Cloning And Functional Study Of The Lymphogenic Tal Of Crassostrea Hongkongensis

Crassostrea hongkongensis belongs to the invertebrate mollusk,which lacks the adaptive immune system,and mainly relies on the innate immune system to resist the infection of external pathogens.The haemolymph of phagocytosis plays a central role in the process of clearing agent,this research through the RACE,q RT-PCR,subcellular localization,prokaryotic expression,determination of blood counts and Edu proliferation,oyster lymphatic factor Tal in C.hongkongensis to analyze the structure and function,discusses their role in the process of oyster immune defense in C.hongkongensis.The main results of the research are as follows: 1.Cloning and bioinformatics analysis of the Tal gene of C.hongkongensis.Tal(T-cell acute lymphocytic leukemia)is an important transcription factor involved in hematopoietic and embryonic angiogenesis,which is essential for biological hematopoietic function.In this study,RACE technique was used to clone the full-length c DNA sequence of the Tal gene of C.hongkongensis,named Ch Tal.The total length of this gene is 812 bp,including 5 ’non-coding region 17 bp,3’ non-coding region 135 bp,open reading frame(ORF)660 bp,encoding 219 amino acids,with the relative molecular weight is 25.11 ku,Theoretical isoelectric point 5.80.Bioinformatics analysis showed that C.hongkongensis Tal is classic b HLH domain structure,Tal proteins for hydrophobic proteins,and is a transmembrane protein,there are 31 serine phosphorylation sites,13 threonine phosphorylation sites,eight tyrosine phosphorylation sites,without the glycosylation sites,secondary structure display,according to the Helix-screw accounted for 27.8 %,Strand-fold accounted for 4 %,Coil random curl accounted for 67.6 %.Multiple sequence alignment and phylogenetic tree results showed that the Ch Tal gene has high homology with C.gigas,C.virginica and Aplysia californica,which proves that it is a member of the Tal family of mollusks.2.Distribution of Tal tissues and expression patterns under bacterial infection in C.hongkongensis.The results of fluorescence quantitative PCR showed that Ch Tal was the most expressed in haemolytic lymphocytes of oyster in C.hongkongensis,followed by heart,gill,digestive gland,adductor muscle,mantle,gonad,suggesting its function in lymphogenesis.After the stimulation of Vibrio alginolyticus and Staphylococcus haemolyticus,the expression levels of Ch Tal were significantly up-regulated,reaching the highest levels at 24 h(P < 0.01)and 12 h(P < 0.01)at 12 h and 6 h,respectively,and then gradually decreased.3.Localization and function of Tal subcells in lymphocyte regeneration of C.hongkongensis.The results of subcellular localization showed that Ch Tal was expressed in the nucleus.In vivo injection of recombinant Ch Tal can significantly increase the number of hemocytes.Edu proliferation assay have further confirmed that Ch Tal can promote the regeneration of hemocytes in C.hongkongensis.In summary,this study revealed that Ch Tal was involved in hemocytes regeneration.These results enriched the current cognition and identification of Tal of C.hongkongensis at the cellular and molecular levels,and provided a good reference for the study of the occurrence,development,immune function and regulatory mechanism of hemolytic cells,providing a basis for further research on the function of th is gene in immune response of oysters.