王浆主蛋白MRJP7基因在意大利蜜蜂体内的表达

王浆蛋白是蜂王浆生物功能的物质基础,是由王浆蛋白基因家族(mrjps)编码合成的。但部分家族成员如MRJP7在王浆中的含量极少甚至检测不到。基因功能与其在生物体内的时空表达特性相关,为探究mrjp7的生物学功能,本研究利用荧光定量PCR技术对mrjp7在不同发育时期的工蜂和成年工蜂、雄蜂和蜂王的不同组织部位的表达进行定量检测。结果显示mrjp7在成年雄蜂体内的表达水平最低,成年蜂王次之,且在它们的各不同组织部位之间的表达量差异较小。该基因在工蜂幼虫和蛹期的表达同样较低,但在羽化后9日龄前后的哺育蜂王浆腺和头部特异性高表达,这与哺育蜂分泌蜂王浆哺育幼虫和蜂王的功能是相适应的,该结果在转录水平上证实了mrjp7的营养功能,为进一步的研究和应用打下了理论基础。     

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。     

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     

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。     

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。     

蜜蜂王浆主蛋白（MRJPs）基因家族结构与功能概述

王浆主蛋白（Major royal jelly proteins,MRJPs）是王浆中的水溶性蛋白,为王浆蛋白的主要组分。因结构与功能的相似性,各王浆蛋白编码基因构成一个基因家族。目前该基因家族已鉴定出9个成员,依次命名为mrjp 1~9。该家族为单系群,拥有共同的祖先—yellow-e3,各成员之间具有较高的同源性。随着进化的进行,该家族逐步进化出营养及其它多种生物学功能。本文从该基因家族成员的鉴定、基因和蛋白质的结构特征、进化、功能以及其表达调控等多个方面进行综述,以期为相关的研究和应用提供帮助。     

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.     

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。     

食物决定蜜蜂身份

新的研究可能对为什么蜜蜂的幼虫在进食了蜂王浆之后会成长为蜂王而不是工蜂作出解释。尽管存在于蜂王浆中的特别成分大体上依然是一个谜,但Robert Kucharksi及其同事现在报道说,进食蜂王浆好像可以通过抑制一种被称为甲基化的过程来影响多种蜜蜂基因的表达。这种对某个基因DNA的化学修饰会降低该基因的表达,而这一过程有部分是由酶Dnmt3所驱动的。当研究人员将蜜蜂幼虫体内的Dnmt3活性破坏了以后,这一改变会出现拟似蜂王浆的效果。使得发育中的蜜蜂幼虫更像蜂王而不像工蜂。     

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.     

Proteomic analysis of honeybee (Apis mellifera L.) pupae head development

The honeybee pupae development influences its future adult condition as well as honey and royal jelly productions. However, the molecular mechanism that regulates honeybee pupae head metamorphosis is still poorly understood. To further our understand of the associated molecular mechanism, we investigated the protein change of the honeybee pupae head at 5 time-points using 2-D electrophoresis, mass spectrometry, bioinformatics, quantitative real-time polymerase chain reaction and Western blot analysis. Accordingly, 58 protein spots altered their expression across the 5 time points (13-20 days), of which 36 proteins involved in the head organogenesis were upregulated during early stages (13-17 days). However, 22 proteins involved in regulating the pupae head neuron and gland development were upregulated at later developmental stages (19-20 days). Also, the functional enrichment analysis further suggests that proteins related to carbohydrate metabolism and energy production, development, cytoskeleton and protein folding were highly involved in the generation of organs and development of honeybee pupal head. Furthermore, the constructed protein interaction network predicted 33 proteins acting as key nodes of honeybee pupae head growth of which 9 and 4 proteins were validated at gene and protein levels, respectively. In this study, we uncovered potential protein species involved in the formation of honeybee pupae head development along with their specific temporal requirements. This first proteomic result allows deeper understanding of the proteome profile changes during honeybee pupae head development and provides important potential candidate proteins for future reverse genetic research on honeybee pupae head development to improve the performance of related organs.     

意蜂王浆生产性能形态学遗传标记的研究

测定19只工蜂头部咽下腺的两条腺体长度和重量,经生统t检验,发现同一工蜂的左、右两条咽下腺长度、重量差异都不显著（P＜0.05）。测定11群 "浙农大1号"意蜂(Apis mellifera ligustica)的330只工蜂的头重、体重、头体重比、咽下腺小囊数和咽下腺长度。在2001年3月30日至4月26日浙江淳安的油菜和紫云英花期测定试验蜂群的王浆产量,分析工蜂头重、头体重比、咽下腺小囊数和咽下腺长度与每千克蜂的王浆产量之间的相关性,发现咽下腺长度与王浆产量的相关性最大,小囊数次之,咽下腺长度可作为意蜂王浆生产性能较理想的形态学遗传标记。 Abstract:The lengths of hypopharyngeal glands (HG) from the left and right side were determined in 19 workers of honeybee(Apis mellifera ligustica).There were no significant differences (P＜0.05) in length between the left and the right in one worker′s hypopharyngeal gland.Three hundred and thirty workers were collected from eleven colonies of "ZND No.1" Italian honeybee(Apis mellifera ligustica) respectively.Head weight,body weight,ratio between head weight and body weight,bursa number and length of hypopharyngeal gland(HG)were tested in these samples.Royal jelly productions were determined during the flowing period of rape and Chinese milk retch from March 30 to April 26 in Chun′an County of Zhejiang Province in 2001.The correlation analysis between royal jelly production and head weight,ratio between head weight and body weight,bursa number of HG,and length of HG were conducted.The correlation coefficient between royal jelly production and length of HG was the largest.The correlation coefficient between royal jelly production and bursa number was the second.It was suggested that the length of HG could be used as one of genetic markers for the production performance of royal jelly.     

Proteome comparison of hypopharyngeal gland development between Italian and royal jelly producing worker honeybees (Apis mellifera L.)

The hypopharyngeal gland (HG) of the honeybee (Apis mellifera L.) produces royal jelly (RJ) that is essential to feed and raise broods and queens. A strain of bees (high royal jelly producing bee, RJb) has been selected for its high RJ production, but the mechanisms of its higher yield are not understood. In this study, we compared HG acini size, RJ production, and protein differential expressions between the RJb and nonselected honeybee (Italian bee, ITb) using proteomics in combination with an electron microscopy, Western blot, and quantitative real-time PCR (qRT-PCR). Generally, the HG of both bees showed age-dependent changes in acini sizes and protein expression as worker behaviors changed from brood nursing to nectar ripening, foraging, and storage activities. The electron microscopic analysis revealed that the HG acini diameter of the RJb strain was large and produced 5 times more RJ than the ITb, demonstrating a positive correlation between the yield and HG acini size. In addition, the proteomic analysis showed that RJb significantly upregulated a large group of proteins involved in carbohydrate metabolism and energy production, those involved in protein biosynthesis, development, amino acid metabolism, nucleotide and fatty acid, transporter, protein folding, cytoskeleton, and antioxidation, which coincides with the fact that the HGs of the RJb strain produce more RJ than the ITb strain that is owing to selection pressure. We also observed age-dependent major royal jelly proteins (MRJPs) changing both in form and expressional intensity concurrent with task-switching. In addition to MRJPs, the RJb overexpressed proteins such as enolase and transitional endoplasmic reticulum ATPase, protein biosynthesis, and development proteins compared to the ITb strain to support its large HG growth and RJ secretion. Because of selection pressure, RJb pursued a different strategy of increased RJ production by involving additional proteins compared to its original counterpart ITb. To our knowledge, this morphological and proteomic comparison study on the HG of the two strains of worker honeybees associated with their age-dependent division of labor is the first of its kind. The study provided not only the quantity and quality differences in the HG from the RJb and the ITb, but also addressed the cellular and behavioral biology development question of how the RJb strain can produce RJ more efficiently than its wild type strain (ITb).