Study On Immune Factors And Intestinal Microflora Response To High Alkalinity Environment In Leuciscus Waleckii

The Amur ide(Leuciscus waleckii),high alkalinity-resistant fish,generally survive in water with carbonate alkalinity up to 53.6 mmol/L(p H 9.6).Due to long-term geographical isolation,two alkali-water and freshwater species have been differentiated in Amur ide,an ideal model fish to explore the adaptive evolution and alkali-saline tolerance mechanism.The relevant research has been carried out in genome resource mining,ion transport,and functional analysis of alkali-saline tolerant genes.However,the immune function and high carbonate alkalinity adaptation mechanism are not precise.Thus,this thesis focuses on the relevant immune genes and the response mechanism of the intestinal microflora of Amur ide in adaptation to high carbonate alkalinity.In further,the use of alkali-saline tolerant bacteria to improve the adaptability of fish to high carbonate alkalinity environment provides data reference for the analysis of fish alkali-saline tolerance mechanism and the cultivation of excellent varieties,which is of great significance to the utilization of alkali-saline water fisheries.Firstly,the differential genes related to high carbonate alkalinity stress in alkali-water species and freshwater species were found by transcriptome analysis.Secondly,the significantly differentially expressed immune genes were selected to verify their functions related to high carbonate alkalinity stress.Thirdly,the activity changes of immune enzymes under high carbonate alkalinity stress were studied from the perspective of the enzyme system on immune function.Further,intestinal microflora changes in the alkaline water and freshwater populations were scanned from an intestinal microflora standpoint.Finally,the directional enrichment of alkalisaline tolerant bacteria to the intestinal microflora can improve its adaptability to the environment with high carbonate alkalinity based on the study of intestinal microflora.(1)The transcriptome data of gill and kidney tissues and freshwater species under high carbonate alkalinity were analyzed.The results showed that the differentially expressed genes could be enriched in GO entries and the KEGG pathway related to the immune system.The differential gene analysis showed that the transcripts of transferrin(Tf)and copper metabolism Murr1 domain 9(commd9)increased significantly in the gills and kidneys under high carbonate alkalinity.The cDNA sequence characteristics of the Tf gene and commd9 gene were identified.The mRNA expressions of the Tf gene and commd9 gene were analyzed by Quantitative Realtime PCR(q RT-PCR).It was found that the presentation of the Tf gene and commd9 gene were closely related to high carbonate alkalinity stress.(2)Under the stress of 50 mmol/L carbonate alkalinity,the activities of immune enzymes such as Acid phosphatase(ACP)and Alkaline phosphatase(AKP)in liver tissue of the freshwater population increased significantly,and the changes in ACP and AKP enzyme activities in liver tissue of alkali-water species were considerably less than those of freshwater species.On the other hand,the movement of Superoxide dismutase(SOD)in liver tissue of freshwater species increased significantly on the third day,and the activity of Glutathione peroxidase(GSH-PX)in kidney tissue of freshwater species increased considerably than that of alkali-water species.(3)The composition of intestinal microflora in alkali-water species and freshwater species was compared and analyzed to clarify the correlation between the intestinal microflora in the longterm evolution and the high carbonate alkalinity environment.The results showed significant differences in intestinal microbial composition,abundance,and diversity between the alkali-water and freshwater species,but there was little difference in microbial community composition in the alkali-water and freshwater environment.Moraxella osloensis,Psychrobacter maritimus,and Psychrobacter faecalis are significantly enriched in the intestinal tract of the alkali-water species.Therefore,some strains of Psychrobacter and Bacillus may play a role in adapting high carbonate environment.(4)Further,the alkali-saline tolerant bacteria were enriched and screened from the high carbonate in alkali-saline pond environment and the intestines of Amur ide,and an alkali-saline tolerant beneficial bacteria strain,Bacillus firmus CT-SL8-3,was obtained.Finally,liquid fermentation prepared liquid microbial agents and applied them to small indoor water bodies and outdoor alkali-saline aquaculture ponds.The results show that the bacterial agent can improve the composition of beneficial microflora in the intestine of Amur ide,improve the growth of Amur ide,promote the adaptation of Amur ide to a high carbonate alkalinity environment,and lay an essential foundation for the directional transformation of the intestinal microflora of high carbonate alkalinity tolerant fish.In conclusion,this thesis analyzed the response mechanism of immune genes,immune enzymes,and antioxidant enzymes in the adaptation to high carbonate alkalinity,clarified the main intestinal functional microflora of Amur ide in the transformation to high carbonate alkalinity environment and obtained the effective B.firmus CT-SL8-3 strain and microbial agent applied to improve the adaptation to high carbonate alkalinity environment.