合浦珠母贝<i>PfCLEC17A</i> 基因的克隆及表达分析

doi:10.7668/hbnxb.20193071

摘要/Abstract

摘要：

为了探究C型凝集素(C-type lectin)家族成员在合浦珠母贝机体内的作用及表达模式,为进一步解析合浦珠母贝PfCLEC17A的免疫应答机制,通过 RACE 技术克隆获得合浦珠母贝C型凝集素基因PfCLEC17A;利用生物信息学方法分析了PfCLEC17A的分子特征,并通过荧光定量PCR检测PfCLEC17A基因在合浦珠母贝的鳃、外套膜、闭壳肌、性腺、血液、足和肝胰腺等组织及溶藻弧菌感染后在肝胰腺中的表达情况。结果显示,从合浦珠母贝中克隆获得新的C型凝集素基因PfCLEC17A,cDNA全长为699 bp,其中开放阅读框(ORF)长534 bp,编码178个氨基酸残基,含有一个CTL结构域。系统进化树分析表明,合浦珠母贝与其他贝类的PfCLEC17A基因聚集在一个分支。实时荧光定量 PCR 结果显示,PfCLEC17A在合浦珠母贝的鳃、外套膜、闭壳肌、性腺、血液、足和肝胰腺等各个组织中均有表达,其中在肝胰腺中的相对表达量最高且显著高于在其他组织中的相对表达量。肝胰腺作为软体动物重要的免疫器官,暗示PfCLEC17A基因可能参与机体免疫过程。藻弧菌感染 1 h 后,合浦珠母贝PfCLEC17A基因表达水平显著升高并达到最大值,说明该基因参与弧菌等微生物刺激的早期反应。综上所述,PfCLEC17A基因参与合浦珠母贝的免疫应激反应,具有一定的免疫功能。

关键词: 合浦珠母贝, C型凝集素, PfCLEC17A, 基因克隆, 基因表达

Abstract:

To explore the immune function and expression pattern of C-type lectin family members in Pinctada fucata,a cDNA sequence of C-type lectin gene PfCLEC17A in P.fucata was cloned through RACE technique.The molecular characteristics of PfCLEC17A were determined by bioinformatics analysis and its mRNA expression level was detected in different tissues including gill,mantle,adductor muscle,gonad,blood,foot and hepatopancreas,as well as expression level in hepatopancreas after Vibrio alginolyticus challenged using the Real-time fluorescent quantitative PCR.The results showed that a new C-type lectin gene PfCLEC17A was successfully cloned from P.fucata,the full cDNA was 699 bp in length with an open reading frame(ORF)of 534 bp,which encoded 178 amino acids.The deduced amino acid sequence was predicated containing a CTL domain.Phylogenetic tree analysis showed that the PfCLEC17A of P.fucata and other shellfish proteins were clustered together in a branch.The Real-time fluorescent quantitative PCR results showed that PfCLEC17A was expressed in all tissues detected in P.fucata,and the highest was found in hepatopancreas,which was significantly higher than the other tissues.The high expression level in hepatopancreas,one of the most important immune tissues in mollusc,suggested that PfCLEC17A gene might be involved in the body's immune reaction.The expression level of PfCLEC17A increased significantly and reached the peak at 1 h after V.alginolyticus challenge,indicating that the gene expression was induced by microbial stimulation at early stage.Therefore,based on the above findings,it could be concluded that the PfCLEC17A gene participates in the immune activation response of P.fucata and functions in immune system.

Key words: Pinctada fucata, C-type lectin, PfCLEC17A, Gene cloning, Gene expression

郑玉斯, 王培, 郭颖, 白丽蓉, 喻达辉, 赵森. 合浦珠母贝PfCLEC17A 基因的克隆及表达分析[J]. 华北农学报, 2022, 37(5): 230-238. doi: 10.7668/hbnxb.20193071.

ZHENG Yusi, WANG Pei, GUO Ying, BAI Lirong, YU Dahui, ZHAO Sen. Molecular Cloning and Expression Analysis of PfCLEC17A in Pearl Oyster Pinctada fucata[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(5): 230-238. doi: 10.7668/hbnxb.20193071.

http://www.hbnxb.net/CN/Y2022/V37/I5/230

图/表 8

表1 PfCLEC17A扩增及表达所用引物

Tab.1 Primers used in amplification and expression of PfCLEC17A

引物 Primer	引物序列(5'—3') Prime sequence(5'—3')	用途 Application
PfCLEC17A-F	ATGATTTCTGCAGATAAGAGAACGTG	RT-PCR
PfCLEC17A-R	TTTTGCCGAAGACTACATCACCAGTG
PfCLEC17A 3'-GSP	GATTACGCCAAGCTTTCTGCAGATAAGAGAACGTGGTATAG	RACE
PfCLEC17A 3'-NGSP	GATTACGCCAAGCTTTTGGATTGGTGGAAGGGACAGCGTAA
PfCLEC17A 5'-GSP	GATTACGCCAAGCTTCAAAAGTTCCTGTAGAAGTCCATTCG
PfCLEC17A 5'-NGSP	GATTACGCCAAGCTTCGAATCTACCCCAAATTACGCTGTCC
UPM	CATGGCTACATGCTGACAGCCTA
UPM-Short	CGCGGATCCACAGCCTACTGA TGATCAGTCGATG
PfCLEC17A-q-F	GCAGCAGAATGTCAGTCTTGGA	qRT-PCR
PfCLEC17A-q-R	CTAAGCTTACCTGAAGATGTCC
18S-F	GAGAAACGGCTACCACATCC
18S-R	CACCAGACTTGCCCTCCAA

图1 合浦珠母贝PfCLEC17A基因扩增产物电泳 DNA Marker为DL2000。

Fig.1 Electrophoresis result of amplified product of PfCLEC17A in P.fucata The DNA Marker is DL2000.

图2 合浦珠母贝PfCLEC17A cDNA 序列及其编码区氨基酸序列双下划线.CLECT结构域;阴影部分.信号肽区域;黄色阴影部分.二硫键;蓝色阴影部分.糖基化位点。

Fig.2 cDNA sequence and deduced amino acid sequence of PfCLEC17A in P.fucata Double underlined.CLECT domain;Shaded part.Signal peptide region;Yellow shaded part.Disulfide bond;Blue shaded part.Glycosylation sites.

图3 合浦珠母贝PfCLEC17A生物信息学分析 A.蛋白质信号肽预测;B.蛋白质结构域预测;C.蛋白质三级结构预测;D.蛋白质亲水性/疏水性预测。

Fig.3 PfCLEC17A bioinformatics prediction of Pinctada fucata A.Protein signal peptide prediction;B.Protein domain prediction;C.Protein tertiary structure prediction;D.Protein hydrophilicity/hydrophobicity prediction.

图4 PfCLEC17A氨基酸序列比对

Fig.4 PfCLEC17A amino acid sequence alignment

图5 CLEC17A氨基酸序列系统进化树

Fig.5 Phylogenetic tree analysis of CLEC17A

图6 合浦珠母贝中pfCLEC17A在不同组织的相对表达量 M.外套膜;B.血细胞;GO.性腺;F.足;A.闭壳肌;GI.鳃;HE.肝胰腺。不同字母表示在不同组织中的相对表达量差异性显著(P<0.05)。

Fig.6 Relative expression of pfCLEC17A among tissues in P.fucata M.Mantle;B.Blood cell;GO.Gonad;F.Foot;A.Adduct muscle;GI.Gills;HE.Hepatopancreas.Different letters indicate significant differences in relative expression among tissues (P<0.05).

图7 溶藻弧菌感染后合浦珠母贝PfCLEC17A mRNA在肝胰腺中的相对表达量不同字母表示溶藻弧菌不同感染时间下的相对表达量有差异性显著(P<0.05)。

Fig.7 Relative expression of PfCLEC17A of P.fucata in hepatopancreas after challenged with V.alginolyticus Different letters indicate significant differences in relative expression at different time after challenged with V.alginolyticus(P<0.05).

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	Li M, Zhou S M, Liu L, Peng D, Wang G L. Molecular clone and expression of C-type lectin in Meretrix meretrix[J]. Oceanologia et Limnologia Sinica, 2015, 46(5):1186-1192.

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合浦珠母贝PfCLEC17A 基因的克隆及表达分析

Molecular Cloning and Expression Analysis of PfCLEC17A in Pearl Oyster Pinctada fucata

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