克氏原螯虾PcCRCN-L基因的克隆鉴定及在低氧胁迫下的表达响应

Molecular characterization and expression response under hypoxic-reoxygenation stress of a crustacyanin-like gene in Procambarus clarkii

中文关键词: 克氏原螯虾  类虾青蛋白  组织表达  性腺不同发育时期  低氧胁迫

英文关键词: Procambarus clarkii  Crustacyanin-like  Tissue expression  Different developmental stages of gonad  Hypoxia stress

中文摘要:

      虾青蛋白(Crustacyanin, CRCN)在甲壳动物脂类代谢及低氧胁迫应激调控等方面具有重要的功能。为获取虾青蛋白在克氏原螯虾(Procambarus clarkii)性腺发育和低氧胁迫应答中的作用，本研究在克氏原螯虾肝胰腺组织中鉴定出1个类虾青蛋白PcCRCN-L基因的cDNA序列，分析了PcCRCN-L基因的结构特征和进化模式，研究了PcCRCN-L基因在不同组织与性腺不同发育时期的表达特征，探讨了PcCRCN-L在低氧–复氧胁迫下的表达响应模式。结果显示，PcCRCN-L基因cDNA序列长2 700 bp，其开放阅读框(ORF)长度为1 587 bp，编码528个氨基酸残基；DNA序列长6 130 bp，位于克氏原螯虾基因组的第12号染色体，包含5个外显子和4个内含子，内含子/外显子剪接方式符合GT-AG规则。PcCRCN-L具有1个完整的lipocalin结构域，包含典型的序列保守区Ⅰ(SCR1)序列G-X-W、保守区Ⅱ(SCR2)序列T-D-Y和保守区Ⅲ(SCR3)序列精氨酸R。多序列比对与系统进化分析结果显示，PcCRCN-L独立于传统虾青蛋白A和C亚族，单独聚为一支。PcCRCN-L在克氏原螯虾多个组织中均有表达，在肝胰腺中表达量最高；在性腺不同发育时期，肝胰腺以及卵巢组织中的PcCRCN-L基因表达量显著降低；低氧胁迫下，肝胰腺组织中PcCRCN-L表达量显著降低，复氧后显著升高。研究结果表明，PcCRCN-L与克氏原螯虾性腺发育密切相关，并参与了克氏原螯虾的低氧–复氧胁迫应激调控。

英文摘要:

      Crustacyanin (CRCN), a family of lipocalin proteins specific to crustaceans, was primarily found in the exoskeleton of crustaceans. By binding with astaxanthin to form the astaxanthin-binding protein complex, it resets the proton at the end of astaxanthin and modifies the acidic site in astaxanthin, thereby regulating crustacean shell color. In addition to regulating shell colors, crustacyanin is involved in the transport of small molecule lipids such as steroid hormones and pheromones and has important biological functions in moult growth, gonadal and nervous system development, resistance to heavy metal lipid metabolism, and hypoxic stress. It was first discovered that the pigment in the blue shell of lobster could be extracted with ammonium chloride via a complex combination of organic bases and lipid pigments. It was later proposed that the pigment was a combination of astaxanthin and multimeric protein complex known as α-CRCN. This complex is composed of an octomer of dimeric β-CRCN subunits, with this dimer formed by two types of CRCN A and C in association with two astaxanthin molecules. Natural crustacyanins are all α-CRCN, comprising a total of 16 molecules. Procambarus clarkii, a member of the order Crustacea (Decapoda, Crayfish), is native to North America and is an important freshwater crustacean in China. It had been established that P. clarkii is susceptible to low oxygen stress during the culture process. The low oxygen environment inhibits the metabolic rate of P. clarkii, leading to increased susceptibility to pathogens; therefore, the ability of P. clarkii to tolerate and physiologically regulate stress due to other environmental factors was also affected, which can lead to irreversible damage and even death in severe cases. To understand the role of crustacyanin-like genes in gonadal development and hypoxic-reoxygenation stress in P. clarkii, a cDNA sequence of the PcCRCN-L gene was isolated from the hepatopancreas of this organism. The structural characteristics and evolutionary patterns of the PcCRCN-L gene were analyzed, and the expression characteristics of the PcCRCN-L gene in different tissues and gonad development stages were investigated. The expression response pattern of PcCRCN-L under hypoxic-reoxygenation stress was investigated. The DNA sequence of the PcCRCN-L gene was 6 130 bp long and located on chromosome 12 of the P. clarkii genome. The cDNA sequence was 2 700 bp, and its open reading frame (ORF) length was 1 587 bp. It contained five exons and four introns, encoded 528 amino acid residues, had a theoretical isoelectric point of 5.71 and a relative molecular weight of 55 613.55, and was a hydrophilic protein. The intron/exon splicing pattern was in accordance with the GT-AG rule. The PcCRCN-L protein had a complete lipocalin domain, which included the typical sequences G-X-W of conserved region I (SCR1), T-D-Y of conserved region II (SCR2), and arginine R of conserved region III (SCR3). The results of multiple sequence alignments and phylogenetic analyses showed that PcCRCN-L, as well as the crustacyanin A and crustacyanin C subgroups, were separately clustered into a branch. The expression characteristics of the PcCRCN-L gene in different tissues showed that PcCRCN-L was expressed in all the tissues; however, the highest expression level was found in the hepatopancreas. The expression trend of the PcCRCN-L gene in the ovary and the hepatopancreas was similar in different stages of gonadal development. The expression level of the PcCRCN-L gene was significantly decreased in stages Ⅰ~Ⅱof ovarian development (P<0.05), but no significant difference was found in stages Ⅱ~Ⅴ (P>0.05). The expression of the PcCRCN-L gene was significantly decreased under hypoxia stress for 1 h (P<0.05), but there was no significant difference between hypoxia stress for 1 h and hypoxia stress for 6 h. After reoxygenation for 1 h, the expression of the PcCRCN-L gene was significantly up-regulated (P<0.05). Compared with reoxygenation for 1 h, the expression of the PcCRCN-L gene was significantly increased at 12 h (P<0.05). The results showed that PcCRCN-L should be closely involved in the regulation of gonadal development and hypoxia-reoxygenation stress in P. clarkii.