Efficient and stable generation of transgenic silkworm, Bombyx mori with the lepidopteran derived transposon piggyBac

Transposable elements, such as P element, have be-come important tools in the study of gene function in Drosophila melanogaster. Their applications are manifold, serving as mutagens and molecular tags for identification and isolation of new genes, and as vehicles for introducing custom-made gene sequences into organism’s genome[1]. However, the use of P elements appears to be only re-stricted to Drosophila[2,3]. Recently, foreign genes have been successfully introduced into several other gr…     

Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination

A 'rescuable' plasmid containing globin gene sequences allowing recombination with homologous chromosomal sequences has enabled us to produce, score and clone mammalian cells with the plasmid integrated into the human beta-globin locus. The planned modification was achieved in about one per thousand transformed cells whether or not the target gene was expressed.     

家蚕促咽侧体素受体基因克隆及发育表达

文章克隆得到家蚕(Bombyx mori)神经肽促咽侧体素受体基因(Allatotropin receptor,BommoATR),开放阅读框全长为1 254 bp,编码416个氨基酸.Bommo-ATR氨基酸序列的二级结构预测为7次穿膜蛋白,符合GPCR家族成员的典型特征.实时荧光定量PCR结果表明,家蚕脑-咽下神经节复合体中的ATR mRNA在5龄幼虫期表达量最高,其次为蛹后期,蛹前期和成虫阶段表达量最低.其中5龄幼虫第2天表达量最高,1～5 d持续维持较高的表达水平,这可能与保幼激素的合成有关.     

^3H—GLY对家蚕蛋白质生物合成参入活性的研究

氚标记甘氨酸（＾３Ｈ－Ｇｌｙ）对家蚕蛋白质生物合成参入试验的研究结果表明,＾３Ｈ－Ｇｌｙ对家蚕５龄幼虫的标记剂量和标记时间以每头蚕５μＣｉ和３０ｍｉｎ为宜;＾３Ｈ－Ｇｌｙ对家蚕几个主要器官的蛋白质参入活性有很大差异,进入蚕体的＾３Ｈ－Ｇｌｙ大部分参入到后部丝腺蛋白质合成中。     

同源重组法敲除酵母snoRNA基因的初步探讨

以3个粟酒裂殖酵母核仁小RNA为对象,通过同源重组法构建相应的基因缺失株,并对影响同源重组效率的因素进行分析.结果显示:载体骨架的切除可以显著提高同源重组效率;增加两侧同源片段的长度也能提高同源重组效率;另外,利用粟酒裂殖酵母野生型单倍体菌株进行snoRNA 基因敲除是可行的.     

Functional analysis of the larval serum protein gene promoter from silkworm <Emphasis Type="Italic">Bombyx mori.</Emphasis>

The regulation region of larval serum protein gene, Bombyx mori. (BmLSP), consisting of the first intron, the first exon, the central promoter region and 5′-upstream region, is cloned from genomic DNA from the silkworm va-riety of Suju譓inghu. Using PCR and restriction endonu-clease methods, a series of luciferase reporter plasmids, driven by different length of BmLSP promoters, are con-structed. Via the transient expression system in BmN cells, the effects of the regulation elements and foreign insect hor-mones on the BmLSP promoter activity are investigated. The results demonstrate that the promoter activity of BmLSP is 5.8- or 4.4-fold higher than that of BmLSPs whose first in-tron or the element in 5′-upstream region harboring the homologous sequence with the first intron of light-chain fib-roin gene (EHIF) is deleted, respectively, suggesting that both the first intron and EHIF contain the main positive cis-acting elements. However, the inactive mariner transposable ele-ment (MTE) in 5′-upstream region presents a negative effect. Furthermore, the effects of juvenile hormone analogue (JHA) on the BmLSP promoter activity show a typical dose-dependent manner, that is, low concentration treat-ments increase the BmLSP promoter activity and high con-centration treatments decrease it. Meanwhile, insect ecdy-sone (MH) treatments present no significant effect.     

同源重组敲除三角褐指藻基因的研究

为探索应用基因敲除技术研究三角褐指藻基因功能的研究体系,本文以三角褐指藻甘油激酶基因作为靶基因,构建了同源重组基因敲除载体,利用微弹轰击法将该载体成功转化至三角褐指藻中,经100μg/mL Zeocin筛选和PCR验证获得了34个阳性转基因藻株;并进一步对三角褐指藻甘油激酶基因敲除转基因藻株的甘油激酶表达量和生长两方面进行分析,结果显示胞外甘油兼养不影响转基因藻细胞生长,甘油激酶不表达或表达量降低.本文通过构建敲除载体,完成了遗传转化,筛选获得阳性转基因藻,并进一步研究了转基因藻的性状,最终建立了应用同源重组基因敲除技术靶向研究三角褐指藻基因功能的研究体系.     

Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination

The bglS gene encoding endo-1,3-1,4-β-glucanase from Bacillus subtil＆ was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone a-factor （MFals）, and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction （PCR）-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae （SC-βG） was preliminarily screened by the clearing hydrolysis zone formed after the barley β-glucan was hydrolyzed in the plate and no proteinase A （PrA） activity was measured in fermenting liquor. The results of PCR analysis of genome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-1,3-1,4-β-glucanase assay methods showed that the recombinant strain SC-βG had high endo-1,3-1,4-β-glucanase expression level with the maximum of 69.3 U/（h·ml） after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-1,3-1,4-β-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.     

3H-GLY对家蚕蛋白质生物合成参入活性的研究

氚标记甘氨酸(3H-Gly)对家蚕蛋白质生物合成参入试验的研究结果表明,3H-Gly对家蚕5龄幼虫的标记剂量和标记时间以每头蚕5μCi和30min为宜;3H-Gly对家蚕几个主要器官的蛋白质参入活性有很大差异,进入蚕体的3H-Gly大部分参入到后部丝腺蛋白质合成中.     

基于青藤碱的抗风湿性关节炎分子构建的研究进展

利用PCR技术扩增出BmDNV-1结构蛋白vp4基因,并将该基因与原核表达载体pMALc2X进行连接,转化大肠杆菌DH10B.获得重组质粒经IPTG诱导表达得到大小为96 000的融合蛋白,融合蛋白经Amylose柱纯化,使用其免疫新西兰大白兔,制备出多克隆抗体.从而为进一步研究该病毒结构蛋白基因的转录和翻译机制提供可靠的工具.     

His-猪生长激素融合基因在Bm-N细胞和蚕体内的表达与纯化

用构建的带有His—Tag pgh融合基因的重组杆状病毒Bm—BacPAK6—pgh研究了Bm—BacPAK6—pgh在家蚕细胞(Bm—N)和蚕体内的表达，并对蚕体表达产物进行了纯化．SDS—PAGE电泳分析显示，重组病毒Bin—BacPAK6—pgh在Bin—N细胞、蚕体中得到了融合表达，Western blot分析表明，Bm—N细胞、蚕体中表达的融合蛋白与大肠杆菌表达的猪生长激素具有相同的抗原性．Bm-BacPAK6-pgh在Bin—N细胞内的表达始于24h，而蚕体中的表达始于72h，二者的表达峰分别在96h和120h．经40％饱和度硫酸铵盐析和Ni—NTA Agarose亲和柱二步纯化可获得SDS—PA(正电泳纯的具有抗原性的重组猪生长激素融合蛋白。     

保幼激素和蜕皮激素对家蚕翅原基生长分化的影响

翅原基（Wing disc）是昆虫幼虫体内成虫原基的一种,经生长分化后最终形成成虫翅。翅原基是研究昆虫变态发育过程的一个理想系统。为探究发育过程中翅原基的生长变化,本文以家蚕翅原基为材料,通过离体和石蜡切片的方法观察了翅原基从5龄第3天至蛹期0天的形态变化,并通过注射外源蜕皮激素活性物质20E和保幼激素类似物Methoprene探究了昆虫激素对家蚕翅原基生长分化的影响。结果显示,翅原基在幼虫阶段发育缓慢,从5龄第6天起生长分化逐渐加快,且翅原基的形态发生了显著变化,原本附着于翅原基腔口处且呈团状的造血器官逐渐分散至消失,而由气管组成的翅脉逐渐形成。外源激素处理的结果显示,2μg剂量的20E可促进翅原基的生长分化,而2μg 的Methoprene抑制了翅原基的生长分化。上述结果暗示了,蜕皮激素和保幼激素共同调控了翅原基的生长分化,并最终实现了翅原基的变态发育。     

家蚕碱性磷酸酶的分离纯化与部分性质

经10％～50％硫酸铵分级沉淀分离，DEAE—Sepharose离子交换柱层析,Sephacryl S-200凝胶过滤纯化,从家蚕（Bombyx mori）肠液中分离纯化出电泳纯的碱性磷酸酶、该酶提纯倍数为464倍，比活力为3936U／mg、酶学性质和动力学性质研究表明，该酶催化磷酸苯二钠的水解反应，最适pH值为10.5，pH小于7.5和大于11均不稳定；最适温度为40℃，温度高于50℃不稳定；米氏常数Km值为1．25mmo1／L.     

家蚕BmFKBP45基因的表达和功能分析

以家蚕（Bombyx mori）为研究对象,对果蝇DmFKBP39的同源蛋白BmFKBP45进行了表达和初步的功能分析. 对DmFKBP39和BmFKBP45进行氨基酸序列比对分析发现,它们都含有酸性氨基酸区域、碱性氨基酸区域、核定位信号及FKBP结构域. 将BmFKBP45的第2个碱性区域与核蛋白HMG2的DNA结合位点进行比对,相似性达到21%,推测BmFKBP45可通过其第2个碱性区域与DNA结合,但EMSA的结果显示重组BmFKBP45不与果蝇的JHRE1元件结合. RT-PCR和Western blot结果显示,BmFKBP45在各个发育时期的家蚕翅原基中都有表达,在蛹期的表达量逐渐下降. 激素处理实验结果显示,BmFKBP45的表达并不受20E和JH的影响. 利用Pull-down、Far-western blot和Co-IP实验鉴定出一个与BmFKBP45相互作用的蛋白Bm6G1. 这些研究结果为进一步探究BmFKBP45的功能提供了线索.