通过RNA干扰研究亚洲玉米螟乙酰己糖胺酶的潜在靶标性

Investigation of β-N-acetyl-D-hexosaminidase as a potential target with RNA interference in Ostrinia furnacalis

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-acetyl-D-hexosaminidase（EC 3.2.1.52） catalyzes the release of N-acetyl-D-hexosamine from glycoconjugates,playing important roles in insect growth and development.The OfHEX2 gene encoding a lysosomal β-N-acetyl-D-hexosaminidase was previously obtained from Ostrinia furnacalis.In order to understand whether OfHex2 could be a potential insecticide target,the physiological importance of OfHex2 was investigated by RNA interference.And two chemicals,2-acetamido-1,2-dideoxynojirimycin（2-ADN）and imidazole,were evaluated for their inhibitory activities toward OfHex2.The injection of dsOfHEX2 into the fifth instar larvae efficiently induced gene silencing of OfHEX2 at the larval stage but not at the pupal stage.RNA interference resulted in the transcription level dropping to 7.1% on the third day of the 5th instar larvae compared to the control,and caused abnormalities of larval abdomen,pupa and adult appendages.The mortality of abnormal pupae was around 15%.These results showed that OfHex2 deficiency in Ostrinia furnacalis led to the aberrant and indirect lethal phenotypes,suggesting that OfHex2 was physiologically important and might be a potential target for insecticide designing.The results of inhibition experiment showed that 2-ADN competitively inhibited the activity of OfHex2 with a Ki value of 0.94±0.035 μmol/L.