Selective Breeding Of Disease-resistant Families And Screening Of Related Genes Of Noble Scallop Chlamys Nobilis

The noble scallop Chlamys nobilis Reeve is an important mariculture shellfish in southern coastal areas of China,which brings huge economic benefits to the industry.However,frequent outbreaks of mass mortality have caused huge economic losses to the scallop breeding industry.Bacterial diseases caused by Vibrio parahemolyticus(V.p.)infection are an important cause of mass mortality.Although this problem can be alleviated by improving breeding methods and optimizing breeding structure,breeding excellent varieties with strong disease resistance is the fundamental way to solve the disease problem.In this study,“Nan’ao Golden Scallop” of noble scallop Chlamys nobilis was selected as the research object.The surviving individuals were selected as the parents through Vibrio infection stress to construct the families,and the control population was set up.Vibrio challenge tests were carried out at different stages of their growth and development to compare the difference in survival rate between each family and the control population,and the indexes related to growth such as shell length were determined.Transcriptome sequencing analysis was performed on the infected larvae to screen out genes with significant differential expression,and function verification and related metabolic pathway analysis were performed on these genes.BAG3 gene was cloned and analyzed by bioinformatics means,combined with RNAi technology and Western Blot to verify the function of the gene.The main results are as follows:1.Construction of breeding families and comparison of traits: Eight resistant families and one control population were constructed,and Vibrio challenge stress and growth indexes were measured at larval stage,juvenile stage and adult stage,respectively.The results showed that the survival rate of 5 families in larval stage was higher than that of the control population,among which the survival rate of family 1and 4 was significantly higher than that of the control population(P < 0.05).The survival rate of three families of juvenile stage was higher than that of control group,and the survival rate of family 3 was significantly higher than that of control group(P< 0.05),After Vibrio infection,SOD activity in serum of the three selected families was lower than that of the control group,and CAT activity was the first to increase to the peak level compared with the control group.The survival rate of the two families was higher than that of the control population in adult stage,and there were significant differences(P < 0.05)and extremely significant differences(P < 0.001)between family 1 and 4 and the control population.In addition,the measurement results of growth data showed that the growth rate of family 4 was significantly faster than that of control population(P < 0.01).2.Screening of differentially expressed genes: Transcriptome data showed3,428 differentially expressed genes were found between the selected disease-resistant families and the control population,including 1,874 up-regulated genes and 1,554down-regulated genes.Combined with the database analysis,it was found that the differentially expressed genes were mainly concentrated in phagocytosis,PUFA metabolism,Toll and Imd signaling pathways,ubiquitination,and CYP450-mediated metabolism of exogenous substances,all of which were part of the immune system or energy metabolism system of noble Scallop Chlamys nobilis.Several candidate genes,such as BAG3,CYP450,HSP70,IFI27,TNFSF10,LZ,E3 and Apo D,were screened and identified.3.Cloning and functional verification of BAG3 gene: Bcl-2 associated athanogene 3(BAG3)is an important member of BAG family and a highly conserved multifunctional protein.As a cochaperone of HSP70,it interacts with the ATpase domain of HSP70 through its Conserved BAG domain at the C-terminal.The cDNA length of CnBAG3 gene in noble scallop Chlamys nobilis is 4,667 bp,and its ORF length is 1,626 bp,encoding 541 amino acids.CnBAG3 gene express in all tissues of noble Scallop Chlamys nobilis,and the expression level in intestinal and gonad are significantly higher than those in other tissues(P < 0.01).Under the stress of Vibrio,BAG3 expression was significantly up-regulated,which promoted autophagy and improved survival rate.In conclusion,this study was the first to screen possible disease-resistant genes in noble scallop Chlamys nobilis by constructing disease-resistant families and control populations,and the functional analysis and validation of candidate genes with significant differentially expressed genes were conducted.This study enriched the genetic resources of noble scallop Chlamys nobilis,and the results could provide basic data for the breeding of resistant new varieties and immunity research.