于线粒体载体家族（mitochondrial carrier family）,这个家族可以进行各种各样的跨线粒体内膜的转运活动
［3,4］。 ant 家族的蛋白是由核基因编码的,表达之后定位到线粒体内膜中。营自由生活的细菌缺少与ant 相似的分子,所以认为ant 蛋白是由真核起源的广泛特异的载体家族进化来的
［5］。大多数真核生物, 包括单细胞真核生物,都具有多个ant 基因。例如, 出芽酵母（Saccharomyces cerevisiae）有3 个ant 基因（ant1,ant2 和ant3）可以编码蛋白质
［6］。其中, ant2 是最主要的同工型,无论是呼吸过程还是厌氧发酵过程,都是广泛表达的
［7］。ant1 和ant3 分别特异的在有氧和厌氧的条件下表达
［6,8］。此外,ant2 和ant3 在丢失线粒体基因组的情况下仍能转运ATP 来维持酵母的生存,而ant1 则无此功能
［9］。因此, 不同的ant 基因可能是被用来输入和输出ATP 以有效地应对外部的营养和氧气条件的变化。有趣的是啤酒酵母（S. cerevisiae）的ant1 基因具有比ant2 和 ant3 更高的碱基累计替换率
［10］,这与ant1 基因在复制之后发生了功能上的变化的观点一致。多细胞生物体中,在不同类型的组织,不同的发育时期以及不同的细胞增殖状态下,ant 基因的表达都是不同的。大多数脊椎动物都可以表达3 个不同的旁系同源的ant 基因,它们表现出较高程度的序列相似性。其中,ant1（Slc25a4）主要在心脏和骨骼肌中表达,可以假定它可以适应心脏和骨骼肌中ATP 的快速代谢
［11］。ant2（Slc25a5）和ant3 （Slc25a6）在躯体组织中广泛表达,然而,ant2 在哺乳动物快速生长的细胞中表达量较高,是有变化的,而ant3 似乎是在所有组织中组成性的表达,其表达量无太多变化
［12,13］。啮齿类动物缺少ant3 基因, 其ant2 基因很可能同时承担了人类上ant2 和ant3 基因的功能
［14,15］。在哺乳动物中,还存在第4 种ant 家族基因ant4（Slc25a31）,它是在人和小鼠中首先被发现的
［16-18］。它可以选择性地在成熟哺乳动物的睾丸和精子中表达,且在小鼠（mouse）精子发生过程中是必需的,但在鸟类、鱼类、蛙中却是缺少的
［19］。 ant4 可以在成熟小鼠睾丸中的生殖细胞中特异表达, 且在减数分裂时期,其表达量特别的高
［20］。鳞的形成是个极其复杂的生物学过程,可能需要成千上万的基因参与其中。ant 作为ATP/ADP 转运载体,可以为各个细胞的活动运送能量,它在鳞形成过程中可能发挥必不可少的作用。为了研究鳞形成的分子机制,我们利用Genefishing 技术特异地钓取了鲤皮肤组织中的ant 基因,经过克隆测序鉴定并进行初步的生物信息学分析来解析ant 基因的基本特征,并通过原位杂交技术对该基因在鳞被形成过程中的表达进行分析,以此证明ant 基因与鳞被发育的相关性。 1 材料与方法 1.1 材料用于提取RNA 的鲤鱼样品以及用来做原位杂交的幼鱼都来自于中国水产科学研究院黑龙江水产研究所松浦实验基地和无锡淡水研究中心。提取RNA 的鲤鱼样品用新鲜的鲤鱼皮肤组织,Trizol 试剂购自 4 different amino acid sequences and that of the zebrafish ant2 using blastp on NCBI, found that the percent identities were 97%, 97%, 96%, 98%, respectively. A phylogenetic tree of ants isoforms from common carp and other species located in different taxonomy status was constructed, and showed that these 4 protein products were obviously classified as ant2 with a 93/100 reproducibility, so the ant gene expressed in skin are likely to ant2. The result of Wholemount in situ hybridization revealed that ant2 is intensively expressed specifically in epiboly（10 hours after fertilization）, somite（24 hours after fertilization）and the regions where the scales were developing in skin, but in other regions weak and dispersed expression were detected. According to the above results, we can infer that ant2 likely subtly regulated the development of the scales via different transcripts（isoforms）.

Key words: ant, Scale, Isoforms, Genefishing in situ hybridization

Cheng Anda, Jiang Li, Zhang Baoyong, Wang Shu, Zhang Yan, Liu Yongxin, Fang Ping, Li Hengde, Sun Xiaowen, . Ants Gene in Common Carp（Cyprinus carpio,Cyprinidae）and Impact on Scale Development[J]. Biotechnology Bulletin, 2013, 0(3): 120-128.

References

[1] Klingenberg M. Molecular aspects of the adenine nucleotide carrier from mitochondria[J]. Arch Biochem Biophys, 1989, 270(1):1-14.
[2] Nelson DR, Felix CM, Swanson JM. Highly conserved charge-pair networks in the mitochondrial carrier family[J]. J Mol Biol, 1998, 277(2):285-308.
[3] Fiore C, Trezeguet V, Le Saux A, et al. The mitochondrial ADP/ATP carrier :structural, physiological and pathological aspects[J]. Biochimie, 1998, 80(2):137-150.
[4] Palmieri L, Lasorsa FM, Vozza A, et al. Identification and functions of new transporters in yeast mitochondria[J]. Biochim Biophys Acta, 2000, 1459(2-3):363-369.
[5] Amiri H, Karlberg O, Andersson SG. Deep origin of plastid/parasite ATP/ADP translocases[J]. J Mol Evol, 2003, 56(2):137-150.
[6] Kolarov J, Kolarova N, Nelson N. A third ADP/ATP translocator gene in yeast[J]. J Biol Chem, 1990, 265(21):12711-12716.
[7] Betina S, Gavurnikova G, Haviernik P, et al. Expression of the AAC2 gene encoding the major mitochondrial ADP/ATP carrier in Saccharomyces cerevisiae is controlled at the transcriptional level by oxygen, heme and HAP2 factor[J]. Eur J Biochem, 1995, 229(3): 651-657.
[8] Sabova L, Zeman I, Supek F, et al. Transcriptional control of AAC3 gene encoding mitochondrial ADP/ATP translocator in Saccharomyces cerevisiae by oxygen, heme and ROX1 factor[J]. Eur J Biochem, 1993, 213(1):547-553.
[9] Smith CP, Thorsness PE. The molecular basis for relative physiological functionality of the ADP/ATP carrier isoforms in Saccharomyces cerevisiae[J]. Genetics, 2008, 179(3):1285-1299.
[10] Mentel M, Piskur J, Neuveglise C, et al. Triplicate genes for mitochondrial ADP/ATP carriers in the aerobic yeast Yarrowia lipolytica are regulated differentially in the absence of oxygen[J]. Mol Genet Genomics, 2005, 273(1):84-91.
[11] Huizing M, Ruitenbeek W, van den Heuvel LP, et al. Human mitochondrial transmembrane metabolite carriers :tissue distribution and its implication for mitochondrial disorders[J]. J Bioenerg Biomembr, 1998, 30(3):277-284.
[12] Stepien G, Torroni A, Chung AB, et al. Differential expression of adenine nucleotide translocator isoforms in mammalian tissues and during muscle cell differentiation[J]. J Biol Chem, 1992, 267(21): 14592-14597.
[13] Lunardi J, Hurko O, Engel WK, et al. The multiple ADP/ATP translocase genes are differentially expressed during human muscle development[J]. J Biol Chem, 1992, 267(22):15267-15270.
[14] Ellison JW, Salido EC, Shapiro LJ. Genetic mapping of the adenine nucleotide translocase-2 gene(Ant2)to the mouse proximal X chromosome[J]. Genomics, 1996, 36(2):369-371.
[15] Ceci JD. Mouse chromosome 8[J]. Mamm Genome, 1994, 5 : 生物技术通报 Biotechnology Bulletin 2013年第3期128 S124-S138.
[16] Rodic N, Oka M, Hamazaki T, et al. DNA methylation is required for silencing of ant4, an adenine nucleotide translocase selectively expressed in mouse embryonic stem cells and germ cells[J]. Stem Cells, 2005, 23(9):1314-1323.
[17] Dolce V, Scarcia P, Iacopetta D, et al. A fourth ADP/ATP carrier isoform in man :identification, bacterial expression, functional characterization and tissue distribution[J]. FEBS Lett, 2005, 579(3):633-637.
[18] Kim YH, Haidl G, Schaefer M, et al. Compartmentalization of a unique ADP/ATP carrier protein SFEC(Sperm Flagellar Energy Carrier, AAC4)with glycolytic enzymes in the fibrous sheath of the human sperm flagellar principal piece[J]. Dev Biol, 2007, 302(2): 463-476.
[19] Lim CH, Hamazaki T, Braun EL, et al. Evolutionary genomics implies a specific function of Ant4 in mammalian and anole lizard male germ cells[J]. PLoS One, 2011, 6(8):e23122.
[20] Brower JV, Rodic N, Seki T, et al. Evolutionarily conserved mammalian adenine nucleotide translocase 4 is essential for spermatogenesis [J]. J Biol Chem, 2007, 282(40):29658-29666.
[21] Westerfield M. The Zebrafish Book[M]. Eugene: University of Oregon Press, 1995.
[22] Jowett T. Double in situ hybridization techniques in zebrafish[J]. Methods, 2001, 23(4): 345-58.
[23] Harris MP, Rohner N, Schwarz H, et al. Zebrafish eda and edar mutants reveal conserved and ancestral roles of ectodysplasin signaling in vertebrates[J]. PLoS Genet, 2008, 4(10):e1000206.
[24] Kondo S, Kuwahara Y, Kondo M, et al. The medaka rs-3 locus required for scale development encodes ectodysplasin-A receptor[J]. Curr Biol, 2001, 11(15):1202-1206.
[25] Laforest L, Brown CW, Poleo G, et al. Involvement of the sonic hedgehog, patched 1 and bmp2 genes in patterning of the zebrafish dermal fin rays[J]. Development, 1998, 125(21):4175-4184.
[26] Nohno T, Kawakami Y, Ohuchi H, et al. Involvement of the Sonic hedgehog gene in chick feather formation[J]. Biochem Biophys Res Commun, 1995, 206(1):33-39.