马氏珠母贝PmLec-8基因的克隆与表达分析

Clone and Expression Analysis of Pm Lec-8 Gene from Pinctada fucata martensii

Lec-8(C-type lectin 8)属于C-型凝集素超家族,在机体抵抗微生物的感染过程起重要的作用。本研究采用RACE技术克隆出了PmLec-8基因cDNA全长序列,并且运用实时荧光定量PCR(qRT-PCR)技术检测了PmLec-8基因在马氏珠母贝(Pinctada fucata martensii)不同组织中的表达模式。结果显示:PmLec-8基因序列全长737 bp,其中5′UTR长为36 bp,3′UTR长为113 bp,开放阅读框(ORF)为588 bp,编码195个氨基酸,预测其分子质量约为22.90 ku,等电点为5.17;PmLec-8具在一个糖类识别结构域(Carbonhydrate-recognition domain,CRD),符合典型的C型凝集素(C-type lectin)家族特征;多序列比对结果表明物种间Lec-8的同源性较低,4个半胱氨基酸残基高度保守;qRT-PCR数据分析表明,PmLec-8在马氏珠母贝肝胰腺、性腺、闭壳肌、鳃、血细胞和外套膜中均有表达,其中肝胰腺中表达量最高,其次是鳃,差异具统计学意义(P<0.05)。

Lec-8(C-type lectin8)belongs to the family of C-type lectin and plays an important role inresisting microorganism infection.A full length of PmLec-8was obtained using rapid amplification ofcDNA ends(RACE)technology from Pinctada fucata martensii.The expression patterns of PmLec-8inall tissues were further tested by Quantitative Real-Time PCR technology.Results showed that the totallength of PmLec-8cDNA was737bp,including a5′UTR of36bp,a3′UTR of113bp and an openreading frame(ORF)of588bp which encodes195amino acids.The predicted molecular mass was22.90ku,and isoelectric point was5.17.The protein encoded by PmLec-8has acarbonhydrate-recognition domain(CRD),conforming to the characteristics of C-type lectin family.Multiple sequence alignment indicated that Lec-8was less conservative among species with fourconserved cysteine residues.qRT-PCR data revealed that PmLec-8was expressed in all tested tissues,including hepatopancreas,gonads,adductor muscle,gill,hemocytes and mantle,with the highestexpression in hepatopancreas,higher in gill(P<0.05).These results indicated that PmLec-8may play animportant role in resisting microorganism infection of Pinctada fucata martensii.