红螯光壳螯虾组织蛋白酶L基因的克隆及维生素C对其表达的影响

为深入了解红螯光壳螯虾组织蛋白酶L基因的表达特性及维生素C对其表达的影响,实验利用RACE-PCR技术及荧光定量PCR技术,从红螯光壳螯虾肝胰腺中克隆得到组织蛋白酶L基因cDNA全长序列,命名为CqCatL(GenBank登录号:KJ913663),同时检测了该基因在红螯光壳螯虾各个组织及添加了不同浓度维生素C的组别中的表达。结果显示,该基因全长1 810 bp,开放阅读框长度为1 026 bp,编码341个氨基酸残基,预测的分子量和等电点(pI)分别为37.63 ku和5.17。同源性分析结果显示,该基因编码的蛋白与其他虾蟹类有较高的相似性,说明组织蛋白酶L基因在甲壳动物具有较高的保守性。组织荧光定量PCR结果显示,CqCatL基因在红螯光壳螯虾的多个组织中均有表达,其中肝胰腺中表达量最高,其次为血细胞,在肠及触角腺中也有一定量的表达。在基础饲料中添加不同水平的维生素C后,CqCatL基因的表达量也存在明显差异,其中维生素C添加量为400 mg/kg的组别中该基因的表达量最高。研究表明,组织蛋白酶L基因在红螯光壳螯虾的生长发育过程中有重要的作用,且其表达量受维生素C的影响。

Cloning and expression of cathepsin L in Cherax quadricarinatus and its regulation by dietary vitamin C

Australian redclaw crayfish, Cherax quadricarinatus, belongs to Decapoda order, and fParastacidae family. Because it grows rapidly and can reach commercial size in a few months at optimal conditions, it has been one of the most important economy shrimps in China. However, the physiological mechanisms of redclaw crayfish have not been well studied, especially the basic information of key nutrients and the related enzymes or proteins are missing. Cathepsin L (CatL) is an important enzyme related to digesting progress in crustaceans, however, its molecular structure and correlation with dietary nutrients has not been investigated. In the present study, the CatL gene of Cherax quadricarinatus was cloned firstly. The tissue-specific CatL gene expression was also performed. In order to obtain insights on the regulatory effects of key dietary nutrients on expression of CatL gene, the Cherax quadricarinatus were fed with artificial diets containing different levels of vitamin C (0, 100, 200, 400, 800, 1600, 3200 mg/kg) for 8 weeks, and the expression of CqCatL mRNA in different tissues and different groups were examined. The CatL cDNA was isolated from the hepatopancreas of Cherax quadricarinatus using RACE-PCR methods, which was designated CqCatL (Genbank accession number: KJ913663). The full-length cDNA of CqCatL obtained from the present study was 1810 bp in length with an open reading frame of 1026 bp, encoding a putative peptide of 341 amino acids, and the predicted molecular mass and isoelectric point were 37.63 kDa and 5.17. By alignment, the amino acid sequence of CqCatL of Cherax quadricarinatus showed high homology with those of other animals. For examples, the deduced amino acids sequence of Cherax quadricarinatus CqCatL shared 79% homology with Penaeus monodon, and 74%, 59%, 59%, 58% and 58% with Macrobrachium nipponense, Eriocheir sinensis, Metapenaeus ensis, Metapenaeus ensis and Litopenaeus vannamei, respectively. This suggested that CqCatL was highly conservative. The tissue-specific expression pattern study indicated that CqCatL mRNA was widely expressed in hemocytes, gill, hepatopancreas, muscle, ovary, intestine, stomach, heart, brain and antennal gland, indicating CqCatL may play important roles in many tissues of the Cherax quadricarinatus. The highest expression was observed in hepatopancreas, and the second was seen in hemocytes, while only very low expressions of CqCatL mRNA were found in intestine and antennal gland. After feeding by seven level of vitamin C, the mRNA expression of CqCatL was the highest in the 400 mg/kg dietary vitamin C group and significantly higher than the other groups., Our results showed that CqCatL may plays an important roles in the physiological progress of Cherax quadricarinatus, and its gene expression and functions is likely regulated by nutrients, such as vitamin C.