Molecular Cloning,Characterization And Expression Analysis Of Cyclic GMP-AMP Synthase (cGAS) In Pelodiscus Sinensis

Pelodiscus sinensis is the traditional freshwater aquaculture varieties raised in China.In recent 20 years,the aquaculture industry has developed rapidly and the scale of aquaculture has gradually expanded.However,with the impact of intensive aquaculture development,its various diseases occur frequently in recent years,which seriously restricts the healthy development of the aquaculture industry.At present,the diseases of P.sinensis are mainly controlled by chemical medicine,and the irregular use of the chemical medicine in the breeding process will lead to greater food safety and environmental safety problems.Immune control is an environmental and safe means of prevention and control of animal diseases,and has gradually become a research hotspot of disease prevention and control of aquatic animals.However,there are few research materials about immune control of the turtle diseases at present,which limits the application of immune control technology in its aquaculture.The research on immune related genes of P.sinensis will help us to understand its immune mechanism against pathogens,then prevent diseases and ensure the healthy and sustainable development of the breeding industry.Cyclic GMP-AMP(cGAMP)synthase(cGAS)has the function of DNA receptor,which can recognize the DNA from viruses and bacteria.It can promote the production of cGAMP,and activate stimulator of IFN genes(STING),and subsequently promote the secretion of type Ⅰ interferon(IFN-I)and other cytokines,and regulate the immune function of vertebrates.1.The full-length cGAS gene(1716 bp)was cloned by RT-PCR,which encoding 519 amino acids incluing a Mab-21 conserved domain.The predicted molecular mass of the cGAS protein was 57.09 kDa,with an estimated PI of 9.73.This protein had no transmembrane region and was an intracellular protein.It had the closest relationship with reptiles and the furthest relationship with birds.We constructed a prokaryotic expression vector of cGAS gene of P.sinensis,which expressed protein weights 55 kDa.We also immunized New Zealand white rabbits to prepare polyclonal antibodies.2.The qRT-PCR results showed that the cGAS gene was expressed in spleen,intestine,kidney,muscle,liver,stomach,skin,lung,blood cells and heart of P.sinensis.Its expression level in different tissues of P.sinensis from high to low was:spleen,intestine,kidney,muscle,liver,stomach,skin,lung,blood cells and heart.Post the stimulation of Aeromonas hydrophila,Poly(I:C)and LPS,the expression of cGAS increased first and then decreased.However,its expression level was different in different tissues post different stimulation.3.Western Blot test found that the cGAS protein increased significantly in the spleen and the intestine tissues post stimulation by A.hydrophila.Its expression level reached the highest value in the spleen at 48 h and the highest value in the intestine at 6 h.Post Poly(I:C)stimulation,the expression level of cGAS reached the highest value in both the spleen and the intestine at 24 h.Post LPS stimulation,its expression level was highest in the intestine at 12 h.The results showed that cGAS protein in P.sinensis has strong immune response to A.hydrophila,Poly(I:C)and LPS.4.Immunofluorescence results showed that the expression of cGAS protein in the intestine of P.sinensis significantly increased after 24 h of stimulation by A.hydrophila.