三疣梭子蟹几丁质酶基因克隆鉴定及在低盐胁迫和蜕皮周期中的表达分析

CLONING AND EXPRESSION OF CHITINASE UNDER LOW SALINITY STRESS DURING MOLTING IN PORTUNUS TRITUBERCULATUS

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c DNA序列,命名为Pt Cht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113.4k Da,理论等电点为6.289。生物信息学分析表明,Pt Cht基因含有2个催化结构域和1个几丁质结合结构域Cht BD2,催化结构域具有几丁质酶第18家族的保守基序MotifⅠ、Ⅱ、Ⅲ、Ⅳ;同源性和系统进化分析表明,三疣梭子蟹Pt Cht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,Pt Cht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。Pt Cht基因在蜕皮周期中的表达规律表明,Pt Cht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Pt Cht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Pt Cht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Pt Cht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。

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 Pt Cht, according to established expressed sequence tag（EST） information using Rapid Amplification of the c DNA Ends（RACE） technique. The full-length c DNA of Pt Cht 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 k Da 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 Pt Cht, containing a signal peptide, two glycoside hydrolase catalytic domains that contain conserved MotifⅠ, Ⅱ, Ⅲ, and Ⅳ, a serine/threoninerich linker, and a chitin-binding domain. Homologous analysis showed that Pt Cht has the highest homology to chitinase of Ostrinia furnacalis. The quantitative real-time RT-PCR showed that Pt Cht 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 Pt Cht gene in cuticle, fell first, then rose; in eyestalk, kept rising. After challenged by low salinity, the expressions of Pt Cht 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 Pt Cht played pivotal roles during moulting in osmotic regulation. Future investigations shall focus on the functions of chitinase in P. trituberculatus and other crustaceans.