【作者】 冯毛；

【导师】 李建科；

【作者基本信息】 中国农业科学院 ， 特种经济动物饲养， 2010， 博士

【摘要】 咽下腺是一对位于工蜂头部的外分泌腺,是合成、分泌蜂王浆的主要腺体。咽下腺分泌的蜂王浆不仅是蜂王、幼虫的主要食物,对蜜蜂的营养和级型分化有至关重要的作用,而且也是一种天然的营养食品和保健品。为了提高王浆的产量,我国从原种意大利蜜蜂(原意)(Apis mellfera L.)中成功选育出了王浆高产蜜蜂(浆蜂)(Apis mellfera L.)。作为目前世界上王浆产量最高的蜂种,浆蜂王浆高产的分子机理尚不清楚。浆蜂是从原意选育而来的,两者可以利用蛋白质组进行比较分析。本试验采用形态研究和蛋白质组相结合的研究方法,分析了原意和浆蜂工蜂咽下腺的发育过程,建立了工蜂咽下腺发育的蛋白质组图谱。利用MALDI-TOF/MS和MALDI-TOF-TOF/MS对蛋白进行鉴定,表达量分析及生化通路分析,了解咽下腺发育过程中蛋白表达调控的特点。并利用比较蛋白质组的方法分析原意和浆蜂咽下腺发育过程中蛋白质组的差异,探讨浆蜂王浆高产的机理。扫描电镜分析结果显示,各日龄浆蜂咽下腺小囊直径均大于原意。6-12日龄的工蜂咽下腺小囊直径最大。从1、3、6、12、15和20日龄原意工蜂咽下腺发育图谱上分别检测到175、189、209、190、168和151个蛋白质点。蛋白鉴定和差异蛋白表达量聚类分析结果显示:34个蛋白在咽下腺发育前期(1-3日龄)高表达。其中代谢相关类9个,蛋白质生物合成相关类10个,蛋白质折叠相关的6个,发育调节类3个,抗氧化类3个,转运相关1个,细胞骨架类2个。1日龄咽下腺中鉴定出了王浆主蛋白。31个蛋白在咽下腺发育中期(6-12日龄)中期高表达。参与蛋白质折叠的有4个,代谢相关的有3个,参与发育调节有2,蛋白质合成相关的1个,抗氧化相关蛋白质2个,骨架蛋白1个,王浆主蛋白18个。13个蛋白质在咽下腺发育后期(15-20日龄)高表达,除王浆主蛋白外,主要是葡萄糖氧化酶、α-葡萄糖苷酶和α-淀粉酶。浆蜂和原意咽下腺发育比较蛋白质组分析结果显示,参与物质和能量代谢的相关蛋白,参与蛋白质合成、转运、抗氧化的蛋白质,以及用于维持细胞形态和结构的骨架蛋白等蛋白在浆蜂中上调表达的数目均多于原意。王浆主蛋白在浆蜂中上调表达的蛋白数和蛋白丰度都高于原意。Western blot分析结果显示,浆蜂和原意1日龄工蜂的咽下腺中存在王浆蛋白,而且王浆蛋白的量随咽下腺的发育逐渐增多,6-12日龄左右达到顶峰,而后逐渐减少,与图谱分析结果一致。实时荧光定量PCR分析结果显示,王浆主蛋白1、2和3的mRNA在1日龄工蜂的咽下腺中就开始表达。基因表达趋势和蛋白表达趋势有差异。研究结果表明,浆蜂工蜂咽下腺细胞分化比原意快,发育比原意迅速,咽下腺小囊变大,咽下腺体积同时也变大。意蜂咽下腺分泌王浆的高峰在6-12日龄,1日龄的工蜂咽下腺就可分泌王浆,3日龄咽下腺已具备较强的分泌能力,为保证王浆的分泌活动,咽下腺前期和中期参与碳水化合物代谢和能量生成、蛋白折叠、发育调节、蛋白质合成、抗氧化、细胞骨架和转运等蛋白质高于发育后期,来完成此时的高分泌活动,咽下腺发育后期表达的蛋白主要与蜂蜜转化有关。这首次在蛋白质组水平上阐明了咽下腺的发育机理。与原意相比,浆蜂咽下腺不仅产浆量提高,而且王浆主蛋白的丰度也相应提高,这种进化策略是为了给蜜蜂幼虫提供平衡的营养保证幼虫的正常发育。为了实现王浆的高产,咽下腺不仅形态增加,而且细胞内代谢能需求增加,大量能量代谢蛋白质高表达,细胞骨架蛋白质高表达以保证腺体细胞的形态,蛋白质生物合成和折叠蛋白质的高表达既满足腺体发育的需要又满足大量王浆主蛋白的合成和分泌,由于王浆分泌量的增加,通过抗氧化蛋白的高表达来消除代谢产生的活性氧等有害物质的危害,保证腺细胞正常行使生理功能。这是首次从分子水平解释了王浆高产的分子机理。对原意咽下腺的功能蛋白质进行蛋白质互做网络分析发现了35个蛋白质在网络中起关键作用,这些关键的节点蛋白,为今后的基因操作如基因敲除、RNA干扰等方法进行分子改良提供靶向蛋白。更多还原

【Abstract】 The hypopharyngeal gland , sited in the head of the honeybee workers, is a paired of long tuberous organ connected to many acini, can secrete a proteinaceous substance named royal jelly which is fed to larvae, queens and drones and plays a key role in honeybee nutrition and caste differentiation. Royal jelly is becoming more and more popular for its nutritional, health, and pharmacological functions.High royal jelly producing bees (RJb) (Apis mellifera L.) which is selected from native Italian bees (ITb) (Apis mellifera L.) for increased royal jelly production in 1980’s, has become the best royal jelly producer in the world. But the mechanism for its high royal jelly production still keeps unclear. To better understanding the characteristic during the development of hypophargngeal gland, the hypopharyngeal gland was studied by the proteomic approach.The 1, 3, 6, 12, 15 and 20-days-old honeybee workers were selected in this study according to their age-related function polymorphism. The results of scanning electron microscopy showed that the acini diameters of hypopharyngeal gland of RJb were larger than that of ITb on each development stage, respectively. And the acini diameter both reached the highest level during 6-12 days.With the combination of two-dimensional gel electrophoresis, mass spectrometry, and protein engine identification tools that were applied to protein search engines and biological network software, a total of 175, 189, 209, 190, 168 and 151 protein spots were detected in the protein profiles on each development stage of ITb. Proteins were identified by MALDI-TOF/MS and MALDI-TOF-TOF/MS and the differentially expressed proteins were classified based on their biological functions and analyzed with hierarchical cluster software.The results showed that, among the 78 differentially expressed proteins, 34 proteins were up-regulated in the early development stage (1-3 days after eclosion), 9 proteins were involved in metabolism of carbohydrate and energy, 10 proteins were related to protein biosynthesis and 6, 3, 3, 1 and 2 proteins were assigned to protein folding, development regulation, antioxidant, transporter, and cytoskeleton, respectively. It was very interesting to identified major royal jelly proteins on 1-day-old hypopharyngeal gland.Thirty-one proteins were up-regulated on the medial stage (6-12 days), including 3 proteins involved in metabolism of carbohydrate and energy, 4 proteins related to protein folding, 1 protein for protein biosynthesis, 2 proteins for development regulation, 2 proteins for antioxidant, 1 protein acted as cytoskeleton, and 18 major royal jelly proteins.Thirteen proteins were up-regulated in the late stage (15-20 days), including 7 major royal jelly proteins and glucose oxidase,α-glycosidase andα-amylse. The comparative proteomic analysis between RJb and ITb showed that the amounts of proteins showed up-regulated trends in the hypopharyngeal of RJb on each development stage were much higher than that of the ITb, including proteins related to metabolism of carbohydrate and energy, protein biosynthesis, transporter, antioxidant, cytoskeleton, respectively.Real-time PCR analysis showed that the mRNA coded for major royal jelly protein 1, 2, 3 were expressed since the eclosion of honeybee workers. The expression trends between protein level and gene level were difference.The results indicate that the acini differentiation of RJb is faster than that of ITb, the volume and acini diameter are becoming big during the development of the hypopharyngeal gland.The protein abundance analysis certified that the workers can secrete royal jelly since eclosion, the hypopharyngeal glands are becoming more active and reach to the peak level on 6-12 days during the development. In order to ensure the high amounts of secretion, proteins related to metabolism of carbohydrate and energy, protein biosynthesis, development regulation, antioxidant, cytoskeleton and transporter were expressed more in early and medial development period than that of late stage. The proteins up-regulated on the late development stage were enzymes involved in the convertion of honey. This gives an explanation for the development of hypopharyngeal gland in protein level for the first time.The comparative proteomic analysis between RJb and ITb indicated that the protein abundance of major royal jelly proteins were higher than that of ITb. So the higher royal jelly production and higher protein abundance of major royal jelly proteins might be an evolution strategy to provide the balance nutrition for the development of larvae. For the purpose of high royal jelly production, proteins involving in energy metabolism and cytoskeleton proteins are in higher expression level for the cell differentiation and growth of hypopharyngeal gland, and proteins related to protein biosynthesis and protein folding are expressed more to ensure the synthesis and secretion of large amounts of major royal jelly proteins and the development of hypopharyngeal gland itself at the same time. Proteins with antioxidant functions are expressed in higher level to protect the gland from the oxidative damage occurred during the metabolism of the gland. For the first time we supply an explanation for the mechanism of high royal jelly production for high royal jelly producing bees from molecular level.Biological network analysis of all the identified proteins and differentially regulated proteins showed that 35 proteins were the key node proteins, these key node proteins probably play an important role in the development of hypopharyngeal gland and provide us some target proteins for further genetically manipulation of the hypopharyngeal gland.更多还原