Cloning And Functional Analysis Of Cuticular Protein Genes From Apis Cerana Cerana

Insect molting and metamorphosis is one of the hot topics in the field of developmentalbiology. The insect cuticle is mainly composed of cuticular protein and chitin. The structure ofchitin is clear, which is polymerized of N-acetyl glucosamine, while the structure andcharacteristics of cuticular protein is various. The nature of the insect cuticle is mainlydetermined by the nature of the cuticular protein. Cuticular protein and cuticular protein gene,which are considered to be an important model for the study of regulation mechanism ofinsect molting and metamorphosis, have received more and more attention. So far,researches about cuticular protein gene are mainly focus on the model speices, such as D.melanogaster and Bombyx mori, and information in honeybee is relatively limited. TheChinese honeybees which are the excellent indigenous bee species are of great significance tothe maintenance of biological diversity of plant resources in China, and to promote thesustainable development of agriculture. The research of cuticular protein family genes mayprovide scientific basis of elucidating the developmental mechanisms of Chinese honeybeesand protecting the indigenous excellent germplasm resource.In this research, we selected the Chinese honeybee as the experiment material, and twogenes, AccCPR1, AccCPR2, have been cloned by RT-PCR and RACE-PCR. Then, a seriesof research have been managed on the isolation, sequence and expression profile analysis, andfunctional identification of the two genes. The main results are as follows:(1) The first cuticle protein R&R gene named AccCPR1was identified from A. ceranacerana. The full-length cDNA (GenBank accession number: JQ282798) has a length of822nt,which contains an open reading frame (ORF) of573nt encoding a190amino acidpolypeptide with a predicted molecular mass of21.3kDa and an isoelectric point of6.50. Theprotein contains a chitin binding region and is a typical cuticle R&R-2protein. The analysisof the secondary structure indicated thatβ-sheets occurred frequently (42.5%) throughout thestructure of the region, whereas there were none in other regions. The phylogenetic treeindicated that AccCPR1has a greater level of sequence identity with Hymenoptera CPRs thanwith Diptera or Hemiptera CPRs. The strict R&R consensus: G-x8-G-x6-(Y/F) was found inthis region. But AccCPR1and its orthologs are exception for the most conservative sequencemotif. Five putative E74binding sites and four BR-C binding sites were predicted in the5’-flanking region, which suggests a potential function in molting and metamorphosis.RT-qPCR showed that AccCPR1transcript occurred as the ecdysteroid titer decreased after reaching a peak, which suggests AccCPR1expression requires a “pulse” regimen ofecdysteroids. This hypothesis was tested using different experimental strategies.20E is amajor component of ecdysone. When larvae were reared with different concentrations of20Ein their diet, the ecdysteroid peak repressed AccCPR1expression. Exposure of the thoracicintegument of the pupae in vitro to different concentration of20E repressed AccCPR1expression, which recovered after the removal of20E. Tissue-specific expression andimmunohistochemical localization showed AccCPR1expressed in the epidermis and in themidgut. These results suggest that AccCPR1is a typical cuticle R&R-2protein that plays animportant role in development, and an ecdysteroid pulse is critical for high AccCPR1geneexpression.(2) We cloned and characterized another cuticle protein R&R gene, referred to AccCPR2,from the honeybee (A. cerana cerana). The full-length cDNA (GenBank accession number:KJ502287) has a length of1134nt, which contains an open reading frame (ORF) of705ntencoding a234amino acid polypeptide with a predicted molecular mass of26.3kDa and anisoelectric point of6.90. The protein contains a chitin binding region and the strict R&Rconsensus: G-x8-G-x6-(Y/F) was found in this region. The first intron interrupts the signalpeptide after five coding amino acids in the AccCPR2genomic sequence, which is a typicalcharacteristic of CPR genomic sequences in insects. Three BR-C, five CdxA, one HB andthree DFD binding sites were predicted in the5’-flanking region, suggesting a potentialfunction in molting and metamorphosis.Bioinformatics analysis indicated that AccCPR2contains a chitin binding region and is atypical cuticle R&R-2protein, while its expression pattern has a relatively large differenceswith "pulse" mechanismin that have been found in some insects. Expression profile of thedifferent developmental stages showed that the expression level of AccCPR2increased withthe increase of ecdysone concentration during the pupal stage, indicating that AccCPR2transcription needs ecdysone persist. Exposure of the integument of the pharte pupae in vitroto different concentration of20E, the expression of AccCPR2was identified with the increaseof20E concentration. Environmental stressors, such as heavy metals and pesticides, alsoinfluenced gene expression. In addition, a disc diffusion assay showed that AccCPR2enhanced the ability of bacterial cells to resist multiple stresses. We infer from our results thatAccCPR2acts in honeybee development and in protecting these insects from abiotic stresses.The results of this study found that, AccCPR1and AccCPR2belong to R&R-2typecuticle protein genes. There are structural differences of the most conserved motifs betweenthe two polypeptide chains and the two genes are regulated by ecdysone titer with different expression patterns, suggesting different cuticle protein genes in Apis cerana cerana play avariety of roles in metamorphosis mechanisms.