转植酸酶基因家蚕的制作及表达检测

家蚕Bombyx mori丝腺具有高效合成蛋白质的特性,开发在丝腺特异表达外源蛋白质的生物反应器具有重要的意义。本研究利用piggyBac来源的两种载体pPIGA3GFP和pBac{3×P3-EGFPaf},建立了稳定的家蚕转基因技术体系; 然后,利用一株黑曲霉来源的植酸酶基因,构建了在家蚕后部丝腺特异表达的融合表达载体pBac [3×P3-EGFP+ FibLphyADsRed],注射蚕卵后,在53个G1蛾区中检测到3个有荧光蚕的蛾区。经Southern blot和反向PCR验证,转基因表达盒整合到家蚕染色体上。RT-PCR结果显示,植酸酶基因特异性地在后部丝腺表达,其表达模式与家蚕轻链丝素基因一致。结果表明我们成功获得了在后部丝腺特异表达植酸酶融合蛋白的转基因蚕,这为进一步开发家蚕生物反应器,利用转基因蚕生产各种重组蛋白具有积极的促进作用。     

家蚕转基因技术中若干因素对转基因效率的影响

建立高效、稳定的家蚕Bombyx mori转基因技术对于推进家蚕功能基因组研究, 解决蚕丝产业重大问题以及向非绢丝产业拓展等具有重要意义。本文在已建立的基于piggyBac的家蚕转基因技术基础上, 探索了多个影响转基因效率的因素。结果显示：以家蚕品种大造 (P50) 为供试材料、pBac［GOI］为供体质粒、pHA4PIG为辅助质粒, 以眼睛和神经组织特异启动子3×p3启动的红色荧光蛋白基因DsRed为报告基因, 在蚕卵产下后2～3 h进行注射,综合效果最佳, 孵化率和转化率分别达到62.7%和34.8%;荧光筛选的最佳时期在胚胎发育第5到第8天;在2 000～8 000 bp之间时, 外源片段的长度对转化率并无太大影响。本研究建立的技术体系, 有望为家蚕功能基因研究、品种分子改良和家蚕生物反应器的开发奠定基础, 并为其他鳞翅目昆虫转基因技术的建立提供参考。     

饲料中转基因成分对家蚕生长发育的影响

采用家蚕（Bombyx mori）H9品种作为实验动物,分别用转基因和非转基因大豆粉制作的人工饲料进行饲育。通过饲育试验,比较分析了不同处理后家蚕的龄期经过时间、全茧量、茧层量、茧层率、蛹体重、疏毛率、死亡率、每龄眠起体重等经济性状指标。同时采用PCR方法对转基因饲料饲育后家蚕组织中外源基因的表达情况进行了检测。结果显示,含转基因大豆粉的人工饲料对家蚕的生长发育并无显著影响,且不存在外源基因的污染。     

家蚕Bms3a基因真核表达载体的构建及其在家蚕细胞和蚕体中的表达

Bms3a基因可能在家蚕Bombyx mori抗病或细胞凋亡中有一定的作用。将融合有绿色荧光蛋白的Bins3a基因克隆到杆状病毒转移载体pFastBac1中获得了pFastBac-IE1-Bms3a-EGFP真核表达载体,利用杆状病毒(Bac-to-Bac)表达系统筛选重组杆状病毒,以重组病毒感染家蚕BmN细胞和五龄幼虫,分别在感染24h和48h检测到有绿色荧光蛋白的表达,Western blot证明表达的融合蛋白在相对分子量约57kD处出现特异条带,与预计的蛋白理论值相符。结果表明BmS3A-EGFP融合蛋白在家蚕细胞BmN及幼虫体中得到高效表达。研究结果为进一步研究BmS3A蛋白的功能奠定了基础。     

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。     

家蚕核型多角体病毒BmNPV泛素基因缺失显著降低病毒增殖效率北大核心

【目的】泛素是一类进化上高度保守的、由76个氨基酸组成的多肽,是蛋白质泛素化修饰过程中所必需的底物分子,蛋白质泛素化修饰异常会影响宿主的生长和发育。家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)基因orf26是一个编码病毒泛素的基因,其在病毒增殖中的具体功能尚不清楚。【方法】本研究利用同源重组的方法将氯霉素(chloramphenicol)基因(Cm)表达盒替换家蚕杆状病毒泛素基因序列3’端的50个碱基对序列,构建携带Cm表达盒的重组BmNPV病毒基因组(Bm-Bacmid^(Ub-KO)),利用转座原理将绿色荧光蛋白基因(green fluorescent protein,gfp)表达盒插入Bm-Bacmid^(Ub-KO)和野生型Bm-Bacmid^(WT)转座位点,构建重组质粒Bm-Bacmid^(Ub-KO-GFP)和Bm-Bacmid^(WT-GFP),以及异位互补型的质粒Bm-Bacmid^(GFP-Ub Rep)。这些重组病毒分别转染家蚕卵巢细胞(Bombyx mori ovarian cell,BmN)后,对转染后的细胞进行绿色荧光观察。【结果】泛素基因缺失后不影响感染性病毒粒子的产生,但与野生型相比,泛素缺失型重组病毒明显降低了感染的细胞中产生的绿色荧光数量。免疫印迹分析表明,泛素缺失后降低重组病毒结构蛋白GP64和VP39在细胞中的表达水平,显著降低病毒增殖效率。生物分析表明,泛素缺失的重组病毒能延缓家蚕半致死时间15 h。【结论】BmNPV泛素基因缺失不影响感染性病毒粒子的产生,但显著降低病毒增殖效率。本研究为阐明泛素在BmNPV增殖中的具体作用提供了实验依据。     

piggyBac转座子介导的转基因叉尾斗鱼的构建

为探讨piggyBac转座子在鱼类动物中应用的可能性,以包含家蚕(Bombyx mori)肌动蛋白3启动子驱动的增强型绿色荧光蛋白(enhance green fluorescent protein,EGFP)基因的piggyBac质粒为载体,以及一个包含piggyBac转座酶的辅助质粒,采用显微注射的方法将其导入叉尾斗鱼(Macropodusopercularis)受精卵中,利用PCR技术证实了piggyBac转座子能够介导EGFP基因进入叉尾斗鱼基因组,并能够稳定遗传到下一代,符合孟德尔遗传规律。EGFP基因遗传到G1代的阳性鱼占交配鱼比率,即外源基因整合率为12.30%。实验证明,piggyBac质粒有可能成为水产动物转基因实验的新型载体。     

基于RNA-Seq分析Ras1~(CA)在家蚕后部丝腺过表达对细胞周期通路基因的影响

【目的】本文旨在挖掘野生型家蚕Bombyx mori和Ras1CA过表达转基因家蚕后部丝腺中的转录本差异,分析和验证细胞周期通路中的差异表达基因,从而探讨Ras1CA过表达转基因家蚕提高蚕丝产量的分子机制。【方法】利用转录组学比较野生型和Ras1CA过表达转基因家蚕后部丝腺中的基因转录本差异,经实时荧光定量PCR(q RT-PCR)验证。【结果】野生型家蚕和Ras1CA过表达转基因家蚕后部丝腺组织中有2 636个差异表达基因,其中细胞周期信号通路中有42个差异表达基因,包括细胞周期依赖性激酶(CDK)、细胞周期素(Cyclin)以及转录因子等。通过q RT-PCR检测cdk1、cyclin D1、cyclin D2、cdc7、cdh1、dp-1,2等6个基因在野生型和Ras1CA过表达转基因家蚕后部丝腺组织中的相对表达量,发现转基因家蚕中的表达量均显著高于野生型(P<0.05),其中cyclin D2的表达差异极显著(P<0.01)。q RT-PCR结果与转录本差异一致,表明Ras1CA过表达后,能够促进细胞周期通路基因的表达。【结论】野生型家蚕和Ras1CA过表达转基因家蚕后部丝腺中有大量差异表达基因,且Ras1CA能够在转录水平上调控细胞周期通路,影响后部丝腺组织的细胞分裂和器官生长,从而促进蚕丝蛋白的合成。     

转座子介导的多角体基因表达及提高病毒粒子的感染效率

现行的杆状病毒表达外源基因的方法是将外源基因取代病毒中的多角体基因,因而得到的重组杆状病毒感染活体时不能经口感染,只能进行针刺注射,效率低且易引起活体感染其他疾病。将家蚕核型多角体病毒(Bombyx mor inucleopolyhedrovirus,BmNPV)中的多角体基因(polyhedrin,poly)及其启动子片段克隆到转座子载体pigA3GFP中,将其与辅助质粒pHA3PIG利用脂质体介导法导入家蚕细胞中,经过多次筛选获得稳定的转基因家蚕细胞。之后先将BmPAK6(含LacZ)及BmGFP(含GFP)重组病毒分别感染转基因细胞,再将得到的重组病毒经口感染5龄家蚕幼虫。结果显示,重组杆状病毒可以经口感染家蚕幼虫。这些研究表明来自于转基因家蚕细胞的poly基因表达产物可以提高重组杆状病毒经口感染家蚕率,为解决杆状病毒表达系统中重组病毒不能经口感染家蚕幼虫的问题提供新思路。     

利用CLSM检测GFP基因在家蚕的瞬时表达

将绿色荧光蛋白(GFP)基因导入农蚕蛹(G0)的睾丸,利用激光共聚焦扫描显微镜(CLSM)系统检测,得到GFP基因在家蚕刚孵出未进食的幼虫(蚁蚕)(G1)瞬时表达的荧光图像。 Abstract:Plasmid DNA containing the GFP(Green Fluorescent Protein)reporter gene was injected into the testis of silkworm(Bombyx mori L.)pupa(G0),we observe the fluorescence imaging of expression of GFP gene in silkworm newly-hatched larvae(G1)examined by Confocal Laser Scanning Microscope.     

Analysis of transgenic silkworms producing insulin-like growth factor-I in Bombyx mori

Silkworms contain a powerful and effective fibroin promoter, which controls the expression of fibroin, a silk protein. The fibroin promoter and well-known characteristics of silkworm, the application of transgenic technique to silkworm will provide an excellent opportunity to mass-produce biomolecules. In this study, the production of recombinant human insulin like growth factor-I (rhIGF-I) in the silkworm system was designed. The method makes use of the microinjection technique and P element vector to transfer foreign genes into the chromosomes. We constructed the expression vector using the fibroin gene promoter and P element vector containing IGF-I gene (pFpIGF-I). We then microinjected this vector into eggs, and through PCR screening, transgenic silkworms were selected. We isolated and purified rhIGF-I from silkworm cocoons, returning a concentration of rhIGF-I of about 1,300 ng/g from transgenic silkworm cocoons. In a comparison of transgenic silkworm rhIGF-I and colostral IGF-I on cell proliferation, colostral IGF-I was better able to increase the proliferation rate of the cell line relative to the transgenic silkworm rhIGF-I, and showed a similar cell proliferation pattern. The anti-cancer effects of transgenic silkworm rhIGF-I were higher than that of colostral IGF-I on HeLa and SNU-C1 cancer cells. These results confirmed the construction of new transgenic silkworm strains producing rhIGF-I.     

Transgenic breeding of anti-Bombyx mori L. nuclear polyhedrosis virus silkworm Bombyx mori

Silkworm strains resistant to Bombyx mori L. nuclear polyhedrosis virus were obtained through transgenic experiments. piggyBac transposon with an A3 promoter were randomly inserted into the silkworm, driving the enhanced green fluorescent protein (EGFP) reporter gene into the silkworm genome. Polymerase chain reaction results verified the insertion of the extraneous EGFP gene, and fluorescence microscopy showed that the EGFP was expressed in the midgut tissue. The morbidity ratio of the nuclear polyhedrosis decreased from 90% in the original silkworm strain to 66.7% in the transgenic silkworm strain. Compared with the resistance to the Bombyx mori L. nuclear polyhedrosis virus in the Qiufeng strain, which is commonly used in the production, there was an increase of 33 centesimal points in the transgenic silkworms. The antivirotic character in the Chunhua x Qiuyue strain, which was bred from a different transgenic family, was about 10 centesimal points higher than that in the Qiufeng x Baiyu, another crossbreed used in production. Our results indicated a good application value of the transposon-inserted mutation in the breeding of anti-BmNPV silkworm strain.     

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。     

非转座子载体介导的转基因家蚕表达hIL-28A

为了探讨非转座子载体介导转基因家蚕表达外源基因的可能性,将hIL-28A克隆进昆虫细胞表达载体pIZT/V5-His,构建了重组载体pIZT/V5-His-hIL-28A.利用精子介导法将该重组载体导入家蚕卵,通过绿色荧光筛选并结合PCR、DNA杂交等分子鉴定,证实成功获得了转基因家蚕.Western blotting结果显示,转基因家蚕表达重组hIL-28A的分子质量为25 ku,ELISA检测结果显示,hIL-28A在G3代转基因蚕、后部丝腺、脂肪组织冻干粉中的含量分别为0.198、0.320和0.238 ng/g.表明通过非转座子载体介导可以将外源基因导入家蚕基因组并实现外源基因的表达.     

Knockdown of ecdysis-triggering hormone gene with a binary UAS/GAL4 RNA interference system leads to lethal ecdysis deficiency in silkworm

Ecdysis-triggering hormone (ETH) is an integration factor in the ecdysis process of most insects, including Bombyx mori (silkworm). To understand the function of the ETH gene in silkworm, we developed an effective approach to knockdown the expression of ETH in vivo based on RNA interference (RNAi) and a binary UAS/GAL4 expression system that has been successfully used in other insect species. Two kinds of transgenic silkworm were established with this method: the effector strain with the ETH RNAi sequence under the control of UAS and the activator strain with the GAL4 coding sequence under the control of Bombyx mori cytoplasmic actin3. By crossing the two strains, double-positive transgenic silkworm was obtained, and their ETH expression was found to be dramatically lower than that of each single positive transgenic parent. Severe ecdysis deficiency proved lethal to the double-positive transgenic silkworm at the stage of pharate second instar larvae, while the single positive transgenic or wild-type silkworm had normal ecdysis. This UAS/GAL4 RNAi approach provides a way to study the function of endogenous silkworm genes at different development stages.     

利用精子介导法向蚕卵导入外源基因的研究

为建立家蚕转基因中切实可行、操作简便的外源基因导入方法,进行了精子介导法探索,以精子介导法的三种方式向家蚕导入所构建质粒pFbGFP,并通过PCR扩增和DNA印迹等手段,已连续两代从基因组DNA检测到导入外源基因GFP的存在,其中的一种导入方式到第二代阳性率约30% .结果表明该法可有效进行家蚕转基因的外源基因导入.     

Efficient soluble protein production on transgenic silkworms expressing cytoplasmic chaperones

Baculovirus expression systems (BES) are widely used for recombinant protein production in lepidopteran cells or larvae. However, even in BES, the insolubility of recombinant proteins sometimes makes their expression difficult. In this study, to improve the solubility and yield of foreign proteins, we constructed transgenic silkworms using silkworm heat-shock proteins, Hsp70 and Hsp40, or Hsc70 and Hsp90 co-chaperone Hop. In these transgenic silkworms, the expression levels of the transgenes were under the control of a UAS·hsp mini-promoter driven by a Gal4NFkBp65 activator. When the transgenic silkworm with HSP70 and 40 (TGS-HSP70/40) was infected with BmNPV carrying mC3d and Gal4NFkBp65 under the control of baculovirus polyhedrin or p10 promoters, respectively, the soluble fraction of the His- or His·GST-tagged mC3d increased significantly. Similarly, the transgenic silkworm with HSC70 and HOP (TGS-HOP7) was effective for the expression of a steroid hormone receptor, USP2. In conclusion, the His-tagged baculovirus expression system featuring the chaperone effect TGS-HSP70/40 and TGS-HOP7 silkworms is effective for increasing the yields of soluble and functional foreign gene products.     

Introduction of foreign genes into silkworm eggs by electroporation and its application in transgenic vector test

Up to now, the most practical method for introducingforeign gene into insect eggs is the microinjection as thatwas utilized in Drosophila melanogaster [1–3]. The dis-covery of transposable elements including: P, Minos,Hermes, mariner, hobo, piggyBac, etc…     

Development of a heat shock inducible and inheritable RNAi system in silkworm

A heat shock inducible and inheritable RNA interference (RNAi) system was developed in the silkworm (Bombyx mori). RNAi transgenic silkworms were generated by injecting silkworm eggs with a piggyBac transposon plasmid carrying RNAi sequence against target gene driven by the Drosophila heat shock protein 70 (HSP70) promoter and the helper plasmid expressing piggyBac transposase. The transgenic EGFP gene and the endogenous eclosion hormone (EH) gene were chosen respectively as the target genes. In the RNAi transgenic silkworms, heat shock at 42 degrees C significantly and specifically reduced the expression of EGFP or EH gene in silkworms according to the corresponding RNAi targeting sequence but not in silkworms with the irrelevant RNAi sequence demonstrating the efficiency and specificity of the RNAi effect. Heat shock in the pupal stage hampered pupal-adult eclosion and reduced egg fertility in EH RNAi transgenic silkworms but not in the wild type or EGFP RNAi transgenic silkworms. The establishment of this heat inducible and inheritable conditional RNA interference system in silkworms provided an approach for the first time to dissect the functions of target genes in silkworms at different stages.