Cloning And Expression Of Grass Carp GPATCH3 Gene And Its Role In Innate Immunity

G-patch domain widely exists in proteins related to RNA binding,with about 48 amino acid residues,mainly characterized by glycine(Gly)rich.These proteins containing g-patch domain are widely expressed in different tissues,and are involved in innate immunity,post-transcriptional processing of mRNA,development,tumor proliferation and other life activities.GPATCH3 is a protein widely expressed in various tissues in the human body.In recent years,studies have shown that human GPATCH3 protein plays a negative role in regulating the RLR signaling pathway.In this pathway,RIG-I and MDA5 recognize viral RNA and stimulate a series of downstream signal transduction,so that IRF3 is phosphorylated and incorporated into the nucleus,and finally induce the massive expression of type I interferon(IFN-I).Afterwards,IFN,as a signal molecule,can induce the expression of several ISGs and play an antiviral role.In the study of fish,reports related to GPATCH3 are still rare and mostly exist in the genome prediction results.In the study of zebra fish,it is found that individuals with GPATCH3 patch3 may develop cranial deformity and eye deformity.The function of fish GPATCH3 in innate immunity has not been reported.In this experiment,grass carp was used as the material,and with the help of prediction software,the mRNA sequence of CiGPATCH3 was successfully cloned and obtained in the cDNA library established with the spleen as the template.The mRNA length of the gene was 1646 nt,in which the CDS region was 1221 nt,encoding 407 amino acids.The 5 ’UTR contained 303 nt,and the 3’ UTR had 122 nt.Compared with the g-patch domain of many species,grass carp GPATCH3 has a sequence that is very consistent with the g-patch domain,with a total of 48 amino acid residues,located at 299-347,containing 8 Gly conserved amino acid sites and 2 low-complexity segments at the n-terminal.Compared with g-patch-containing protein structure of other species,other sequences are different except conservative sites.The result of phylogenetic tree analysis shows that among most bony fish,GPATCH3 is closely related to carp and crucian carp.Then,qRT-PCR method was used to analyze the tissue template.It was found that grass carp GPATCH3 is expressed in brain,eye,heart,gill,intestine,liver,spleen and kidney.The expression in liver and brain was higher.After poly(I:C)stimulation,the gene was significantly upregulated in eye,intestine,liver and kidney tissues at 12 h and 48 h,respectively.The expression of brain,heart and gill tissue decreased first and then increased.Compared with other tissues,the mRNA level of GPATCH3 in the spleen tissue shows no significant change.The same method was used to analyze the CIK cell template stimulated by poly(I:C)gradient time.The expression of GPATCH3 is basically consistent with the tissue quantitative analysis.RNA viruses were simulated to invade cells by transfection with poly(I:C),and GPATCH3 was overexpressed in CIK cells.After 24 h,GPATCH3 was transfected into poly(I:C),and total RNA was extracted for detection after 12 h.Quantitation indicated that compared with cells transfected with poly(I:C)only,mRNA levels of IFN and ISG15 in cells with overexpression of GPATCH3 were significantly higher.However,the mRNA levels of IFN and ISG15 in the two experimental groups that incubated poly(I:C)showed only a similar upregulation no matter whether GPATCH3 was overexpressed or not.Results of cell activity experiment are consistent with qtr-pcr results.Overexpression of GPATCH3 can significantly enhance the activity of cells under endogenous poly(I:C)stress.It is believed that GPATCH3 may be involved in the RLR signaling pathway.IRF3 was selected as the key protein to observe the changes of its karyoplasmic distribution.GPATCH3 was overexpressed in CIK cells and transfected into poly(I:C)24h later.Different time gradients were used as checkpoints.The results show that the amount of IRF3 protein in the nucleus of the test group with overexpression of GPATCH3 is relatively large.Further experiments showed that overexpression of GPATCH3 could activate IRF3 into the nucleus earlier after being stimulated by poly(I:C).