The Cloning And Immune Function Analysis Of Akirin In Antheraea Pernyi

Antheraea pernyi is an important economic insect.Studying its innate immunity has important reference value for finding ways to improve the disease resistance of Antheraea pernyi.As a nuclear factor,Akirin can participate in the immune response,and its research in Antheraea pernyi has not been reported yet.This paper mainly the expression and analysis of Akirin gene in Antheraea pernyi,and studies the effect of Akirin on the immune response of Antheraea pernyi.At the same time,the expression and analysis of Akirin interaction protein 14-3-3 clone,in vitro interaction verification,and 14-3-3 in the immune response of Antheraea pernyi.The results are as follows:1.The Akirin and 14-3-3 genes of Antheraea pernyi were cloned.The ORFs of Akirin and 14-3-3 were 588 bp and 744 bp,respectively,encoding 195 and 247 amino acids.Sequence analysis showed that there were nuclear localization signals(KRRRC)and 14-3-3 protein sites(SPP)in Akirin protein;Akirin of Antheraea pernyi had higher similarity with Akirin of Helicoverpa armigera;14-3-3 of Antheraea pernyi and Bombyx mori of 14-3-3 has a high degree of similarity.Simultaneously,By constructing a recombinant prokaryotic expression vector,the corresponding recombinant protein was successfully induced and purified and polyclonal rabbit anti-preparation was carried out.2.Far-Western blot showed that the recombinant Akirin protein and 14-3-3 protein can interact in vitro.3.Tissue distribution showed that Akirin and 14-3-3 were expressed in the fat body,epidermis,midgut,silk gland,hemocyte and malpighian tube of the third day of the fifth instar larvae,among which Akirin was highly expressed in the midgut,14-3-3 is highly expressed in fat bodies.4.Escherichia coli,Micrococcus luteus,Beauveria bassiana,and Antheraea pernyi nuclear polyhedrosis virus(NPV)were used to stimulate the third day of the fifth instar larvae.The results showed that the expression of Akirin was up-regulated after stimulation by different pathogenic microorganisms.The expression of 14-3-3 was down-regulated after stimulation by different pathogenic microorganisms.5.After Akirin was knocked down in the Antheraea pernyi pupa,the expression levels of four antibacterial peptides(Attacin-1,Cecropin B,Gloverin,Lebocin-2)and Relish and Dorsal showed a downward trend,indicating that Akirin was involved in the positive regulation of Antheraea pernyi immunity.After 14-3-3 was knocked down,the expression levels of the four antimicrobial peptides were up-regulated,indicating that 14-3-3 was involved in the negative regulation of Antheraea pernyi immune.