Cloning of the McNF-κB gene of Mytilus coruscus and its role in development
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摘要: 核因子κB（Nuclear Factor kappa-B，NF-κB）具有免疫、炎症、凋亡、细胞增殖和发育的调节作用，目前NF-κB在脊椎动物和果蝇中的研究较为丰富，在贝类中的报道较少。为进一步探究NF-κB在厚壳贻贝（Mytilus coruscus）免疫和发育中的作用，本研究克隆了厚壳贻贝McNF-κB基因的序列全长，其全长为4 087 bp，开放阅读框为2 613 bp，编码871个氨基酸，具有典型的锚蛋白重复序列（ankyrinrepeat, ANK）结构域和死亡结构域。氨基酸序列分析结果发现，该基因与欧洲贻贝（Mytilus edulis）和地中海贻贝（Mytilus galloprovincialis）分别具有72.76%和66.58%同源性，且在系统进化树中与欧洲贻贝和地中海贻贝聚为一支。经实时荧光定量PCR（qRT-PCR）技术检验表明，McNF-κB基因于厚壳贻贝各组织均有分布，在鳃中表达最高；McNF-κB基因在厚壳贻贝眼点幼虫阶段和稚贝阶段均有表达，且在稚贝阶段表达量显著高于眼点幼虫阶段。利用RNA干扰技术沉默眼点幼虫McNF-κB基因后幼虫变态率显著下降，推测McNF-κB基因调控厚壳贻贝幼虫变态过程。本研究为探究McNF-κB基因如何调控厚壳贻贝发育奠定了基础。Abstract: Nuclear factor κB (NF-κB) can regulate immunity, inflammation, apoptosis, cell proliferation, and organism development. At present, NF-κB has been well studied in vertebrates and fruit flies, while its role in shellfish is still elusive. In order to further explore the role of NF-κB in the immunity and development of mussel Mytilus coruscus, the full length McNF-κB cDNA sequence was cloned from M. coruscus. McNF-κB gene was 4087 bp long, and the open reading frame was 2 613 bp, encoding 871 amino acids and had a typical ankyrinrepeat (ANK) domain and DEATH domain. The results of amino acid sequence analysis showed that the gene had 72.76% homology with M. edulis and 66.58% with M. galloprovincialis, respectively, and was clustered with M. edulis and M. galloprovincialis in the phylogenetic tree. The real-time PCR (qRT-PCR) technology showed that the McNF-κB gene was expressed distributed in all tissues of M. coruscus, and the expression was the highest in the gill. McNF-κB gene was expressed in both the pediveliger larvae stage and the juvenile stage of M. coruscus, and the expression was significantly higher in the pediveliger larvae stage than that in the juvenile stage. After using RNA interference technology to silence the McNF-κB gene of pediveliger larvae, the larval metamorphosis rate decreased significantly, indicating that this gene was involved in regulating the metamorphosis process of M. coruscus. This study provides a basis for exploring how McNF-κB gene regulates development of M. coruscus.
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Key words:
- Mytilus coruscus /
- NF-κB /
- gene cloning /
- larval metamorphosis
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图 1 McNF-κB基因cDNA全长及推导的氨基酸序列
下划线表示起始密码子ATG；“*”表示终止密码子；灰色区域表示ANK结构域；黑色方框表示死亡结构域
Fig. 1 Full-length cDNA and deduced amino acid sequences of McNF-κB gene
The start codon ATG is underlined; the stop codon is indicated by an asterisk；the gray area represents the ANK domain; the black box represents the DEATH domain
图 3 不同物种NF-κB氨基酸序列多重比对分析
绿色框线表示具有多个ANK结构域；红色框线表示死亡结构域；深色背景中的氨基酸序列具有100%的同源性；粉色背景中氨基酸具有大于75%的同源性；绿色背景中氨基酸具有大于50%的同源性
Fig. 3 Multiple alignment analysis of NF-κB amino acid sequences of different species
Green borders indicate multiple ANK domains; red borders indicate DEATH domain; the amino acids in dark gray boxes indicate conserved residues with 100% homology; the amino acids in pink boxes show conservation of residues with > 75% homology; the amino acids in green boxes show conservation of residues with > 50% homology
图 4 不同物种NF-κB氨基酸序列系统进化树
Fig. 4 Phylogenetic tree of NF-κB amino acid sequences in different species
图 5 McNF-κB基因在厚壳贻贝不同组织的表达
不同字母表示各组间存在显著差异（p < 0.05）
Fig. 5 Expression of McNF-κB gene in different tissues of Mytilus coruscus
Bars with different letters are significantly different （p < 0.05）
图 6 McNF-κB基因在厚壳贻贝幼虫变态前后阶段的表达
不同字母表示各组间存在显著差异（p < 0.05）
Fig. 6 Expression of McNF-κB gene in the pre- and post-metamorphosis stages of Mytilus coruscus larvae
Bars with different letters are significantly different (p < 0.05)
图 7 RNA干扰McNF-κB基因后的幼虫变态率（A）和存活率（B）
不同字母表示各组间存在显著差异（p < 0.05）
Fig. 7 Larval metamorphosis rate (A) and survival rates (B) after RNA interference with McNF-κB gene
Bars with different letters are significantly different (p < 0.05)
表 1 厚壳贻贝NF-κB基因cDNA全长克隆和mRNA表达分析所用的引物序列
Tab. 1 Primers sequences used for full-length cDNA cloning and Mytilus coruscus NF-κB gene mRNA expression analysis
引物名称 序列 用途 McNF-κB-1-3′ RACE TGACAGAAAAGGCAATACCCCG 3′ RACE McNF-κB-2-3′ RACE GAGGTGACCCTGAGATGGAA 3′ RACE McNF-κB-1-5′ RACE GAGGTTGGCAAAAGTGACAG 5′ RACE McNF-κB-2-5′ RACE CTTCAGAACATTTGCCCCC 5′ RACE McNF-κB-RT-F GTATACCCAGACCCCAATC qRT-PCR McNF-κB-RT-R TCTTCTACCGTCACCACC qRT-PCR EF-1α-RT-F CACCACGAGTCTCTCCCTGA qRT-PCR EF-1α-RT-R GCTGTCACCACAGACCA TTCC qRT-PCR 
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