东方蜜蜂微孢子虫N6-腺苷特异性甲基化转移酶基因的克隆、分子特性及表达模式

Cloning,molecular characterization,and expression pattern of N6-adenine-specific DNA methyltransferase gene in Nosema ceranae

【目的】东方蜜蜂微孢子虫(Nosme ceranae)专性侵染成年蜜蜂导致微孢子虫病,给养蜂生产造成很大损失。目前,东方蜜蜂微孢子虫的N6-腺苷特异性甲基化转移酶(N6-adenine-specific methyltransferase,N6AMT)基因NcN6AMT的研究仍然缺失。本研究对NcN6AMT的编码序列(coding sequence,CDS)区进行克隆,并解析NcN6AMT蛋白的理化性质和分子特性,进而测定东方蜜蜂微孢子虫侵染意大利蜜蜂(Apis mellifera ligustica)和中华蜜蜂(Apis cerana cerana)工蜂过程中NcN6AMT的相对表达量,以期丰富NcN6AMT的信息,并为探究东方蜜蜂微孢子虫侵染过程NcN6AMT的功能及表观调控机制提供基础。【方法】采用Protparam和ProtScale软件对NcN6AMT进行等电点和亲水性分析。通过SignalP 5.0、NetPhos 3.1、TMHMM-2.0、SOPMA和SWISS-MODEL等软件分别预测NcN6AMT的信号肽、磷酸化位点、跨膜结构域、二级结构和三级结构。使用WoLF PSORT II软件预测NcN6AMT的亚细胞定位。根据N6AMT氨基酸序列,通过TBtools软件对智人(Homo sapiens)、小鼠(Mus musculus)、褐飞虱(Nilaparvata lugens)、兔脑炎微孢子虫(Encephalitozoon cuniculi)、肠脑炎微孢子虫(Encephalitozoon intestinalis ATCC 50506)、蚱蜢脑炎微孢子虫(Encephalitozoon romaleae SJ-2008)、美洲思普雷格孢虫(Spraguea lophii 42_110)、家蚕微孢子虫(Nosema bombycis CQ1)、隐生菱形藻(Nitzschia inconspicua)和东方蜜蜂微孢子虫(Nosema ceranae)的N6AMT进行结构域预测和分析。利用MEME软件和MEGA 11.0软件进行东方蜜蜂微孢子虫和其他物种N6AMT的保守基序预测及进化树构建。采用实时荧光定量聚合酶链式反应(real-time fluorescence quantitative polymerase chain reaction,RT-qPCR)检测NcN6AMT在东方蜜蜂微孢子虫侵染意大利蜜蜂和中华蜜蜂工蜂过程的相对表达量。【结果】通过PCR扩增出大小约500 bp的目的片段,克隆测序结果显示其与GenBank数据库收录的预测序列一致;NcN6AMT蛋白的分子量约为18.7 kDa,分子式为C845H1374N214O249S6,理论等电点为5.88,脂溶系数是119.76,不稳定系数为37.47,平均亲水系数为0.025,含166个氨基酸和15个磷酸化位点,不含典型的跨膜结构域和信号肽,可同时定位于细胞质、线粒体、细胞核和液泡膜;NcN6AMT含1个甲基转移酶小结构域(methyltransferase small domain,MTS),该结构域同样存在于家蚕微孢子虫和兔脑炎微孢子虫等8个其他物种的N6AMT;在东方蜜蜂微孢子虫、兔脑炎微孢子虫、肠脑炎微孢子虫和蚱蜢脑炎微孢子虫的N6AMT中均预测到5个相同的保守基序;NcN6AMT与家蚕微孢子虫、肠脑炎微孢子虫、兔脑炎微孢子虫和蚱蜢脑炎微孢子虫的N6AMT序列一致性达到70.92%;东方蜜蜂微孢子虫和家蚕微孢子虫的N6AMT在系统进化树上聚为一支;东方蜜蜂微孢子虫接种后1–4 d,NcN6AMT在意大利蜜蜂和中华蜜蜂工蜂中肠内均呈现先上升后下降的表达趋势。【结论】成功克隆到NcN6AMT基因的CDS区,明确了NcN6AMT蛋白的理化性质和分子特性,并揭示东方蜜蜂微孢子虫和家蚕微孢子虫的N6AMT蛋白具有较高的保守性,NcN6AMT在东方蜜蜂微孢子虫侵染意大利蜜蜂和中华蜜蜂工蜂的第一个增殖周期(1–4 dpi)内动态表达且均呈上升-下降的表达模式。

[Objective]Nosema ceranae exclusively infects adult honey bees and causes nosemosis,resulting in great losses for the beekeeping industry.Little is known about the N.ceranae N6-adenine-specific methyltransferase gene NcN6AMT.This study cloned the coding sequence(CDS)region of NcN6AMT,investigated the physicochemical properties and molecular characteristics of NcN6AMT,and then determined the relative expression level of NcN6AMT during the infection process of N.ceranae in Apis mellifera ligustica and Apis cerana cerana workers.This study aim to enrich the information about NcN6AMT and lay a foundation for exploring the function and epigenetic regulation mechanism of NcN6AMT during the infection process of N.ceranae.[Methods]Protparam and ProtScale were used to analyze the isoelectric point and hydrophilia of NcN6AMT.The signal peptide,phosphorylation site,transmembrane domain,secondary structure,and tertiary structure of NcN6AMT were predicted by SignalP 5.0,NetPhos 3.1,TMHMM-2.0,SOPMA,and SWISS-MODEL,respectively.WoLF PSORT II was employed to predict the subcellular localization of NcN6AMT.TBtools was employed to predict the domains in the amino acid sequences of N6AMTs in Homo sapiens,Mus musculus,Nilaparvata lugens,Encephalitozoon cuniculi,Encephalitozoon intestinalis ATCC 50506,Encephalitozoon Romaleae SJ-2008,Spraguea lophii 42_110,Nosema bombycis CQ1,Nitzschia inconspicua,and N.ceranae.MEME and MEGA 11.0 were employed to predict the conserved motifs and build the phylogenetic tree of N6AMTs.Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)was performed to determine the relative expression level of NcN6AMT during the N.ceranae infection of A.m.ligustica and A.c.cerana workers.[Results]The target fragment with a size of about 500 bp was amplified via PCR,and the cloning and sequencing results showed that it was consistent with the predicted sequence in GenBank.The deduced NcN6AMT protein was composed of 166 residues and had the molecular weight of 18.7 kDa,the formula of C845H1374N214O249S6,the theoretical isoelectric point of 5.88,the lipid solubility coefficient of 119.76,the instability coefficient of 37.47,the average hydrophilic coefficient of 0.025,and 15 phosphorylation sites,with no typical transmembrane domain or signal peptide.NcN6AMT was located in cytoplasm,mitochondria,nucleus,and vacuole membrane.It had one structural domain MTS,which also existed in the N6AMTs of other eight species such as N.bombycis CQ1 and E.cuniculi.Five same conserved motifs were predicted in the N6AMTs of N.ceranae,E.cuniculi,E.intestinalis ATCC 50506,and E.romaleae SJ-2008.The sequence identity of N6AMTs in N.ceranae,N.bombycis CQ1,E.cuniculi,E.intestinalis ATCC 50506,and E.romaleae SJ-2008 was 70.92%.The N6AMTs in N.ceranae and N.bombycis CQ1 were grouped into one clade.The expression of NcN6AMT was first up-regulated and then down-regulated within 1–4 days post inoculation(dpi)of N.ceranae in A.m.ligustica and A.c.cerana workers.[Conclusion]The CDS region of NcN6AMT was successfully cloned,and the physiochemical properties and molecular characteristics of NcN6AMT protein were clarified.The N6AMT proteins in N.ceranae and N.bombycis were highly conserved.During the first proliferation cycle(1–4 dpi)of N.ceranae in A.m.ligustica and A.c.cerana workers,the expression of NcN6AMT exhibited a bell-shaped pattern.