摘要
【目的】当前农作物害虫对高湿等逆境环境适应能力增强,水通道蛋白(aquaporin,AQP)是一种膜蛋白,是昆虫维持体内水分平衡的必要蛋白。本研究通过高湿度胁迫豌豆修尾蚜(Megoura crassicauda),经转录组测序筛选出两条McAQP序列结构,结合实时荧光定量PCR(qRT-PCR)验证,明确豌豆修尾蚜AQP特征及响应高湿度胁迫表达变化,为深入挖掘AQP基因功能提供理论依据。【方法】设置环境相对湿度(RH)为60%、75%和90%,培养豌豆修尾蚜。观察其生长发育情况并检测体内相关物质含量及基因相对表达量,基于从豌豆修尾蚜比较转录组测序中获得的两条McAQP序列(分别命名为McAQP X1、McAQP X2),通过生物信息学方法分析其理化性质和序列结构,以及与其他昆虫间的同源性。最后采用qRT-PCR技术测定长期稳定高湿度胁迫和24 h紧急高湿度胁迫下AQP X1、AQP X2、Vg、VgR的相对表达量。【结果】与RH 60%和RH75%相比,RH90%条件下豌豆修尾蚜繁殖力极显著降低,发育历期与存活率却无显著差异。随着相对湿度的升高,豌豆修尾蚜体内含水率下降,同时出现体色变化、长翅等特殊表型现象。McAQP X1、McAQP X2序列分子量分别为33.89和28.94 kDa,理论等电点为5.36和5.32,蛋白长度分别为308、272 aa。在多级结构上,McAQP由6个长α螺旋经逆时针旋转排布形成一个桶状通道,而4个单体形成一个四聚体行使功能。qRT-PCR分析表明,McAQP X1和McAQP X2均随相对湿度升高而表达水平上调,与转录组测序结果基本一致。在长期稳定高湿度胁迫下豌豆修尾蚜的Vg和VgR均在RH 75%表达水平上调,在RH 90%表达水平下调。在24 h紧急高湿度胁迫下Vg表达水平随湿度升高呈现下调,而VgR在RH 90%表达水平上调。【结论】高湿度环境影响豌豆修尾蚜的繁殖、含水率、体型及体色;定位于亚家族Ⅱ的水通道蛋白序列结构相对保守,没有形成差异性强烈的物种特异分化;NPA位点对于水通道蛋白的功能至关重要,其中的天冬酰胺残基对于水分子在中心通道的通过起着结构性的作用;McAQP表达量变化可能是豌豆修尾蚜应对湿度变化的重要手段。
【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.
作者
孙思思
马伍
司惠茹
王贤忠
柳强
罗炎琳
陈孝玉龙
唐斌
SUN SiSi;MA Wu;SI HuiRu;WANG XianZhong;LIU Qiang;LUO YanLin;CHEN XiaoYuLong;TANG Bin(College of Agriculture/College of Life Sciences,Guizhou University,Guiyang 550025;College of Life and Environmental Sciences,Hangzhou Normal University,Hangzhou 311121;Guizhou Mountainous Meteorological Science Research Institute,Guiyang 550002;Guizhou Tobacco Company Qiandongnan Tobacco Branch Company,Kaili 556000,Guizhou)
出处
《中国农业科学》
CAS
CSCD
北大核心
2024年第20期4057-4070,共14页
Scientia Agricultura Sinica
基金
贵州省科学技术基金基础研究项目(黔科合基础-ZK[2021]一般210)
贵州省“百层次”创新性人才支持计划(GCC[2023]070)
中国烟草总公司贵州省公司科技项目(中烟黔科[2021]1号2021XM16)。
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
Megoura crassicauda
high relative humidity stress
reproduction
aquaporin(AQP)
protein structure
bioinformatics
