Expression Profile Of Cuticular Protein Genes And Function Study Of Cuticular Protein BmorCPT1in Silkworm, Bombyx Mori

Insect cuticle is one of the most solid structures in the nature, which is an important protective organ for insects to adapt to complex external environments. Cuticular proteins, as well as chitin, are essential structural components for insect cuticle. During molting and metamorphosis controlled by ecdysone and juvenile hormones, the most dramatic changes occur in cuticle. The coding genes of cuticular proteins, which are the main component of cuticle, are definitely regulated by insect hormones. Therefore, the cuticular protein gene is an ideal target for insect hormone study. However, the hormonal regulation of cuticular protein genes is unclear until now. The lack of expression profiles of cuticular protein genes is the major obstacle to understanding this scientific issue. Cuticular protein genes have a large gene family revealed by multiple insect genome data. In the case of silkworm, it employs more than1%of genome to encode over200cuticular protein genes. Recent studies are focused on identification and activity analysis of transcription factors of them in response to hormonal signal. Little is known about the function of insect cuticular proteins.Insects exploit the innate immune system to resist the microbial infections. Pattern recognition proteins are used to distinguish the "self" with "non-self" and to activate the immune responses. Various pattern recognition proteins are needed for different microbes. Previous studies have published dozens of proteins for pattern recognition and microbial binding proteins, including peptidoglycan recognition proteins (PGRPs), glucan recognition proteins (GNBP/βGRP), LPS binding proteins and lectins. It is not yet identified for other proteins capable of binding to microbes and activating the immune response. In Drosophila, three proteins of PGRP-SA, PGRP-SD and GNBP1are necessary for the activation of TOLL pathway triggered by Gram positive bacteria, which indicates that a variety of molecular participations are needed for microbial recognition. This mode probably exists in other insect species.Bombyx mori is one of lepidopteran insects with most well-known genome including abundant genomic sequences, EST data and platform of microarray, which provide convenience for identification and analysis of expression profile of cuticular protein genes. Compared with Drosophila, weak basic immunological research and lack of mutants are the major obstacles for immune studies in Bombyx mori. However, dominant performance in Bombyx mori is its larger body size that provides convenience for collection of enough hemolmph and the tissues involved in innate immunity. Based on these works, we identified the cuticular protein genes by bioinformatic criteria and utilized microarray to perform their stage expression profile. Meanwhile, we carried out the bioinformatic analysis and expression profile of silkworm Tweedle genes. Therefore, we exploited the regulation and function of BmorCPTl gene with the combination of the methods of luciferase-reporter system, Northern blot, Western blot, chromatin immunoprecipitation, immunofluorescence, Co-immunoprecipitation, LC-MS/MS and piggyBac-mediated transgenesis. The main results are as follow:1. The expression profile of cuticular protein genes of Bombyx moriIn Bmobyx mori, based on the bioinformatic methods, two-hundred and fifty-five of cuticular protein genes are identified, including151CPR genes,5CPF/CPFL genes,4Tweedle genes,51CPG genes, and44CPH genes. Results of comparative analysis among11insect species showed that dipteran insects have more cuticular protein genes than others, and lepidopteran insects are at an intermediate level.We investigated the developmental expression profile of genes in epidermal tissues using the microarrays bearing23134probes. A total of6676genes were transcribed in at least one of11selected stages. From results of hierarchical clustering, eleven stages were divided into two groups, first group mainly including the food-eating stages and second mainly for molting stages. K-means clustering showed that6676genes possessed three expression patterns containing2450,1853and2373genes, respectively. Therefore, Gene Ontology (GO) annotation indicated that significant differences were observed in genes of the three clusters. It is suggested that cuticular genes exhibit distinct patterns between food-eating stages and molting stages. By comparison of expression ratios of IVM to IV4and W3to W2, ninety-four up-regulated and two down-regulated genes were identified.Among255cuticular protein genes, two-hundred and forty-four probed were designed for representing249cuticular protein genes. ESTs for6genes without probes were shown. Two-hundred and twenty-seven genes (93%) were detected in at least one stage. Northern blot experiments were employed to confirm the microarray expression profile. From the results of hierarchical clustering, sixteen developmental stages were divided into two groups in which the food-eating stages, larval molting stages and late pupal stages were included, respectively. Significant differences shown between these two groups suggested that cuticular protein genes were influenced by developmental fluctuations caused by insect hormones. K-means clustering yielded five gene clusters with distinct expression patterns, named as cluster A to E. In detail, genes in cluster A were widely expressed in all16stages. Genes in cluster B and C were mainly expressed at larval molting stages, late wandering stage and late pupal stages, and genes in cluster B possessed higher signal at pupal stages than cluster C. Cluster D was composed of the genes mainly expressed in pupal stages. Cluster E showed various expression patterns. Moreover, no correlation was found between the expression patterns of genes and the bearing motifs. Among the101cuticular protein genes in Cluster B and C, forty-nine CPR genes located in chromosome22. Furthermore, twenty-six CPR genes were co-expressed along the larval and wandering stages. Three common regulatory elements were identified at upstream region of these26CPR genes, suggesting the3elements might be involved in the regulation of these CPR genes.2. Bioformatic analyses and expression profile of Tweedle genes of Bmobyx moriComparative analysis among11species exhibited the different numbers of Tweedle gene of insect, ranging from3of Acyrthosiphon pisum to27of Drosophila melanogaster. Four Tweedle genes were identified in each lepidopteran insect. The Tweedle genes from Bmobyx mori were named as BmorCPT1-4. Analysis of eighty-one amino acid sequences form11species showed that although significant differences were found between different insects, certain sites in Tweedle motif were conserved within4BLOCKs:KxxY/F. YK/RxxxFKAP, KTxxYVL. KPEVY/FFxKY (x representing hydrophobic residues V,I, L). The phylogenetic analysis indicated Tweedle genes of lepidoptera are conserved. Fourteen Drosophila Tweedle genes were clustered separately from others, supporting the perspective of gene duplication. Comparative and phylogenetic analysis of25Tweedle amino acid sequences from6lepidopteran insects revealed that BmorCPT1homologs are significantly divergent from others. Also, the BmorCPTl homologs showed various gene structures, indicating the divergence of nucleotide level. In addition, a novel motif was identified at the C-terminal of BmorCPTl, which beared18amino acid residues. The next analyses revealed that this new motif was only found at the homologs of BmorCPT1of lepidopteran insects, named as LSM (Lepidoptera Specific Motif).The results of RT-PCR showed that BmorCPT1gene was highly expressed in epidermal tissues, testis, and head and weakly expressed in trachea and fatbody at day3of the fifth instar larva. BmorCPT2was only detected with weak signal in testis and head. BmorCPT3was not expressed in all ten tissues. BmorCPT4had weak signal in trachea, testis, malpighian tubule, and midgut. At embryonic stage, BmorCPT1expression reached a peak at96h after hatching. BmorCPT2and BmorCPT3were both expressed at96h and120h. Additionally, BmorCPT4was strongly transcribed at120h. During post-embryonic stages, BmorCPT1had high level along all the developmental stages except for weak signal at molting stages of the third and the fourth instar, day4to6of pupal stages. Besides, an obvious pattern occurred to BmorCPT1:it was highly expressed at early period of each larval instar and weakly expressed at each molting stage, especially for stages from the third to the fifth instar. In contrast to BmorCPT1, the peak of BmorCPT2-4genes appeared at larval molting stages, late wandering stage, and mid or late pupal stages. The expression of BmorCPT2-4genes was undetectable at other stages. The differences of expression profile suggested they might possess divergent functions.3. The regulation and function study of BmorCPTlIn this study, we cloned the2002bp (-1992to+10. translation initiation site as+1) upstream region of BmorCPT1gene, and identify a NF-κB factor binding site at-1442bp. We then constructed the transfection vector using pGL3-basic plasmid. The results of luciferase reporter assay and Western blot analysis revealed that the promoter of BmorCPTl was activated by LPS (lipopolysaccharide), and this effect of LPS was inhibited by PDTC. a specific inhibitor of NF-κB signal. Furthermore, the result of chromatin immunoprecipitation provided the direct evidence that NF-κB factor can bind to the NF-κB binding region lying upstream of BmorCPT1gene. Taken together, we proved that the activation of BmorCPT1by LPS was mediated by NF-κB factor. Meanwhile, the result of luciferase reporter assay and Western blot experiment indicated that the promoter of BmorCPT1was activated by juvenile hormone analog JHA (fenoxycarb) but not by the ecdysone analog EA (tebufenozide). In addition, JHA and EA suppressed the activation of BmorCPT1by LPS. The phosphorylation level of IKKa/β was analyzed by Western blot, which was nacessary for activation of NF-κB signaling, and the results showed that EA but not JHA caused the down-regulation of phosphorylation level of IKKa/β, suggesting that JHA and EA blocked the activation of BmorCPT1by LPS at different positions. All above results of luciferase reporter assay were testified with significant differences by T-test.The BmorCPT1ORF with removal of signal peptide coding region was cloned into the pET-28(a) vector. Then, the recombinant plasmid was transformed into BL21strain. IPTG was used to induce the expression of recombinant BmorCPT1protein. The inclusion body including recombinant protein was dissolved in8M urea and the target proteins were purified by histidine affinity column. After ultrafiltration, the purified fraction was examined by SDS-PAGE stained with silver nitrate. The result of silver staining showed that the recombinant BmorCPTl protein was purified with the predicted molecular weight. The recombinant proteins were used to inject the rabbit to make the antibody. The titer of BmorCPT1antibody was about1:32000.Western blot results revealed that BmorCPTl protein was highly expressed in epidermal tissues and head and mildly in trachea and testis. Furthermore, the expression level of BmorCPT1protein in epidermal tissue along developmental stages was analyzed. The results showed that the level of BmorCPT1was higher at day2to3and lower at molting stage of the fourth instar. After ecdysis to day1of the fifth instar, the BmorCPT1was sharply increased to the peak and gradually decreased from day2to6of the fifth instar. Besides, at day2and3of wandering stage, the expression of BmorCPTl was at high level and quickly decreased when entering the pupal stages, then increased from day2to6of pupal stages. At the epidermal tissues of day7to8of pupa and adult. BmorCPTl was undetectable. These results revealed that the weight of silkworm was increasing while the expression of BmorCPTl in epidermal tissues was declining, suggesting the negative correlation between the expression of BmorCPTl and the changes of silkworm larval body shape.The result of microbial induction experiment proved that BmorCPTl protein was detected only in the hemolmph of microbial-challenged silkworm. After injection of Beauveria bassiana and Bacillus bombysepticus, BmorCPT1was detected in hemolmph at3h,6h, and12h with weak expression. However, from6h to24h after injection of Escherichia coli, the expression of BmorCPTl was increasing and reached to the peak at24h. The result indicated that BmorCPT1gene is more sensitive to E.coli than to B. bassiana and B. bombysepticus. After infection by E.coli, Northern blot was used to analyze the expression changes of BmorCPT1gene. In epidermal tissues, no obvious changes were detected. However, a mild up-regulation was observed from fatbody. Obviously, only the infected hemacytes produced the BmorCPTl mRNA. Compared with the epidermal tissues, a shift band of BmorCPTl protein was detected from the hemolmph after challenged by E.coli, suggesting BmorCPT1from infected hemolmph might be modified. Western blot results showed that BmorCPT1was mildly increased in fatbody after E.coli infection. Surprisingly, the same shift BmorCPTl band was detected from infected hemacytes, suggesting that the hemacytes accounted for the post-translation modification of BmorCPTl. Based on previous references, we speculated that BmorCPTl identified from hemolmph might be glycosylated. The results from bioinformatic prediction revealed that no N-glycosylation sites were found, while18O-glycosylated sites were predicted, with the top score at Thr434.The hemolmph from E.coli challenged silkworm was used for bacterial binding and immunofluorescence experiments, which revealed that BmorCPT1in hemolmph strongly bound to E.coli. Then, the result was confirmed by Western blot analysis. In order to investigate the function of BmorCPT1, the Co-IP and LC-MS/MS were performed to identify the binding partners of BmorCPT1in hemolmph. Sixty-one proteins matched by429unique peptides were identified. The results suggested that BmorCPT1in hemolmph can bind to peptidoglycan recognition protein BmPGRP-S5and lipopolysaccharide binding protein BmLBP, which were involved in the recognition and binding of bacterial peptidoglycan and lipopolysaccharide, respectively. The results were confirmed by the subsequent co-immunoprecipitation and Western blot experiments.4. Transgenic study of BmorCPT1We constructed the piggyBac-based transgenic vector, piggyBac {3xP3-DsRed-pDmHSP70-BmorCPT1_ORF-SV40}, abbreviated as PBHT, in which the BmorCPTl gene was driven by DmHSP70promoter, and the DsRed gene driven by3xP3 promoter was set as screening marker. Thirty-two G1positive silkworms were obtained with28%positive rate. The PBHT silkworm genomic DNA was isolated. It was determined by reverse PCR that the transgenic vector was inserted at the position chr12:6278159-6278000.Through heat shock at42℃for90min, the silkworms were collected after4h and8h for Northern blot analyses. The results indicated that massive BmorCPT1mRNA was detected in transgenic silkworms (PBHT-AHS). Conversely, no BmorCPT1is detected from three control groups (non-heat shock transgenic PBHT-CON, heat shock non-transgenic silkworm WT-AHS and non-heat shock non-transgenic silkworm WT-CON). The results indicated that heat shock activated the expression of BmorCPTl driven by DmHSP70promoter. Meanwhile, we determined whether the hatching rate of silkworms were influenced by heat shock treatment. The hatching rates from four groups PBHT-AHS, PBHT-CON, WT-AHS and WT-CON showed no significant differences. It indicated that one time heat shock treatment had no effects on the development process and physiological status of Bombvx mori.Furthermore, Northern blot results revealed that the Bombyx mori peptidoglycan recognition protein BmPGRP-S5gene and lipopolysaccharide binding protein BmLBP gene from PBHT silkworms were up-regulated at8h after heat shock treatment, whereas the control groups were at background levels. Additionally, antimicrobial peptide genes BmGlv1and BmGlv4were activated in PBHT silkworm at8h, and the mRNA was not detected from control groups. These results indicated that without any microbial infections, the immune-related genes were activated by ectopic expression of BmorCPT1. Then, it was proved that the ectopic expression of BmorCPTl could activate the expression of immune-related genes in fatbody. including BmPGRP-S5and BmLBP. IMD pathway genes Bmlmd, BmDredd and BmRelish, as well as antimicrobial peptide genes BmGlv1and BmGlv4.Considering the results of bacterial binding experiments and Co-IP, we proposed that the modified BmorCPTl in hemolmph bound to BmPGRP-S5and BmLBP to recognize and bind to E.coli. via IMD pathway, to activate the expression of BmGlv1and BmGlv4. Taken together. Bombyx mori might employ post-translational modification to transform the Tweedle motif containing protein BmorCPTl to one of pattern recognition receptor-like factors.