鳞翅目昆虫病原微孢子虫研究进展

微孢子虫广泛存在于鳞翅目昆虫中,是一类重要的病原微生物。微孢子虫病一方面影响野外昆虫种群的自然平衡,另一方面对家蚕、柞蚕等经济和资源昆虫造成了严重的危害。微孢子虫分子生物学研究基础相对薄弱,再加上微孢子虫表面坚厚的孢壁,无疑增加了研究难度。随着核酸、蛋白质等生物大分子分离制备方法和高通量测序技术的不断更新发展,基于各种组学(Omics)研究微孢子虫的工作方兴未艾,并且有了一些重要的发现。本文综述了微孢子虫与鳞翅目昆虫寄主的相互作用及寄生于鳞翅目昆虫的病原微孢子虫基因组、转录组和蛋白质组进展情况,以期为微孢子虫的深入研究提供参考。这些昆虫微生物研究将为鳞翅目害虫生物防治提供新的思路,并对家蚕等经济昆虫微粒子病的诊断、防控及治疗产生积极影响。     

家蚕丝腺蛋白质组学研究方法的建立

通过高精度的双向电泳技术对家蚕中部丝腺组织的蛋白质进行分离,采用基质辅助激光解析电离飞行时间质谱(matrix-assistedlaserdesorption/ionizationtimeofflightmassspectrometry,MALDI-TOF-MS)对其中一些表达量较高的蛋白点进行鉴定,并利用GPMAW(GeneralProtein/MassAnalysisforWindows)软件结合家蚕基因组预测的蛋白质数据库构建本地的肽质量指纹图谱数据库,对所得到的肽质量指纹图谱进行分析。研究发现,经过双向凝胶电泳及其图象分析技术,硝酸银染色和考马斯亮蓝染色分别能分离出500个以上和100个以上的蛋白点。这些蛋白质点主要集中在分子量15~90kD区域,等电点pH3·5~7之间。MALDI-TOF-MS鉴定的25个考染蛋白点中有60%以上的PMF(PeptideMassFingerprint)的信号峰较强。在数据库检索过程中,利用家蚕肽质量指纹数据库所得检索结果与在Mascot的检索结果相比,前者不仅能够准确鉴定出一些已有研究报道的蛋白,从而验证检索方法的可行性,而且还能够对一些已经被家蚕基因组数据库所预测但未曾报道的新蛋白质进行鉴定,从而建立了一整套适合于家蚕蛋白质组研究的方法,并为其它绢丝昆虫蛋白质组研究提供了重要参考。     

五龄家蚕若干组织器官蛋白质数据库构建

为构建家蚕蛋白质数据库,达到从蛋白质水平研究基因的表达及表达产物的加工修饰的目的,采用五龄家蚕休体壁、中肠和脂肪为材料获取蛋白质样品,用蛋白质双向电泳技术分离、纯化蛋白质,对其中的40个蛋白质斑点进行了氨基酸序列分析及同源性检索,发现其中58.5％的蛋白质与果蝇的有关蛋白质具有较高同源性,36.5％与其他生物的蛋白质具有较高同源性,只有5％与家蚕已知蛋白拷贝产高同源性,研究发现有27个蛋白质的N－末端氨基酸序列在家蚕中是第一次被检测到,并通过互联网向SWISS－PROT数据库进行了登录。研究证明利用蛋白质数据库的结果,可以得到基因表达及其产物修饰的信息。     

家蚕体液低分子蛋白Apolipophorin Ⅲ的质谱鉴定及表达分析

利用SDS-PAGE电泳对家蚕不同时期的血淋巴蛋白进行了研究。发现在家蚕羽化前,血淋巴液中有一个分子量约20kD的蛋白表达量明显上调。为了探究该蛋白的功能,本研究通过双向电泳分离和肽质量指纹图谱分析对该蛋白进行了鉴定。结果表明,此羽化前后高表达的蛋白为Apolipophorin Ⅲ蛋白,该类蛋白被普遍认为与昆虫的飞行能力有关。利用RT-PCR对该蛋白编码的基因进行了组织表达分析,结果表明该基因除了在马氏管几乎不表达外,其它组织均有表达,这也暗示了该蛋白除了与昆虫的飞行活动有关以外,还参与了幼虫及蛹期的其他重要的生命活动。     

粘虫中肠总蛋白质双向电泳体系的优化方法

为了今后更好的利用双向电泳技术研究不同处理后粘虫Mythimna separata中肠蛋白表达差异,本研究比较了不同的蛋白提取方法、IPG胶条、二硫苏糖醇(DTT)浓度和等电聚焦条件,建立了适用于粘虫中肠总蛋白质的双向电泳体系。结果表明:采用Tris-饱和酚法提取蛋白质,选取p H为5-8的线性IPG胶条进行第一向等电点分离,按照DTT浓度I和等电聚焦程序II进行双向电泳分离,获得了蛋白质点数多、背景清晰、分辨率高的蛋白质双向电泳图谱。     

家蚕性别及其决定基因的研究进展

家蚕作为模式生物和鳞翅目昆虫的典型代表,性别决定的分子机制是近年来的研究热点,其分子机制的阐明将为家蚕的雄蚕饲养和害虫的生物防治打下基础.主要对国内外家蚕性别决定、性染色体、家蚕性别决定基因的研究进展进行了综述,并对家蚕性别调控研究中存在的问题进行了分析和展望.     

鳞翅目昆虫中肠Bt杀虫蛋白受体研究进展

杀虫晶体蛋白(insecticidal crystal proteins,ICPs;含有Cry和Cyt 2大家族)和营养期杀虫蛋白(vegetative insecticidal proteins,Vips)等Bt杀虫蛋白可有效防治鳞翅目害虫,其中Cry应用最广泛。然而,一些地区的鳞翅目害虫已对Bt杀虫蛋白产生了抗性。目前,普遍认为鳞翅目昆虫中肠受体与Bt杀虫蛋白结合能力的改变是导致其对Bt杀虫蛋白产生抗性的最主要因素。在鳞翅目昆虫中,Cry受体是研究得最为透彻的Bt受体,已经被证实的有氨肽酶N、钙黏蛋白、碱性磷酸酶和ABC转运蛋白等。Vips杀虫蛋白类与鳞翅目昆虫中肠受体的结合方式与Cry杀虫蛋白相似,但结合位点与Cry杀虫蛋白不同。本文从结构特点、作用机制及不同鳞翅目昆虫间的表达差异等角度对以上4种鳞翅目昆虫中肠Bt受体进行了综述,并提出如下展望:(1)以棉铃虫或小菜蛾等鳞翅目昆虫为农业害虫模式生物进行深入研究,阐明其对Bt杀虫蛋白产生抗性的机制,为研究其他鳞翅目农业害虫对Bt杀虫蛋白产生抗性的机制提供理论借鉴;(2)鉴于在不同鳞翅目昆虫间,中肠Bt受体与Bt杀虫蛋白结合存在差异,且同一Bt杀虫蛋白与鳞翅目昆虫Bt受体并不专一性结合,Bt杀虫蛋白多基因组合策略是较为有效的田间鳞翅目昆虫防治策略,是今后一段时间内Bt杀虫蛋白应用的发展方向。     

家蚕热激蛋白的研究进展

热激蛋白在家蚕的生长发育过程中发挥着重要的生物学作用。该文对国内外学者近几年在家蚕热激蛋白上的研究进展进行了综述,分析了家蚕热激蛋白的分类,概括了家蚕热激蛋白的基本性质,介绍了家蚕热激蛋白在对温度的响应、家蚕抗逆、抗病与滞育中的作用及其他生物学功能等方面的研究现状,提出了在家蚕热激蛋白研究上存在的问题,展望了家蚕热激蛋白研究的发展趋势,以期为利用热激蛋白提高家蚕品种的抗逆性和深入研究家蚕以及其他鳞翅目昆虫热激蛋白的生物学功能提供参考。     

高低温催青温度敏感期家蚕卵的差异蛋白筛查

滞育（Diapause）是昆虫发育停滞或减缓的生理状态,家蚕Bombyx mori作为卵滞育的代表,其滞育过程已得到广泛研究,但诱导滞育发生的分子机制尚不清楚。二化性家蚕滞育性由遗传和母系在胚胎期所处的环境条件决定,25℃催青蚕卵孵化后的家蚕产滞育卵,15℃低温催青则诱导家蚕产下非滞育卵。本研究分别用25℃和15℃催青蚕卵,在发生滞育诱导的温度敏感期取样,抽提蛋白质通过非标（Label-free）蛋白质组定量技术进行质谱测序。筛选出具有明显表达差异的蛋白104个,其中56个蛋白上调,48个蛋白下调。通过生物信息学对差异蛋白进行GO分类和KEGG功能富集分析,结果显示差异蛋白主要参与生长发育、物质代谢和胁迫应答等生物过程;同时差异蛋白主要参与胰岛素信号通路、乙醛酸和二羧酸代谢等相关途径。分别选取上调基因和下调基因进行qRT-PCR验证,其趋势与蛋白组学结果一致。该研究将为进一步解析家蚕滞育诱导发生机制提供靶标蛋白和数据参考。     

家蚕后丝腺亚细胞组分中核酸和蛋白质的分布

丝心蛋白是家蚕后丝腺的分泌产物。在家蚕五龄早期,后丝腺细胞中迅速形成丝心蛋白合成所必需的一些亚细胞结构,在后丝腺细胞质中粗糙内质网迅速增多,平滑内质网迅速形成空泡状或管状。而且生化测定表明,在五岭早期后丝腺中DNA,RNA、蛋白质和脂肪的含量皆迅速增加,这些为在五龄后期丝心院的合成,即为丝心蛋白信息的转录和翻译,提供亚细胞结构和生化代谢的物质基础(Tashiro等1968;赤井弘1973)。因此有必要对家蚕后丝腺亚细胞组分如细胞核、线粒休、微粒体和105,000g上清液中核酸和蛋白质代谢进行研究。本文初步建立一种分离家蚕后丝腺亚细胞组分的差级离心方法,并测定各亚细胞组分中核酸和蛋白质的含量。     

Proteome analysis of silk gland proteins from the silkworm, Bombyx mori

The silk gland of Bombyx mori is an organ specialized for the synthesis and secretion of silk proteins. We report here the resolution of silk gland proteins by 2-DE and the identification of many of those proteins. This was accomplished by dissecting the glands into several sections, with each exhibiting more than 400 protein spots by 2-DE, of which 100 spots were excised and characterized by in-gel digestion followed by PMF. Ninety-three proteins were tentatively identified. These were then categorized into groups involved in silk protein secretion, transport, lipid metabolism, defense, etc. Western blotting of a 2-DE gel using an antibody of the carotenoid binding protein confirmed the presence of this protein in the silk gland. Proteins including fibroin L-chain and P25 were found as multiple isoforms, some of which contained differential amounts of phosphate residues as analyzed by on-probe dephosphorylation. The current analysis contributes to our understanding of proteins expressed by the silk gland not only of the model lepidopteran B. mori, but also to proteins from other silk-producing insects such as Philosamia cynthia ricini.     

Insights into the regulatory characteristics of silkworm fibroin gene promoters using a modified Gal4/UAS system

Transgenic Research - The silkworm Bombyx mori is a valuable insect that synthesizes bulk amounts of fibroin protein in its posterior silk gland (PSG) and weaves these proteins into silk cocoons....     

不同提取方法及上样量对牡丹试管苗茎基部蛋白双向电泳的影响

为建立一套适合于牡丹试管苗茎基部蛋白的双向电泳技术,以便更好地利用蛋白质组技术研究牡丹试管苗不定根的发生机理,本研究比较了三种不同蛋白质提取方法对双向电泳结果的影响,并在蛋白质上样量方面进行了比较。结果表明,乙酸铵/甲醇酚提取法所得2-DE图谱的蛋白点很少,仅检测到45个,且较模糊,有明显的拖尾现象,分辨率很低;乙醇/乙醚丙酮法所得的蛋白点也较少(101个),较模糊,且横竖纹干扰较大;三氯乙酸/丙酮法所得蛋白点数较多,可检测到434个清晰的蛋白点,且形状规则,重复性好,适合后续分析,操作也较为简便。用三氯乙酸/丙酮法提取蛋白,采用800μg、1000μg和1200μg三个不同的上样量进行双向电泳,在上样量为1200μg时(IPGpH3～10,24cm),蛋白质在12%SDS-PAGE胶上得到了较好的分离,在2-DE图谱上可分辨出562个蛋白点。因此,三氯乙酸/丙酮法是较适合于牡丹试管苗茎基部蛋白质提取的方法,1200μg是较为合适的上样量。     

莲种子蛋白质提取方法比较与双向电泳分析

莲(Nelumbo nucifera Gaertn.)不仅是重要的水生蔬菜作物之一,而且是进行基础研究的好材料。本文采用4种蛋白质提取方法(新型TCA/丙酮法、传统TCA/丙酮法、改良的Tris-HCl法、Tris-饱和酚法)并结合双向电泳技术,对莲子蛋白质提取方法进行筛选与优化。双向电泳实验结果显示,所得蛋白质图谱与莲种子蛋白质组成分布特点一致。通过PDQuest软件分析表明,新型TCA/丙酮法适用于莲子叶和胚芽组织的双向电泳蛋白质提取,而传统TCA/丙酮法则适用于莲胚轴组织双向电泳的蛋白质提取。研究结果为进一步利用质谱进行莲子蛋白质组研究奠定了基础。     

Strategies for the enrichment and identification of basic proteins in proteome projects

Two-dimensional gel electrophoresis (2-DE) is currently the method of choice for separating complex mixtures of proteins for visual comparison in proteome analysis. This technology, however, is biased against certain classes of proteins including low abundance and hydrophobic proteins. Proteins with extremely alkaline isoelectric points (pI) are often very poorly represented using 2-DE technology, even when complex mixtures are separated using commercially available pH 6-11 or pH 7-10 immobilized pH gradients. The genome of the human gut pathogen, Helicobacter pylori, is dominated by genes encoding basic proteins, and is therefore a useful model for examining methodology suitable for separating such proteins. H. pylori proteins were separated on pH 6-11 and novel pH 9-12 immobilized pH gradients and 65 protein spots were subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry, leading to the identification of 49 unique proteins. No proteins were characterized with a theoretical pI of greater than 10.23. A second approach to examine extremely alkaline proteins (pI > 9.0) utilized a prefractionation isoelectric focusing. Proteins were separated into two fractions using Gradiflow technology, and the extremely basic fraction subjected to both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and liquid chromatography (LC) - tandem mass spectrometry post-tryptic digest, allowing the identification of 17 and 13 proteins, respectively. Gradiflow separations were highly specific for proteins with pI > 9.0, however, a single LC separation only allowed the identification of peptides from highly abundant proteins. These methods and those encompassing multiple LC 'dimensions' may be a useful complement to 2-DE for 'near-to-total' proteome coverage in the alkaline pH range.     

Carbonic anhydrase generates a pH gradient in Bombyx mori silk glands

Silk is a protein of interest to both biological and industrial sciences. The silkworm, Bombyx mori, forms this protein into strong threads starting from soluble silk proteins using a number of biochemical and physical cues to allow the transition from liquid to fibrous silk. A pH gradient has been measured along the gland, but the methodology employed was not able to precisely determine the pH at specific regions of interest in the silk gland. Furthermore, the physiological mechanisms responsible for the generation of this pH gradient are unknown.In this study, concentric ion selective microelectrodes were used to determine the luminal pH of B. mori silk glands. A gradient from pH 8.2 to 7.2 was measured in the posterior silk gland, with a pH 7 throughout the middle silk gland, and a gradient from pH 6.8 to 6.2 in the beginning of the anterior silk gland where silk processing into fibers occurs. The small diameter of the most anterior region of the anterior silk gland prevented microelectrode access in this region. Using a histochemical method, the presence of active carbonic anhydrase was identified in the funnel and anterior silk gland of fifth instar larvae. The observed pH gradient collapsed upon addition of the carbonic anhydrase inhibitor methazolamide, confirming an essential role for this enzyme in pH regulation in the B. mori silk gland. Plastic embedding of whole silk glands allowed clear visualization of the morphology, including the identification of four distinct epithelial cell types in the gland and allowed correlations between silk gland morphology and silk stages of assembly related to the pH gradient.B. mori silk glands have four different epithelial cell types, one of which produces carbonic anhydrase. Carbonic anhydrase is necessary for the mechanism that generates an intraluminal pH gradient, which likely regulates the assembly of silk proteins and then the formation of fibers from soluble silk proteins. These new insights into native silk formation may lead to a more efficient production of artificial or regenerated silkworm silk fibers.     

家蚕V型ATP酶B亚基的克隆及表达特征

V型ATP酶(Vacuolar-type ATPase)是一种定位于细胞膜和细胞器膜上的氢离子转运酶。它利用ATP水解的能量将氢离子转运到液泡、囊泡或者胞外,从而维持细胞内正常的酸碱环境。V型ATP酶B亚基(V-ATPase B)作为ATP的催化位点,也有着非常重要的作用。为了探讨家蚕V-ATPase B(Bm V-ATPase B)的功能,首先从家蚕五龄幼虫的中肠c DNA中克隆了Bm V-ATPase B基因并构建原核表达载体进行原核表达,获得了重组蛋白,经质谱鉴定正确后,通过镍柱亲和层析的方法纯化了该蛋白并制备了多克隆抗体;最后分析了该蛋白在家蚕丝腺中的表达特征并利用免疫荧光对其在丝腺中的表达位置进行了定位。结果显示Bm V-ATPase B基因序列全长1 473 bp,预测蛋白分子量55 k Da,预测等电点5.3。通过Western blotting对家蚕5龄第3天和上蔟第1天幼虫丝腺的不同区段进行Bm V-ATPase B蛋白的表达特征分析,发现在两个时期该蛋白均在前部丝腺高量表达,而在中部丝腺和后部丝腺表达量相对较低。进一步对两个时期丝腺的不同区段进行免疫荧光定位,发现该蛋白在两个时期的前部丝腺、中部丝腺和后部丝腺均定位于细胞层。利用激光共聚焦显微镜对该蛋白进行进一步的定位,发现该蛋白主要在丝腺的细胞膜表达。研究结果明确了该蛋白在丝腺中的表达模式,为深入研究该蛋白在蚕丝纤维形成中的作用奠定了基础。     

Ecdysteroid as a mediator in the regulation of silk protein synthesis and its influence on silkworm (Bom., Lepidoptera) genome

Abstract:  Ecdysteroid is one of the two most important insect hormones, which controls growth and developmental activities of insect. In silkworm, Bombyx mori L., basically ecdysteroid induces molting and metamorphosis in larvae and exogenous administration hastens the silk synthetic activity and cocoon spinning process but it strictly depends on the time of administration. In the present study, we administered phytogenous ecdysteroid to the 48-h-old 5th instar silkworm, B. mori at a single dose of 2  μ g per larva to study its effects on the larval growth, cocoon and silk variables. The possible role of ecdysteroid in altering the developmental sequence of silkworm and thereby its effect on cocoon and silk production at molecular level have been tried to get elucidated. The genomic DNA was isolated from the posterior silk gland on day 5 and 7 of the 5th instar from the ecdysteroid treated and the control larvae and was randomly amplified with arbitrary primers. The result presented notable variation in the amplified product suggesting the participation of ecdysteroid in regulating the silk gene. The feeding period of treated larvae was unaffected while the cocoon characters exhibited considerable improvement. The filament traits also were improved notably in the treated larvae. The participation of ecdysteroid in the silk biosynthetic process with its physiological and molecular implications was discussed.     

Proteomic analysis of silk gland programmed cell death during metamorphosis of the silkworm Bombyx mori

The silk gland of the silkworm Bombyx mori undergoes programmed cell death (PCD) during pupal metamorphosis. On the basis of their morphological changes and the occurrence of a DNA ladder, the tissue cells were categorized into three groups: intact, committed, and dying. To identify the proteins involved in this process, we conducted a comparative proteomic analysis. Protein expression changes among the three different cell types were examined by two-dimensional gel electrophoresis. Among approximately 1000 reproducibly detected protein spots on each gel, 43 were down-regulated and 34 were up-regulated in PCD process. Mass spectrometry identified 17 differentially expressed proteins, including some well-studied proteins as well as some novel PCD related proteins, such as caspases, proteasome subunit, elongation factor, heat shock protein, and hypothetical proteins. Our results suggest that these proteins may participate in the silk gland PCD process of B. mori and, thus, provide new insights for this mechanism.