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溶酶体β-N-乙酰己糖胺酶(EC 3.2.1.52)催化水解糖复合物,释放单分子乙酰己糖胺,在昆虫生长发育过程中发挥重要作用。OfHEX2基因(GenBank登陆号为EF469203)是已从亚洲玉米螟Ostrinia furnacalis(Guenée)5龄幼虫体内获得的基因,其编码产物是β-N-乙酰己糖胺酶OfHex2。为考察OfHex2能否成为农药设计的分子靶标,研究了RNA干扰沉默OfHEX2基因对亚洲玉米螟生长发育的影响,同时探索了小分子抑制剂2-乙酰氨基-1,2-二脱氧野尻霉素(2-acetamido-1,2-dideoxynojirimycin,2-ADN)和咪唑对OfHex2的体外抑制效果。结果表明:向5龄第1天的亚洲玉米螟幼虫注射dsOfHEX2,能够显著干扰OfHEX2基因在幼虫期的正常表达,但不能干扰其在蛹期的表达;RNA干扰导致OfHEX2基因在亚洲玉米螟5龄第3天幼虫中的表达水平仅为对照组的7.1%,并导致预蛹的腹部无法正常收缩,蛹和成虫的附件发育畸形,其中15%畸形蛹因发育不正常而死亡。研究结果表明,OfHex2在昆虫发育中具有重要的生理意义,其功能缺失具有致畸和间接致死效果,因此可能成为新型农药设计的潜在靶标。抑制作用研究结果表明,小分子2-ADN是OfHex2的竞争性抑制剂,酶活力抑制常数Ki值为0.94±0.035μmol/L。
Lysosomal β-N-acetylhexosaminidase (EC 3.2.1.52) catalyzes the hydrolysis of sugar complexes to release the monohexylhexosamine, which plays an important role in insect growth and development. The OfHEX2 gene (GenBank accession number EF469203) is a gene that has been obtained from the 5th instar larva of Ostrinia furnacalis (Guenée) and its coding product is β-N-acetylhexosaminidase OfHex2. In order to investigate whether OfHex2 can be used as a molecular target for pesticide design, the effect of OfHEX2 gene silencing RNAi on the growth and development of Asian corn borer, Ostrinia furnacalis, was also investigated. The effects of small molecule inhibitor 2-acetamido-1,2-dideoxynojirimycin 2-acetamido-1,2-dideoxynojirimycin, 2-ADN) and the inhibitory effect of imidazole on OfHex2 in vitro. The results showed that injection of dsOfHEX2 on the first instar larvae of the 5th instar larvae of A. furnacalis could interfere with the normal expression of OfHEX2 gene at larval stage but could not interfere with its expression at pupal stage. RNA interference led to the occurrence of OfHEX2 gene at the 5th instar stage The expression level in larvae on the third day was only 7.1% of that in the control group, which resulted in the normal contraction of the prepupalaemal abdomen, malformation of the appendages of pupae and adults, and 15% of the deformed pupae died due to abnormal development. The results show that OfHex2 has important physiological significance in insect development, and its lack of function has teratogenic and indirect lethal effects and therefore may be a potential target for designing new pesticides. Inhibition The results showed that the small molecule 2-ADN is a competitive inhibitor of OfHex2, the enzyme activity inhibition constant Ki value of 0.94 ± 0.035μmol / L.