| 摘要: |
| 甲壳动物高血糖激素家族在调控甲壳动物的变态发育、生殖、蜕皮、血糖平衡及体内的渗透压平衡等方面起着重要的作用，甲壳动物高糖激素（CHH）是甲壳动物高血糖激素家族中的重要成员．通过ＴａｉｌＰＣＲ方法克隆获得拟穴青蟹（Ｓｃｙｌｌａｐａｒａｍａｍｏｓａｉｎ）ｃｈｈ基因的近端启动子区序列，从翻译起始位点（ATG）起，共６５７ｂｐ．生物信息学分析结果显示，转录起始位点位于翻译起始位点（ATG）上游第７４ 个碱基（Ａ），在转录起始位点（Ａ）上游31bp处存在ＴＡＴＡｂｏｘ；核心启动子区位于－41～＋9bp之间， 包含在线软件预测的转录起始位点；潜在的转录因子结合位点包括Ｏｃｔ１、Ｃ／ＥＢＰａｌｐ、ＧＡＴＡ１、Ｓｐ１、Ａｐ１、ＮＦκａｐｐａＢ、ＮＦ１、ＲＡＰ１、ＨＮＦ３等；目前获得的ｃｈｈ基因启动子区中没有ＣｐＧ岛．成功构建了ｃｈｈ基因４个启动子不同长度片段Ｃ１—Ｃ４，分别位于－583～＋205、－388～＋205、－286～＋205、－46～＋205bp．含启动子不同长度片段的表达载体瞬时转染HEK293T细胞和Sf9细胞后的活性分析表明，在－286～－46、－583～－388bp区域可能存在负调控基因表达的转录因子结合位点，在－388～－286bp区域可能存在正调控基因表达的转录因子结合位点． |
| 关键词: 海洋生物学 拟穴青蟹 chh基因 近端启动子 瞬时转染 |
| DOI： |
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| 基金项目:国家自然科学基金资助项目（41176152）；集美大学创新团队基金资助项目（2010A001） |
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| Functional analysis of the proximal promoter of chh gene
in mud crab (Scylla paramamosain) |
| HUANG Danping,ZHANG Ziping,JIA Xiwei,ZOU Zhihua,TU Chuandeng,WANG Yilei |
| (Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture, College of Fisheries, Jimei? University, Xiamen 361021, China;College of Animal Science, Fujian Agriculture and? Forestry University, Fuzhou 350002, Chiaa) |
| Abstract: |
| Crustacean hyperglycemic hormone ( CHH) family plays an important role in the regulation of metamor-phosis development, reproduction, molting, blood glucose balance and internal osmotic pressure balance in crusta-
cean. CHH is an impoitant member of CHH family. In this study, the proximal promoter sequence of chh gene was cloned by Tail-PCR from the mud crab, Scylla paramamosain. The sequence is 657 bp starting from the translation start site ( ATG). Forecast analysis results by the bioinformatics software showed that the transcription start site is
located at 74 bp upstream of start codon ATG, and TATA box is located at 31 bp upstream of transcription start site. The core promoter region is located between -41 bp and +9 bp, including the forecasted transcription start site. Potential transcription factor binding sites include Oct-1, C/EBPalp, GATA-1, Spl, Ap-1, NF-KappaB, NF-1, RAP1, HNR3, and so on.There is no CpG island in the proximal promoter sequence of chh gene. Four dele
tion fragments of chh gene promoter Cl ( -583 ~ +205 bp), C2 ( -388 ~ +205 bp)，C3 ( -286 - +205 bp)and C4 ( -46 - + 205 bp) were cloned and their promoter activities were analyzed using pEGFP-1, pGL3-Basic or
pIEx-Lue2 reporter constructs. Activity analysis of deletion fragments in HEK293T and Sf9 cells all showed that there are binding sites of potential negative transcription factors between -286 - 46 bp and -583-388 bp,and that there are binding sites of potential positive transcription factors between -388-286 bp. |
| Key words: marine biology Scylla paramamosain chh gene proximal promoter transient transfection |
