Construction Of CDNA Library, Cloning And Expression Of Immune-Related Genes From Chinese Mitten Crab Eriocheir Sinensis

Chinese mitten crab (Eriocheir sinensis) is the economical aquatic species of China. In recent years, with the development of scale cultivation and environmental deterioration, various diseases caused by bacterial, viruses, parasites had frequently occurred in cultured E. sinensis populations, which resulted in enormous losses to the crab aquaculture. In the present study, we adopted molecular biology method and constructed cDNA library of E. sinensis challenged by lipopolysaccharide (LPS). Based on it, we screening immune-related genes sequencing of E. sinensis, and conduct experiments to further cloning and expression the immune-related genes by RACE and RT-PCR. The identification and expression of immune-related genes in crab will help our better understanding of crustacean immunity. The results are as follows:1. Based on the SMART technology, a cDNA library of a Chinese mitten crab E.sinensis challenged by LPS was constructed. This library reaches 6.28×106 in capacity, the percentage of recombination is as high as 95.8%. The average inserted cDNA fragments were about 700 bp. From the library, some cDNA fragments was selected to sequence. And a total of 319 successful sequencing reations which include 140 ESTs covered by contig. Basic local Alignment Search Tool (BLAST) analysis showed that 121 ESTs were assigned putative function because they showed similarity to known gene. The results showed that it contained ESTs which are play important role in the E. sinensis for immune reaction. These results have established a basis for the deep study of immune related genes for E. sinensis.2. In invertebrates, C type lectins are a superfamily of carbohybrate-recognition domain which play crucial roles in the innate immunity. In this study, two novel C type lectin genes (EsLecA and EsLecB) from E. sinensis were cloned by RACE approach based on EST analysis. The full-length cDNA of EsLecA 772 bp with a 5'-terminal untranslated region (UTR) of 175 bp, a 3'UTR of 135 bp with a poly (A) tail, and an open reading frame (ORF) of 462 bp encoding a polypeptide of 153 amino acids (aa). And the full length cDNA of EsLecB is 700 bp with a 5'UTR of 24 bp, a 3'UTR of 172 bp with a poly (A) tail, and an ORF of 504 bp encoding a protein of 167 aa. The tissue distribution of EsLecA and EsLecB mRNA were examined by fluorescent quantitative real-time PCR. In the normal crab, the highest expression of EsLecA and EsLecB were observed in the hepatopancreas, which were significantly higher than other tissues. The temporal expression of EsLecA and EsLecB transcript in the intestine, gill, haemolymph and hepatopancreas of E. sinensis challenged by LPS and Poly I:C were recorded by RT-PCR respectively. The results indicated that the mRNA expression of EsLecA and EsLecB were up-regulated in the tissues tested. The results collectively suggested that EsLecA and EsLecB were involved in the immune defense of E. sinensis against pathogen infection.3. In living organisms, ferritin plays a key role in iron storage and detoxification. The full-length cDNA sequence of a ferritin (EsFer) was obtained from the cDNA library of the Chinese mitten crab E. sinensis stimulated with LPS. It has 1118 bp in length and contains a 480 bp ORF encoding 159 aa. The 5'-and 3'-untranslated region (UTR) is 178 bp and 461 bp, respectively. A transmembrane helix was found at the position of 15-37aa. A complete iron-responsive element (IRE) was found at the 5' untranslated region corresponding to the nucleotide sequence at the positions of the 135-162. Quantitative real-time PCR was used to assess the mRNA expression of EsFer in various tissues and its induced expression in haemolymph, intestine, hepatopancreas of crabs challenged with LPS. The results show that EsFer was expressed in all tested organs of the Chinese mitten crab, including haemolymph, muscle, intestine, gills, heart, gonad and hepatopancreas. The highest expression level was observed in hepatopancreas, while the lowest in haemolymph. The expression of EsFer in haemolymph, intestine and hepatopancreas was up-regulated following the challenge with LPS.