家蚕蛾触角蛋白的双向电泳分析

为探讨家蚕Bombyx mori蛾触角发生发育、结构与功能的分子基础和调控机制, 我们采用双向电泳结合基质辅助质量飞行时间质谱(MALDI-TOF/MS)技术初步探讨了家蚕蛾触角蛋白及其雌雄表达差异。采用ImageMast 6.0软件分析电泳图谱, 在家蚕蛾触角中检测到约550个蛋白点, 主要集中在分子量14～70 kD, 等电点4～8之间。从雌雄蛾触角电泳图谱中分别检测到419和489个蛋白点, 其中雌雄匹配蛋白点有326对, 匹配率为71.81%。雌雄间表达差异点有34个, 雌雄分别所特有的特异蛋白点分别为9和20个。对特异和差异点进行MALDI-TOF/MS鉴定获得其中5种蛋白--成虫原基生长因子(imaginal disk growth factor)、表皮蛋白RR-1基序15(cuticular protein RR-1 motif 15)、硫醇过氧化还原酶(thiol peroxiredoxin)、空泡ATP酶B亚基(vacuolar ATPase B subunit)以及gasp前体(gasp precursor), 它们在雄蛾触角中表达量都比雌蛾高。这为家蚕触角蛋白的进一步研究提供了基本信息。     

拟黑多刺蚁成虫头部USP基因的定位与定量分析及其与EcR基因相关性

【目的】超气门蛋白（ultraspiracle protein, USP）是蜕皮激素作用靶标的重要组成部分。本研究拟通过分析在拟黑多刺蚁Polyrhachis vicina Roger USP基因PvUSP在不同品级成虫头部mRNA表达的差异,推测PvUSP对其脑神经功能或行为的影响;拟通过饲喂PvUSP dsRNA对拟黑多刺蚁不同品级成虫PvUSP进行RNA干扰（RNAi）,推测PvUSP对虫体生理功能的影响及其与蜕皮激素受体（ecdysone receptor, EcR）基因PvEcR之间功能的相关性。【方法】利用荧光原位杂交及荧光实时定量PCR技术对拟黑多刺蚁不同品级成虫头部PvUSP mRNA组织分布与表达水平进行检测;通过饲喂PvUSP dsRNA对拟黑多刺蚁不同品级成虫PvUSP进行RNAi,采用荧光实时定量PCR检测拟黑多刺蚁PvUSP与PvEcR表达水平的变化。【结果】PvUSP mRNA广泛表达于拟黑多刺蚁不同品级成虫头部（主要分布于蕈形体）,不同品级成虫头部PvUSP mRNA的表达量不同,工蚁头部表达量最高,雄蚁头部次之,雌蚁头部的表达量最低;RNAi沉默PvUSP后,与对照组相比,PvUSP mRNA在不同品级拟黑多刺蚁成虫体内表达量均明显降低,并存在显著差异(P<0.01);与对照组相比,PvEcR mRNA在各品级表达量均有增加,工蚁和雄蚁表达量变化不显著（P>0.05）,但雌蚁体内PvEcR mRNA表达量显著增加（P<0.05）。【结论】PvUSP基因对拟黑多刺蚁不同品级头部神经系统的构建、功能作用的发挥有关;拟黑多刺蚁PvUSP基因与PvEcR基因可能在异源二聚体形成过程中存在关联性或功能的互补性;PvUSP可能对雌蚁的生殖功能产生重要影响。     

食源性肥胖大鼠下丘脑差异表达蛋白的蛋白质组学分析

建立食源性肥胖大鼠模型,对正常大鼠和肥胖大鼠下丘脑全蛋白进行双向凝胶电泳,产生下丘脑蛋白双向凝胶电泳图谱.对图谱进行比对分析后,从凝胶上切取差异表达的蛋白点,经胶内酶解,通过基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF-MS) 对酶解后的肽段进行分析,再经数据库（NCBInr）检索,对蛋白质进行鉴定.研究发现,正常组表达图谱可检测到1　160±15（n=5）个蛋白点,肥胖组表达图谱可检测到1　070±10 （n=5）个蛋白点,与对照组相比,匹配率大于80％.并且成功鉴定了17种差异表达蛋白质,其中有7 种在肥胖组表达上调,10种表达下调.它们分别属于代谢酶、细胞周期调控因子、抗氧化蛋白、信号传导蛋白、蛋白酶体相关蛋白、细胞骨架蛋白以及未知蛋白等. 与正常对照组相比,肥胖组的蛋白质表达存在着较大差异,通过对差异表达蛋白的分析,提示了在肥胖发生的过程中,下丘脑神经中枢经历了一个非常复杂的信号活动和特定改变,为深入认识肥胖的发病机制奠定了基础.     

快速老化小鼠额叶皮质的蛋白组学研究

目的:探讨快速老化过程中差异蛋白质及其与学习记忆的关系.方法:以13月龄和8月龄快速老化小鼠模型的快速老化亚系SAMP8和抗快速老化亚系SAMR1的额叶为研究对象,经双向电泳技术和考马斯亮兰G250染色,分别获取13月龄和8月龄SAMP8和同龄SAMR1额叶的2-DE考染图谱,用PDQuest 7.40图像分析软件,建立不同月龄蛋白质组的匹配差异图谱,分析图谱中SAMP8与SAMR1蛋白质的表达变化.结果:8月龄SAMP8额叶检测到579个蛋白点,同龄SAMR1额叶检测到612个蛋白点;13月龄SAMP8额叶检测到705个蛋白点,同龄SAMR1额叶检测到621个蛋白点.PDQuest7.40软件匹配差异分析显示,8月龄SAMP8与同龄SAMR1比较,SAMP8表达缺失33个蛋白,两者共有显著差异表达蛋白35个.13月龄SAMP8与同龄SAMR1比较,SAMR1中表达缺失84个蛋白,两者均有但表达有显著变化的蛋白质36个.13月龄和8月龄的SAMP8互相比较,13月龄组新增蛋白126个,二者共有差异表达蛋白58个.13月龄和8月龄的SAMR1比较,13月龄组新增9个蛋白,二者共有差异表达蛋白33个.结论:两组不同月龄小鼠额叶SAMP8和SAMR1存在差异表达蛋白质,进一步研究有助于了解衰老的发生机制,并为研发调节学习记忆蛋白的新药提供依据.     

家蚕蛹期雌雄生殖腺蛋白质双向电泳比较分析

分析家蚕Bombyx mori雌雄生殖腺细胞蛋白质,有利于发现性别分化相关的功能蛋白质,探讨生殖腺发育相关基因的表达调控机理。本研究利用蛋白质双向凝胶电泳和图像分析技术,分析家蚕蛹期第2天的雌雄生殖腺细胞蛋白质。结果表明: 在雄蚕生殖腺蛋白质电泳图谱中共检测到435个蛋白斑点,其中特异性蛋白斑点73个,占总蛋白斑点数的16.8％;雌蚕生殖腺的电泳图谱中有417个蛋白斑点,其中特异性蛋白斑点55个,占总蛋白斑点数的13.2％。雌雄能匹配的蛋白斑点有362对,匹配率达85.0％。     

CREB在拟黑多刺蚁不同品级脑部mRNA水平的定位

为探讨CREB在拟黑多刺蚁(Polyrhachis vicina)不同品级脑部mRNA水平的表达,采用地高辛标记法原位杂交技术对拟黑多刺蚁工蚁、雌蚁、雄蚁3个品级脑部CREB mRNA的表达进行了定位研究。结果显示,CREB mRNA在拟黑多刺蚁不同品级脑部均有广泛表达。阳性反应主要分布在蕈形体冠部的Kenyon细胞、视叶和嗅叶等部位。在3个不同品级蚂蚁的脑部中,工蚁的嗅球和蕈形体内有较明显的CREB mRNA阳性反应,雄蚁的视叶具有较强的阳性反应,与工蚁和雄蚁相比,雌蚁脑部各个部位的阳性表达都较弱。推断CREB可能在视觉和嗅觉信息的获取与整合中起着重要作用,且与不同品级蚂蚁的行为相关。     

鼎突多剌蚁群体结构和生活史的研究

本文报告了鼎突多刺蚁(Polyrhachis vicina Roger)的群体结构和生物史。通过研究表明,鼎突多刺蚁一年发生一代,以蚁后、雄蚁、工蚁、幼虫和卵越冬。卵和幼虫在冬天发育停滞（或极慢）。到了春天,随着气温的上升,卵和幼虫又恢复正常发育。据室内人工饲养观察,卵的发育历期为23.8±2.5天（平均温度26℃）幼虫为20.4±4.4天(26℃),工蚁蛹为19.8±5.5天(27℃)。成长工蚁在5—11月出现;8—11月,雄蚁从蛹中羽化;10月,雌蚁从蛹中羽化。雌蚁分飞交尾后,进入邻近蚁巢或回到原巢,脱翅成为蚁后。蚁巢内存在着多后现象。     

毒蕈碱Ⅱ型乙酰胆碱受体类似物在拟黑多刺蚁三个品级脑中的表达（英文）

昆虫脑内胆碱能系统在中枢神经系统中起着重要作用,其与昆虫的复杂行为密切相关.本文选取有复杂行为的膜翅目社会性昆虫拟黑多刺蚁为研究材料,用免疫组织化学方法,对毒蕈碱Ⅱ型乙酰胆碱受体类似物在拟黑多刺蚁工蚁、雌蚁和雄蚁脑中进行定位检测.结果表明,毒蕈碱Ⅱ型乙酰胆碱受体类似物在拟黑多刺蚁前脑蕈形体、中央体和中腩嗅叶中普遍存在,但不同品级表达区域和强弱存在差异.这意味着毒蕈碱Ⅱ型乙酰胆碱受体类似物在拟黑多刺蚁视觉信息、嗅觉信息的整合输出中起着重要作用.     

红火蚁复眼的扫描电镜观察

了解红火蚁Solenopsis invicta Buren复眼形态结构及其与不同性别、品级的关系,为探索其基于视觉行为习性的、有效的非化学防控措施提供新思路和依据。采用扫描电镜技术,比较研究红火蚁工蚁、有翅雌蚁、雄蚁的复眼形态差异。结果表明:(1)工蚁复眼圆形,略外凸,小眼数约110个;雌蚁复眼长椭圆形,外凸,小眼数约510个;雄蚁复眼近半球形,小眼数约805个;(2)工、雌和雄蚁复眼中心区域小眼排列较紧密,多为较规则的五、六边形,边缘区域小眼排列不紧密,多为不规则的四至六边形,且少量相邻小眼的间距较大。工蚁、雌蚁和雄蚁复眼小眼面积大小依次为500,360,348.61μm2,同品级内小眼面大小相差不大;(3)雌、雄蚁复眼中心区域近背区小眼间着生少量感觉毛,感觉毛长度和直径依次为:雌蚁17.5~90.2,2.16~4.29μm,雄蚁17.5~27.9,1.41~2.52μm。表明雌蚁、雄蚁复眼及视力较发达,工蚁则较弱,不同性别或品级个体复眼的形状、小眼数目和形状、表面被物均有较大差异和区域性分化。     

表皮碳氢化合物在3种火蚁属蚂蚁成虫鉴定中的应用

红火蚁Solenopsis invicta是火蚁属重要的入侵蚂蚁,与其近缘种黑火蚁S. richteri和杂交蚁S. invicta × S. richteri形态相似,难以区分。为了快速准确鉴定3种火蚁属近缘种,本研究利用气相色谱-质谱联用仪（GC-MS）,解析3种火蚁的工蚁、有翅雌蚁、有翅雄蚁的表皮碳氢化合物种类和含量,并进行主成分分析、判别分析及聚类分析。结果表明：3种火蚁共检测到62种表皮碳氢化合物,主要包括一甲基烷烃、二甲基烷烃和正构烷烃等;红火蚁、黑火蚁及杂交蚁不同品级的表皮碳氢化合物种类及含量存在显著的种间差异,红火蚁不同地理种群的表皮碳氢化合物种类及含量相似度较高;建立3种火蚁相应品级的分类判别函数,可准确区分各品级下的3种火蚁。因此,表皮碳氢化合物组成分析可用于红火蚁及其近缘种的分类鉴定,为口岸火蚁属蚂蚁的快速检疫鉴定提供新技术。     

Studies on middle silkgland proteins of cocoon colour sex-limited silkworm (<Emphasis Type="Italic">Bombyx mori</Emphasis> L.) using two-dimensional polyacrylamide gel electrophoresis

Qualitative and quantitative differences in proteins expressed in the middle silkglands of male and female silkworm larvae that differ in silk colour were investigated by high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), followed by computer assisted image analysis. About 1000 protein spots were resolved in both the sexes and most proteins were shown to be distributed in the area from 15 kDa to 70 kDa and pH 4–8. It was found that some proteins displayed higher expression in yellow cocoon, while two proteins were only expressed in female silkworm silkgland tissue through the comparison and analysis by two-D software. These proteins especially existed in female silkworm middle silkgland tissue of yellow cocoon. Furthermore, these proteins might be involved in the expression of cocoon colour phenotype     

家蚕胚胎发育时期的蛋白质变化及构造分析

采用蛋白质双向电泳技术及蛋白质氨基酸序分析技术,从蛋白质水平研究了家蚕胚胎发育时期的基因表达情况。结果表明,从家蚕临界期胚胎直到点青期胚胎的较长一段时间内,蛋白质的双向电泳图变化不大,匹配蛋白质斑点率达６３．０％,卵特异性蛋白质,３０Ｋ蛋白质的含量很大。     

Differential expression of sex-related fat body proteins during the larval–pupal developmental stages of the silkworm (Bombyx mori)

The silkworm fat body is the site of many intermediary metabolic processes, and a source of sustenance for growth throughout the life cycle. Fat body proteins are responsible for storing nutrients, providing energy, and regulating hormones, and they have been identified using proteomic approaches. However, detailed differential expression of sex-related fat body proteins has not previously been evaluated. In the present study, we characterized the differential expression of sex-related fat body proteins, by using 2-dimensional gel electrophoresis (2-DE) followed by mass spectrometry identification and bioinformatics methods. We extracted the fat body proteins from 5-day-old fifth instar larvae (L5), 10-day-old fifth instar larvae (corresponding to the end of spinning [LE]), and 0-day-old pupae (P0) of the multivoltine silkworm variety “Da Zao”. We confirmed the presence of 11 important sex-specific expression proteins and 14 stage-specific expression proteins. We accurately identified 13 of these specific expression proteins, including actin, calponin-like protein, 75 kDa subunit NADH, receptor for activated protein kinase C from Bombyx mori (BmRACK), IMP (inosine monophosphate) cyclohydrolase, tropomyosin 1, β-tubulin, hypothetical protein, antichymotrypsin precursor, and 30 K protein precursor. We showed that BmRACK was differentially expressed between male and female silkworms. We discuss the biological roles of the specific expression proteins during the larval–pupal developmental stages.     

饥饿胁迫下家蚕5龄起蚕中的蛋白表达谱及 BmLp-c 6的转录分析

【目的】分析家蚕Bombyx mori受饥饿胁迫后的蛋白质谱变化,探索其耐受饥饿的机理。【方法】以家蚕品种932为实验材料,利用双向电泳和质谱技术检测5龄起蚕经过24 h饥饿胁迫的蛋白质谱差异变化,利用荧光定量PCR技术分析BmLp-c 6的转录表达。【结果】经比对饥饿蚕和正常取食蚕的血淋巴蛋白谱,饥饿蚕有62个特异蛋白点。蛋白点的等电点在4.22~6.98之间,分子量分布在20.81～144.69 kDa间。选取只在饥饿时出现的特异蛋白点No. 7111进行质谱鉴定,根据其氨基酸序列进行引物设计,获得了目的基因BmLp-c 6,经与载体pET-21d(+)连接重组后,成功获得诱导表达。经实时荧光定量PCR分析,当5龄起蚕受到饥饿胁迫影响时,BmLp-c 6基因在血淋巴中大量转录表达,但在中肠中的转录表达水平却极低。【结论】家蚕5龄起蚕在饥饿胁迫下,血淋巴中的蛋白质谱发生变化,BmLp-c 6会大量转录表达。     

家蚕精巢蛋白质的双向电泳及质谱分析

精巢是雄性家蚕Bombyx mori的生殖腺,它的主要功能是产生精子,全面检测和鉴定精巢器官的蛋白分布将为分析家蚕雄性个体的发育和繁殖奠定基础。本研究利用双向聚丙烯酰胺凝胶电泳和蛋白硝酸银染色技术对家蚕5龄第5天幼虫的精巢组织进行了蛋白检测,利用基质辅助激光解析质量飞行时间质谱（MALDI-TOF-MS）对表达量较高的蛋白点进行了肽质量指纹图谱鉴定。结果表明：家蚕精巢蛋白质可以检测出1 000个以上的蛋白点,这些蛋白点主要集中在分子量为15～90 kD区域,等电点3.5～9之间,其中60个蛋白点得到了成功鉴定,按照已知或推测的蛋白功能,将其分为8类,包括：细胞骨架和细胞结构蛋白,膜蛋白或信号相关蛋白,大量应激反应蛋白（伴侣蛋白）,线粒体和能量产生相关蛋白,转录调控和翻译及DNA/RNA结合相关蛋白,酶和少量血液组成蛋白。其中很多蛋白可能在鞭毛形成、能量代谢及减数分裂过程中有重要作用。这些结果为进一步认识家蚕精子形成过程提供了重要的生物学信息。     

Development-Specific Differences in the Proteomics of Angiostrongylus cantonensis

Angiostrongyliasis is an emerging communicable disease. Several different hosts are required to complete the life cycle of Angiostrongylus cantonensis. However, we lack a complete understanding of variability of proteins across different developmental stages and their contribution to parasite survival and progression. In this study, we extracted soluble proteins from various stages of the A. cantonensis life cycle [female adults, male adults, the fifth-stage female larvae (FL5), the fifth-stage male larvae (ML5) and third-stage larvae (L3)], separated those proteins using two-dimensional difference gel electrophoresis (2D-DIGE) at pH 4–7, and analyzed the gel images using DeCyder 7.0 software. This proteomic analysis produced a total of 183 different dominant protein spots. Thirty-seven protein spots were found to have high confidence scores (>95%) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Comparative proteomic analyses revealed that 29 spots represented cytoskeleton-associated proteins and functional proteins. Eight spots were unnamed proteins. Twelve protein spots that were matched to the EST of different-stage larvae of A. cantonensis were identified. Two genes and the internal control 18s were chosen for quantitative real-time PCR (qPCR) and the qPCR results were consistent with those of the DIGE studies. These findings will provide a new basis for understanding the characteristics of growth and development of A. cantonensis and the host–parasite relationship. They may also assist searches for candidate proteins suitable for use in diagnostic assays and as drug targets for the control of eosinophilic meningitis caused by A. cantonensis.     

Female specific proteins in Triatoma infestans haemolymph

The presence of female specific proteins in triatoma infestans haemolymph, as well as the relationship between the female specific proteins and egg proteins, were analysed. At the same time, the presence of specific female proteins in different instars was studied. Cellulose acetate electrophoresis, polyacrylamide gel electrophoresis, and immunochemical methods were used.No differences between immature female and male haemolymph were established. Female haemolymph obtained from insects with ovary development revealed quantitative differences, with cellulose acetate, with respect to the control males. With polyacrylamide gel electrophoresis, one component that is not detected in control males was detected in mature female haemolymph. With immunochemical assays, at least two antigenic components that were not observed in male haemolymph were detected.Egg extract showed, with cellulose acetate, two bands with a mobility similar to that of the proteins increased in the haemolymph of the mature female; with polyacrylamide gel, two major bands with a mobility similar to that of the specific female haemolymph protein were detected. Egg extract contains at least two components demonstrated by double-diffusion assays and three components by immunoelectrophoresis, with immunological identity to specific mature female haemolymph proteins.The extract obtained from recently hatched insects revealed two components with immunological identity to specific female proteins. Haemolymph from first, second, third, fourth and fifth instars do not appear to contain any femalespecific haemolymph protein.