Cloning and Function Analysis of Peroxiredoxin 3 Gene in Litopenaeus vannamei
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摘要: 【目的】探究凡纳滨对虾（Litopenaeus vannamei）过氧化物还原酶3 （Peroxiredoxin 3，Prx3）基因（LvPrx3）的结构特征、组织分布和抗胁迫能力，为揭示凡纳滨对虾抗环境胁迫的应答机制提供理论依据。【方法】通过RACE克隆技术取得LvPrx3基因的全长cDNA序列，应用Clustal X、EditSeq、ExPASy、MEGA 6.0、SignalP 5.0和TMHMM2.0等多个软件开展Prx3的生物信息学分析。运用半定量PCR和qRT-PCR技术检测分析LvPrx3基因在多种组织和不同胁迫条件下的表达响应情况。【结果】LvPrx3基因cDNA全长序列962 bp，内含45 bp 5′端非编码区（5′-UTR）、233 bp 3′端非编码区（3′-UTR）和684 bp开放阅读框（ORF），可编码227个氨基酸残基。LvPrx3的蛋白分子质量为25.09 ku，理论等电点（pI）为6.22，属于典型的2-Cys Prx，包含高度保守的“FYPLDFTFVCPTE” N端和“GEVCPA” C端。进化树分析显示，LvPrx3与节肢动物门的Prx亲缘关系较近，与斑节对虾（Penaeus monodon）的Prx氨基酸序列相似性最高，为98%。LvPrx3在眼柄中的表达量最高，肠道次之，在其他组织中的表达量无明显区别。脂多糖（LPS）注射后，眼柄中LvPrx3的表达分别在胁迫3 h和12~48 h时显著上调，而肠道中，LvPrx3表达量在胁迫3~24 h时均显著下调；在4-壬基酚（4-NP）胁迫后，眼柄中LvPrx3表达量在胁迫6~24 h后显著上调，24 h时最高，为对照组的4.11倍；肠道中的LvPrx3表达量在胁迫12 h时显著上调，在胁迫48 h时显著下调；以上显著性水平α均为0.05。【结论】LvPrx3基因属典型的2-Cys Prx，主要在凡纳滨对虾眼柄和肠道中高表达。脂多糖LPS刺激和4-NP胁迫可明显诱导凡纳滨对虾LvPrx3基因的表达水平，表明LvPrx3在对虾抵御病原菌感染及抗逆境胁迫的调控中发挥重要作用。
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关键词:
- 凡纳滨对虾 /
- 过氧化物还原酶（Prx） /
- 基因克隆 /
- 脂多糖 /
- 4-壬基酚（4-NP）
Abstract: 【Objective】 The structural characteristics, tissue distribution and stress resistance of peroxiredoxin 3 (Prx3) gene in Pacific white shrimp Litopenaeus vannamei (LvPrx3) were investigated to provide a theoretical basis for revealing the response mechanism of L. vannamei to environmental stress. 【Method】 The complementary DNA (cDNA) sequence of LvPrx3 was obtained by RACE method. The bioinformatics analysis of Prx3 was carried out by several softwares, including Clustal X, EditSeq, ExPASy, MEGA 6.0, SignalP 5.0 and TMHMM 2.0. Semi-quantitative PCR and quantitative real-time PCR were used to detect the expression of LvPrx3 gene in various tissues and under different stress conditions. 【Result】 The full-length cDNA of LvPrx3 was 962 bp, which contained 45 bp 5' non-coding region (5'-UTR), 233 bp 3'-UTR and 684 bp open reading frame (ORF), encoding 227 amino acids. The deduced mature LvPrx3 protein has a calculated molecular mass of 25.09 ku with a theoretical pI of 6.22. It belongs to the typical 2-Cys Prx, including highly conservative "FYPLDFTDVCPTE" in the N-terminal and "GEVCPA" in the C-terminal. Phylogenetic trees based on the similarity of Prx amino acid showed that LvPrx3 was closely related to Prx of Arthropoda. It shared the highest similarity with Prx from Penaeus monodon (98%). The expression of LvPrx3 was high in the eyestalk and intestine, while no significant difference in other tissues. After lipopolysaccharide (LPS) injection, the expression level of LvPrx3 in eyestalk was increased in the 3 h and 12-48 h. By contrast, its expression was downregulated after 3-24 h injection in intestine. In response to 4- nonylphenol (4-NP) stress, LvPrx3 expression in eyestalk was significantly upregulated after 6-48 h exposure, and reached the peak in 24 h, which was 4.11 times that of control group. In intestine, LvPrx3 expression was significantly upregulated after 12 h exposure, but significantly downregulated after 48 h. 【Conclusion】LvPrx3 was a typical 2-Cys Prx, which was highly expressed in eyestalk and intestine. The expression level of LvPrx3 in the eyestalk of L. vannamei was significantly induced by LPS stimulation and 4-NP stress, showing that LvPrx3 played a vital role in the defense mechanism of shrimp against pathogen infection and environmental stress.-
Key words:
- Litopenaeus vannamei /
- peroxiredoxin (Prx) /
- gene cloning /
- lipopolysaccharide /
- 4-nonylphenol (4-NP)
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