为进一步探究KK-42缩短蜕皮周期的分子机制,本研究以日本沼虾幼虾为材料,通过RACE技术克隆了几丁质降解途径中的限速酶基因NAGase cDNA全长序列,定量测定了KK-42处理不同时间对头胸甲表皮组织中NAGase mRNA相对表达量和对应酶活力的影响.序列分析结果显示,NAGase cDNA全长2536 bp,编码617个氨基酸.同源性分析显示,NAGase基因保守性较低,与凡纳滨对虾的相似度最高,仅为68%.系统进化分析显示,日本沼虾、三疣梭子蟹、凡纳滨对虾、中国明对虾聚为一个大类,凡纳滨对虾和中国明对虾亲缘关系更为接近,日本沼虾单列一个分支.Real-time PCR分析表明,NA-Gase相对表达量在蜕皮前D0期达到峰值;KK-42处理后3 h,D4期的相对表达量是对照组的253%,处理后6 h,C期和D0期较对照组分别提高了226%和187%.NAGase酶活力从C期到D4期逐渐提高,KK-42处理能明显提高C和D0期NAGase酶活力,尤其对C期的影响最为显著,在处理后3、6和12 h分别提高了11.26、5.99、7.15倍.结果提示,KK-42对日本沼虾表皮NAGase的诱导效应可能是其缩短蜕皮周期的分子机制之一.%In order to further explore the molecular mechanism that KK-42 shortens the molt cycle of the juvenile prawn Macrobrachium nipponense,the full-length cDNA sequence of NAGase,the rate-limiting enzyme in chitin catabolism,was cloned from carapace by RACE technique.The relative expression of NAGase mRNA and its activity in cuticular tissue were determined before and after KK-42 treatment. Sequence analysis showed that the full length NAGase cDNA was 2 536 bp,encoding 617 amino acids.Homology analysis indicated that NAGase was less conserved and had the highest similarity of 68% to that from Litopenaeus vannamei.Phylogenetic analys-is showed that the amino acid sequence of NAGase from M.nipponense was clustered into one major group with that from Portunus trituberculatus,L.vannamei,and Fenneropenaeus chinensis.The NAGase sequence from L. vannamei and F.chinensis shared more similarities with each other,M.nipponense belonged to a separate branch. The mRNA concentration of cuticular NAGase in control group peaked at premolt D0stage.Once treated by KK-42, the mRNA content increased significantly, with a 253% rise in D4phase at 3 h, as well as a 226% or 187% rise in C or D0phase at 6 h. The activity of NAGase rose gradually from C to D4. KK-42 treatment could cause signi-ficant increase of NAGase activity in C and D0stages, especially in C stage, during which the activity increased by 11.26, 5.99 and 7.15 folds, respectively, at 3, 6 and 12 h. The results above suggest that the induction effect of KK-42 on the cuticular NAGase of M.nipponense may be one of the molecular mechanisms to shorten the molt cycle.
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