西方蜜蜂(Apis mellifera L.)体内王浆蛋白MRJP6基因的表达

为探究蜜蜂王浆MRJP6基因在意大利蜜蜂体内的时空表达情况,为进一步的功能研究提供依据。本研究利用实时荧光定量PCR技术对西方蜜蜂不同级型、发育时期和组织内的mrjp6表达量进行检测和分析。结果表明mrjp在蜜蜂幼虫和蛹期的表达水平较低,与对照基本一致,但在成年蜜蜂体内表达量显著提高,约为对照的2~5~2~8倍(p0.05);在成年三型蜂体内,其在雄蜂体内表达量低,约为对照的2~5倍,而在雌性的工蜂和蜂王体内表达量明显较高,可达28倍(p0.05)。这说明mrjp6的表达与蜜蜂的性别有关。但其在工蜂和蜂王之间的表达没有差异,显示其表达与蜜蜂级型无关;进一步的研究表明,成年工蜂体内,mrjp6在哺育蜂和采集蜂体内表达量很高且比较稳定,通过对不同组织的检测表明该基因主要是在蜜蜂的头部特异性高表达。作为王浆蛋白家族的一员,mrjp6在成年雌性蜜蜂头部高水平表达,但又与典型的一般王浆蛋白在9日龄前后哺育蜂体内高表达、其他阶段低表达的模式不同,说明其具有不同于一般王浆蛋白的功能,分析其可能与蜜蜂神经系统的功能以及成年雌性蜜蜂复杂的行为发育模式有关。     

蜜蜂体内王浆主蛋白基因mrjp9的转录表达研究

蜜蜂体内有9种王浆蛋白基因(major royal jelly protein,MRJPs1～9),其中MRJPs1～5在蜂王浆中含量较高,是蜂王浆生物学功能的基础。MRJPs6～9在王浆中没有或含量极少,且功能未知。为研究非王浆蛋白组分的MRJP9的生物学功能,本研究用RT-PCR的方法对意大利蜜蜂Apis mellifera ligustica Spinola不同组织,不同部位,不同级型样本中mrjp9的转录水平进行检测和定量。结果发现mrjp9在蜜蜂的幼虫、蛹和成年蜜蜂的各组织部位均广泛转录表达,但其在幼虫、蛹和刚出房的成年蜜蜂体内表达水平较低,而在成年采集蜂体内表达水平则较高,其表达与蜜蜂的发育时期有关。通过对在成年蜜蜂体内各组织部位的表达水平进行检测的结果显示该基因主要在蜜蜂的头、胸和王浆腺等组织部位的表达较高,其他组织部位表达较少。此外,该基因也在雄蜂和蜂王体内广泛表达,不受蜜蜂性别和级型的影响。这些结果说明mrjp9是一与蜜蜂发育有关的基因,可能与蜜蜂的行为发育和分工调控有关。     

西方蜜蜂不同级型王浆主蛋白MRJP8基因的表达差异

王浆主蛋白在蜜蜂的级型分化中具有重要的功能。为探究mrjp8在西方蜜蜂Apis mellifera不同级型的表达模式及功能差异。【方法】 利用荧光定量PCR技术对西方蜜蜂工蜂、 雄蜂和蜂王不同发育时期和不同组织的mrjp8表达水平进行检测。【结果】 工蜂体内mrjp8在9日龄前后的毒腺组织内特异性高表达, 为参照基因表达量的上万倍, 在其他发育时期和组织的表达量则明显较低, 其表达具有明显的时空特异性; 在雄蜂体内其表达量与对照相当; 在蜂王体内表达量可达参照的近1 000倍, 没有组织特异性。【结论】 mrjp8的这种表达模式提示其在工蜂防御及维系蜂王长寿命方面有积极作用, 这为进一步研究该基因乃至整个王浆蛋白基因家族的进化和功能分化提供了依据。     

本期重点推介

正蜜蜂Apis spp.是具有级型分化的社会性昆虫。揭示其级型分化及调控机理,对认识社会性昆虫的演化形成机制、不同级型的发育和维持机理以及对其更好地加以应用均有重要的参考价值。已有研究表明,蜂王浆的主要蛋白成分———王浆主蛋白(major royal jelly proteins,MRJPs)在蜜蜂的级型分化中具有重要的功能。mrjp8是mrjps家族中较晚发现的一个成员。为了进一步明确mrjp8基因表达与蜜蜂级型分化的关系,福建农林大学蜂学学院李江红等对西方蜜蜂Apis mellifera不同发育龄期工蜂体内,以及成年工蜂、新出房蜂王和雄蜂不同组织中的mrjp8表达水平进行了检测,发现     

意大利蜜蜂血淋巴HP19蛋白的表达及功能

昆虫血淋巴蛋白HP19主要参与蜕皮激素调控昆虫的变态及发育进程,蜂群内的工蜂、蜂王和雄蜂具有不同的变态和发育历期,为探究hp19在调控意大利蜜蜂Apis mellifera ligustica三型蜂变态发育中的作用,本研究利用实时荧光定量PCR技术对hp19在不同发育时期的工蜂、雄蜂和蜂王体内的转录水平进行检测。结果表明,该基因在工蜂和雄蜂3龄幼虫体内表达量分别为参照基因的104和105倍,在5龄幼虫体内的表达则分别降为参照的10和102倍,蛹期表达量又都显著提高。hp19在蜂王整个幼虫期的表达保持在参照的102倍,封盖后逐渐上升,至化蛹前达到参照的105倍。在成年工蜂体内的表达为参照的104倍,而在新羽化蜂王和雄蜂体内表达水平为参照的107倍,但在性成熟的产卵蜂王和雄蜂体内的表达显著降低至参照的104倍。hp19在三型蜂体内这种不同的表达模式说明其不仅与三型蜂差异的变态发育历期有关,还可能与雄蜂和蜂王的生殖有关,具有多样的生物学功能。     

西方蜜蜂(Apismellifera)不同级型蜕皮激素早期应答因子ecr和usp的表达及功能分析

为探究蜕皮激素早期应答因子ecr和usp在不同级型的西方蜜蜂体内的表达及其他相关生物学功能和作用,本研究通过荧光定量PCR技术对不同级型、发育时期的西方蜜蜂中的ecr与usp表达水平进行检测。结果表明,ecr、usp在工蜂小幼虫体内的表达与参照相同,随后稍有增加,但在变态期急剧降至最低水平,而后的蛹期又逐步增加,在成年蜂时期的表达持续维持在较高水平;蜂王体内的变化趋势与工蜂基本类似,所不同的是各阶段的表达量都显著高于工蜂,且在产卵王体内的表达显著高于处女蜂王;雄蜂体内ecr的表达在小幼虫时期稍高,但此后至蛹期都一直处于较低的表达水平,至蛹后期才稍有增加,但羽化后水平较低,老龄雄蜂体内又稍有提高。但usp在雄蜂体内的表达却基本呈现出与发育时期无关的高表达,大致为参照的283倍。ecr与usp在三型蜂体内的这种表达模式揭示了MH不仅调控蜜蜂生长发育、蜕皮变态,还可能与蜜蜂的生殖有关,而usp在雄蜂体内的表达说明USP除了与Ec R组成活性复合体参与MH调控蜕皮与生殖外,还可能具有其他的生物学功能。该结果为进一步研究蜕皮激素及其效应因子的生物学功能提供了依据。     

短舌熊蜂及红光熊蜂耐寒力的测定

以过冷却点和冰点为评价指标,对短舌熊蜂Bombus terrestris的幼虫,蛹,成年工蜂,成年雄蜂,处女蜂王,越冬后的蜂王及红光熊蜂Bombus ignitus的成年工蜂,成年雄蜂,处女蜂王的耐寒力进行检测。结果显示,短舌熊蜂幼虫期耐寒力最低,其次是蛹,工蜂和雄蜂,处女王较高。耐寒力最高的是越冬后的蜂王,其含水量也最低。将短舌熊蜂与红光熊蜂的成年工蜂,成年雄蜂和处女蜂王三型蜂分别进行对比,结果显示,红光熊蜂成年雄蜂与处女蜂王的耐寒力要比短舌熊蜂高。样本的湿重和含水量与过冷却点和冰点无显著相关性。     

影响中华蜜蜂工蜂王浆腺活性的因素

王浆腺是一对位于工蜂头部的泡状腺体,是分泌王浆的主要腺体。青年工蜂的王浆腺在春夏肥大,分泌王浆,饲喂幼虫及蜂王。采集蜂的王浆腺呈可逆的萎缩状态(Free,1961)。越冬工蜂的王浆腺虽肥大,但无泌浆活性(Brouwers,1983)。本试验为了寻找诱发工蜂王浆腺活性的主要因素,为王浆生产提供理论依据,故研究了工蜂王浆腺活性与幼虫、蜂王、花粉、温度之间的关系。     

蜂王浆蛋白生物学功能的研究

蜂王浆是哺育蜂咽下腺与上颚腺分泌的供3日龄以内蜜蜂幼虫和蜂王食用的浆状物质,具有多种生物活性。王浆含有丰富的蛋白质,通过直接分离纯化王浆中的蛋白质分子,或通过王浆蛋白基因克隆表达以获得表达产物,进而研究王浆蛋白的功能。研究发现王浆中含有多种具有特定功能的蛋白质,如MRJP3具有免疫调节作用,Jelleine-I-IV及Royalisin具有抗菌作用,MRJP1具有抗肿瘤功能及可能的蜜蜂行为调节功能,57kDa蛋白及Apisimin的促细胞生长功能,57kDa蛋白具有抗疲劳功能等。随着王浆蛋白组分分离、基因克隆表达及其功能研究的深入,王浆蛋白将被应用到生物医药、细胞培养、组织工程等更多的研究领域。     

短舌熊蜂耐热性研究

【目的】测定高温对短舌熊蜂Bombus terrestris L.个体存活率和体内总蛋白含量的影响,为熊蜂授粉应用提供理论依据。【方法】以高温暴露致死率为耐热性的评价指标,对短舌熊蜂的工蜂幼虫,成年工蜂,成年雄蜂进行检测并对成年工蜂进行亚致死高温驯化。同时考察不同处理温度下熊蜂体内蛋白质含量变化。【结果】结果显示,短舌熊蜂的耐热性能从高到低依次是成年工蜂、成年雄蜂、工蜂幼虫,亚致死高温驯化可显著提高成年工蜂的耐热性能。熊蜂体内蛋白质含量随着温度的升高,呈先增加后减少的趋势,熊蜂体重与体内蛋白质含量无显著相关性。【结论】熊蜂不同虫态对高温的耐受性不同,可通过亚致死高温驯化措施来提高熊蜂的耐热性能和授粉效率。     

More than royal food - <Emphasis Type="Italic">Major royal jelly protein</Emphasis> genes in sexuals and workers of the honeybee <Emphasis Type="Italic">Apis mellifera</Emphasis>

Background

In the honeybee Apis mellifera, female larvae destined to become a queen are fed with royal jelly, a secretion of the hypopharyngeal glands of young nurse bees that rear the brood. The protein moiety of royal jelly comprises mostly major royal jelly proteins (MRJPs) of which the coding genes (mrjp1-9) have been identified on chromosome 11 in the honeybee’s genome.

Results

We determined the expression of mrjp1-9 among the honeybee worker caste (nurses, foragers) and the sexuals (queens (unmated, mated) and drones) in various body parts (head, thorax, abdomen). Specific mrjp expression was not only found in brood rearing nurse bees, but also in foragers and the sexuals.

Conclusions

The expression of mrjp1 to 7 is characteristic for the heads of worker bees, with an elevated expression of mrjp1-4 and 7 in nurse bees compared to foragers. Mrjp5 and 6 were higher in foragers compared to nurses suggesting functions in addition to those of brood food proteins. Furthermore, the expression of mrjp9 was high in the heads, thoraces and abdomen of almost all female bees, suggesting a function irrespective of body section. This completely different expression profile suggests mrjp9 to code for the most ancestral major royal jelly protein of the honeybee.

     

The MRJP/YELLOW protein family of Apis mellifera: identification of new members in the EST library

Major royal jelly proteins (named MRJP1-5) of honeybee (Apis mellifera), yellow proteins of Drosophila, together with putative proteins found in several bacteria, form a protein family termed the MRJP/yellow family. Members of the family exert diverse physiological functions and amongst eukaryotes appear to be restricted to the order Insecta. MRJPs constitute about 90% of total protein of royal jelly, which is secreted by nurse bees to feed the queen and growing larvae. We looked for mrjp and yellow homologues in a honeybee brain expressed sequence tags (EST) library. In addition to the five mrjp cDNAs previously characterized, we found three additional cDNAs encoding novel MRJPs and importantly, two cDNAs coding for orthologues of Drosophila yellow proteins. One yellow cDNA and all three cDNAs coding for the novel MRJPs were assembled completely, the sequence of the other yellow homologue was partially assembled. The data we present here supports the view that repeated duplications and functional divergence occurred during the evolution of MRJPs in honeybees, with even closely related MRJPs appearing to perform diverse physiological functions. Conversely, yellow protein orthologues appear to be conserved and thus candidates for maintaining the former function(s) of yellow proteins.     

Behavior and molecular physiology of nurses of worker and queen larvae in honey bees (Apis mellifera)

In a honey bee colony, worker bees rear a new queen by providing her with a larger cell in which to develop and a large amount of richer food (royal jelly). Royal jelly and worker jelly (fed to developing worker larvae) differ in terms of sugar, vitamin, protein and nucleotide composition. Here we examined whether workers attending queen and worker larvae are separate specialized sub-castes of the nurse bees. We collected nurse bees attending queen larvae (AQL) and worker larvae (AWL) and compared gene expression profiles of hypopharyngeal gland tissues, using Solexa/Illumina digital gene expression tag profiling (DGE). Significant differences in gene expression were found that included a disproportionate number of genes involved in glandular secretion and royal jelly synthesis. However behavioral observations showed that these were not two entirely distinct populations. Nurse workers were observed attending both worker larvae and queen larvae, and there was no evidence of a specialized group of workers that preferentially or exclusively attended developing queens. Nevertheless, AQL attended larvae more frequently compared to AWL, suggesting that nurses sampled attending queen larvae may have been the most active nurses. This study serves as another example of the relationship between differences in gene expression and behavioral specialisation in honey bees.     

LncRNA在蜜蜂级型分化中的功能研究

蜜蜂的级型分化被证实是由蜂王浆中的Royalactin决定,工蜂和蜂王幼虫在级型分化时编码基因的表达差异也被广泛研究.我们发现,在蜜蜂幼虫的级型分化过程中,lncRNA也有着显著的表达差异,因此认为,lncRNA也参与了蜜蜂的级型分化过程.进一步的分析显示,lncRNA可能通过影响上下游基因的转录和功能执行的方式,在蜜蜂早期发育的多细胞组织发育、神经系统发育和转录调控的过程中起到重要的调控作用.     

蜂王浆主蛋白研究进展

作为社会性昆虫,蜜蜂是研究社会行为和学习记忆的理想模式生物。王浆主蛋白（Major royal jelly protein, MRJP）是蜂王浆中蛋白质的主要成分,该家族一共有9个成员,MRJP1~MRJP9。所有mrjps均以串联排列的形式位于蜜蜂11号染色体上一个大约60 kb的DNA片段上。mrjp的同源体也存在于其他的膜翅目昆虫,均是通过yellow进化而来的。随着不断地进化,MRJPs家族进化出许多重要功能,其中最主要的就是营养功能。本文从MRJPs家族的基因及蛋白质结构、mRNA表达情况、进化和功能等方面进行综述,为今后开展相关研究提供理论支持。     

A comparison of honeybee (Apis mellifera) queen,worker and drone larvae by RNA‐Seq

Honeybees (Apis mellifera) have haplodiploid sex determination: males develop from unfertilized eggs and females develop from fertilized ones. The differences in larval food also determine the development of females. Here we compared the total somatic gene expression profiles of 2-day and 4-day-old drone, queen and worker larvae by RNASeq. The results from a co-expression network analysis on all expressed genes showed that 2-day-old drone and worker larvae were closer in gene expression profiles than 2-day-old queen larvae. This indicated that for young larvae (2-day-old) environmental factors such as larval diet have a greater effect on gene expression profiles than ploidy or sex determination. Drones had the most distinct gene expression profiles at the 4-day larval stage, suggesting that haploidy, or sex dramatically affects the gene expression of honeybee larvae. Drone larvae showed fewer differences in gene expression profiles at the 2-day and 4-day time points than the worker and queen larval comparisons (598 against 1190 and 1181), suggesting a different pattern of gene expression regulation during the larval development of haploid males compared to diploid females. This study indicates that early in development the queen caste has the most distinct gene expression profile, perhaps reflecting the very rapid growth and morphological specialization of this caste compared to workers and drones. Later in development the haploid male drones have the most distinct gene expression profile, perhaps reflecting the influence of ploidy or sex determination on gene expression.     

Effect of juvenile hormone treatment on caste differentiation in the honeybee, Apis mellifera

Synthetic juvenile hormone (methyl trans,trans,cis-10-epoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate, 1 μg/μl acetone per animal) (JH) was topically applied to 2- to 3-day-old worker honeybee larvae in the hive. Eighty per cent of the hormone-treated larvae were removed from their brood cell before pupation. Only 1 out of 42 adults showed characteristics of an intercaste. Fifty per cent of the control larvae (1 μl acetone) developed to adults, all of which were workers.After topical application of JH and feeding on royal jelly under in vitro conditions, the rate of survival is high (up to 85 per cent adults), but up to 67 per cent of queens and 44 per cent of workers exhibit eye malformations with characteristics of somatic mutation. Formation of a more solid web by the spinning larvae, shortening of the diapause by 1 to 2 days, and unusual shapes of mandibles, legs, and abdomen are a consequence of hormone treatment. The effects are less marked after application of 0·1 instead of 1 μg hormone or after its addition to the food (2 μg/g royal jelly). Treatment of the 2- to 3-day-old worker larvae and subsequent rearing on royal jelly is followed by a shift in caste differentiation from queens and workers to intercastes. In no case, are more queens developed after juvenile hormone treatment.Queen bee determinator, partially purified from royal jelly, induces a concentration-dependent shift from workers to queen differentiation. A threefold increase in the natural determinator concentration of royal jelly results in an almost exclusive (98 per cent) queen formation from 2- to 3-day-old worker larvae. In contrast to this direct effect, the influence of JH is explained as an indirect morphogenetic effect not directly coupled with honeybee caste differentiation.