| 斑节对虾PmML1和PmML2基因的鉴定及表达分析 |
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| 引用本文： | 符丹凤,刘洪涛,杨明秋,何玉贵.斑节对虾PmML1和PmML2基因的鉴定及表达分析[J].热带生物学报,2022,13(5):440-450. |
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| 作者姓名： | 符丹凤 刘洪涛 杨明秋 何玉贵 |
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| 作者单位： | 1.海南省水产技术推广站，海口 570226 |
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| 基金项目： | 2017年海南省本级部门预算项目（琼财预［2017］233号）；财政部和农业农村部“国家现代农业产业技术体系项目”(CARS-48)；2021年海南省创新能力建设计划省重点实验室建设补助经费项目（琼科［2021］295号） |
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| 摘 要： | 为进一步了解ML家族基因在斑节对虾先天免疫中的作用，采用PCR和RACE方法克隆到斑节对虾(Penaeus monodon)2个ML家族基因PmML1和PmML2，并对其进行生物信息学分析，同时通过实时荧光定量PCR技术分析2个ML家族基因在斑节对虾各组织的表达分布。结果显示:PmML1的开放阅读框(Open Reading Frame, ORF)为462 bp，编码153个氨基酸；PmML2的ORF为471 bp，编码156个氨基酸。2个基因都具有1个典型的ML家族蛋白结构域。多重比对结果显示，2个基因的ML结构域与组成3对二硫键的半胱氨酸残基位置都相对保守。氨基酸序列对比结果显示，PmML1和PmML2之间的相似性仅为42.37%。PmML1与日本囊对虾(Marsupenaeus japonicus)的ML蛋白MjML4的相似性最高，比例高达91.5%，PmML2与凡纳滨对虾(Litopenaeus vannamei)的ML蛋白相似性最高，比例高达88.46%。系统进化分析结果显示，PmML1和PmML2存在于2个不同的分支，说明2个基因的进化相对独立，证明本研究中鉴定到的是2个不同的基因。实时荧光定量PCR结果显示，PmML1在对虾眼柄中的表达量最高，其次为肠、胃、心、鳃、肌肉和肝胰腺，在血细胞的表达水平最低；PmML2在眼柄中表达量最高，其次为肠、鳃、胃、血细胞、心、肝胰腺和肌肉，在肌肉中的表达水平最低。眼柄是重要的神经内分泌调节器官，推测PmML1和PmML2可能在斑节对虾神经内分泌调控等过程中发挥重要作用。
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| 关 键 词： | 斑节对虾 ML基因 克隆 组织表达 |
| 收稿时间： | 2022-01-20 |
Identification and expression analysis of PmML1 and PmML2 in Penaeus monodon |
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| Affiliation: | 1.Hainan Provincial Fishery Technical Extension Station, Haikou, Hainan 5702262.Hainan Provincial Key Laboratory of Tropical Maricultural Technologies, Hainan Academy of Ocean and Fisheries Sciences, Haikou, Hainan 571126, China |
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| Abstract: | MD-2-related Lipid-recognition proteins (ML) are a class of proteins that play an important role in the innate immunity and lipid recognition and metabolism. Two ML family genes PmML1 and PmML2 were cloned from shrimps (Penaeus monodon) by using PCR and RACE methods for bioinformatics analysis, and their expression profiles in various tissues of the shrimps were analyzed by using the quantitative real-time PCR (qRT-PCR). The results showed that the open reading frame (ORF) length of PmML1 was 462 bp, encoding 153 amino acids and that the ORF length of PmML2 was 471 bp, encoding 156 amino acids. There is a typical ML family protein structural domain in these two genes. Multiple alignments revealed that the ML domain and the cysteine position in the PmML1 and PmML2 were relatively conserved. Alignment of the amino acid sequences showed that the similarity between PmML1 and PmML2 was only 42.37%. Moreover, the similarity between PmML1 and the MjML4 of Marsupenaeus japonicus was the highest (91.5%), and the similarity between PmML2 and the ML protein of Litopenaeus vannamei was the highest (88.46%). Phylogenetic analysis showed that PmML1 and PmML2 were found in two different branches, indicating that the evolution of two genes is relatively independent, which proved that two genes were identified different. Furthermore, the qRT-PCR results showed that PmML1 expressed highest in the eye stalk, followed by in the intestine, stomach, heart, gill, muscle and hepatopancreas, and the lowest expression level was in the blood cells. PmML2 expressed highest in the eye stalk, followed by in the intestine, gill, stomach, blood cells, heart, hepatopancreas and muscle, and the lowest expression level was in the muscle. As the eyestalk is an important neuroendocrine regulatory organ, it is speculated that PmML1 and PmML2 may play a role in the neuroendocrine regulation in P. monodon. This result might provide basic data for further study of ML family genes and their biological functions in shrimps. |
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