Isolation And Characterization Of Cuticle Protein Genes And Their Differential Functional Identification In Beet Armyworm Spodoptera Exigua

Insect cuticle is a multi-layered structure with three functional regions,the epicuticle,exocuticle,and endocuticle,which differ in protein composition,physiological structural and functions.The class and amount of cuticular proteins determine the structure of the cuticle.Spodoptera exigua(Hiibner),is a polyphagous pest spread throughout the world.It is extensively distributed in a number of economic crops,including cotton,celery,cabbage,tomato,lettuce,and alfalfa.The beet armyworm shows resistance to many important synthetic insecticides,such as chlorinated hydrocarbons,organophosphates,carbamates,pyrethroids,and benzoyl phenyl ureas.Exploring new target for pesticide is an important way to control the beet armyworm.In this study,three cuticular protein genes CPG316,CPG860 and CPG4855 have been cloned from Oh pupal integument of S.exigua through RACE PCR strategy.CPG316 has 722 bp in total with a 585 bp open reading frame(ORF)encoding 194 amino acids.CPG860 has 710 bp with a 585 bp ORF region encoding 194 amino acids.CPG4855 has 863 bp with a 630 bp ORF encoding 209 amino acids.The amino acid sequences were blasted to find similar sequences,which were used to construct phylogenetic trees for each.The deduced amino acid sequences of CPG316,CPG860 and CPG4855contain the RR-2 consensus region,which has the conserved domains among other insect cuticular proteins belonged to the CPR family.Using quantitative RT-PCR,the developmental expression of these three cuticular protein genes through 3rd instar larvae to 24h pupa were studied.CPG316 was expressed at low levels in the 3rd instar larvae,at higher levels through the 4th and 5th larval instars and the early pupa then fell to low levels in the 24 h pupa.Both CPG860 and CPG4855 were highly expressed in the 3rd instar larvae,then expression decreased to low levels in 5th instar larvae and early pupae to very low levels in 24 h pupa.CPG316 is 10 fold less then both CPG860 and CPG4855 at third instar stage.Based on the structural composition,all the three genes may contribute to the endocuticle although CPG316 may have a different role from the other two genes.Furthermore,the functions of these three genes were determined by RNAi through direct injection of the dsRNA into early 5th instar larvae.Functional analysis results show that it effects on the cuticle in both larval and pupal stages,and necessary for cuticle development,flexibility,and metamorphosis.Further studies are necessary to determine whether these particular cuticular proteins have any role in tissues other than the integument.