关键词: 豌豆修尾蚜, 高湿度胁迫, 繁殖, 水通道蛋白, 蛋白结构, 生物信息学

Abstract:

【Objective】At present, crop pests are more adaptable to high relative humidity. Aquaporin (AQP) is a membrane protein that is necessary for insects to maintain water balance in the body. The objectives of this study are to screen two McAQP sequence structures by transcriptome sequencing through high relative humidity stress, clarify the AQP characteristics of Megoura crassicauda and the expression changes in response to high relative humidity stress combined with real-time fluorescence quantitative PCR (qRT-PCR) verification, and to provided a theoretical basis for further exploring the gene function of AQP.【Method】The environmental relative humidity (RH) of 60%, 75% and 90% were set to cultivate M. crassicauda. The growth and development were observed, the relative expression of genes and the content of related substances in the body were detected. Based on two McAQP sequences (named as McAQP X1 and McAQP X2, respectively) obtained from comparative transcriptome sequencing of M. crassicauda, the physicochemical property, sequence structure, and the homology with other insects were analyzed by bioinformatics methods. Finally, qRT-PCR was used to determine the relative expression of AQP X1, AQP X2, Vg and VgR under long-term stable high relative humidity stress and 24 h emergency high relative humidity stress.【Result】Compared with RH 60% and RH 75%, the fecundity of M. crassicauda under RH 90% was significantly reduced, but there was no significant difference in development duration and survival rate. With the increase of relative humidity, the water content of M. crassicauda decreased, and the special phenotype phenomena such as body color change and long wing appeared. The molecular weights of McAQP X1 and McAQP X2 are 33.89 and 28.94 kDa, the theoretical isoelectric points are 5.36 and 5.32, and the protein lengths are 308 and 272 aa, respectively. On the multistage structure, McAQP is arranged by six long alpha helices via counterclockwise rotation to form a barrel channel, whereas four monomers form a tetramer to exercise function. The relative expression of McAQPs quantified by qRT-PCR showed that both McAQP X1 and McAQP X2 were up-regulated with increasing relative humidity, which was generally consistent with the transcriptome sequencing results. Meanwhile, under long-term stable high humidity stress, both Vg and VgR were up-regulated under RH 75% and down-regulated under RH 90%. Under 24 h emergency high humidity stress, Vg expression level was down-regulated with increasing humidity, while VgR was up-regulated under RH 90%.【Conclusion】High humidity environment affects the reproduction, water content and body color of M. crassicauda. The aquaporin sequence structure in subfamily II is relatively conservative, without strong species-specific differentiation. NPA site is important for the function of aquaporin, and the asparagine residue plays a structural role for water molecules through the central channel. It is speculated that the expression change of AQPs may be an important means for M. crassicauda to cope with humidity changes.

Key words: Megoura crassicauda, high relative humidity stress, reproduction, aquaporin (AQP), protein structure, bioinformatics