A Preliminary Study On The Molecular Mechanism Of Integrin ?1 Regulating Carps Against CyHV-3 Infection

Carp herpesvirus 3?Cy HV-3?is a viral disease that spreads rapidly and can cause up to 90%mortality.It is a common and fatal disease among carp?Cyprinus carpio?and koi?Cyprinus carpio?Pathogens,causing immeasurable serious economic losses to the carp breeding industry.Therefore,it is imminent to study the genetic regulation mechanism of fish disease resistance from the perspective of genetic genes,and to carry out disease resistance breeding.At present,research has been carried out on the breeding of mirror carp anti-herpes virus?Cy HV-3?,and F₄has been bred,and its disease-resistant traits have reached stability.In this study,the integrin gene was combined with the mirror carp anti-herpes virus?Cy HV-3?and its related genes?TK,ORF72?.The mirror carp anti-herpes virus?Cy HV-3?F4 was used as the experimental material.Real-time quantitative PCR was used.Technology and RACE cloning kit technology to analyze the expression levels of F4 generation virus of mirror carp anti-herpes virus?Cy HV-3?,clone the full length of carp integrin?1 gene,expression of integrin?1 gene in various tissues of carp,integrin?1 gene The expression of Cy HV-3in carp virus,further study the molecular mechanism of integrin?1 gene regulation of carp resistance to Cy HV-3 infection.The findings are as follows:Cy HV-3 virus expression comparison:In this study,F₄was used as the experimental material,and the virus TK and ORF72 genes were used as virus marker genes to analyze the mirror carp anti-herpes virus?Cy HV-3?for breeding resistant strain F₄The expression level in different stages of viral infection,so as to explore the effect of F₄resistance breeding and virus expression pattern from the perspective of virus expression.?1?The expression of spleen and kidney tissues of the two genes at different stages of infection are consistent,showing an upward trend from 24 to 144 hours and a downward trend from 144 to 288 hours.The spleen of the breeding group was significantly different from other periods at 144h?P<0.05?;the spleen,kidney and kidney of the non-selected group were significantly different from other periods at 144h?P<0.01??2?The expression levels of each gene was lower than that of kidney tissue.The TK and ORF72 in the breeding group and the non-breeding group were significantly different between 96 and 144 hours,and TK and ORF72 in the breeding group were significantly different at 96 hours and the ORF72 gene at 144 hours?P<0.05?,and the other periods were significantly different?P<0.01?..?3?The expression levels of the TK and ORF72 in the breeding group were lower than those in the non-breeding group.The TK and ORF72 in the spleen tissue were significantly different between 96 and 168 h?P<0.01?,and the ORF72 gene was also significantly different in192 h and 216 h?P<0.01?;the TK and ORF72 in the kidney tissue were significantly different between 96 h and 168 h?P<0.01?.;ORF72 gene was also significantly different at 192h?P<0.01?.The above results show that the disease resistance of the breeding group is stronger than that of the non-breeding group,in which the kidney tissue has better disease resistance than the spleen,and the immune resistance of the selected kidney tissue is greatly improved.Cloning of carp integrin?1 gene:The sequencing results of this study showed that the full-length c DNA of integrin?1 in common carp was 3835bp.Using the DNAstar?Editseq?software,we suspected integrin?1 contained a 5'-UTR of 223bp,3'-UTR of1236bp with the putative polyadenylation consensus signal?AATAAA?,an open reading frame?ORF?of 2394bp.The ORF was encoding polypeptides of 796 amino acids with a predicted molecular mass of 88.42k Da and theoretical PI of 6.018?Figure 1A?.The integrin?1 protein included 92 strongly basic?+?amino acids?K,R?,104 strongly acids?-?amino acids?D,E?,219 hydrophobic amino acids?A,I,L,F,W,V?,257 polar amino acids?N,C,Q,S,T,Y?.Carp integrin?1 is highly conserved in evolution and forms clusters with zebrafish integrin?1?.Expression of integrin?1 gene in various tissues of carp:This experiment analyzed the expression of integrin?1 gene in ten tissues:intestine,gill,kidney,liver,heart,eye,brain,spleen,testes and ovary.The results showed that the integrin?1 gene was widespread in ten carp tissues,and the relative expression of the gene was higher in carp intestine and gill tissues.In contrast,the expression levels of integrin?1 in the testis and ovary is relatively low,and it is significantly different from the five tissues of liver,heart,eye,brain,and spleen?P<0.001?.There were also significant differences in expression levels between the two tissues?P<0.001?.Expression of integrin?1 in Cy HV-3 virus of carp:In this experiment,the gene expression of integrin?1 gene in spleen and kidney tissues was analyzed by fluorescent quantitative PCR.The results showed that the relative expression of the integrin?1 gene was highest in the early 48 hours of infection,and reached a very significant difference compared with the control group?P<0.01?.In addition,it was found that the relative expression of the integrin?1 gene gradually decreased with the extension of the infection time.In addition,in the early stage of Cy HV-3 infection,the expression of integrin?1 gene in kidney tissue increased significantly,and gradually decreased with the extension of infection time,but its effect in Cy HV-3 carps needs further study.At present,the mapping of the whole genome sequence of carp has been completed,but the function of carp integrin?1 has not been fully elucidated.Therefore,the full-length sequence of the carp integrin?1 gene was cloned and the structural characteristics of the carp integrin?1 gene were predicted and analyzed.The high expression of integrin?1 in the intestines and gills and kidneys of carps suggests that it may play an important role in the immune response of carps.In addition,it was determined that the integrin?1 gene was differentially expressed in the spleen and kidney tissues of the carp during Cy HV-3 infection,and it was confirmed that the integrin?1 gene expression was significantly increased in the spleen and kidney tissues of the carp before the Cy HV-3 virus infection.Research on the molecular mechanism and disease resistance mechanism of carp integrin?1 provided data support.