Cloning And Analysis Expression Of Squaliobarbus Cunrriculus Mx1 CDNA And ScMx-Transgenic Grass Carp Test

Myxovirus resistance(Mx)gene is a factor of antiviral activity induced by interferon(IFN),which exists in the biological world.The antiviral effect of Mx protein is closely related to the typical structure.There are also many types of fish Mx which can play an antiviral role in the natural immune response of fish.In this study,one another Mx genotype was cloned from Squaliobarbus curriculus,called ScMx1 by using rapid-amplification of cDNA ends(RACE)technology.Expression characteristics of ScMx1 and ScIFN-I were investigated after GCRV infection by using Real-time PCR technology.Furthermore,transgenic grass carp study was carried out initially by using the recombination plasmid containing the code region of ScMx(ScMxORF-pTgf2-L-R)and Tgf2 efficient transposing system,The main results of this study are as follows:(1)cDNA cloning and sequence analysis of ScMx1 gene: The full-length cDNA of ScMx1 is 3 000 bp,including 124 bp 5?-UTR,983 bp 3?-UTR and 1 893 bp ORF,encoding 630 bp amino acid.The protein structure encoded by ScMx1 has the common characteristics of the vertebrate Mx protein,which contains a N-terminal Dynamin GTPase domain(including a triad GTP binding region and a power protein signal),a Dynamin central domain,and a C-terminal Dynamin GTPase effector domain(including Leucine zipper area).The result of percentage identities in amino acid sequence of ScMx1 with other species found that,ScMx1 have high identities with black carp bcMx1,grass carp CiMx1 and CiMx3,97%,95% and 97%,respectively.What is noteworthy is that ScMx have less identities with ScMx1,grass carp CiMx2,crucian Mx2 and other fishes,only 49%~50%.The result of phylogenetic tree showed that ScMx1 is closed to bcMx,CiMx3,CiMx1 and other cyprinidae,while ScMx is closed to CiMx2,CaMx and other cyprinidae.The evolutionary relationships between fish Mx and other reptiles,birds and mammalian Mx is consistent with branches.(2)The expression analysis of ScMx1: Expressions of ScMx1 had detected in all 10 healthy tissues(gill,liver,spleen,trunk kidney,head kidney,brain,skin,muscle,intestine and heart).Among them,the expression of ScMx1 was the highest in spleen,followed by heart,and low expressions in the intestine and liver.The expression characteristics of ScMx1 and ScIFN-I in the liver,spleen,trunk kidney and head kidney were investigated further after GCRV infection(6 h,12 h,24 h,72 h).The result showed that ScMx1 and ScIFN-I were significantly up-regulated in liver,spleen,trunk kidney and head kidney after GCRV infection,but ScMx and ScIFN-I had different expression characteristics in different tissues.In liver and trunk kidney,the expression of ScMx1 had been up-regulated to 168 h,but the expression of ScIFN-I was down-regulated after 72 h.In spleen and head kidney,both ScMx1 and ScIFN-I peaked at 72 h.(3)The preliminary result of ScMx gene transferred to grass carp: The recombinant plasmid ScMxORF-pTgf2-L-R and auxiliary plasmid pCS2 CMV-gfTP were injected into the fertilized eggs of grass carp in breeding season,and about 6 000 eggs.There were 206 transgenic grass carp cultured after six months,survival rate is 3.4%.In addition,PCR detection was conducted for all the surviving samples,and the positive rectification rate is 37.9%,which was finally screened for the positive group of transgenic grass carp.