家蚕MLP基因的克隆及其结构分析

利用生物信息学的方法快速获得家蚕MLP (Muscle LIM protein, MLP)基因cDNA电子序列, 经RT-PCR生物验证正确, 登录GenBank (No. DQ311195)。MLP基因cDNA长2 327 bp, ORF全长1 485 bp, 编码产生494个氨基酸。该MLP基因组DNA含有11个外显子, 10个内含子, 所有内含子/外显子边界都符合典型的GT/AG剪切模式。MLP基因编码的蛋白富含Gly (14.4%), 分子量约为53.03 kDa, 等电点(PI)为8.29。通过BLAST分析发现该基因编码的家蚕肌肉LIM蛋白, 含有5个保守的LIM结构域, 家蚕的另一种LIM蛋白(AAR23823)含一个LIM结构域, 两者可能是通过可变剪切产生; 后者可能通过竞争作用调节前者在肌细胞中的功能。MLP的克隆为进一步研究其体内功能奠定了基础。     

一个新的家蚕LIMonly蛋白基因的序列分析和原核表达

LIM蛋白是一类含有LIM结构域的真核蛋白家族,它可介导细胞分化和发育,转录调控和激活、参与细胞骨架形成等多种重要的生物调节过程。从家蚕蛹cDNA文库获得一个新的编码LIM only蛋白的基因,我们将其命名为BmLIMO（BombyxmoriLIMonly protein）。该基因ORF长度为561 bp,编码长度为186个氨基酸残基的蛋白质,预测分子量为21.6 kDa,等电点为9.14。对BmLIMO基因进行了生物信息学方面的分析,并用DNA重组技术将Bm-LIMO片段克隆到原核表达载体pET-28α中,在大肠杆菌BL21（DE3）中成功表达了可溶性BmLIMO蛋白,这为进一步研究该蛋白功能奠定了基础。     

cDNA cloning and mRNA expression of LIM protein gene homologue from the silkworm, Bombyx mori

LIM protein cDNA, from Bombyx mori that contains an open reading frame of 622 bp encoding 94 amino acids, was identified and characterized. The B. mori LIM protein homologue is classified into group 2 LIM proteins that contain glycine-rich LIM domain. B. mori LIM protein mRNA is up-regulated at late embryogenesis and detected in the mid-gut of 5th instar larvae.     

家蚕瘫痪肽结合蛋白基因克隆与分析

ENF肽家族具有保守的N末端结构（Glu—Asn—Phe-）。该家族成员肽大多具有重叠功能活性,在鳞翅目昆虫的免疫反应,生长调控和自体调节等方面都发挥着重要的作用。在昆虫的免疫反应中,血细胞尤其是淋巴液的黏附性是针对外来侵入物的免疫应答过程中的重要因素。家蚕瘫痪肽（paralytic peptide）是ENF肽家族的一种,其具有多种的生物学活性,包括致瘫痪性及在家蚕血细胞免疫反应中的促吞噬细胞扩散活性。ENF肽家族的另一成员,粘虫（Pseudaletia separata）的生长阻抑肽（Growth-blocking peptide）,同家蚕瘫痪肽一样能够在粘虫的血细胞免疫反应中起到调节吞噬细胞的功能。目前,关于昆虫细胞免疫应答的终端调控分子机制的研究还比较少,有文献报道粘虫的生长阻抑肽结合蛋白（GBP—BP）能够起到沉默生长阻抑肽活性的功能,从而可能参与调节细胞免疫应答的终端调控。在本研究中,利用荧光差异显示技术（FDD）分析了家蚕感染BmNPV病毒后基因表达差异情况,在血淋巴中获得了一条差异条带G12782＊通过5＇-RACE技术,首次在家蚕中克隆得到了该基因的全长cDNA序列。通过同源性分析得知,该基因所编码的蛋白质与粘虫的生长阻抑肽结合蛋白具有很大的同源性,并被命名为家蚕瘫痪肽结合蛋白（Bmori paralytic peptide binding protein,PP-BP）。通过RT-PCR研究发现,该蛋白基因在血淋巴中大量表达。同时,利用实时荧光定量PCR（Real-time quantitative PCR）技术分析了该基因在正常饲养家蚕与添食BmNPV病毒的家蚕中的表达差异,结果显示该基因在家蚕添食BmNPV病毒后的表达量大大增强,这就暗示该基因可能与BmNPV病毒刺激后所引起的家蚕血液细胞免疫反应相关。利用生物信息学方法对该基因的结构进行了分析,发现该基因具有两个外显子和一个内含子。这个基因已经登入GenBank数据库,收入号为DQ306881。     

Behavior of silkworm yolk protein on phospholipid membranes

During early development, the plasma membrane of silkworm (Bombyx mori) eggs undergoes a superficial cleavage that separates the blastodermal protoplasm and the yolk. To test whether the blastoderm absorbs yolk through the plasma membrane in B. mori, we studied the interaction of phospholipid membranes and yolk using a phospholipid planar bilayer membrane (PBM) and liposomes. In addition, egg-specific protein (ESP; 225 kDa), a yolk protein that is specific to B. mori eggs, was collected by fractionating the eggs. Liposomes were mixed with either B. mori yolk or ESP, and observed under an electron microscope. This showed that the phospholipid membrane was spanned by fine particles 10-20 nm in diameter. Both yolk and ESP caused the PBM to become extraordinarily leaky, with a membrane potential of -70 mV for yolk and -198 mV for ESP. These results suggest that although it is a water-soluble protein, ESP permeates the phospholipid membrane without the help of enzymes.     

家蚕核糖体蛋白L7基因cDNA序列的克隆和分析

为了研究家蚕孤雌生殖的调节机制,应用二维凝胶电泳(2DE)技术分离正常生殖的家蚕卵与孤雌生殖家蚕卵的差异蛋白质,在蛋白质水平上筛选与家蚕孤雌生殖过程相关的重要蛋白质.利用MALDI-TOF-TOF MS分析这些差异蛋白,获得了大量小肽的序列特征.BLAST搜索本实验室构建的cDNA文库,获得了1个与家蚕孤雌生殖相关的核糖体蛋白L7基因.根据已有的cDNA文库,采用RACE方法克隆得到该核糖体蛋白基因的全长cDNA.利用生物信息学的方法和工具,对这个基因在核酸水平和蛋白质水平分别作了详细的分析和讨论并进行蛋白结构预测.结果表明,核糖体L7基因的cDNA全长为858 bp,编码区包含6个外显子,共编码268个氨基酸残基,蛋白的疏水性平均值为-0.586,分子量大小为30 kD,极性的最大值为 39.616,最小值为0.451,等电点为10.52.分子系统分析显示,该蛋白与Apis, Lysiphlebus 和Meladema中的核糖体蛋白L7具有较高的同源性.     

Phosphorylation of Rab proteins from the brain of Bombyx mori

Rab proteins play fundamental roles in the regulation of membrane traffic. Previously, from the brain of Bombyx mori we isolated two cDNA clones (BRab1 and BRab14), each of which encoded a different member of Rab-protein family and was expressed in Escherichia coli and purified using an affinity chromatography. In this study, one cDNA clone (BRab8) was isolated from a cDNA library from the brain of B. mori. The recombinant protein was expressed in E. coli and purified. Next, the phosphorylations of these three purified BRab proteins were examined, using mammalian protein kinases in vitro. Protein kinase C (PKC) phosphorylated BRab8 and BRab14 proteins. Protein kinase A faintly phosphorylated BRab8 and BRab14 proteins. Calcium/calmodulin-dependent protein kinase faintly phosphorylated BRab8 protein. Next, brains of B. mori were dissected and homogenized. The homogenate showed a calcium-dependent protein kinase activity of BRab8 and BRab14 proteins. So PKC from the brain of B. mori was partially purified by a sequence of chromatographies on DEAE-Cellulofine and affinity chromatography. This PKC phosphorylated BRab8 and BRab14 proteins. These results suggest that the function of Rab proteins in the brain of B. mori is regulated by calcium-dependent protein kinases.     

家蚕抗真菌因子BmSPI39对球孢白僵菌入侵的表达响应

[目的]制备家蚕Bombyx mori抗真菌因子BmSPI39的多克隆抗体,分析BmSPI39对球孢白僵菌Beauveria bassiana入侵的表达响应.[方法]利用原核表达和固定化镍离子亲和层析技术获得高纯度的BmSPI39重组蛋白,利用胶内活性染色检测重组蛋白BmSPI39对枯草杆菌Bacillus subti...     

家蚕生殖细胞相关基因nanos的克隆表达及在胚胎发育中的表达模式

【目的】探讨nanos基因在家蚕Bombyx mori胚胎发育中的表达模式,为进一步研究该基因在家蚕胚胎发育中的功能奠定基础。【方法】根据本实验室提交到GenBank中家蚕nanos的cDNA序列(登录号EF647589)设计引物,扩增出了一条长684 bp的编码片段,对该片段进行了克隆和表达,亲和纯化表达的蛋白并免疫新西兰大白兔制备抗体。Western blot检测家蚕早期胚胎nanos的表达情况,荧光定量PCR检测nanos在整个家蚕胚胎发育中的表达情况。【结果】克隆并表达了一条长684 bp的编码片段,得到了分子量约33 kD的融合蛋白。用制备的抗血清对家蚕早期胚胎蛋白的Western blot检测表明,nanos在此阶段基本是恒定表达。荧光定量PCR结果显示刚产的卵中nanos的表达量最大,第2天开始急剧下降,此后到第10天表达量几乎没有变化。【结论】本实验克隆的nanos是家蚕中的一个同源物,该基因在家蚕胚胎发育中的表达模式与蜜蜂等有很大的不同,反映了昆虫生殖细胞形成机制的多样性。     

双向电泳法在家蚕蛋白质分离中的应用

家蚕Bombyxmori(L.)既是重要的经济昆虫,又是鳞翅目昆虫研究的典型模式生物。开展家蚕蛋白质组研究,将有助于阐明家蚕绢丝蛋白的分泌机理,也是研究鳞翅目昆虫及其他生物生命本质的需要。双向电泳是蛋白质分离的关键技术。为探讨适宜家蚕蛋白质组研究的双向电泳条件,以家蚕丝腺、丝腺内容物、蚕卵和血液为材料,在不同条件下进行双向电泳,并对分离的蛋白点进行质谱分析。结果表明:通过改进的蛋白质裂解液辅以超声破碎制备的蛋白质,双向电泳后能够得到较好的2-DE图,也能满足进行MALDI-TOFMS分析的需要。因此本研究方法适用于家蚕不同组织中蛋白质的提取和双向电泳。     

Small GTPases of the Rab family in the brain of Bombyx mori

Small GTPases of the Rab family are key regulators of membrane trafficking. We produced antibodies against the Rab7 protein of Bombyx mori (BRab7) in rabbits, and against the Rab11 protein of B. mori (BRab11) in mice. The antibodies recognized BRab7 and BRab11 proteins, but did not recognize other Rab proteins. Immunoblotting of samples from brain tissue of B. mori revealed a single band for each antibody. Rab11 was expressed in most tissues, whereas Rab7 was expressed in the brain, ovary, and testis. Immunohistochemical reactivity of Rab7 and Rab11 in the brain of B. mori was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Double-labeling experiments demonstrated that immunohistochemical reactivity of Rab7 co-localized with that of Rab11 and partially with that of Rab8. Immunohistochemical reactivity of Rab11 and Rab8 co-localized with that of PERIOD, one of the proteins associated with circadian rhythm. These findings suggest that Rab7, Rab8, and Rab11 are involved in protein transport in the neurons of the brain of B. mori and might play a role in the control of circadian rhythm.     

30K蛋白对家蚕细胞及幼虫存活的作用

利用杆状病毒表达系统在家蚕BombyxmoriL.细胞BmN中表达家蚕30K蛋白,以亲本病毒BmPAK6为对照,将重组病毒Bm/r-30K分别感染BmN细胞及家蚕幼虫,观察其感染后不同时间的凋亡及存活率。与对照相比,重组病毒感染后的BmN细胞的存活率明显高于对照组,并且感染的家蚕幼虫存活时间也较长,表明体内过量表达家蚕30K蛋白有助于延长其细胞及幼虫的存活。     

hhLIM is a novel F-actin binding protein involved in actin cytoskeleton remodeling

Human heart LIM protein (hhLIM) is a newly cloned protein. In vitro analyses showed that green fluorescent protein (GFP)-tagged hhLIM protein accumulated in the cytoplasm of C2C12 cells and colocalized with F-actin, indicating that hhLIM is an actin-binding protein in C2C12 cells. Overexpression of hhLIM-GFP in C2C12 cells significantly stabilized actin filaments and delayed depolymerization of the actin cytoskeleton induced by cytochalasin B treatment. Expression of hhLIM-GFP in C2C12 cells also induced significant changes in the organization of the actin cytoskeleton, specifically, fewer and thicker actin bundles than in control cells, suggesting that hhLIM functions as an actin-bundling protein. This hypothesis was confirmed using low-speed co-sedimentation assays and direct observation of F-actin bundles that formed in vitro in the presence of hhLIM. hhLIM has two LIM domains. To identify the essential regions and sites for association, a series of truncated mutants was constructed which showed that LIM domain 2 has the same activity as full-length hhLIM. To further characterize the binding sites, the LIM domain was functionally destructed by replacing cysteine with serine in domain 2, and results showed that the second LIM domain plays a central role in bundling of F-actin. Taken together, these data identify hhLIM as an actin-binding protein that increases actin cytoskeleton stability by promoting bundling of actin filaments.     

Diversity in copy number and structure of a silkworm morphogenetic gene as a result of domestication

The carotenoid-binding protein (CBP) of the domesticated silkworm, Bombyx mori, a major determinant of cocoon color, is likely to have been substantially influenced by domestication of this species. We analyzed the structure of the CBP gene in multiple strains of B. mori, in multiple individuals of the wild silkworm, B. mandarina (the putative wild ancestor of B. mori), and in a number of other lepidopterans. We found the CBP gene copy number in genomic DNA to vary widely among B. mori strains, ranging from 1 to 20. The copies of CBP are of several types, based on the presence of a retrotransposon or partial deletion of the coding sequence. In contrast to B. mori, B. mandarina was found to possess a single copy of CBP without the retrotransposon insertion, regardless of habitat. Several other lepidopterans were found to contain sequences homologous to CBP, revealing that this gene is evolutionarily conserved in the lepidopteran lineage. Thus, domestication can generate significant diversity of gene copy number and structure over a relatively short evolutionary time.