厚壳贻贝McCaspase 3-4基因的克隆及其在幼虫变态中的作用

为进一步了解细胞凋亡及其效应基因caspase-3在海洋贝类厚壳贻贝(Mytilus coruscus)变态中的作用,研究通过RACE技术克隆并鉴定了一个厚壳贻贝caspase-3基因的cDNA全长,并对该基因在幼虫变态中的作用进行了研究。碱基序列和氨基酸序列特征分析显示,该基因编码的蛋白质具有典型的caspase P20和P10结构域,但核心半胱氨酸活性位点QACXG五肽保守序列发生了突变。系统进化分析结果显示该基因与无脊椎动物Caspase-3聚在一起,并与地中海贻贝(Mytilus galloprovincialis)caspase 3/7-4相似度最高,因此将其命名为McCaspase 3-4。随后,通过实时荧光定量PCR技术对该基因在成贝不同组织及幼虫变态不同时间点的表达水平进行了分析。结果显示, McCaspase 3-4在成贝外套膜和唇瓣中的表达量显著高于其他组织。肾上腺素诱导眼点幼虫变态12h后, McCaspase 3-4的表达水平出现显著上升,并在刺激24h后达到最高,表明该基因可能在幼虫变态早期发挥作用。利用RNA干扰技术敲降眼点幼虫McCaspase 3-4基因...

MOLECULAR CLONING OF MCCASPASE 3-4 AND ITS FUNCTIONS IN MYTILUS CORUSCUS LARVAL METAMORPHOSIS

As an essential way to remove redundant tissues or cells, apoptosis plays a crucial role in regulating the metamorphosis of vertebrates and insects. However, little is known about the function of apoptosis and its executioner genes caspase-3 in the metamorphosis of marine mollusks. In the present work, a full-length caspase-3 cDNA had 1269 bp full length and 855 bp sequences of Open Reading Frame (ORF), which coded a polypeptide of 1486 amino acids, was cloned from the Mytilus coruscus. It encodes a predicted protein containing conserved caspase p20 and p10 domains and has the caspase family cysteine active site (KPKIFIFQCSRR) in the p20 domains. However, compared with conserved caspase 3 in other species, three amino acids sites of the pentapeptide active motif (QACXG, where X is R, Q, or D) at the end of the cysteine active site is mutated. Multi-sequence alignment and phylogenetic analysis showed that this gene had the highest similarity with the Caspase 3/7-4 gene in Mytilus galloprovincialis and was named McCaspase 3-4. The mRNA expression level of McCaspase 3-4 in different tissues of the adult and larval metamorphosis were analyzed by real-time quantitative PCR. Results showed that McCaspase 3-4 mRNA expression in the mantle and labial palp was significantly higher than that in other tissues and was 9.34 and 7.37 times higher than that in the adductor muscle, which had the lowest expression level, suggesting this gene might be involved in host immune defense or tissue renewal and repair of labial palp. The McCaspase 3-4 mRNA expression also increased at 12 to 24h after the epinephrine inducing and peaked at 24h after inducing when the expression level was 3.0 times higher than that at 0h, indicating the McCaspase 3-4 might play a role in the early stage of larval metamorphosis (larval metamorphosis usually begin at 48 to 72h after epinephrine inducing). Furthermore, after the pediveligers were transfected with the specific McCaspase 3-4 siRNA by electroporation, the metamorphosis rate of pediveligers at 48h induced by epinephrine was decreased to 5%, which were significantly lower than the epinephrine inducing after non-target gene siRNA transfected group (25.7%). These results indicated that although the pentapeptide active motif of McCaspase 3-4 is mutated, this molecular still has the function of metamorphosis regulation and plays a role in the early stage of larval metamorphosis. However, we do not know whether the active site mutation will affect its metamorphosis regulation effect. These results will contribute to understanding the role of apoptosis in mussel larval metamorphosis and the molecular mechanism of marine mollusks’ metamorphosis.