| Chinese giant salamander（Andrias davidianus）is an unique large-scale amphibian animal in China and has important economic value.It has now become a rare aquaculture species.However,with the rapid development of the salamander aquaculture,the number and density of cultures have soared,together with the deterioration of the growing environment,which has led to frequent occurrence of diseases.Therefore,the research on the pathology and immunology of A.davidianus becomes particularly important.Lysozyme is an alkaline protease that breaks the beta-1,4 glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine in the bacterial cell wall peptidoglycan.The polysaccharide breaks down into soluble glycopeptides,which disintegrates the bacteria.As an immune defense factor of the body,lysozyme can resist the infection of the external pathogenic bacteria and plays a very important role in the non-specific immune process of A.davidianus.Therefore,we have screened the c-type and g-type lysozyme gene fragments from A.davidianus’s transcriptome database and designed primers to clone and identify the full-length cDNA fragments of the c-type and g-type lysozyme（AdlysC and AdlysG）.The bioinformatics analysis,expression characteristics,gene expression changes after pathogenic stimulation,and in vitro prokaryotic expression and product activity studies were performed on the AdlysC and AdlysG.The results as follows:（1）The cDNA sequence of AdlysC was of 873 bp,consisting of 56 bp 50-terminal UTR,456 bp open reading frame and 352 bp 30-terminal UTR.The deduced protein was composed of 152 amino acids,with an estimated molecular mass of 16.86 kDa and a predicted pI of8.66.The cDNA sequence of AdlysG was of 948 bp,consisting of 270 bp 50-terminal UTR,558 bp open reading frame and 120 bp 30-terminal UTR.The deduced protein was composed of 185 amino acids,with an estimated molecular mass of 20.48 kDa and a predicted pI of8.41.（2）Real time quantitative PCR（qRT-PCR）analysis indicated that AdlysC gene was widely expressed in each tissue tissues,with the highest level in the stomach and liver,and higher expression in the intestine,spleen,blood and lungs,and the lowest level in muscle,kidney,heart and brain.AdlysG was mainly distributed in the liver,spleen and lung,and much lower expression level was detected in muscle,kidney,skin and intestines.The transcription level of AdlysC and AdlysG were upregulated after Aeromonas hydrophila infection in liver and spleen,indicating its important role in the immune response.（3）The pET32a/AdlysC and pET32a/AdlysG recombinant expression vectors were constructed and used to express rAdlysC and rAdlysG proteins in E.coli BL21 using a prokaryotic expression system.The expression of rAdlysC and rAdlysG was obtained by IPTG-induced expression,affinity purification of Ni2+,and inclusion body renaturation.The activities of recombinant proteases were measured at different pH gradients and temperature gradients in vitro.It was found that the activity of lysozyme was highest at rAdlysC and rAdlysG at pH 6.0 and 30°C.The minimal inhibitory concentration（MIC）test showed that rAdlysC and rAdlysG proteins had certain antibacterial activities against gram-negative bacteria and gram-positive bacteria,and the antibacterial activity of rAdlysC was higher than that of rAdlysG.The results of the study not only have a certain degree of understanding of the functional roles and activity characteristics of Euphorbia lysozyme genes,but also provide the basis for further understanding of the functional genes in the immune system of A.davidianus. |
