关键词: 暗黑鳃金龟, 几丁质, HpUAP, 原核表达, 组织表达谱, RNAi

Abstract:  【Aim】 The aim of this study is to elucidate the sequence characteristics and function of UDP-N-acetylglucosamine pyrophosphorylase (UAP) gene HpUAP in the dark black chafer, Holotrichia parallela. 【Methods】 The full-length cDNA sequence of HpUAP was amplified from the 2nd instar larvae of H. parallela by PCR and analyzed with bioinformatics. The recombinant expression vector pET30a-HpUAP was constructed and transformed into Escherichia coli BL21(DE3) to express the protein after IPTG induction. The expression levels of HpUAP in different larval stages (the 1st-3rd instar larva) and tissues of the day-2 3rd instar larvae (integument, midgut, ileum, rectum, Malpighian tubules, and fat body) of H. parallela were analyzed by qRT-PCR. After the HpUAP gene in the 2nd instar larvae of H. parallela was silenced by RNAi, their growth, development and survival were observed, and the expression level of HpUAP and the chitin content in their integument were determined at 72 h after RNAi. 【Results】 The full-length cDNA sequence of HpUAP (GenBank accession no.: MW676788) of H. parallela was obtained by PCR amplification. Its open reading frame is 1 461 bp in length, encoding 486 amino acid residues with the molecular weight of about 53.9 kD. The result of phylogenetic analysis showed that the amino acid sequences of HpUAP and Onthophagus taurinus UAP were clustered into one clade with high confidence. The HpUAP protein of 53.9 kD induced by IPTG was consistent with the expected size. Developmental expression profile revealed that HpUAP was highly expressed in the day-1 1st and 3rd instar larvae, and tissue expression profile showed that HpUAP was highly expressed in the midgut and integument of the day-2 3rd instar larvae. RNAi of HpUAP resulted in the slow growth and movement of the 2nd instar larvae of H. parallela, and their body surface became deeper and shriveled. At 72 h after RNAi, the expression level of HpUAP in the dsHpUAP injection group was decreased by 93.06%, the mortality rate was increased by ~40%, and the chitin content in the integument was decreased by 29% as compared with those of the control group (dsGFP injection group). 【Conclusion】 The results suggest that HpUAP is involved in chitin metabolism and plays a key role in the growth and development of H. parallela larvae.

Key words: Holotrichia parallela, chitin; HpUAP, prokaryotic expression, tissue expression profile, RNAi