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

【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。     

东方蜜蜂微孢子虫侵染意大利蜜蜂工蜂过程中nce-miR-10660及其靶基因表达谱

【目的】本研究旨在为探究nce-miR-10660调控东方蜜蜂微孢子虫Nosema ceranae侵染的作用机制提供理论和实验依据。【方法】采用Stem-loop RT-PCR对前期鉴定到的东方蜜蜂微孢子虫nce-miR-10660进行表达验证，再通过Sanger测序验证nce-miR-10660的序列。利用相关生物信息学软件预测和分析nce-miR-10660的靶基因。通过RT-qPCR检测nce-miR-10660及其靶基因在东方蜜蜂微孢子虫侵染意大利蜜蜂Apis mellifera ligustica工蜂过程中中肠中的表达谱。【结果】Stem-loop RT-PCR和Sanger测序结果分别证实了nce-miR-10660在东方蜜蜂微孢子虫孢子中的表达和真实存在。靶向预测结果显示nce-miR-10660共靶向RRDRP和RCDP 42等9个基因；分别有2和6个靶基因可被分别注释到KEGG数据库中的4条通路和GO数据库中的23个条目。RT-qPCR结果显示，相较于东方蜜蜂微孢子虫侵染后1 d时意大利蜜蜂工蜂中肠中nce-miR-10660的表达量，东方蜜蜂微孢子虫侵染后2 d时意...     

东方蜜蜂微孢子虫侵染意大利蜜蜂工蜂过程的nce-miR-23928及其靶基因表达谱

【背景】东方蜜蜂微孢子虫(Nosema ceranae)专性侵染成年蜜蜂中肠上皮细胞而导致的微孢子虫病给养蜂业造成严重损失。【目的】检测东方蜜蜂微孢子虫nce-miR-23928及其靶基因在侵染意大利蜜蜂(Apis mellifera ligustica)工蜂过程的表达谱,为深入探究nce-miR-23928在东方蜜蜂微孢子虫侵染中的功能及调控机制提供依据。【方法】通过RNAhybrid、miRanda和TargetScan软件预测nce-miR-23928的靶基因。使用BLAST工具将上述靶基因比对到基因本体论(geneontology,GO)、京都基因和基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG)、Nr和Swiss-Prot数据库以获得相应注释。采用实时荧光定量PCR(realtimequantitativePCR,RT-qPCR)技术检测nce-miR-23928及其靶基因在东方蜜蜂微孢子虫侵染意蜂工蜂过程中的相对表达量。【结果】相较于接种后1 d (1 day post infection, 1 dpi),nce-...     

东方蜜蜂微孢子虫类甲基转移酶样蛋白5基因NcMettl5的克隆、分子特性与表达模式

N⁶-甲基腺苷(N⁶-methyladenosine,m⁶A)是真核生物中分布广泛、含量丰富且作用关键的一类RNA修饰。东方蜜蜂微孢子虫Nosemaceranae专性侵染成年蜜蜂导致微孢子病,给养蜂业造成很大损失。本研究对东方蜜蜂微孢子虫的甲基转移酶样蛋白5(Methyltransferase-likeprotein5,Mettl5)基因NcMettl5进行克隆,通过生物信息学预测NcMettl5蛋白的理化性质和分子特性,并通过RT-qPCR检测NcMettl5在东方蜜蜂微孢子虫侵染意大利蜜蜂Apis mellifera ligustica工蜂过程的相对表达量,以期丰富NcMettl5的信息,并为探究东方蜜蜂微孢子侵染中NcMettl5的功能及调控机制提供基础。结果表明,扩增出约600bp的目的片段,与GenBank数据库收录的预测出的NcMettl5序列一致;NcMettl5的分子量约为22.64kDa,分子式为C₁₀₃₉H₁₆₄₅N₂₆₉O_2...     

东方蜜蜂微孢子虫侵染过程中nce-miR-34537和靶基因PIP5KI的表达谱

【目的】本研究旨在对前期鉴定到的nce-miR-34537进行表达和序列验证,预测nce-miR-34537的靶基因并明确其分子特性,进而检测nce-miR-34537及其靶基因在东方蜜蜂微孢子虫(Nosema ceranae)侵染意大利蜜蜂(Apis mellifera ligustica)工蜂过程的表达谱,为进一步探究nce-miR-34537调控东方蜜蜂微孢子虫侵染的功能和作用机制提供基础。【方法】通过Stem-loop-RT-PCR和Sanger测序验证nce-miR-34537的表达和序列。通过生物信息学软件预测nce-miR-34537的靶基因PIP5KI(I型磷脂酰肌醇4-磷酸-5-激酶基因)的理化性质等分子特性和保守基序,并构建基于氨基酸序列的系统进化树。采用RT-qPCR检测nce-miR-34537及其靶基因的表达谱。【结果】nce-miR-34537在东方蜜蜂微孢子虫孢子中真实存在和表达。nce-miR-34537共靶向PIP5KI等151个基因。PIP5KI蛋白的分子式为C₈₈₂H_{1 364}N₂₂₆     

参与调控意大利蜜蜂工蜂中肠基因表达的东方蜜蜂微孢子虫miRNA的组学解析及其调控网络

[目的]本研究结合前期已获得的miRNA和mRNA组学数据对东方蜜蜂微孢子虫Nosema ceranae 的差异表达 miRNA(differentially expressed miRNA,DEmiRNA)靶向意大利蜜蜂 Apis mellifera ligustica 工蜂中肠的 mRNA 和差异表达 mRNA(d...     

蜜蜂微孢子虫在中国的自然种系构成初探

蜜蜂微孢子虫(Nosema apis及Nosema ceranae)是微孢子虫的典型代表之一,由它寄生蜜蜂所产生的疾病,称为蜜蜂微孢子虫病。通过目前国际上普遍使用的克隆16srRNA基因(16srDNA)的片段并测序的方法来进行蜜蜂微孢子虫在我国主要养蜂地区的分布情况。结果表明,在我国主要养蜂地区,造成大量西方蜜蜂患蜜蜂微孢子虫病的是东方蜜蜂微孢子虫Nosema ceranae,至今为止未发现过去我们广泛认定的西方蜜蜂微孢子虫Nosema apis。     

Calcofluor White M2R与Sytox Green双重染色法鉴别蜜蜂微孢子虫

东方蜜蜂微孢子虫(Nosema ceranae)是一种广泛寄生于东方蜜蜂Apis cerana,西方蜜蜂Apis mellifera和熊蜂Bombus Latreille上的寄生虫,对蜜蜂和熊蜂的危害较大,进而影响养蜂业的发展。本实验采用荧光染色试剂Calcofluor White M2R与核酸染料Sytox Green双重染法来鉴别蜜蜂或熊蜂体内的N.ceranae及孢子的存活状态。结果得出,在荧光显微镜下可见死孢子被染上黄绿色荧光,活的呈现蓝白色荧光,而寄主细胞、细菌、病毒等不被染色。这是一种快速有效鉴别N.ceranae及其死活的方法,从而判定蜜蜂或熊蜂体内的微孢子虫在是否具有侵染活性,对微孢子虫的研究及药物防治具有重要作用。     

东方蜜蜂微孢子虫传染病学分析

东方蜜蜂微孢子虫病是一种由东方蜜蜂微孢子虫(Nosema ceranae)引起的蜜蜂传染病,已经蔓延到全球。蜜蜂感染东方蜜蜂微孢子虫后会导致早衰、哺育能力下降、生产力和繁殖能力降低,严重时可直接导致蜂群瓦解。本文从传染病学角度出发,对近10年东方蜜蜂微孢子虫病原学、流行病学和防治方法等方面进行总结,以此提高对微孢子虫的认识,为微孢子虫防治提供新思路。     

东方蜜蜂微孢子虫LCFAL 2基因的分子特性及系统进化分析

本研究旨在解析东方蜜蜂微孢子虫Nosema ceranae的长链脂肪酸辅酶A连接酶(long chain fatty acid COA ligase)基因LCFAL 2的分子特性,鉴定东方蜜蜂微孢子虫和其他物种的LCFAL 2蛋白的保守基序和结构域并进行系统进化分析,以期丰富东方蜜蜂微孢子虫LCFAL 2的信息,为深入开展相关功能研究提供基础。通过Expasy网站上的相关软件预测和分析LCFAL 2的理化性质、跨膜螺旋域、信号肽、磷酸化位点、二级结构和三级结构。使用MEME软件预测东方蜜蜂微孢子虫和其他物种LCFAL 2蛋白的保守基序。采用TBtools软件预测东方蜜蜂微孢子虫和其他物种LCFAL 2的结构域。通过Mega 11.0软件构建东方蜜蜂微孢子虫和其他物种的LCFAL 2的系统进化树。结果表明,东方蜜蜂微孢子虫LCFAL 2含有1 836个核苷酸,可编码611个氨基酸,分子式为C₃₁₄₅H₄₉₈₅N₈₁₃O₉₀₄S₂₃,分子量约为69.39 kDa,脂溶系数为96.8...     

Nosema ceranae, a new parasite in Thai honeybees

Adult workers of Apis cerana, Apis florea and Apis mellifera from colonies heavily infected with Nosema ceranae were selected for molecular analyses of the parasite. PCR-specific 16S rRNA primers were designed, cloned, sequenced and compared to GenBank entries. The sequenced products corresponded to N. ceranae. We then infected A. cerana with N. ceranae spores isolated from A. florea workers. Newly emerged bees from healthy colonies were fed 10,000, 20,000 and 40,000 spores/bee. There were significant dosage dependent differences in bee infection and survival rates. The ratio of infected cells to non-infected cells increased at 6, 10 and 14 d post infection. In addition, hypopharyngeal glands of bees from the control group had significantly higher protein concentrations than infected groups. Bees infected with 40,000 spores/bee had the lowest protein concentrations. Thus, N. ceranae isolated from A. florea is capable of infecting another bee species, impairing hypopharyngeal gland protein production and reducing bee survival in A. cerana.     

家蚕微孢子虫感染对家蚕BmN细胞凋亡以及凋亡蛋白抑制因子IAPs表达的影响

【目的】在无任何外界凋亡因素诱导条件下,探究家蚕微孢子虫感染对家蚕卵巢细胞-BmN凋亡的影响,以及凋亡蛋白抑制因子IAPs实相表达的变化情况。【方法】显微镜下观察家蚕微孢子虫感染BmN细胞后不同时间段宿主细胞的变化情况,以及利用荧光定量PCR方法检测家蚕促凋亡基因——细胞色素C(BmCyt c)表达水平的变化,随后检索家蚕基因组与蛋白质家族数据库搜寻家蚕凋亡蛋白抑制因子IAPs基因信息,并通过荧光定量PCR方法对这些基因的实相表达情况进行定量分析。【结果】家蚕微孢子虫感染BmN细胞的前5 d,细胞状态未见明显变化。感染后7 d,BmN细胞的生长受到了一定程度的影响。第12天时,对照组中几乎所有细胞出现空泡化或细胞死亡的现象,而感染家蚕微孢子虫的BmN细胞未见空泡的出现,并且大量细胞形态完整,细胞核清晰可见。同时,BmCyt c基因的表达几乎一直处于被抑制状态,特别是感染后的第10天与第12天,该基因的表达量显著性降低(P0.01)。通过数据库检索共得到4个家蚕凋亡蛋白抑制因子:BmIAP-1、BmIAP-2、BmSurvivin-1与BmSurvivin-2。荧光定量PCR结果表明:BmIAP-1和BmSurvivin-1基因在感染后期(10 d与12 d)表达量有上升趋势,尤其是感染后的12 d,表达量显著上升(P0.01)。然而,BmIAP-2与BmSurvivin-2基因的表达在大多数时间段均处于下调状态。【结论】当无任何外界凋亡因素诱导条件下,家蚕微孢子虫感染BmN细胞后可影响宿主细胞的生长,并可抑制细胞的正常生理凋亡。依据荧光定量PCR结果,我们推测在家蚕微孢子虫感染BmN细胞时,BmIAP-1和BmSurvivin-1蛋白可能在调节细胞凋亡的过程中起一定作用。     

Infections of Nosema ceranae in four different honeybee species

The microsporidium Nosema ceranae is detected in honeybees in Thailand for the first time. This endoparasite has recently been reported to infect most Apis mellifera honeybee colonies in Europe, the US, and parts of Asia, and is suspected to have displaced the endemic endoparasite species, Nosema apis, from the western A. mellifera. We collected and identified species of microsporidia from the European honeybee (A. mellifera), the cavity nesting Asian honeybee (Apis cerana), the dwarf Asian honeybee (Apis florea) and the giant Asian honeybee (Apis dorsata) from colonies in Northern Thailand. We used multiplex PCR technique with two pairs of primers to differentiate N. ceranae from N. apis. From 80 A. mellifera samples, 62 (77.5%) were positively identified for the presence of the N. ceranae. Amongst 46 feral colonies of Asian honeybees (A. cerana, A. florea and A. dorsata) examined for Nosema infections, only N. ceranae could be detected. No N. apis was found in our samples. N. ceranae is found to be the only microsporidium infesting honeybees in Thailand. Moreover, we found the frequencies of N. ceranae infection in native bees to be less than that of A. mellifera.     

Phylogenetic analysis of Nosema ceranae isolated from European and Asian honeybees in Northern Thailand

Nosema ceranae was found to infect four different host species including the European honeybee (A. mellifera) and the Asian honeybees (Apis florea, A. cerana and Apis dorsata) collected from apiaries and forests in Northern Thailand. Significant sequence variation in the polar tube protein (PTP1) gene of N. ceranae was observed with N. ceranae isolates from A. mellifera and A. cerana, they clustered into the same phylogenetic lineage. N. ceranae isolates from A. dorsata and A. florea were grouped into two other distinct clades. This study provides the first elucidation of a genetic relationship among N. ceranae strains isolated from different host species and demonstrates that the N. ceranae PTP gene was shown to be a suitable and reliable marker in revealing genetic relationships within species.     

Asymmetrical coexistence of Nosema ceranae and Nosema apis in honey bees

Globalization has provided opportunities for parasites/pathogens to cross geographic boundaries and expand to new hosts. Recent studies showed that Nosema ceranae, originally considered a microsporidian parasite of Eastern honey bees, Apis cerana, is a disease agent of nosemosis in European honey bees, Apis mellifera, along with the resident species, Nosema apis. Further studies indicated that disease caused by N. ceranae in European honey bees is far more prevalent than that caused by N. apis. In order to gain more insight into the epidemiology of Nosema parasitism in honey bees, we conducted studies to investigate infection of Nosema in its original host, Eastern honey bees, using conventional PCR and duplex real time quantitative PCR methods. Our results showed that A. cerana was infected not only with N. ceranae as previously reported [Fries, I., Feng, F., Silva, A.D., Slemenda, S.B., Pieniazek, N.J., 1996. Nosema ceranae n. sp. (Microspora, Nosematidae), morphological and molecular characterization of a microsporidian parasite of the Asian honey bee Apis cerana (Hymenoptera, Apidae). Eur. J. Protistol. 32, 356-365], but also with N. apis. Both microsporidia produced single and mixed infections. Overall and at each location alone, the prevalence of N. ceranae was higher than that of N. apis. In all cases of mixed infections, the number of N. ceranae gene copies (corresponding to the parasite load) significantly out numbered those of N. apis. Phylogenetic analysis based on a variable region of small subunit ribosomal RNA (SSUrRNA) showed four distinct clades of N. apis and five clades of N. ceranae and that geographical distance does not appear to influence the genetic diversity of Nosema populations. The results from this study demonstrated that duplex real-time qPCR assay developed in this study is a valuable tool for quantitative measurement of Nosema and can be used to monitor the progression of microsprodian infections of honey bees in a timely and cost efficient manner.     

Comparative Analysis of Codon Usage Bias Patterns in Microsporidian Genomes

The sub-3 Mbp genomes from microsporidian species of the Encephalitozoon genus are the smallest known among eukaryotes and paragons of genomic reduction and compaction in parasites. However, their diminutive stature is not characteristic of all Microsporidia, whose genome sizes vary by an order of magnitude. This large variability suggests that different evolutionary forces are applied on the group as a whole. In this study, we have compared the codon usage bias (CUB) between eight taxonomically distinct microsporidian genomes: Encephalitozoon intestinalis, Encephalitozoon cuniculi, Spraguea lophii, Trachipleistophora hominis, Enterocytozoon bieneusi, Nematocida parisii, Nosema bombycis and Nosema ceranae. While the CUB was found to be weak in all eight Microsporidia, nearly all (98%) of the optimal codons in S. lophii, T. hominis, E. bieneusi, N. parisii, N. bombycis and N. ceranae are fond of A/U in third position whereas most (64.6%) optimal codons in the Encephalitozoon species E. intestinalis and E. cuniculi are biased towards G/C. Although nucleotide composition biases are likely the main factor driving the CUB in Microsporidia according to correlation analyses, directed mutational pressure also likely affects the CUB as suggested by ENc-plots, correspondence and neutrality analyses. Overall, the Encephalitozoon genomes were found to be markedly different from the other microsporidians and, despite being the first sequenced representatives of this lineage, are uncharacteristic of the group as a whole. The disparities observed cannot be attributed solely to differences in host specificity and we hypothesize that other forces are at play in the lineage leading to Encephalitozoon species.     

Prevalence and infection intensity of Nosema in honey bee (Apis mellifera L.) colonies in Virginia

Nosema ceranae is a recently described pathogen of Apis mellifera and Apis cerana. Relatively little is known about the distribution or prevalence of N. ceranae in the United States. To determine the prevalence and potential impact of this new pathogen on honey bee colonies in Virginia, over 300 hives were sampled across the state. The samples were analyzed microscopically for Nosema spores and for the presence of the pathogen using real-time PCR. Our studies indicate that N. ceranae is the dominant species in Virginia with an estimated 69.3% of hives infected. Nosema apis infections were only observed at very low levels (2.7%), and occurred only as co-infections with N. ceranae. Traditional diagnoses based on spore counts alone do not provide an accurate indication of colony infections. We found that 51.1% of colonies that did not have spores present in the sample were infected with N. ceranae when analyzed by real-time PCR. In hives that tested positive for N. ceranae, average C_T values were used to diagnose a hive as having a low, moderate, or a heavy infection intensity. Most infected colonies had low-level infections (73%), but 11% of colonies had high levels of infection and 16% had moderate level infections. The prevalence and mean levels of infection were similar in different regions of the state.     

意大利蜜蜂工蜂幼虫nkd基因的生物信息学分析及功能研究

【目的】意大利蜜蜂(Apis mellifera ligustica,简称意蜂)是西方蜜蜂(Apis mellifera)的亚种之一。蜜蜂球囊菌(Ascosphaera apis)侵染意蜂幼虫导致白垩病。本研究对西方蜜蜂裸表皮蛋白(naked cuticle, Nkd)进行保守基序预测和系统进化分析,并通过RNAi明确nkd基因对意蜂工蜂幼虫体重及宿主响应蜜蜂球囊菌胁迫的免疫应答的影响,以期丰富西方蜜蜂基因nkd的信息,并揭示意蜂幼虫nkd的功能。【方法】通过MEME软件预测西方蜜蜂和其他9个物种Nkd蛋白的保守基序。采用MEGA X软件对西方蜜蜂及其他9个物种的Nkd蛋白进行系统进化分析。通过饲喂dsRNA对意蜂幼虫肠道内的nkd进行RNAi。使用电子天平对幼虫进行称重。利用RT-qPCR检测nkd基因的干扰效率及免疫基因的相对表达量。【结果】西方蜜蜂与东方蜜蜂、柑橘凤蝶、家蚕和金凤蝶的Nkd蛋白均含有3个保守基序(motif 1、motif 2 和motif 3),说明上述5个昆虫物种的Nkd具有较高的保守性。西方蜜蜂与东方蜜蜂的Nkd蛋白聚为一支,说明二者的亲缘关系近。与dsRNA-egfp组相比,dsRNA-nkd组5日龄和6日龄幼虫肠道内nkd的表达量均极显著下调(P<0.001),干扰效率分别为49.60%和56.40%。另外,dsRNA-nkd组幼虫体重较dsRNA-egfp组显著下降,说明nkd显著影响幼虫体重。RT-qPCR结果显示,4日龄幼虫肠道内abaecin、apidaecin、birc5、defensin-1和PGRP-S2均被激活表达;5日龄幼虫肠道内abaecin、apidaecin、birc5和defensin-1均被激活表达,PGRP-S2的表达受到抑制;6日龄幼虫肠道内abaecin被激活表达,而apidaecin、birc5、defensin-1和PGRP-S2的表达均受到抑制,说明上述5个免疫基因在宿主响应胁迫的过程中呈不同的表达趋势,均参与宿主的免疫应答,nkd与abaecin和apidaecin的表达存在负向调控关系。【结论】西方蜜蜂的Nkd蛋白含有3个保守基序(motif 1、motif 2和motif 3),西方蜜蜂与东方蜜蜂的Nkd蛋白亲缘关系最近,通过饲喂dsRNA能有效干扰意蜂工蜂幼虫肠道内nkd表达,nkd影响意蜂工蜂幼虫体重及宿主对蜜蜂球囊菌胁迫的免疫应答。