棒络新妇体重与其拖牵丝机械性能的关系研究

利用YG001A单纤维电子强力仪测定了15只不同体重棒络新妇的拖牵丝机械性能,分析了蜘蛛体重与拖牵丝机械性能的相互关系,并利用SEM及计算机图象处理技术分析了拖牵丝的直径,发现不同个体的拖牵丝平均直径和断裂强度随着体重的增加而增加,而屈服点应力、屈服点拉伸率、断裂点拉伸率与体重都没有相关性。     

兔抗仿蜘蛛牵丝蛋白抗体的制备及应用

将仿蜘蛛牵丝基因s6 0 0克隆到GST融合蛋白表达质粒pGEX KG中 ,利用大肠杆菌表达系统表达并纯化了仿蜘蛛牵丝蛋白S6 0 0 ,以之作为抗原制备了兔抗血清。S6 0 0的氨基酸组分分析与理论值相吻合。蛋白质免疫印迹发现该抗血清能与天然蜘蛛丝反应 ,表明设计的仿蜘蛛丝与天然蜘蛛丝有相似的免疫原性。为了定量检测仿蜘蛛牵丝蛋白在转基因家蚕丝腺中 (或茧壳中 )的表达 ,建立了用ELISA方法定量检测茧壳中仿蜘蛛牵丝蛋白量的工作系统     

家蚕转基因方法的初步研究

为建立家蚕转基因研究中切实可行的外源基因导入方法、分别用显微注射法、精子介导法、脂质体法和压力渗透法将含有绿色荧光蛋白(gfp)基因的转座子载体和辅助质粒转入到家蚕的受精卵中。在后代中检测到发绿色荧光的蚕茧,用PCR方法检测到后代个体染色体中含有gfp基因,并比较了上述几种方法的优缺点,为进一步进行转基因家蚕的研究奠定了基础。     

悦目金蛛拖牵丝力学性能的变异

蜘蛛丝作为一种具有优良机械性能的天然动物蛋白纤维,其特有的结构和机械性能与其生物学功能密切相关。由大壶状腺纺出的拖牵丝在蜘蛛的行走、建网、捕食、逃生、繁殖等多种生命活动中均发挥了重要的功能,其机械性能会受到多种内外因素相互作用的影响。本文对在不同体重、不同猎物饲养和不同营养状态3种条件下人工抽出的悦目金蛛(Argiope amoena)拖牵丝与其不同单丝间的力学性能进行了比较研究。结果表明,悦目金蛛拖牵丝的力学性能在组间、组内不同个体,以及同一个体不同丝纤维间变异都较大。随着蜘蛛个体的增大,蛛丝横截面直径逐渐增大,这会使得蛛丝的力学性能更好,便于作为救命索的拖牵丝在遇到危险时承受蜘蛛体重;蜘蛛在经过1个月的饥饿后,蛛丝在屈服点附近的力学性能并未发生显著变化,而断裂点应变和断裂能均显著减小,同时也表明无论对于作为救命索还是网丝,拖牵丝的弹性形变性能在与蛛丝相关的微观进化中要优先于塑性形变。这是蜘蛛在能量摄入受到限制时对拖牵丝的投入权衡的结果。     

转基因家蚕的研究进展及应用前景

转基因家蚕Bombyx mori是指利用分子生物学手段,将外源基因转移到家蚕染色体中, 使之出现先前不具有的性状和产物,并且可以保持传代,在个体水平可以体现外源基因的功能,使外源基因获得大量表达。目前转基因家蚕研究主要以piggyBac转座系统为常用载体, 绿色荧光蛋白(green fluorescent protein, GFP)基因为常用报告基因,经显微注射法获得转基因家蚕的成功率可达40%。通过转基因家蚕技术已经探明了家蚕外源导入核受体基因BmFtz-F1,调控家蚕体壁半透明的BmBLOS2基因、蜕皮启动激素(ecdysis-triggering hormone, ETH)基因以及家蚕抗菌肽CecB(cecropin B)基因的功能;获得了具有高品质、高细纤度、高拉伸强度和高弹性丝品种,能吐带绿色荧光或粉红色荧光的蚕丝品种, 抗家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus, BmNPV)品种及抗藤黄微球菌的品种;成功表达纯化了人的Ⅲ型前胶原蛋白、 人碱性纤维生长因子、人血清蛋白、人脑源性神经营养因子、人胰岛素生长因子Ⅰ、猫干扰素、单克隆抗体等生物活性蛋白、疫苗及特殊的生物材料。随着家蚕转基因技术的深入研究, 转基因家蚕产物将在国防、 军工、 航天、 医药等方面有着更为广阔的应用前景。     

日本通过转基因蚕制出荧光丝

据日本共同社报道,日本农业生物资源研究所与群马县蚕丝技术中心等研究组成功大量培养出可结出荧光色蚕茧的转基因蚕,并研发出能保持发光特性而将蚕茧转为生丝的方法。     

利用基因工程使家蚕分泌绿色荧光蚕丝

《ＮａｔｕｒｅＢｉｏｔｅｃｈｎｏｌｏｇｙ》１９９９年１７卷５期４１２页报道：日本京都工艺研究所的科学家茂理及其同事利用基因工程使杆状病毒将编码家蚕丝心蛋白的基因与编码绿色荧光蛋白的基因相互融合。再用这种病毒感染家蚕。这就使病毒中的上述工程基因通过同源重组过程而替代了家蚕的内源丝心蛋白基因。观察表明,含有这种融合工程基因的家蚕丝腺和蚕丝都可发出绿色的光泽。这种技术的主要优点是可将异源ＤＮＡ特异地导入家蚕本身的丝心蛋白基因部位,使其可接受家蚕内源启动基因的调控。而利用转座子诸如Ｐ因子的方法,则是将外…     

利用同源重组改变家蚕丝心蛋白重链基因

在家蚕丝心蛋白重链基因5‘和3‘端序列之间插入以绿色荧光蛋白（green fluorescent protein,GFP）基因（gfp）与人工合成丝心蛋白样基因的融合基因,利用电穿孔方法导入蚕卵中,卵孵化、发育和结茧后,用紫外灯检查,在约5400个茧中有73个“亮茧”,茧蛋白在ELISA应中可以与GFP的多克隆抗体反应。“亮茧”对应的蚕蛾进行交配、制种。对其后代进行了基因鉴定,Southern杂交的结果表明,gfp基因和人工合成丝心蛋白样基因都存在于家蚕基因组DNA中且发生了预期的同源重组事件。上述结果说明“亮茧”这一表型能用于筛选转基因蚕,融合基因已通过同源重组进入家蚕基因组。     

能发荧光的转基因家蚕

利用同源重组的方法研究了家蚕丝心蛋白链基因的定点替代。将ＩＥ启动子带动的ＧＦＰ基因插入蚕丝心慢白质链基因的上、下游序列之间,构民 重组质粒。将该质粒线性化后导入早期受精卵。当家蚕饲养至五龄期时用紫外灯检测,在约５０００条蚕中发现３条有绿色荧光斑块。ＰＣＲ检测证明ＧＦＰ已整合到家蚕基因组中,对其中一条蚕的Ｓｏｕｔｈｅｒｎ杂交分析表明丝心蛋白重链基因已被成功敲除并被ＧＦＰ报告基因所替代。这条转基因蚕能     

哺乳动物N-糖基化途径中关键酶唾液酸合酶和CMP-唾液酸合成酶基因在转基因家蚕中的表达

对昆虫的N-糖基化途径进行修饰改变是扩展昆虫蛋白表达系统应用范围的重要途径。本研究利用基于piggyBac转座子的家蚕Bombyx mori转基因技术表达昆虫所缺乏的哺乳类糖基化途径中的关键基因, 构建了可以同时表达小鼠Mus musculus唾液酸合酶和小鼠CMP-唾液酸合成酶两个基因的piggyBac表达载体, 选用家蚕肌动蛋白A3启动子控制基因的表达, 并导入3×P3启动子控制下的增强绿色荧光蛋白EGFP作为分子标记。在得到的G1代转基因家蚕中对转入的基因进行了分子水平的鉴定和分析, 为在家蚕这种模式昆虫中模拟哺乳类糖基化途径奠定了基础。     

Engineering Silkworms for Resistance to Baculovirus Through Multigene RNA Interference

Bombyx mori nucleopolyhedrovirus (BmNPV) that infects the silkworm, B. mori, accounts for >50% of silk cocoon crop losses globally. We speculated that simultaneous targeting of several BmNPV essential genes in transgenic silkworm would elicit a stable defense against the virus. We introduced into the silkworm germline the vectors carrying short sequences of four essential BmNPV genes in tandem, either in sense or antisense or in inverted-repeat arrangement. The transgenic silkworms carrying the inverted repeat-containing transgene showed stable protection against high doses of baculovirus infection. Further, the antiviral trait was incorporated to a commercially productive silkworm strain highly susceptible to BmNPV. This led to combining the high-yielding cocoon and silk traits of the parental commercial strain and a very high level of refractoriness (>75% survival rate as compared to <15% in nontransgenic lines) to baculovirus infection conferred by the transgene. We also observed impaired infectivity of the occlusion bodies derived from the transgenic lines as compared to the wild-type ones. Currently, large-scale exploitation of these transgenic lines is underway to bring about economic transformation of sericulture.     

High-Toughness Silk Produced by a Transgenic Silkworm Expressing Spider (Araneus ventricosus) Dragline Silk Protein

Spider dragline silk is a natural fiber that has excellent tensile properties; however, it is difficult to produce artificially as a long, strong fiber. Here, the spider (Araneus ventricosus) dragline protein gene was cloned and a transgenic silkworm was generated, that expressed the fusion protein of the fibroin heavy chain and spider dragline protein in cocoon silk. The spider silk protein content ranged from 0.37 to 0.61% w/w (1.4–2.4 mol%) native silkworm fibroin. Using a good silk-producing strain, C515, as the transgenic silkworm can make the raw silk from its cocoons for the first time. The tensile characteristics (toughness) of the raw silk improved by 53% after the introduction of spider dragline silk protein; the improvement depended on the quantity of the expressed spider dragline protein. To demonstrate the commercial feasibility for machine reeling, weaving, and sewing, we used the transgenic spider silk to weave a vest and scarf; this was the first application of spider silk fibers from transgenic silkworms.     

A fibroin secretion-deficient silkworm mutant, Nd-sD, provides an efficient system for producing recombinant proteins

The silkworm Nd-s(D) mutant is silk fibroin-secretion deficient. In the mutant, a disulfide linkage between the heavy (H) and light (L) chains, which is essential for the intracellular transport and secretion of fibroin, is not formed because of a partial deletion of the L-chain gene. To utilize the inactivity of the mutant L-chain, we investigated the possibility of using the Nd-s(D) mutant for the efficient production of recombinant proteins in the silkworm. A germ line transformation of the mutant with a normal L-chain-GFP fusion gene was performed. In the transgenic mutant, normal development of the posterior silk gland (PSG) was restored and it formed a normal cocoon. The biochemical analysis showed that the transgenic silkworms expressed the introduced gene in PSG cells, produced a large amount of the recombinant protein, secreted it into the PSG lumen, and used it to construct the cocoon. The molar ratio of silk proteins, H-chain:L-chain-GFP:fibrohexamerin, in the lumen and cocoon in the transgenic silkworm was 6:6:1, and the final product of the fusion gene formed about 10% of the cocoon silk. This indicates that the transgenic mutant silkworm possesses the capacity to produce and secrete the recombinant proteins in a molar ratio equal to that of the fibroin H-chain, contributing around half molecules of the total PSG silk proteins.     

基于超高效液相色谱-质谱技术对野蚕茧和家蚕茧化学成分进行比较

鉴定并比较野蚕茧与家蚕茧的化学成分对于理解家蚕的驯化具有重要的意义。利用高温高压结合甲醇-水提取的方法获得蚕茧中的化学成分,利用UHPLC-MS技术对野蚕、家蚕大造品种和皓月品种3种蚕茧丝中的小分子成分进行鉴定和比较分析。通过阳离子模式和阴离子模式的UHPLC-MS获得了野蚕、大造和皓月蚕茧丝的代谢指纹图谱,对鉴定到的高丰度化合物进行注释,发现其中包括了氨基酸、黄酮、生物碱、萜类、有机酸和木脂素等成分。PLS-DA的得分图表明,野蚕、家蚕大造品种和皓月品种的3种蚕茧的代谢组存在显著差异。发现脯氨酸、亮氨酸/异亮氨酸和苯丙氨酸在大造茧中的含量显著高于在野蚕和皓月茧中的含量,黄酮类植物次生代谢物在大造茧中的含量显著提高,包括槲皮素、异槲皮素、槲皮素-3-O-槐糖苷、槲皮素-3-O-L-鼠李糖苷、槲皮素-3-O-芸香糖苷和山奈酚;而神经碱、白桥楼碱、毛果芸香次碱、美洲豚草内酯、线叶泽兰素和中缅木莲素等生物碱、萜类和木脂素类的植物次生代谢物在野蚕茧中的含量显著高于在家蚕茧中的含量。在紫外光的激发下观察黄酮的绿色荧光发现家蚕大造茧中的黄酮含量最高,家蚕皓月茧中的黄酮含量最低,而野蚕茧中的黄酮含量居中。生物碱和有机酸是良好的抗虫抗菌剂,它们在野蚕茧中的含量较高,能够提高野蚕茧的防护能力。黄酮类物质在家蚕大造茧中的含量较高,是导致家蚕大造茧呈黄绿色的主要原因。     

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.     

A new method for the modification of fibroin heavy chain protein in the transgenic silkworm

We constructed a new plasmid vector for the production of a modified silk fibroin heavy chain protein (H-chain) in the transgenic silkworm. The plasmid (pHC-null) contained the promoter and the 3' region of a gene encoding the H-chain and the coding regions for the N-terminal domain and the C-terminal domain of the H-chain. For the model protein, we cloned a foreign gene that encoded EGFP between the N-terminal domain and the C-terminal domain in pHC-null and generated transgenic silkworms that produced a modified H-chain, HC-EGFP. Transgenic silkworms produced HC-EGFP in the posterior part of silk gland cells, secreted it into the lumen of the gland, and produced a cocoon with HC-EGFP as part of the fibroin proteins. N-terminal sequencing of HC-EGFP localized the signal sequence cleavage site to between positions A((21)) and N((22)). These results indicate that our new plasmid successfully produced the modified H-chain in a transgenic silkworm.     

Recognition of signal peptide by protein translocation machinery in middle silk gland of silkworm Bombyx mori

To investigate the functions of signal peptide in protein secretion in the middle silk gland of silkworm Bombyx mori, a series of recombinant Autographa californica multiple nucleopolyhedroviruses containing enhanced green fluorescent protein （egfp） gene, led by sericin-1 promoter and mutated signal peptide coding sequences, were constructed by region-deletions or single amino acid residue deletions. The recombinant Autographa californica multiple nucleopolyhedroviruses were injected into the hemocoele of newly ecdysed fifth-instar silkworm larvae. The expression and secretion of EGFP in the middle silk gland were examined by fluorescence microscopy and Western blot analysis. Results showed that even with a large part （up to 14 amino acid residues） of the ser-1 signal peptide deleted, the expressed EGFP could still be secreted into the cavity of the silk gland. Western blot analysis showed that shortening of the signal peptide from the C-terminal suppressed the maturation of pro-EGFP to EGFP. When 8 amino acid residues were deleted from the C-terminal of the signal peptide （mutant 13 aa）, the secretion of EGFP was incomplete, implicating the importance of proper coupling of the h-region and c-region. The deletion of amino acid residue（s） in the h-region did not affect the secretion of EGFP, indicating that the recognition of signal peptide by translocation machinery was mainly by a structural domain, but not by special amino acid residue（s）. Furthermore, the deletion ofArg^2 or replacement with Asp in the n-region of the signal peptide did not influence secretion of EGFP, suggesting that a positive charge is not crucial.     

Transgenic silkworms (<Emphasis Type="Italic">Bombyx mori</Emphasis>) produce recombinant spider dragline silk in cocoons

Spider dragline silk is a unique fibrous protein with a combination of tensile strength and elasticity, but the isolation of large amounts of silk from spiders is not feasible. In this study, we generated germline-transgenic silkworms (Bombyx mori) that spun cocoons containing recombinant spider silk. A piggyBac-based transformation vector was constructed that carried spider dragline silk (MaSp1) cDNA driven by the sericin 1 promoter. Silkworm eggs were injected with the vector, producing transgenic silkworms displaying DsRed fluorescence in their eyes. Genotyping analysis confirmed the integration of the MaSp1 gene into the genome of the transgenic silkworms, and silk protein analysis revealed its expression and secretion in the cocoon. Compared with wild-type silk, the recombinant silk displayed a higher tensile strength and elasticity. The results indicate the potential for producing recombinant spider silk in transgenic B. mori.