Isolation And Identification Of A Chitin-degrading Bacterium,Cloning And Expression Of Chitinase Genes,and Its Effect On The Growth And Intestinal Microbiota Of Penaeus Vannamei

Firstly,potential probiotics with chitinase activity were isolated and screened from the intestinal tract of Penaeus vannamei in this study.Then,the chitinase genes were cloned and expressed based on genome sequencing and analysis of the target strain.Finally,the effects of the target strain on the growth and intestinal microbiota of P.vannamei were evaluated through culture experiment.These studies provide a reference for the potential application of chitin-degrading bacteria in shrimp aquaculture.The main research contents and results are as follows:1.Chitin-degrading bacteria were isolated from the intestines of P.vannamei in Zhanjiang by using selective medium,and identified by 16 S r DNA sequencing.Potential probiotic strains were screened based on extracellular enzyme activity and safety to Artemia larvae.The results showed that,among the 36 preliminarily identified chitinase-producing strains,Vibrio was the most dominant genus,followed by Aeromonas,and Pseudoalteromonas C923 and Shewanella C813 presented high safety and strong chitinase activity,which could be used as potential probiotics for shrimp.2.The whole genome of strain C923 was sequenced by Prometh ION platform,and was annotated and analyzed.Then,the chitinase genes A(chi A)and D(chi D)of C923 were cloned and expressed,and the enzyme domains were analyzed.The results showed that the genome size of C923 was 5.37 Mb,the GC content was 43.34%,and the average nucleotide identity was most similar to the type strain of Pse.piscicida,thus it was identified as this species.The genome of C923 encodes 4576 complete CDS,including 10 genes related with chitin-degradation.The chi A and chi D were cloned and expressed with lengths of 2484 bp and 3036 bp respectively,and the encoded protein sizes were 108 k Da and 129 k Da,which belonged to the glycosidase family 18.3.The effects of Pseudoalteromonas C923 and chitin on growth,immune enzyme activity and intestinal microflora of P.vannamei were investigated by 8-week culture experiment with self-made formulated feeds containing 0%,4% and 8% chitin.The results showed that,compared with the control group,adding chitin and / or C923 could significantly reduce the feed coefficient,and both the 4% chitin and C923 groups could significantly increase the weight gain rate and specific growth rate of shrimp(P < 0.05),while the combined effect of 4% chitin and C923 group was the best.The activities of chitinase in shrimp hepatopancreas and acid phosphatase in serum were significantly increased by adding 4% and 8% chitin respectively,while the activities of superoxide dismutase,acid phosphatase and alkaline phosphatase in hepatopancreas were significantly increased by adding C923.The difference of intestinal microbiota structure increased among groups after adding 4% chitin or C923 for 8 weeks,and C923 significantly increased the relative abundance of Pseudoalternariaceae.Compared with the control,the abundance of Vibrionaceae of shrimp intestines increased significantly in the 4% chitin group,but it was not significantly changed under the combination of 4% chitin and C923.