Silencing of Chitin-Binding Protein with PYPV-Rich Domain Impairs Cuticle and Wing Development in the Asian Citrus Psyllid, Diaphorina citri.
Silencing of Chitin-Binding Protein with PYPV-Rich Domain Impairs Cuticle and Wing Development in the Asian Citrus Psyllid, Diaphorina citri.
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蜕皮对昆虫的生长发育至关重要，它伴随着旧角质层的降解和新角质层的合成。甲壳素和蛋白质是昆虫表皮的主要组成部分，维持着昆虫外骨骼的刚性。几丁质结合蛋白的功能尚未在柑桔木虱中得到鉴定。在本研究中，我们确定了角质层蛋白（DcCP 64）根据甲壳素柱纯化和LC-MS/MS分析。DcCP 64的沉默诱导了异常表型，并增加了腹部和翅膀的通透性。此外，DcCP 64抑制后，死亡率和畸形率显著增加，蜕皮率下降。转录组测序分析显示，DEGs表达上调主要与氧化磷酸化有关，而表达下调主要与MAPK和FoxO信号通路有关。这些结果为进一步研究DcCP 64在D.柑橘。甲壳素是节肢动物外骨骼的主要组成部分，它与甲壳素结合蛋白共同维持外骨骼的功能。在本研究中，我们确定了角质层蛋白64从柑桔木虱几丁质结合试验。生物信息学分析表明，DcCP 64含有8个保守的PYPV基序，但缺少Rebers-Riddiford（R-R）共有结构域和其他几丁质结合结构域。RT-qPCR分析表明，DcCP 64在翅期和5龄若虫中的表达量最高。通过RNA干扰（RNAi）敲低DcCP 64导致翅畸形表型、更高的死亡率和降低的蜕皮率。转录组学分析显示，与dsDcCP 64组和dsGFP组相比，1244个差异表达基因（DEG）表达上调，580个DEG表达下调。KEGG富集分析显示，DEG的上调主要与氧化磷酸化有关，而DEG的下调主要与MAPK和FoxO信号通路有关。此外，抑制DcCP 64显著影响角质层表面，并增加腹部和翅膀的渗透性。进一步的几丁质和纤维素结合试验证实了重组DcCP 64在体外的几丁质结合特性。这些结果表明，DcCP 64可能在黑腹果蝇的角质层和翅发育中起重要作用。柑橘。
Molting is extremely important for insect growth and development, which is accompanied the degradation of old cuticle and synthesis of new cuticle. Chitin and proteins, as major components of insect cuticle, maintain the rigidity of the exoskeleton. The functions of chitin-binding proteins have not, to date, been characterized in Diaphorina citri. In the current study, we identified a cuticle protein (DcCP64) according to chitin column purification and LC-MS/MS analysis. Silencing of DcCP64 induced an abnormal phenotype and increased the permeability of the abdomen and wings. Additionally, the mortality and malformation rate significantly increased, and the molting rate decreased after inhibition of DcCP64. Transcriptome sequencing analysis revealed that up-regulated DEGs were mainly related to oxidative phosphorylation, whereas down-regulated DEGs were mainly involved in MAPK and FoxO signaling pathways. Our results provide a basis for further functional research on DcCP64 in D. citri. Chitin is a major component of the arthropod exoskeleton, always working together with chitin-binding proteins to maintain the functions of extracellular structures. In the present study, we identified a cuticle protein 64 from Diaphorina citri using a chitin-binding assay. Bioinformatics analysis revealed that DcCP64 contained eight conserved PYPV motifs but lacked a Rebers–Riddiford (R–R) consensus and other chitin-binding domains. RT-qPCR analysis suggested that DcCP64 had the highest expression level in the wing and fifth-instar nymph stage. Knockdown of DcCP64 by RNA interference (RNAi) resulted in a malformed-wing phenotype, higher mortality and decreased molting rate. Furthermore, transcriptomics analysis revealed that 1244 differentially expressed genes (DEGs) were up-regulated and 580 DEGs were down-regulated, compared with dsDcCP64 groups and dsGFP groups. KEGG enrichment analysis revealed that up-regulated DEGs were mainly related to oxidative phosphorylation, whereas down-regulated DEGs were mainly involved in the MAPK and FoxO signaling pathways. Moreover, inhibition of DcCP64 significantly affected the cuticle surface, and increased the permeability of the abdomen and wings. Further chitin- and cellulose-binding assay confirmed the chitin-binding properties of recombinant DcCP64 in vitro. These results indicate that DcCP64 might play an important role in the cuticle and wing development of D. citri.
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