| 摘要: |
| 几丁质酶在甲壳动物中参与多个生物学进程, 发挥重要作用。本研究采用RACE技术, 克隆获得总长为3694 bp的三疣梭子蟹(Portunus trituberculatus)几丁质酶基因全长cDNA序列, 命名为PtCht基因。该基因5'和3'非编码区分别为200bp和455bp, 开放阅读框为3039bp, 推测编码1012个氨基酸, 预测分子量为113.4kDa, 理论等电点为6.289。生物信息学分析表明, PtCht基因含有2个催化结构域和1个几丁质结合结构域ChtBD2, 催化结构域具有几丁质酶第18家族的保守基序Motif Ⅰ、Ⅱ、Ⅲ、Ⅳ;同源性和系统进化分析表明, 三疣梭子蟹PtCht基因与亚洲玉米螟(Ostrinia furnacalis)Cht的同源性最高;荧光定量RT-PCR分析表明, PtCht基因在各组织中均有表达, 而在眼柄和表皮中的相对表达量较高。PtCht基因在蜕皮周期中的表达规律表明, PtCht基因在不同蜕皮时期存在明显变化, 在表皮中先下调后上调;在眼柄中, 呈整体上调趋势。通过分析PtCht基因在低盐胁迫进程中的表达规律发现, 低盐胁迫可显著改变PtCht基因在鳃和肝胰腺中的表达模式, 整体呈先下降后上升最后下降的表达趋势。该研究结果表明PtCht基因在三疣梭子蟹蜕皮发育和渗透压调节中发挥重要作用, 为深入研究三疣梭子蟹和其它甲壳动物几丁质酶的功能提供了重要信息。 |
| 关键词: 几丁质酶 蜕皮 低盐胁迫 三疣梭子蟹 几丁质外骨骼 基因表达 |
| DOI：10.11693/hyhz20140800220 |
| 分类号: |
| 基金项目:国家高技术研究发展计划(863计划),2012AA10A409号;农业科技成果转化资金项目,2013GB23260589号;山东省自主创新专项,2013CX80202号;国家自然科学基金,41306177号。 |
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| CLONING AND EXPRESSION OF CHITINASE UNDER LOW SALINITY STRESS DURING MOLTING IN PORTUNUS TRITUBERCULATUS |
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ZHANG Feng1,2, LV Jian-Jian1, LIU Ping1, GAO Bao-Quan1, LI Jian1, CHEN Ping1
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1.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China;2.College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China
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| Abstract: |
| Chitinase is an essential enzyme in crustaceans involved in several biological processes, including digestion, moulting, and defense against viruses.We identified a chitinase gene in swimming crab Portunus trituberculatus, named PtCht, according to established expressed sequence tag (EST) information using Rapid Amplification of the cDNA Ends (RACE) technique.The full-length cDNA of PtCht was 3694 bp, containing a 5'-untranslated region (UTR) in 200 bp, 3'-UTR in 455 bp with a poly (A) tail, an open reading frame (ORF) in 3039 bp, encoding a 1012 amino-acid polypeptide;molecular weight was predicted for 113.4 kDa and isoelectric point estimated for 6.289.Sequence analysis revealed that the conserved domains in chitinases family 18 were predicted in the amino acid sequence of PtCht, containing a signal peptide, two glycoside hydrolase catalytic domains that contain conserved Motif Ⅰ, Ⅱ, Ⅲ, and Ⅳ, a serine/threonine-rich linker, and a chitin-binding domain.Homologous analysis showed that PtCht has the highest homology to chitinase of Ostrinia furnacalis.The quantitative real-time RT-PCR showed that PtCht gene could be detected in all tested tissues of P.trituberculatus, in higher expression level in cuticle and eyestalk.During the moulting cycle, the expression of PtCht gene in cuticle, fell first, then rose;in eyestalk, kept rising.After challenged by low salinity, the expressions of PtCht gene in gill and hepatopancreas of P.trituberculatus fell first, then rose.Analysis of the moulting cycle and low-salinity (at 11) stimulation provided further evidence that PtCht played pivotal roles during moulting in osmotic regulation.Future investigations shall focus on the functions of chitinase in P.trituberculatus and other crustaceans. |
| Key words: Chitinase moulting low salinity stress Portunus trituberculatus chitin exoskeleton gene expression |
