CDNA Cloning And Expression Analysis Of Two Novel C-type Lectins From The Silkworm

In insect immunity, the identification of proteins takes part in the identification of the pathogen. C-type lectins are innate immune pattern recognition receptor, which contains one or more carbohydrate-recognition domains(CRD). Ca2+ plays an essential role in these lectins, so they are called C-type lectins. After recognizing the G- molecules which located in the microbial surface by C-type lectin, it will activate the immune system to clear invaded microbes.In this paper, we used silkworm as experimental material. First, we have identified two new C-type lectins, which named BmLectin-10 and BmLectin-11, then we researched their function in the immunization through the method of injection micro-organisms. The results are as follows:1. Gene identification and sequence analysis:we predicted two new C-type lectins from the silkworm genome, and obtained the two full-length genes cDNA sequences by RT-PCR. BmLectin-10 has 990bp, with EPD and the EPN motifs, and we find that BmLectin-10 has two carbohydrate-recognition domains by analysising the amino acid sequence. BmLectin-10 has both a short-type structure CRD from 16 to 150 amino acids and a long-type structure CRD from 162 to 301 amino acids. Its amino acid sequence is similar with those lectins which contain two CRDs.The amino acid sequence of BmLectin-10 with the Manduca sexta's immulectin-2 have 96%,with the Helicoverpa armigera's lectin have 95% similarity.BmLectin-11 to 810bp, with QPD motif, the amino acid sequence analysis BmLectin-11 found that the protein contains a signal peptide, located 1-21 amino acids; There is also a CRD structure, located from 45 to 203 amino acids, which is similar to the short-type CRD. The similarity of BmLectin-11 amino acid sequence is 96% compared with Drosophila melanogaster, and 95% compared with Anopheles gambiae.2. Tissue-specific analysis:This paper selected silkworm midgut, head, fat body, epidermis, Malpighian tubules, hemolymph and silk gland as the material, using RT-PCR method to analyze the BmLectin-10 and BmLectin-11's expression profile in these tissues. We found that BmLectin-10 had the highest expression level in the fatbody and was much less in the epidermis. The expression level of BmLectin-11 in the fat body is higher than that of the epidermis. There are not any expression in other tissues between BmLectin-10 and BmLectin-11.3.C-type lectins on the role of microbial recognition:The expression of BmLectin-10 and BmLectin-11 is analysed by RT-PCR. The results showed that after 24h of the E. coli invasion BmLectin-10 increased dramatically; after 6h of yeast invasion the relative expression of BmLectin-11 was significantly increased. It shows BmLectin-10 may have the role to specific identification G-, and BmLectin-11 has specific roles to indentify the fungi, but BmLectin-10 and BmLecti-11 had no significant effect on indentification of G+.4.Prokaryotic expression:Recombinant plasmid BmLectin10-pET32a and BmLectinll-pET32a transformed into expression strain BL21 (DE3), obtained BmLectin-10 and BmLectin-11 recombinant protein. The analysis shows that the recombinant protein present in the precipitate, denatured in urea used a nickel ion affinity chromatography purification of filler, obtained a soluble recombinant protein.In summary, this paper sequenced two new C-type lectins and the similarity of BmLectin-10 sequence is high compared with other C-type lectins which containing two domains. For another thing, the similarity of BmLectin-11 is high compared with other C-type lectinc which containing one domain. Therefore, it indicates that BmLectin-10 and BmLectin-11 belong to C-type lectin family, and may differ in pattern recognition. Injection of different micro-organisms found BmLectin-10 has a specific recognition role in identifing the G-, but BmLectin-11 with specific identification of fungi. We also got BmLectin-10 and BmLectin-11 recombinant protein in this paper, and it is useful to analyze the function of BmLectin-10 and BmLectin-11 and reveal the relationship between pattern recognition proteins and insect immunity.