三疣梭子蟹Spook基因克隆及其表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2016.05.017

摘要/Abstract

摘要： 甲壳动物蜕皮激素（Ecdysteroids）主要在Y器官（YO）中合成与分泌，参与调控蜕皮、繁殖等多项生理活动。Spook基因编码的细胞色素P450（CYP）307a1是蜕皮激素合成通路早期的关键酶。为了研究其在三疣梭子蟹蜕皮过程中的调控作用，采用反转录PCR（RT-PCR）和cDNA末端快速扩增（RACE）技术，克隆得到了三疣梭子蟹Spook基因的全长cDNA序列（Gen-Bank 登录号：KM030021）。该序列全长为2 200 bp，包含一个长度为1 563 bp的开放阅读框，编码520个氨基酸；比对分析显示该氨基酸序列含helix-C、helix-K、helix-I、PERF、heme-binding 5个P450特征保守区域；系统进化树分析发现推导的三疣梭子蟹Spook蛋白与其他物种Spook聚为一支，而其他Halloween基因则分别聚为一支，表明推导的氨基酸确实是三疣梭子蟹Spook的蛋白序列。采用实时荧光定量PCR（qPCR）技术分析了其在不同组织中的表达情况，结果显示Spook基因主要在三疣梭子蟹的YO中表达。在三疣梭子蟹的蜕皮周期中，Spook基因的表达水平自蜕皮后期（A、B期）逐渐上升，并在蜕皮间期（C期）上升至最大，随后在蜕皮前期逐渐下降至D3、D4亚期最低。研究结果表明Spook基因可能参与调控三疣梭子蟹的蜕皮过程。

关键词: 三疣梭子蟹, Spook, 蜕皮周期, 基因克隆, 表达水平

Abstract: Ecdysteroids in crustaceans are mainly synthesized and secreted in the Y-organs（YO），and involve in the many essential physiological activities such as molting，reproduction，etc. The cytochrome P450（CYP）307a1 encoded by Spook is the key enzyme in early steps of ecdysteroids biosynthesis. To study the regulatory role of Spook in molting process，the full-length cDNA of gene was cloned and characterized from Portunus trituberculatus（GenBank accession number：KM030021）by reverse transcription PCR（RT-PCR）and RACE，designated as Pt-Spook. The cDNA of Pt-Spook was 2 200 bp in length and contained an opening reading frame（ORF）of 1 563 bp encoding 520 amino acid residues. Multiple alignment revealed that the Pt-Spook contained 5 conserved domains of helix-C，helix-K，helix-I，PERF and heme-binding. The analysis by phylogenetic tree discovered that the Pt-Spook clustered in the same branch with other species，however，Halloween genes were in another branch，indicating that the deduced amino acid sequence was truly the protein sequence of P. trituberculatus Spook. The tissue distribution of Pt-Spook was detected by qPCR，and the results showed that Pt-spook expression was highly specific to YO. During the molt cycle，the expression of Pt-spook gradually increased from post-molt（stage A and B），and reached the maximum in intermolt（stage C），then gradually decreased during pre-molt to the minimum in sub-stage D3 and D4. The results suggested that the Pt-spook involved in the regulation of molt cycle in P. trituberculatus.

Key words: Portunus trituberculatus, Spook, molt cycle, gene clone, expression level

周彦琦, 朱冬发. 三疣梭子蟹Spook基因克隆及其表达分析[J]. 生物技术通报, 2016, 32(5): 131-139.

ZHOU Yanqi,ZHU Dong-fa. Cloning and Expression Analysis of Spook Gene in Portunus trituberculatus[J]. Biotechnology Bulletin, 2016, 32(5): 131-139.

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