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通过c DNA末端快速扩增(Rapid amplification of c DNA ends,RACE)技术克隆得到魁蚶(Scapharca broughtonii)C型凝集素(C-type lectin,Sb-Lec1)基因,该基因全长为700 bp,其中,5′-UTR为29 bp,3′-UTR为167 bp,开放阅读框长度为504 bp,编码167个氨基酸,包括长度为23个氨基酸的信号肽序列,129个氨基酸的糖识别结构域(CRD)以及参与二硫键形成的6个半胱氨酸。预测蛋白分子量为19.11 kDa,理论等电点为4.74。多序列比对结果显示,Sb-Lec1基因CRD编码的氨基酸序列与长牡蛎(Crassostrea gigas)、紫贻贝(Mytilus galloprovincialis)、海湾扇贝(Argopecten irradians)C型凝集素的同源性分别为38%～40%、34%～35%、38%～39%,Sb-Lec1基因编码的氨基酸序列与其他物种的凝集素基因具有相似的结构,均含有形成二硫键的4个保守半胱氨酸。系统进化分析结果显示,魁蚶先与贝类聚为一支,再与脊椎动物聚在一起,表明,魁蚶Sb-Lec1在进化树上的位置与其传统分类所处位置一致。采用荧光定量PCR技术,检测了Sb-Lec1在组织中的表达情况,发现其在肝胰腺、血淋巴、鳃、外套膜、闭壳肌、斧足中均有表达,其中肝胰腺表达量最高。同时,分析了Sb-Lec1基因在鳗弧菌(Vibrio anguillarum)刺激下的mRNA表达量变化情况。结果显示,与对照组相比,菌刺激组Sb-Lec1基因mRNA在各检测组织中的表达量均显著上调(P<0.05),随着刺激时间的延长,表达量呈先升高后降低的趋势。本研究表明,魁蚶Sb-Lec1基因在机体免疫防御方面发挥重要功能。
The gene encoding C-type lectin (Sc-Lec1) of Scapharca broughtonii was cloned by rapid amplification of c DNA ends (RACE) Among them, the 5’-UTR was 29 bp, the 3’-UTR was 167 bp, the open reading frame was 504 bp, encoding 167 amino acids including the signal peptide of 23 amino acids in length and the 129 amino acid sugar recognition domain (CRD) and six cysteines involved in disulfide bond formation. The predicted protein molecular weight was 19.11 kDa, and the theoretical isoelectric point was 4.74. Multiple sequence alignment showed that the deduced amino acid sequence of CRD of Sb-Lec1 was 38% homologous with C-type lectins of Crassostrea gigas, Mytilus galloprovincialis and Argopecten irradians, ~ 40%, 34% ~ 35%, 38% ~ 39%. The Sb-Lec1 gene encodes a similar sequence of lectin genes to other species and contains four conserved cysteines . Phylogenetic analysis showed that Kui 蚶 first clustered with shellfish and then with vertebrates, indicating that the position of Kui 蚶 Sb-Lec1 in the phylogenetic tree is consistent with its traditional classification. The expression of Sb-Lec1 was detected by real-time PCR. The results showed that the expression of Sb-Lec1 in hepatopancreas, hemolymph, gill, mantle, adductor muscle and axillary was the highest. At the same time, the change of mRNA expression of Sb-Lec1 gene stimulated by Vibrio anguillarum was analyzed. The results showed that compared with the control group, Sb-Lec1 mRNA expression was significantly up-regulated in all groups (P <0.05). The expression of Sb-Lec1 increased first and then decreased with the stimulation time trend. This study shows that Kui-bao Sb-Lec1 gene plays an important role in the body’s immune defense.