两种泥鳅芳香化酶基因的克隆与时空表达

MOLECULAR CLONING AND DEVELOPMENTAL EXPRESSION OF AROMATASES GENE IN TWO SPECIES OF LOACHES

鱼类的性别分化易受发育环境的影响。向性成熟的泥鳅和大鳞副泥鳅个体注射绒毛膜促性腺激素,获得卵子和精子进行人工授精。把胚胎分别置于20℃、25℃和30℃条件下,使其发育。经性腺检查发现随着温度的升高两种泥鳅中雄性个体所占的比例明显升高,获得明显的偏雄比率群体。根据已知细胞色素P450芳香化酶CYP19 b基因序列设计嵌套简并引物用巢式PCR扩增并克隆出了两种泥鳅的CYP19 b的DNA片段。MaCYP19 b片段和Pd-CYP19 b片段分别长1337bp和1473bp。在此基础上用各自的特异引物克隆出两种泥鳅CYP19 b的相应cDNA片段。通过基因组DNA和cDNA序列的比较证明两种泥鳅的CYP19 b基因均包含三个内含子和四个外显子,编码的蛋白质序列长145氨基酸残基。以GAPDH基因为对照,分别对两种泥鳅成体组织和不同发育阶段的胚胎的CYP19 b进行了半定量RT-PCR表达分析,结果表明泥鳅CYP19 b基因只在成体泥鳅卵巢、肾以及原肠胚和神经胚中表达。大鳞副泥鳅CYP19 b基因在成体的脑、卵巢和肾以及神经胚和卵黄吸收期表达。这些结果为揭示细胞色素P450芳香化酶基因与环境性别决定机制的关系奠定了基础。

Fishes serve as a key link on the view of the vertebrate systematic evolution. Its sex determination is primitive and plastically compared with the other advanced vertebrates. Sexual differentiation of gonad is sensitive to environmental factors during a critical period of embryogenesis in some fish. The cytochrome P450 aromatase, which converts androgens to estrogens, is a key enzyme in sex differentiation of gonad. Therefore, the cytochrome P450 aromatase （CYP19） genes control the pathway of gonadal sex differentiation radically. Misgurnus auguillicaudatus and Paramisgurnus dabryanus are common Cypriniformes fishes in China. They have a very close relationship and were classified to the same genus by some taxonomists. Heteromorphic sex chromosomes have been found only in P. dabryanus. To detect the effect of temperature to sex differentiation, Molecular cloning and developmental expression of aromatases gene in two species of loaches were studied. The zygotes of Misgurnus anguillicaudatus and Paramisgurnus dabryanus were incubated separately at 20℃, 25 ℃ and 30℃, until that the sex could be distinguished. The results show that high temperature （25--30℃） can promote male-biased sex ration in both species loach. And with the temperature rising, the male sex rate gets higher. A pair of nested degenerate consensus primes was designed basing on the sequence of cytochrome P450 aromatase gene（ CYP19b）. Using nest-PCR method, the part genomic sequences of CYP19b in two species loach were amplified and cloned from genomic DNA. The DNA fragments of MaCYP19b and PdCYP19b are 1337bp and 1473bp respectively. The gene-specific primers were designed based on the genomic DNA sequence. Then using RT-PCR method, the corresponding cDNA of CYP19b in both species loach were amplified and cloned from total RNA that was isolated from ovary tissue. By comparing their genomic DNA sequence and cDNA sequence, we know that the cloned CYP19b of two species loach all include 3 introns and 4 extrons and encode 145 amino acids. With the control gene of GAPDH, the expression patterns of the two genes were studied in adult tissues and different stages of embryo of two species loach separately with semi-quantitive RT-PCR. The results showed that M. anguillicaudatus CYP19b gene expressed only in ovary and kidney of adult tissues and gastrulae and neurula of embryo. The P. dabsryanu CYP19b expressed in brain, ovary and kidney of adult tissues and neurula and yolk-sac absorption phase of embryo. Those results lay the foundations of clarifying the relation between cytochrome P450 aromatase gene and the mechanism of ESD to a certain extent.