两蜂种王浆蛋白质组分比较分析

王浆高产蜜蜂(Apis mellifera L.浆蜂)是我国20世纪90年代从意大利蜜蜂培育成功的新蜂种,产浆性能明显高于未经选育的意大利蜜蜂.中华蜜蜂(A. cerana cerana F.中蜂)是我国土生土长的蜂种,其蜂王浆产量低于以意大利蜜蜂为代表的西方蜜蜂.两蜂种所产蜂王浆的蛋白质组研究还未见报道.本研究利用双向电泳技术对浆蜂和中蜂新鲜蜂王浆全蛋白质组进行分析比较,为鉴定和判断蜂王浆的来源和蜂王浆功能成分的开发利用提供科学依据.     

家蚕茧色限性品种雌雄绢丝腺组织蛋白质组双向凝胶电泳分析

采用双向凝胶电泳和计算机辅助分析方法，分别对家蚕(Bombyx mori)限性黄茧品种雌蚕(黄茧)和雄蚕(白茧)的中部丝腺组织细胞蛋白质进行分离和比较分析。用银染的方法，两张图谱均可见到分离出600个以上的点，这些蛋白主要集中在20-70kD区域，在pH4-7上几乎均匀分布。比较分析发现，结黄茧的雌蚕中部丝腺组织细胞一些蛋白质的表达水平明显高于结白茧的雄蚕，有两种蛋白质只在雌蚕中表达，但表达量不高。这些差异蛋白质可能与有色茧丝的形成有关，或是与W染色体的易位片段上其它未知基因有关。     

两种剂量重离子辐照绵羊精子差异蛋白质组学研究

为检测和分析0.5和3.0Gy 2种剂量重离子辐照绵羊精子差异表达的蛋白质,用二维凝胶电泳分离精子蛋白,凝胶用硝酸银染色,PDQuest8.0检测分析差异表达蛋白斑点并经高效液相色谱串联离子阱质谱鉴定,结果显示:2种剂量凝胶图谱分析后得到8个共同差异蛋白斑点,鉴定为3种表达上调的蛋白质(谷氧还蛋白1、转录因子AP-2α...     

南方型紫花苜蓿叶片响应盐胁迫蛋白质组和转录组关联分析

为了从基因和蛋白质水平上揭示南方型紫花苜蓿适应盐胁迫环境的分子机制,以南方型紫花苜蓿Millennium为材料,对正常培养和盐胁迫条件下的2个样品叶片进行转录组和蛋白质组关联分析。结果表明,定量蛋白和基因关联系数为0.2485;变化趋势相反差异蛋白和基因表达的关联系数为-0.2440;变化趋势相同差异蛋白质和基因表达的关联系数为0.8122。鉴定出109个与差异基因表达趋势相同的差异蛋白,其中77个上调,32个下调,这些差异蛋白功能涉及光合作用、抗氧化物、信号传递、翻译后修饰、翻译和分子伴侣、胁迫防御、能量产生与转运、代谢和其它未知功能蛋白等。下调表达的蛋白主要与光合作用相关,而上调表达的蛋白主要参与了抗氧化物、信号传递和胁迫防御等。此外,关联发现了与紫花苜蓿盐胁迫响应相关的III类过氧化物酶、铁蛋白、谷胱甘肽S-转移酶、磷脂酰肌醇特异性磷脂酶C、LRR类受体激酶、ABA反应蛋白、钙联接蛋白2、液泡H+-ATP酶C亚基和NADP-苹果酸酶等差异蛋白。本研究通过高通量多组学数据的关联分析,发现一些可能作为紫花苜蓿耐盐潜在靶标蛋白(基因),这为深入认识紫花苜蓿盐胁迫的应答分子调控机制奠定了坚实的基础。     

小峰熊蜂工蜂蛹期发育蛋白质组分析

本文采用双向凝胶电泳法对小峰熊蜂工蜂蛹期的3个发育期进行蛋白质组研究,结果表明小峰熊蜂工蜂蛹期的白眼期（A期）、褐眼期（B期）和黑眼期（C期）分别检测到77、81和59个蛋白点,特有蛋白分别为7个、6个和1个,共有蛋白为48个,A期到B期显著上调的蛋白有9个,显著下调的蛋白有3个,B期到C期显著上调的蛋白有8个,显著下调的蛋白有3个,A期到C期显著上调的蛋白有15个,显著下调的蛋白有2个。研究还发现有17个蛋白是在A期和B期表达而在C期关闭;有2个蛋白是在A期表达,B期关闭,在C期又表达。本研究初步揭示了小峰熊蜂工蜂从蛹期发育到成蜂过程中,不仅需要一些保守蛋白来调控,而目．还需要一此特异蛋白。     

梅花鹿鹿茸再生干细胞蛋白质组双向电泳条件优化

鹿茸是目前唯一可以完全再生的哺乳动物附属器官,这种再生基于鹿茸再生干细胞。本研究以梅花鹿(Cervus nippon)鹿茸再生干细胞为样品,在处理方法、染色方法、蛋白纯化和等电聚焦条件4个方面对蛋白质组双向电泳进行优化。结果显示,利用Bullet Blender细胞组织破碎仪处理细胞优于超声波破碎;双染法染色能够得到更多且更清晰的蛋白点;等电聚焦总volt-hours在15 000 volt-hours时竖条纹相对较少;通过比较6种不同的蛋白提取方法与纯化方法组合,发现采用自制裂解液与双向电泳纯化试剂盒纯化相结合的方式获得的电泳图谱较好。通过综合优化后的双向电泳技术所得到的蛋白图谱中蛋白点相对较多且圆滑,条纹现象较轻,重复性较好,满足后续软件分析以及数据处理的要求,本研究为不同发育期梅花鹿鹿茸再生干细胞比较蛋白质组学研究提供了基础实验数据。     

中华蜜蜂两种化学通讯相关蛋白基因时空表达分析

利用Real-time PCR技术对中华蜜蜂(Apis cerana cerana)两个化学通讯相关蛋白：气味结合蛋白Ac-ASP2和化学感受蛋白Ac-ASP3基因的时空转录谱进行了研究。结果表明,Ac-ASP2是一个特异表达于中蜂触角的基因,在幼虫及蛹中均不表达;利用2-ΔΔCt的相对定量方法,发现Ac-ASP2在成虫触角中具有几个丰度较高的日龄,即1、9、15、27和30日龄,这几个高表达日龄均较其它时期高出近10倍以上;利用标准曲线的绝对定量方法,Ac-ASP3在其不同器官中的原始拷贝数关系为：翅>腹>胸>足>触角>头,其中翅、腹和胸部模板拷贝数较高（均达到106数量级）,而在足、触角以及头中的表达丰度相对较低（均为105数量级）,表明Ac-ASP3与中蜂翅与腹部的化学感受行为有密切的关系。     

金黄色葡萄球菌乳房炎奶牛血浆的比较蛋白质组研究

为了分析金黄色葡萄球菌(Staphylococcus aureus)自然感染引起的隐性乳房炎奶牛血浆蛋白的表达变化,经细菌培养分离鉴定了乳中金黄色葡萄球菌,选择其感染的奶牛.采用二维凝胶电泳技术分离了临床健康奶牛和乳房炎奶牛的血浆蛋白,考马斯亮蓝G-250染色后PDQuest 8.0软件检测差异表达蛋白点,高效液相色谱串联离子阱质谱鉴定.结果发现,金黄色葡萄球菌乳房炎奶牛血浆中有10个蛋白点的表达量发生改变,其中6个蛋白点经质谱鉴定为结合珠蛋白、转甲状腺素蛋白和α1酸性糖蛋白等4种蛋白.金黄色葡萄球菌感染可造成奶牛血浆结合珠蛋白、α1酸性糖蛋白和血清淀粉样蛋白A的表达量增加.ELISA法测定血浆结合珠蛋白的结果也发现,金黄色葡萄球菌感染奶牛血浆结合珠蛋白水平显著高于健康牛(P＜0.01).结果提示乳房炎奶牛血浆蛋白的变化可为揭示金黄色葡萄球菌感染乳腺炎症的应答提供依据.     

光系统Ⅱ抑制型除草剂Atrazine诱导小麦幼苗叶绿体蛋白质组变化分析

为了研究除草剂作用机理,用光系统Ⅱ抑制型除草剂阿特拉津（Atrazine）处理小麦幼苗,用2-DE技术和生物质谱方法,分析了叶绿体蛋白质组的变化。结果发现,在10mg·L-1浓度处理时,有7个叶绿体蛋白质斑点（斑点1,50kDa/PI8.1;斑点2,41kDa/PI8.4;斑点3,41kDa/PI7.6;斑点4,23kDa/PI7.1;斑点5,31kDa/PI5.0;斑点6,35kDa/PI8.9;斑点7,14kDa/PI8.1）丢失。对7个发生变化的斑点利用MALDI-MS方法,于NCBI进行数据查询,其中,有6个叶绿体蛋白质归属得到鉴别,它们是Calvin循环中,固定CO2的RuBPcase的激活酶（2个同工体和1个β型前体）,在H2O氧化裂解中起重要作用的23kDa氧释放蛋白（psbp protein）,在能量转贮中起重要作用的3-磷酸甘油酸激酶和催化HCO3--CO2水合作用可逆反应的碳酸酐酶。研究表明,叶绿体蛋白质组中丢失的6个蛋白质是Atrazine处理的相关蛋白。     

小麦幼穗蛋白质双向电泳条件的优化

本研究以温光敏小麦为试材,用TCA/丙酮和酚提取法提取小麦幼穗蛋白样品,进行了双向电泳优化分析,并对双向电泳过程中出现的问题进行了讨论。结果表明,用TCA/丙酮法提取小麦幼穗蛋白质其产率（浓度）高于酚提取法。SDS-PAGE电泳显示,用TCA/丙酮提取法提取的蛋白质能获得较清晰条带,分辨率较高,而酚提取法提取的蛋白质其条带模糊,分辨率低。对蛋白质纯化除盐可以提高分辨率,减少横竖纹,获得背景清晰的圆形蛋白点。通过ImageMasterTM 2D Platinum5.0软件分析凝胶图谱,结果显示纯化后可降低噪点,纯化后蛋白点数可从未纯化蛋白点数的216增加到583。显然,采用TCA/丙酮法可获得高浓度高质量的蛋白质,而进一步纯化、除盐离子可进一步获得背景清晰可高重复性的电泳图谱。在双向电泳实验过程中,观察到一些异常缺陷胶的出现,如双向电泳图谱中蛋白点扩散,蛋白聚集形成斑点串,没有点或点很少,出现纵纹横纹及图谱扭曲等影响图谱质量的严重问题,本研究对这些问题做了分析并提出了解决方案。     

Chemistry and bioactivity of royal jelly from Greece

Twenty-five compounds were identified from the dichloromethane and methanol extracts of royal jelly from Greece. Among them, 16 compounds are reported for the first time as royal jelly constituents, whereas 7 of them are isolated for the first time as natural products. The 7 new compounds were fatty acid derivatives: 10-acetoxydecanoic acid (1), trans-10-acetoxydec-2-enoic acid (2), 11-oxododecanoic acid (3), (11S)-hydroxydodecanoic acid (4), (10R,11R)-dihydroxydodecanoic acid (5), 3,11-dihydroxydodecanoic acid (6), and (11S),12-dihydroxydodecanoic acid (7). The structures of the isolated compounds were determined by spectroscopic methods, mainly by the concerted application of 1D and 2D NMR techniques (HMQC, HMBC) and mass spectrometry. The studied sample and the isolated compounds were tested for their antimicrobial activity against Gram-positive and Gram-negative bacteria and fungi and exhibited interesting activities.     

Storage stability assessment of freeze-dried royal jelly by furosine determination

The effect of freeze-drying and the assessment of the storage stability of freeze-dried royal jelly (RJ) were investigated by the determination of furosine and blocked lysine. The level of furosine in the RJ samples collected from cells at different times (1, 2, and 3 days after grafting) showed that the Maillard reaction had already occurred in the hive as indicated by the increase in furosine: from 9.6 to 20.8 mg/100 g of protein. Freeze-dried RJ was more prone to the early stage of the Maillard reaction than fresh RJ, as confirmed by the significantly higher furosine values found after 12 months, both at 4 degrees C (253.4 versus 54.9 mg/100 g of protein) and at room temperature (884.3 versus 332.5 mg/100 g of protein). After 18 months at room temperature, the lyophilized samples reached a furosine level of 1440.4 mg/100 g of protein, which corresponded to the blocked lysine levels, amounting to 24% of total lysine.     

Novel royal jelly proteins identified by gel-based and gel-free proteomics

Royal jelly (RJ) plays an important role in caste determination of the honeybee; the genetically same female egg develops into either a queen or worker bee depending on the time and amount of RJ fed to the larvae. RJ also has numerous health-promoting properties for humans. Gel-based and gel-free proteomics approaches and high-performance liquid chromatography-chip quadruple time-of-flight tandem mass spectrometry were applied to comprehensively investigate the protein components of RJ. Overall, 37 and 22 nonredundant proteins were identified by one-dimensional gel electrophoresis and gel-free analysis, respectively, and 19 new proteins were found by these two proteomics approaches. Major royal jelly proteins (MRJPs) were identified as the principal protein components of RJ, and proteins related to carbohydrate metabolism such as glucose oxidase, α-glucosidase precursor, and glucose dehydrogenase were also successfully identified. Importantly, the 19 newly identified proteins were mainly classified into three functional categories: oxidation-reduction (ergic53 CG6822-PA isoform A isoform 1, Sec61 CG9539-PA, and ADP/ATP translocase), protein binding (regucalcin and translationally controlled tumor protein CG4800-PA isoform 1), and lipid transport (apolipophorin-III-like protein). These new findings not only significantly increase the RJ proteome coverage but also help to provide new knowledge of RJ for honeybee biology and potential use for human health promotion.     

Specific hydroxy fatty acids in royal jelly activate TRPA1

This is the first report of TRPA1 activation by fatty acids. Activation of TRPA1 and TRPV1 induces thermogenesis and energy expenditure enhancement. In this study, we searched for novel agonists of TRPA1 and TRPV1 from a nonpungent food, royal jelly (RJ). We measured the activation of human TRPA1 and TRPV1 by RJ extracts and found that the hexane extract contains TRPA1 agonists. The main functional compounds in the hexane extract were trans-10-hydroxy-2-decenoic acid (HDEA) and 10-hydroxydecanoic acid (HDAA). These are characteristic fatty acids of RJ. Their EC50 values were about 1,000 times larger than that of AITC, and their maximal responses were equal. They activated TRPA1 more strongly than TRPV1. Their EC50 values for TRPV1 were 2 times larger, and the maximal response was less than half of that for TRPA1. Next, we studied the potencies of other lipid components for both receptors. Most of them have higher affinity to TRPA1 than TRPV1. Among them, dicarboxylic acids showed equal efficacy for both receptors, but those are present in only small amounts in RJ. We concluded that the main function of RJ is TRPA1 activation by HDEA and HDAA, the major components of the RJ lipid fraction.     

蜂王浆三针夹取式拣虫机的设计与试验

蜂王浆拣虫是挖浆前必须完成的工序。由于蜜蜂幼虫个体较小、数目庞大,因此人工拣虫过程劳动强度非常大;然而中国养蜂人员老龄化日益严重,且目前蜂王浆拣虫机械化尚未成熟。该文针对中国养蜂现状提出一种以三针夹取的方式将王台中蜜蜂幼虫夹出的方法。以三针夹取的拣虫方式构建蜂王浆拣虫机整机方案。三根针在王台孔内壁同时做向心运动或分离运动,将蜜蜂幼虫夹住或松开。对传动机构中球面槽轮和凸轮的参数进行计算。通过有限元分析得出动力输入端脚踏板受力情况。计算得出球面槽轮圆柱销弧长7.5π,槽深20.68 mm;得出凸轮的理论廓线;有限元分析结果得出动力输入方式可靠。该机器在保留蜂王浆幼虫完整的前提下,一次性将整条王台(64孔)中蜜蜂幼虫全部拣出,可实现连续作业,拣虫效率为10条/min时为人工的10倍,克服了蜂场手工作业时间长和劳动强度大问题。对扩大中国蜂业养蜂规模有着极大的促进作用。     

Furosine: a suitable marker for assessing the freshness of royal jelly

Fifteen commercial samples of royal jelly, consisting of 10 imported samples, and 5 samples of known origin obtained freshly harvested from beekeepers, were analyzed for protein, lysine, and furosine content. In addition, a commercial sample of royal jelly, at the beginning of its commercial shelf life, was stored for 10 months both at 4 degrees C and at room temperature in order to assess the development of the Maillard reaction (furosine) and relative nutritional damage (blocked lysine). The commercial royal jelly products contained different amounts of furosine, ranging from 37.1 to 113.3 mg/100 g protein, evidence of different storage times and conditions. The average furosine content of the royal jelly samples of known origin and harvesting was significantly lower than that of the imported samples (41.7 versus 73.6 mg/100 g protein, respectively). With regard to shelf life, furosine content increased significantly from 72.0 mg/100 g protein to 500.8 mg/100 g protein after 10 months of storage at room temperature, while it increased to a much lower level (100.5 mg/100 g protein) when the royal jelly was stored at 4 degrees C. However, nutritional damage, expressed as blocked lysine (calculated indirectly from the furosine content), was minor or negligible, 11.9 and 2.3% of total lysine, in samples stored at room temperature and at 4 degrees C, respectively. Lysine was determined by an innovative procedure based on high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The results showed that furosine is a suitable index for assessing the quality and freshness of royal jelly.     

Proteomic analysis of royal jelly from three strains of western honeybees (Apis mellifera)

To compare the protein complement of royal jelly (RJ) from high RJ producing honeybees ( Apis mellifera L.), a strain of A. mellifera artificially selected for increased RJ production from Italian honeybees in China for more than two decades was compared to those of native Italian honeybees ( A. mellifera L.) and Carnica honeybees ( A. mellifera C.); the protein in RJ from these three strains of honeybees was partially identified by using a combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS), and a protein engine identification tool applied to the honeybee genome. The results showed that 152, 157, and 137 proteins were detected in the three species of RJ; among which 57, 57, 51 high abundant proteins ere identified, respectively. Most identifited spots, 45, 45, 41, were assigned to major royal jelly proteins (MRJPs). Remarkable differences were found in the heterogeneity of the MRJPs, in particular, MRJP3. Also, 3-glucose oxidase, 1-peroxiredoxin (PRDX), and 1-glutathione S-transferase (GST) S1 were identified in three RJ samples. Furthermore, during the determination of the peptides mass fingerprinting (PMF) of each spot, for the first time, PRDX and GST S1 proteins have been identified in RJ. Thus, the results suggest that the protein complement of high RJ producing honeybees is not different compared to native Italian honeybees, while a difference remains between Carnica honeybees.     

Application of solid/liquid extraction for the gravimetric determination of lipids in royal jelly

Gravimetric lipid determination is a major parameter for the characterization and the authentication of royal jelly quality. A solid/liquid extraction was compared to the reference method, which is based on liquid/liquid extraction. The amount of royal jelly and the time of the extraction were optimized in comparison to the reference method. Boiling/rinsing ratio and spread of royal jelly onto the extraction thimble were identified as critical parameters, resulting in good accuracy and precision for the alternative method. Comparison of reproducibility and repeatability of both methods associated with gas chromatographic analysis of the composition of the extracted lipids showed no differences between the two methods. As the intra-laboratory validation tests were comparable to the reference method, while offering rapidity and a decrease in amount of solvent used, it was concluded that the proposed method should be used with no modification of quality criteria and norms established for royal jelly characterization.     

Fast determination of adenosine 5'-triphosphate (ATP) and its catabolites in royal jelly using ultraperformance liquid chromatography

To obtain insight into the metabolic regulation of adenosine 5'-triphosphate (ATP) in royal jelly and to determine whether ATP and its catabolites can be used as objective parameters to evaluate the freshness and quality of royal jelly (RJ), a rapid ultraperformance liquid chromatography (UPLC) method has been developed for feasible separation and quantitation of ATP and its catabolites in RJ, namely, adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), inosine monophosphate (IMP), inosine (HxR), and hypoxanthine (Hx). The analytes in the sample were extracted using 5% precooled perchloric acid. Chromatographic separation was performed on a Waters Acquity UPLC system with a Waters BEH Shield RP18 column and gradient elution based on a mixture of two solvents: solvent A, 50 mM phosphate buffer (pH 6.5); and solvent B, acetonitrile. The recoveries were in the range of 86.0-102.3% with RSD of no more than 3.6%. The correlation coefficients of six analytes were high (r(2) ≥ 0.9988) and within the test ranges. The limits of detection and quantification for the investigated compounds were lower, at 0.36-0.68 and 1.22-2.30 mg/kg, respectively. The overall intra- and interday RSDs were no more than 1.8%. The developed method was successfully applied to the analysis of the analytes in samples. The results showed that ATP in RJ sequentially degrades to ADP, AMP, IMP, HxR, and Hx during storage.     

2次降雨条件下不同土壤细沟侵蚀分析

不同土壤细沟侵蚀发育对比研究是探讨细沟侵蚀机制的必要内容。通过间隔为24 h的2次人工模拟降雨,在不同降雨强度(1.5,2.0,1.0 mm/min)、不同坡长(5、10 m)的试验条件下,分析坡度为20°时塿土和黄绵土2种土壤细沟侵蚀过程中产流产沙、空间变化的差异。结果表明:1)从坡面产流量来看,2种土壤在2次人工降雨过程中有相似的产流过程,坡长和降雨强度相同时,塿土坡面产流量大于黄绵土;2)从坡面形态看,第1场降雨过程中塿土坡面表面积、细沟侵蚀强度大于黄绵土坡面,但黄绵土坡面一旦发生细沟侵蚀,其体积变化幅度剧烈于塿土坡面;3)塿土在第1场降雨过程中侵蚀速率的变化过程可以反应细沟发育的各个阶段,较大降雨强度使黄绵土发生细沟侵蚀侵蚀,其细沟发育的各个阶段持续时间长于塿土,即黄绵土细沟形态变化缓慢。