东方蜜蜂微孢子虫腺苷酸激酶的生物信息学与基因表达特征

Bioinformatic and gene expression pattern of adenylat kinase gene iin Nosema ceranae

本研究旨在解析东方蜜蜂微孢子虫Nosema ceranae的腺苷酸激酶(Adenylat kinase,ADK)NcADK的理化性质和分子特性,并检测NcADK基因在东方蜜蜂微孢子虫侵染意大利蜜蜂Apis mellifera ligustica工蜂过程中的表达特征,以期丰富NcADK相关信息,并为进一步的功能研究提供依据。利用相关生物信息学软件预测和分析NcADK的理化性质、信号肽、磷酸化位点、二级结构和三级结构。使用MEME软件和Batch CD-Search工具分别预测东方蜜蜂微孢子虫和其它7种微孢子ADK蛋白的保守基序和保守结构域。通过Mega 11.0软件基于ADK氨基酸序列构建进化树。采用RT-qPCR检测东方蜜蜂微孢子虫侵染过程中NcADK的相对表达量。结果表明,NcADK的CDS含有540个核苷酸,可编码179个氨基酸;NcADK的分子量约为20.66 kDa,分子式为C903H1488N258O278S8,脂肪系数为100.61,平均亲水系数为-0.502,等电点为6.83,含29个负电荷氨基酸和29个正电荷氨基酸;NcADK可同时定位于细胞质、线粒体、细胞核、囊泡和过氧化物酶体;NcADK含20个磷酸化位点,不含典型的信号肽;NcADK含88个α-螺旋,24条延长链,17个β-转角,50个无规则卷曲,与模板A0A0F9WEU7.1.A之间的序列同源性为100%;在东方蜜蜂微孢子虫、东方赤孢子虫Hamiltosporidium tvaerminnensis、肠脑炎微孢子虫Encephalitozoon intestinalis、按蚊微孢子虫Anncaliia algerae和角膜条孢虫Vittaforma corneae ADK中均鉴定到1个相同的结构域和5个相同的保守基序;东方蜜蜂微孢子虫与蜜蜂微孢子虫Nosema apis的ADK在进化树上聚为一支。相较于接种后1 d(1 day post inoculation,1 dpi),NcADK的表达量在2 dpi上调但无显著差异(P>0.05),在3 dpi和4 dpi均显著上调(P>0.05)。研究结果明确了NcADK的理化性质和分子特性,并揭示NcADK是潜在的亲水性蛋白和胞内蛋白,不同微孢子虫的ADK具有较强的保守性,东方蜜蜂微孢子虫及其姐妹种蜜蜂微孢子虫的ADK具有高同源性,NcADK在3 dpi和4 dpi被激活表达。

The objective of this study is to analyze the physicochemical property and molecular characteristics of the adenylate kinase(ADK)in Nosema ceranae,and detect the expression feature of NcADK during the N.ceranae infection of Apis mellifera ligustica worker,enriching relevant information of and providing a basis for further functional investigation.The physicochemical properties,signal peptides,phosphorylation sites,and secondary and tertiary structures of NcADK were predicted and analyzed by using related bioinformatics software.MEME software and Batch CD-Search tool were employed to predict the structural domains and conserved motifs in ADK proteins in N.ceranae and 7 other species.The phylogenetic tree was constructed by Mega 11.O software based on the amino acid sequences of ADK proteins.RT-qPCR was performed to determine relative expression level of NcADK in N.ceranae during the infection process.The results indicated that the NcADK CDS contained 540 nucleotides encoding 179 amino acids;the molecular weight of NcADK was approximately 20.66 kDa,the molecular formula was Co HlussN2s OzsSs,the lipophilicity cofficient was 100.61,and the isoelectric point was 6.83;NcADK included 29 negatively charged amino acids and 29 positively charged amino acids;NcADK could be meantime localized in the cytoplasm,mitochondria,nucleus,vesicles,and peroxisomes;NcADK contained 20 phosphorylation sites while lacked typical signal peptide;NcADK included 88α-helices,24 extended strands,17β-turns,and 50 random coils,and it shared 100%sequence homology with the template AOAOF9WEU7.1.A;the same one structural domains and the same 5 conserved motifs were indetified in NcADK and the ADK of Hamiltosporidium tvaerminnensis,Encephalitozoon intestinalis,Anncalia algerae and Vittaforma corneae;NcADK and Nosema apis ADK were clustered into one clade on the evolutionary tree;as compared with that at 1 dpi(day post inoculation),the expression level of NcADK was up-regulated at 2 dpi without significant difference(P>0.05),whereas the expression level of NcADK was significantly up-regulated at 3 dpi and 4 dpi(P>0.05).These findings clarify the physicochemical property and molecular characteristics of NcADK,and suggested that NcADK was a potential hydrophilic and intracellular protein,ADK in different microsporidian species were highly conserved,the homology between NcADK and ADK in N.apis was the highest,and the expression of NcADK was activated at 3 dpi and 4 dpi.