西方蜜蜂Lozenge基因的克隆鉴定及时空表达分析

Lozenge蛋白（Lz蛋白）是昆虫的重要转录因子,在昆虫胚胎发育过程中发挥重要作用。为研究Lozenge在西方蜜蜂Apis mellifera中的作用,本研究克隆了Lozenge基因,并对其进行生物信息学分析,同时基于荧光定量PCR技术检测该基因在西方蜜蜂不同发育时期（卵期、幼虫期、蛹期和成年蜂）和10日龄哺育蜂各组织的表达谱。生物信息学分析结果显示,Lozenge基因的开放阅读框（ORF）为1 554 bp,共编码517个氨基酸,预测分子量为54.63918 kDa,等电点为6.08;结构域预测分析发现Lozenge蛋白含有一个Runt结构域,多物种蛋白序列对比发现该蛋白同源性高。时期表达谱表明,该基因在第1日卵和第2日卵的表达量远高于其他时期,在卵期表达量随时间依次递减,幼虫期表达量极低,蛹期表达量呈先增后减的趋势,而成年蜂中均有表达;组织表达谱显示,该基因在哺育蜂头部、上颚腺中的表达量较高,而在腹部的表达量低。这些结果表明,Lozenge基因可能在西方蜜蜂胚胎期细胞发育过程、哺育蜂蜂王浆合成和分泌过程中发挥重要作用,这些结果为该基因功能的深入研究提供了重要的理论参考。     

西方蜜蜂发育基因AmWnt1的克隆鉴定及时空表达分析

本研究旨在克隆鉴定西方蜜蜂Apis mellifera发育相关基因AmWnt1,分析其在不同发育时期和刚出房工蜂不同组织的表达特征,为进一步研究Wnt1基因功能提供理论参考。根据NCBI中AmWnt1基因序列信息,利用Primer 6.0设计引物,RT-PCR扩增AmWnt1基因完整的CDS序列,进行生物信息学预测,用推导的氨基酸序列构建系统进化树;利用荧光定量PCR检测该基因在卵（1日龄、2日龄和3日龄）、幼虫（1日龄、3日龄和5日龄）、预蛹（1日龄和3日龄）、蛹（0日龄、2日龄、4日龄、6日龄和8日龄）、刚出房工蜂、哺育蜂和采集蜂以及刚出房工蜂8个组织中相对表达量。克隆获得西方蜜蜂的Wnt1基因CDS序列,命名为AmWnt1,上传NCBI,获得GenBank登录号MT993937。全长1 239 bp,编码412个氨基酸,预测等电点为9.48,相对分子质量为46.40313 kDa。序列比对和系统进化树结果表明：AmWnt1蛋白与其它膜翅目昆虫聚为一类,其中和东方蜜蜂Apis cerana亲缘关系最近,序列相似度为99.50%。时空表达谱结果表明：AmWnt1基因在各个时期中均有表达,且在胚胎后期表达量最高,预蛹期和蛹前期表达量相对较高,其它时期表达量相对较低;AmWnt1基因在刚出房工蜂头、胸、触角表达量高于其它组织。AmWnt1可能参与西方蜜蜂胚胎晚期的神经系统发育和化蛹过程中的四肢发育等关键历程,为进一步研究AmWnt1功能提供了理论参考。     

中华蜜蜂气味受体基因AcerOr167的克隆及表达分析

本研究通过RT-PCR获得中华蜜蜂气味受体基因Or167的cDNA序列,并采用多种生物信息学软件对其结构特征进行预测分析;利用荧光定量PCR检测Acer Or167 m RNA在工蜂羽化后不同发育阶段(1、5、10、15、20、25和30日龄)和不同组织(触角、头、胸、腹、足、翅)中的相对表达量。克隆获得中华蜜蜂Or167的c DNA序列,命名为Acer Or167(Gen Bank登录号为KF239369),其全长1311 bp,编码436个氨基酸,预测有5个跨膜结构域;多序列比对结果显示,中华蜜蜂Acer Or167与西方蜜蜂Amel Or167的一致性最高,达94%,而与毕氏粗角蚁Cbir Or4-like的一致性最低,为49%;荧光定量结果显示,Acer Or167 m RNA在成蜂不同发育期的各个组织中均有表达,且触角中的表达量极显著高于其它各个组织(P0.01),在头、胸、腹、足、翅中有少量表达;从触角不同发育阶段的表达情况来看,1日龄表达量最低,5日龄表达量显著升高,20日龄表达量最高,且极显著高于其它各日龄(P0.01)。推测Acer Or167可能在中华蜜蜂的嗅觉识别系统中起着重要的作用,与中华蜜蜂外出采集,识别花香气味有关。本研究为进一步深入研究中华蜜蜂传统气味受体的功能奠定理论基础。     

西方蜜蜂毒蕈碱型乙酰胆碱受体基因的生物信息学分析

利用生物信息学方法分析了西方蜜蜂 Apis mellifera毒蕈碱型乙酰胆碱受体的核酸和氨基酸序列,并对其组成成分、疏水/亲水区、跨膜拓扑结构域、分子系统进化关系进行了预测和推断.结果显示,该受体定位在第8条染色体上,由618个氨基酸组成,分子量69 906.5D,等电点(pI)8.56;是G蛋白偶联型受体,含N-糖基化位点、蛋白激酶C磷酸化位点、cAMP/cGMP依赖蛋白激酶磷酸化位点.     

中华蜜蜂气味受体基因AcOr170的克隆与序列分析

本研究采用自行设计的引物对中华蜜蜂Apis cerana cerana雄蜂触角中气味受体基因(Odorant receptors 170)Ac Or170的c DNA序列进行了克隆和序列分析,以探寻中华蜜蜂雄蜂气味受体Ac Or170基因在近缘种昆虫间的进化差异。结果表明:中华蜜蜂雄蜂气味受体基因Ac Or170的c DNA序列总长度为1356 bp,编码区序列长度为1188 bp,共编码396个氨基酸,其分子量为46.272 k Da,等电点8.96,Genbank登录号:KX264359。结构域的分析结果显示,该蛋白具有7tm-6一个保守结构域。经序列比对后发现,Or170的序列在中华蜜蜂、西方蜜蜂和大蜜蜂间的亲缘性很近。     

中华蜜蜂细胞色素CYP9E2基因克隆及其表达分析

【目的】克隆中华蜜蜂Apis cerana cerana细胞色素CYP9E2基因的完整编码区序列,分析CYP9E2基因在工蜂体内的表达特征,为研究该基因的生物学功能提供理论基础。【方法】以解剖获得的中华蜜蜂采集蜂中肠组织为材料,提取总RNA。利用RT-PCR技术克隆中华蜜蜂CYP9E2基因的编码区。采用多种生物信息学软件分析该基因的核苷酸和氨基酸序列,利用荧光定量PCR技术(quantitative real-time PCR)分析其在中华蜜蜂工蜂成虫期不同阶段(初生蜂、哺育蜂、守卫蜂以及采集蜂)头部和中肠组织中的相对表达量及在饲喂氟氯苯菊酯后工蜂中肠组织中的表达变化。【结果】克隆获得中华蜜蜂CYP9E2基因(命名为Ac CYP9E2)mRNA序列,长度为1 600 bp(Gen Bank登录号:KX394629),编码区长1 494 bp,编码497个氨基酸,其蛋白质分子量为57.026k D,等电点为8.32。系统发育树显示,中华蜜蜂Ac CYP9E2与西方蜜蜂Apis mellifera、小蜜蜂Apis florea CYP9E2基因聚成一支。对中华蜜蜂工蜂成虫期不同阶段头部和中肠组织Ac CYP9E2相对表达量测定发现,该基因在中华蜜蜂工蜂成虫期不同阶段的表达量存在一定差异,其中,采集蜂头部和中肠组织中Ac CYP9E2相对表达量均显著高于初生蜂、哺育蜂以及守卫蜂(P0.05),而且4个阶段工蜂中肠组织中的Ac CYP9E2相对表达量均显著高于其头部(P0.05)。饲喂氟氯苯菊酯后,工蜂中肠组织中Ac CYP9E2的相对表达量显著高于对照组(P0.05)。【结论】推测Ac CYP9E2可能参与了中华蜜蜂机体外源物质的代谢与解毒过程。     

一个新的水稻MADS—box基因的克隆及表达分析

根据ＭＡＤＳ－ｂｏｘ基因保守区结构,设计简并性引物,利用３＇ＲＡＣＥ从水稻（Ｏｒｙｚａ ｓａｔｉｖａ Ｌ．）中克隆了１个新的水稻ＭＡＤＳ－ｂｏｘ基因的ｃＤＮＡ片段,同时利用５＆ＲＡＣＥ获得了全长ｃＤｎＡ命名为ＦＤＲＭＡＤＳＳ。序列分析表明,该ｃＤＮＡ全长１４０６ｂｐ,开放阅读框共编码２３３个到,具有典型的植物ＭＡＤＳ－ｂｏｘ基因的结构。推测的氨基酸序列与拟南芥的ＭＡＤＳ－ｂｏｘ基因,ＡＧＬ１４同     

中华蜜蜂酪胺受体基因克隆及表达分析

酪胺(tyramine)属于生物多聚胺类,是昆虫中枢神经系统内重要的神经递质、神经调质和神经激素,参与调控昆虫的多种行为和生理过程,如酪胺受体基因参与调控动物的学习与记忆。本研究首次克隆获得中华蜜蜂(Apis cerana cerana)酪胺受体基因Actyr1和Actyr2的全长cDNA序列,利用qRT-PCR方法鉴定了Actyr1和Actyr2在中华蜜蜂不同组织器官中的表达谱,采用地高辛原位杂交技术对Actyr1和Actyr2在大脑中的表达进行了定位。中华蜜蜂Actyr1、Actyr2的cDNA全长序列分别为1241 bp(GenBank登录号：KC814693)和1270 bp(GenBank登录号：KC814694),分别编码297、399个氨基酸残基。qRT-PCR分析结果表明,Actyr1和Actyr2在不同组织中的表达量为头部最高,其次是腹部表皮,触角和胸部肌肉的表达量最低,并且头部的表达量显著高于其他组织的表达量;原位杂交结果显示,Acytr1和Actyr2在中华蜜蜂大脑蘑菇体的凯尼恩细胞、触角叶周围的细胞处均有较强阳性着色。这些研究表明,Acytr1和Actyr2基因可能参与了蜜蜂的学习记忆,并且在相同的细胞中互相作用,共同调控蜜蜂的生物学功能。     

中华蜜蜂Malvolio基因 Acmvl 的克隆及组织表达分析

【目的】本研究克隆了中华蜜蜂Apis cerana cerana Malvolio (Mvl)基因的cDNA序列,分析了其编码蛋白的结构特点,并探讨其mRNA在内勤蜂、采蜜蜂和采粉蜂各部位组织中的表达差异,以期为该基因的生物学功能研究提供参考。【方法】利用RT-PCR技术从中华蜜蜂内勤蜂头部组织中扩增和克隆获得Acmvl的全长序列,并采用多种生物信息学软件分析Acmvl蛋白的结构特征;采用Real-time PCR对中华蜜蜂Acmvl在内勤蜂、采蜜蜂和采粉蜂各组织中的表达特征进行分析。【结果】Acmvl基因cDNA全长为2 130 bp（GenBank登录号：KP662686）,编码587个氨基酸,预测该蛋白分子量为65.86 kD,等电点为6.03,无信号肽,存在11个跨膜结构域、9个糖基化位点和14个潜在磷酸化位点;系统发育树分析结果显示,中华蜜蜂Acmvl与其他膜翅目昆虫Malvolio聚为一支,与小鼠Mus musculus和人Homo sapiens Nramp家族的Nramp2聚为另一大分支,且与小鼠、水稻 Oryza sativa 、黑腹果蝇 Drosophila melanogaster 和酵母Saccharomyces cerevisiae的Nramp家族同源体在跨膜区、跨膜区带电残基及转运蛋白特征结构域上有很高的保守性,尤其是与Nramp2。Acmvl 基因在中华蜜蜂各部位组织中均有表达,但高表达于内勤蜂的胸部及采蜜蜂和采粉蜂的腹部和足部,提示该基因表达的差异影响采集行为。【结论】Acmvl 属于Nramp基因家族,可能为Nramp2的同源基因,该基因影响采集行为可能与转运Cu²⁺, Mn²⁺和Fe²⁺（尤其是Fe²⁺）有关。     

p38 MAPK基因在西方蜜蜂越冬期表达模式的研究

[目的]p38 MAPK基因在昆虫的低温响应机制中发挥着重要作用,本研究旨在探究p38 MAPK基因在西方蜜蜂Apis mellifera越冬期内表达规律.[方法]本研究对西方蜜蜂p38 MAPK蛋白序列进行生物信息学分析,并利用荧光定量PCR技术检测该基因在意大利蜜蜂Apis mellifera ligustica、欧洲黑蜂Apis mellifera mellifera、高加索蜂Apis mellifera caucasica和卡尼鄂拉蜂Apis mellifera carnica于不同越冬时期和不同越冬方式下体内的表达量.[结果]西方蜜蜂38 MAPK包含1083 bp的开放阅读框区域,编码360个氨基酸,包含TGY双磷酸化三肽模体序列和磷酸化激活环序列.p38 MAPK在蜜蜂所有组织中均有表达,分别在胸部和腹部处于最高和最低表达丰度.38 MAPK mRNA在不同蜂种不同越冬时期的表达量存在显著差异,4个蜂种于室外越冬时p38 MAPK的表达量均显著高于室内越冬(P＜0.05);随着蜜蜂越冬持续时间的延长,意大利蜜蜂和欧洲黑蜂不同越冬方式下体内p38 MAPK的表达均呈现先上升后下降的表达趋势,高加索蜂在室外与室内越冬时体内该基因的表达量均表现出持续上升的表达趋势,该基因在卡尼鄂拉蜂中的表达趋势与其他3个蜂种呈现一定差异,卡尼鄂拉蜂p38 MAPK在室内越冬方式下表达量保持恒定,但在室外越冬方式下表现出先下降后上升的表达趋势;比较室外越冬情况下4个蜂种于不同越冬时期体内38 MAPK表达量后发现,除在次年的1月外,其他越冬时期4个蜂种体内p38 MAPK的表达量均存在显著差异(P＜0.05).[结论]p38 MAPK基因在西方蜜蜂越冬期内发挥着重要的生理功能,p38 MAPK信号通路可作为蜜蜂抗寒机制研究的候选信号通路.     

Antennal malformations in light ocelli drones of Apis mellifera (Hymenoptera, Apidae)

Malformed antennae of Apis mellifera light ocelli drones were drawn, dissected and mounted permanently on slides containing Canada balsam, in order to count the olfactory discs present in each segment, in comparison with the number of those structures in normal antennae of their brothers. Some drones presented morphological abnormalities in a single segment of the right or left antenna, but others had two or more malformed segments in a same antenna. Drones with malformations in both antennae were also observed. The 4th and 5th flagellum segments were the most frequently affected. In a low number of cases the frequency of olfactory discs in malformed segments did not differ from that one recorded for normal segments. However, in most cases studied, the antennal malformations brought about a significant reduction in the number of olfactory discs from malformed segments.     

Cloning and expression pattern of odorant receptor 11 in Asian honeybee drones,Apis cerana (Hymenoptera,Apidae)

Odorant receptors play a crucial role in the special recognition of scent molecules in the honeybee olfaction system. The odorant receptor 11 (AmOR11) in western honeybee drones (Apis mellifera) has been demonstrated to specifically bind to 9-oxo-2-decenoic acid (9-ODA) of queens. However, little is known regarding the functions of OR11 Asian honeybee drones (Apis cerana) in the context of their mating activities. In this study, the odorant receptor 11 gene (AcOr11) from A. cerana was cloned, and its expression profiles were examined during two developmental stages (immature and sexually mature) and different physiological statuses (flying and crawling). The cDNA sequence of AcOr11 was highly similar to that of AmOr11, and encoded a membrane-coupled protein of 384 amino acids. The results of qRT-PCR indicated that AcOr11 was expressed at higher levels in drone antennae compared to brains, and the expression was significantly up-regulated in sexually mature drone brains compared to immature brains. Interestingly, AcOr11 expression in brains of mature flying drones was dramatically higher than those of mature crawling drones. To our knowledge, this study demonstrate a link between AcOr11 gene expression in the brain of honeybee drones and behavior associated with sexual maturity and mating flight.     

Effect of entomopathogens on Africanized Apis mellifera L. (Hymenoptera: Apidae)

This study aimed to evaluate the effect of commercially used entomopathogens on Africanized Apis mellifera L. (Hymenoptera: Apidae). Four bioassays were performed: 1) pulverized entomopathogens on A. mellifera; 2) entomopathogens sprayed on a smooth surface; 3) entomopathogens sprayed on soy leaves; and 4) entomopathogens mixed with candy paste (sugar syrup). Five treatments were prepared: sterile distilled water (control), distilled water sterilized with Tween^® 80 (0.01%), and the commercial entomopathogens Metarhizium anisopliae E9 (1.0 × 10⁹ conidia mL^?1), Beauveria bassiana PL63 (1.0 × 10⁸ conidia mL^?1) and Bacillus thuringiensis var. kurstaki HD-1 (3.0 × 10⁸ spores mL^?1). Each treatment consisted of five repetitions, with 20 workers per repetition, which were stored in a plastic box and, later, in a biological oxygen demand (B.O.D.) incubator (27 ± 2 °C, RH of 60% ± 10%, 12-h photophase). The mortality of the workers was evaluated from 1 h to 240 h, and the data were analyzed using Bayesian inference. The workers killed by the ingestion of candy paste contaminated with the pathogens (products) were randomly separated and selected for the removal of the midgut. Each midgut was fixed in Bouin's solution and prepared for histology. B. bassiana was verified to reduce the survival of A. mellifera workers in all bioassays. Moreover, M. anisopliae reduced the survival of A. mellifera workers directly sprayed, on a smooth surface and mixed with candy. B. thuringiensis reduced A. mellifera survival on a smooth surface and mixed with candy paste. However, its effects were lower than that observed by B. bassiana. The treatments with the biological products did not induce morphometric alterations in the midgut of A. mellifera.     

Bombus impatiens (Hymenoptera: Apidae): an alternative to Apis mellifera (Hymenoptera: Apidae) for lowbush blueberry pollination.

The pollination effectiveness of the commercially reared bumble bee Bombus impatiens Cresson, was compared in field studies to the honey bee, Apis mellifera L., for lowbush blueberry, Vaccinium angustifolium Ait. A preliminary study indicated that B. impatiens had potential as an alternative pollinator. In a 3-yr study, percentage fruit set, percentage harvested berries, berry weight, and seeds per berry were compared in blueberry fields stocked at 7.5 A. mellifera hives per hectare to 5, 7.5, or 10 B. impatiens colonies per hectare. Percentage of harvested berries (yield) was significantly higher in fields stocked with B. impatiens at 10 colonies per hectare. No other parameters measuring pollinator effectiveness were significantly different at 5, 7.5, or 10 colonies per hectare. Flower handling time was significantly faster for B. impatiens and it more frequently collected blueberry pollen. All parameters of pollinator effectiveness were similar for B. impatiens, A. mellifera, and native wild bees in a follow-up study. Overall, B. impatiens was a suitable alternative to A. mellifera.     

Geographical patterns of mitochondrial DNA variation in Apis mellifera iberiensis (Hymenoptera: Apidae)

An extensive survey of mitochondrial haplotypes in honeybee colonies from the Iberian Peninsula has corroborated previous hypotheses about the existence of a joint clinal variation of African (A) and west European (M) evolutionary lineages. It has been found that the Iberian Peninsula is the European region with the highest haplotype diversity (12 haplotypes detected of the M lineage and 10 of the A lineage). The frequency of A haplotypes decreases in a SW-NE trend, while that of M haplotypes increases. These results are discussed in relation to hypotheses about the African origin of Apis mellifera and an early colonization of west Europe during intermediate Pleistocene glaciation events, followed by a regional differentiation. The extant pattern of haplotype frequency and distribution seems to be influenced at a regional scale by adaptation to local climatic conditions and the mobile beekeeping that has become a large-scale practice during the last decades. Other previous anthropogenic influences (Greek, Roman and Arab colonizations) are thought to be of minor importance in present day populations.     

Crystal structure of peptidoglycan recognition protein SA in Apis mellifera (Hymenoptera: Apidae)

Peptidoglycan recognition protein SA (PGRP‐SA) is a key pattern recognition receptor in the insect innate immune system. PGRP‐SA can bind to bacterial PGN and activate the Toll pathway, which triggers the expression and release of antimicrobial peptides to prevent bacterial infection. Here, we report the first structure of Apis mellifera PGRP‐SA from Hymenoptera at 1.86 Å resolution. The overall architecture of Am‐PGRP‐SA was similar to the Drosophila PGRP‐SA; however, the residues involved in PGN binding groove were not conserved, and the binding pocket was narrower. This structure gives insight into PGN binding characteristics in honeybees.     

Assessment of genetic variation in Apis mellifera jemenitica (Hymenoptera: Apidae) using Cytochrome Oxidase I gene sequences

The Arabian Honeybee Apis mellifera jemenitica is endemic to the Arabian Peninsula. It is highly adapted to temperature extremes and drought dominating the region. In this study, the mitochondrial Cytochrome Oxidase I (COI) was analyzed in 133 specimens of A. m. jemenitica from eight localities along the Red Sea cost of Saudi Arabia. Results revealed 33 synonymous, and 6 non-synonymous mutations within the COI sequences, resulting in change of 4 amino acids. Phylogenetic analysis based on either type of mutations revealed two main haplogroups accounting for 94% of the samples. In total Eighteen new haplotypes were identified and uploaded in the genebank, Fourteen of them are restricted to one/both haplogroups. All haplotypes identified in this study clustered with reference COI sequences of the sub-lineag Z (African Lineage). However one Haplotype (MW428270) represents high COI variability compared to other haplotypes and may resemble different evolutionary sub-lineage. Tajima's Neutrality Test (Ps = 0.025; D = -1.5) indicated population size expansion that took place after selective sweep and/or purifying selection.     

意大利蜜蜂amPFK基因的克隆与表达分析

[目的]本研究旨在明确意大利蜜蜂磷酸果糖激酶(amPFK)基因的序列特征及表达模式,为进一步研究amPFK在生殖和发育中的功能奠定理论基础.[方法]采用RT-PCR技术克隆了amPFK基因,并对其氨基酸序列和蛋白结构进行了分析;通过RT-qPCR检测了amPFK基因在意大利蜜蜂不同品级不同发育时期的表达模式.[结果]克...