THE CLONE OF FOXO1 GENE AND ITS SPATIOTEMPORAL EXPRESSION IN GONADAL COMPLEX OF NEWLY HATCHED ALLIGATOR SINENSIS
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摘要: 研究以初孵扬子鳄(Alligator sinensis)性腺组织为实验材料, 开展Forkhead box家族蛋白-1(FoxO1)基因CDS区克隆和序列特征分析。利用免疫组织化学检测其在胃、肠和肺脏等组织中的表达谱, 结合免疫荧光(IF)、RT-PCR和Western Blot探讨其在不同出壳后日龄(17日龄、63日龄和96日龄)雌性扬子鳄性腺组织中的时空表达规律, 探讨其在雌性扬子鳄性腺发育过程中的生物学功能。研究成功克隆获得FoxO1基因长度为1941 bp的完整编码区及646个预测编码氨基酸序列。与其他物种间进行同源性比对和构建进化树分析发现, 扬子鳄FoxO1基因与鸟类的亲缘关系相较中华鳖(Pelodiscus sinensis)和原矛头蝮(Protobothrops mucrosquamatus)等爬行动物更近。在其他脊椎动物中, 哺乳动物、硬骨鱼和两栖物种也各自聚类称为子群, 表明FoxO1兼具功能保守性和种间特异性; 免疫组织化学结果表明FoxO1在初孵鳄性腺复合体、肺、胃和肠中均有表达, 免疫荧光结果表明17日龄性腺组织中的FoxO1主要表达于肾上腺和中肾区但在皮质部呈微弱表达, 在63日龄性腺皮质部表达逐渐上调且呈无规则随机分布, 在96日龄性腺皮质部中卵母细胞发育程度更高的位置呈强表达; RT-PCR结果表明不同时期扬子鳄性腺组织FoxO1基因mRNA表达量差异不显著, Western Blot结果显示性腺组织中的FoxO1表达量呈先上升后下降的变化模式, 表现为63日龄FoxO1基因表达量显著高于17日龄和96日龄, 相关结果对于丰富扬子鳄卵子发生调控机制提供了重要的研究参考。Abstract: The molecular cloning and sequence characteristic analysis of Forkhead Box protein-1 (FoxO1) CDS region were investigated using the gonad tissues of Alligator sinensis in the resent research. Immunohistochemistry was used to detect its expression patterns in in peripheral tissues such as stomach, intestine, and lung. Immunofluorescence (IF), RT-PCR and Western blot were used to investigate the temporal and spatial expression patterns in the gonadal tissues of Chinese Alligator at different days of age (17, 63 and 96 days of age), and to investigate the biological functions in the gonadal development of Chinese alligator. The complete coding region of FoxO1 gene and predicted coding amino acid were 1941 bp and 646 aa, respectively. Homology comparison and phylogenetic tree analysis showed that FoxO1 of Chinese alligator was more closely to birds than reptiles such as Pelodiscus sinesis and Protobothrips mucrisquamatus. Other vertebrates such as mammals, teleost, and amphibians were also grouped into subgroups, suggesting that FoxO1 is both functionally conserved and species-specific. FoxO1 protein expressed in the newly hatched crocodile gonadal complex, lung, stomach and intestine. FoxO1 expressed significantly higher in the ovarian tissue at the 17th days post-hatched than that at the 63rd and 96th days post-hatched individuals. FoxO1 mainly expressed in the adrenal and mesonephrons but weakly expressed in the cortex of ovarian collected at the 17th days post-hatched, and its irregular and randomly distribution was gradually upregulated in the cortex at the 63rd days post-hatched individual. FoxO1strongly expressed in the cortex of the 96th days post-hatched individual ovarian tissues where oocytes were more developed. FoxO1 mRNA level was no significant difference in the gonadal tissues of Chinese alligator at different periods. These results provide important reference for enriching the regulation mechanism of oogenesis of Chinese Alligator.
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Key words:
- Gonadal complex /
- Oogenesis /
- FoxO1 /
- Tissue expression profile /
- Spatio-temporal expression /
- Alligator sinensis
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图 1 扬子鳄FoxO1基因氨基酸序列系统进化树
Figure 1. Phylogenetic tree of amino acid sequence of FoxO1 gene in Alligator sinensis
图 2 FoxO1在扬子鳄外周组织中的时空表达规律
Figure 2. Temporal and spatial expression of FoxO1 in the peripheral tissues of Alligator sinensis
图 3 FoxO1在扬子鳄性腺组织中的时空表达规律
Figure 3. Temporal and spatial expression of FoxO1 in the ovarian tissues of Chinese Alligator
表 1 引物序列信息
Table 1. Information of primers sequences
引物Primer 引物序列Sequence (5′—3′) 产物大小Product size (bp) CDS-1F CAATGGAACTCAATCCGTCA 427 CDS-1R GATGGAGGGTATGACATAGGG CDS-2F TCAGGCCAGGAAGGAAATGG 588 CDS-2R TGTGGGTAGGAGAATCAGAAGTCAGT CDS-3F ACAAGCACCAGGTTATTCATTT 539 CDS-3R GCATAAGGGTTCATAGTATTCATC CDS-4F TCCTCAGCCCAGTGGTAGAGTATTG 520 CDS-4R AGCCTGACACCCAACTATGTGTGGT 
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