GFP基因在棉花转化中的应用

以绿色荧光蛋白GFP基因为报道基因,用花粉管通道和农杆菌介导的转化方法将外源基因导入棉花(Gossypium hirsutumL.),分别获得转化幼胚、幼苗和转化愈伤组织。用手持紫外灯结合显微镜检术能够快速地对转化子进行活体筛选鉴定,比用GUS检测方法有明显的优越性。本研究不但为花粉管通道转化法的可行性提供了新的证据,同时也建立了GFP用于棉花基因工程研究的检测技术体系。 Abstract：With the Green Fluorescent Protein gene (GFP) as a reporter gene, the transgenic embryos, seedlings and calli of cotton(Gossypium hirsutum L.) were obtained by the method of pollen tube pathway and Agrobacterium-mediated techniques separately. The GFP gene under the control of the 35s Cauliflower Mosaic Virus promoter produced bright?green fluorescence easily detectable and screenable in cotton tissue by fluorescence microscopy and a hand-held ultraviolet lamp. The screenable marker aided and facilated the rapid segregation of individual transformation events, drastically reduced the quantity of tissue to be handled. The GFP can be screened in vivo without destroying the materials, so it is more practical and useful than GUS. The use of GFP could advance the development of cotton gene engineering.     

Recombination with coat protein transgene in a complementation system based on Cucumber mosaic virus (CMV)

In order to study the feasibility of Cucumber mosaic virus (CMV) as an expression vector, the full-length cDNA of RNA 3 from strain SD was cloned and the sequence around the start codon of the coat protein (CP) gene was modified to create an Nsi I site for insertion of foreign genes. The CP gene was replaced by the green fluorescent protein (GFP) gene. The cDNAs of Fny RNAs 1 and 2 and the chimeric SD RNA 3 were cloned between the modified 35S promoter and terminator. Tobacco protoplasts were transfected with a mixture of the viral cDNAs containing 35S promoter and terminator as a replacement vector and expressed GFP. A complementation system was established when the replacement vector was inoculated onto the transgenic tobacco plants expressing SD-CMV CP. GFP was detected in the inoculated leaves in 5 of 18 tested plants and in the first upper systemic leaf of one of the 5 plants ten days after inoculation. However, no GFP could be detected in all the plants one month after inoculation. Recombination be     

PCR一步法构建融合蛋白基因fpg

采用一种不需要限制核酸酶和连接酶的新方法——“PCR一步法”将芳香烃化合物降解的关键基因pheB和绿色荧光蛋白编码基因gfp融合,构建得到融合蛋白基因fpg。该方法在一个PCR反应体系中通过三个引物、两个模板扩增得到一个含有中间柔性肽段-Gly4Ser-的融合基因fpg。本文研究结果表明,PCR一步法是一种快速方便的构建融合基因的方法。Abstract: TP-PCR,a method developed for fusion gene construction without the use of endonuclease and ligase, was performed to construct a fused fpg gene. The TP-PCR reaction system contained three primers and two templates and resulting PCR product, fused fpg gene, consisted of three sections: pheB gene, which was responsible for catechol 2,3-dioxygenase, gfp gene for GFP protein and the intermediate ligation segment which was designed for the correct expression of the fusion gene. The result in this paper showed that the TP-PCR method is one of rapid and convenient methods for fused gene construction.     

A palmitoyltransferase Approximated gene Bm-app regulates wing development in Bombyx mori

The silkworm Bombyx mori is an important lepidopteran model insect in which many kinds of natural mutants have been identified.However,molecular mechanisms of most of these mutants remain to be explored.Here we report the identification of a gene Bm-app is responsible for the silkworm minute wing(mw)mutation which exhibits exceedingly small wings during pupal and adult stages.Compared with the wild type silkworm,relative messenger RNA expression of Bm-app is significantly decreased in the ul 1 mutant strain which shows mw phenotype.A 10 bp insertion in the putative promoter region of the Bm-app gene in mw mutant strain was identified and the dual luciferase assay revealed that this insertion decreased Bm-app promoter activity.Furthermore,clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases-mediated depletion of the Bm-app induced similar wing defects which appeared in the mw mutant,demonstrating that Bm-app controls wing development in B.mori.Bm-app encodes a palmitoyltransferase and is responsible for the palmitoylation of selected cytoplasmic proteins,indicating that it is required for cell mitosis and growth during wing development.We also discuss the possibility that Bm-app regulates wing development through the Hippo signaling pathway in B.mori.     

ZNF230/荧光蛋白融合基因表达载体的构建及其在Cos细胞中的表达与定位

为了用绿色荧光蛋白标记观察人类无精症相关基因ZNF230在Cos7细胞中的蛋白质表达及定位,用PCR方法扩增得到突变的人和小鼠mt-ZNF230和mt-znf230基因,使其3′端的终止密码TGA突变为TGG,并装入T-载体,双酶切后通过定向克隆将其与真核表达载体pEGFP-N1的绿色荧光蛋白（green fluorescence protein,GFP）基因融合,构建了ZNF230—荧光蛋白融合基因表达载体。然后经真核表达质粒－脂质体介导,导入Cos7细胞系。荧光显微镜观察显示：在空白载体pEGFP-N1转染的Cos细胞中荧光布满整个细胞,而在转染阳性载体pEGFP-ZNF230的Cos细胞中荧光主要聚集在细胞核中。表明转染的Cos细胞系能高效表达人ZNF230和小鼠znf230蛋白,ZNF基因表达的蛋白定位于细胞核内。Abstract: To use green fluorescent protein as a marker to studythe localization of the fusion protein, the mutant full length cDNAs of human ZNF230 and mouse znf230 with their stop codon TGA changed to TGG were obtained by PCR amplification., and then cloned into pGEM-Teasy vector. After the double enzyme cutting, the mutated human and mouse ZNF230(znf230) were inserted into mammalian expression plasmid pEGFP-N1.Thus we constructed the plasmid with fusion gene of ZNF230 and green fluorescent protein(GFP).Then the Cos cell was transfected with the fused gene by liposome. Fluorescence microscopy showed that green fluorescence protein expressed over the whole cell when transfected with vector pEGFP-N1.While after the transfection with pEGFP-ZNF230, the fluorescence located mainly on the nuclei of the cells. We demonstrated that the transfected Cos cell line can express human ZNF230 and mouse znf230 with high efficiency.When transfected with the constructed recombinant pEGFP-ZNF230 vector, the ZNF230 protein localizes mainly on the nucleus.     

Analysis of ESTs and gene expression patterns of the poste-rior silkgland in the fifth instar larvae of silkworm, Bom-byx mori L.

The fibroin gene expression pattern and regulation of the posterior silkgland were studied by means of expressed sequence tags (ESTs) using the first and fifth day larvae of the fifth instar of silkworm, Bombyx mori L (strain: C 108). The results showed that there were 911 repetitive ESTs and 1950 single sequences (Singlets) among total 2861 consentient sequences, which were spliced. 1335 sequences were identified and the other 1526 were unknown. 5560 sequences (55.89%) in the posterior silkgland cell of the silkworm were new ESTs without ho-mology with EST data published by Mita et al. The number of repetitive ESTs and single sequences from the first day larvae of the fifth instar was double more than that of the fifth day of the same instar in the silkworms. The unigenes which were more than 50 in repetitive EST size (contig size) came to only about 0.5% in total consentient sequences. There were significant differences between gene expression frequencies, and expressed genes were related to fibroin synthesis and its secretion and fibroin composition. Comparing the fifth day with the first day of the fifth instar, the genes-expressed quantity of fibroin heavy-chain gene was 18 fold higher, fibroin light-chain gene 9 fold and fibroin P52 gene 8 fold. 508 genes functioned for cellular component and 315 for enzyme after function tracing. These results implied that the gene expression of the first day was mainly for preparation for fibroin synthesis except for the growth of silkgland cells, and the gene expression of the fifth day of the fifth instar was mainly for synthesizing and excreting fibroin. Because the ratio of heavy chain, light chain and p25 of fibroin was not 6:6:1 as theoretically expected, or its special H-chain structure, the H-chain gene was not easy to detect through EST technique. Most of genes among total 2861 consentient sequences functioned for fibroin synthesis and secretion. This suggested the fibroin synthesis and secretion procedure of the posterior silkgland was more complex than the knowledge we have.     

尕海孤雌生殖卤虫基因组的特异DNA片段

Artemia is not only valuable for aquaculture but also exhibits unique biological characters. In this study, based on the silkworm Bmdsx gene, a pair of primers was designed. After amplification with these primers, a DNA fragment Apdsx900 from parthenogenesis Artemia genomic DNA was obtained. The following Southern blotting and FISH analysis also proved the fragment was specific for Gahai parthenogenesis Artemia genome. To our knowledge, this is the first report of parthenogenesis genome specific DNA fragments. Apdsxg00 shares little similarity with the silkworm Bmdsx gene. [Acta Zoologica Sinica 50 (3): 470-474, 2004].     

SSR based linkage and mapping analysis of C,a yellow cocoon gene in the silkworm,Bombyx moil

The yellow color of the cocoon of the silkworm Bombyx mori is controlled by three genes, Y （Yellow haemolymph）, 1 （Yellow inhibitor） and C （ Outer-layer yellow cocoon）, which are located on linkage groups 2, 9 and 12, respectively. Taking advantage of a lack of crossing over in females, reciprocal backcrossed F1 （BC1） progeny were used for linkage analysis and mapping of the C gene using silkworm strains C 108 and KY, which spin white and yellow cocoons, respectively. DNA was extracted from individual pupae and analyzed for simple sequence repeat （SSR） markers. The C gene was found to be linked to seven SSR markers. All the yellow cocoon individuals from a female heterozygous backcross （BC1F） showed a heterozygous profile for SSR markers on linkage group 12, whereas individuals with light yellow cocoons showed the homozygous profile of the strain C108. Using a reciprocal heterozygous male backcross （BC1M）, we constructed a linkage map of 36.4 cM with the C gene located at the distal end, and the closest SSR marker at a distance of 13.9 cM.     

Possible effect of 30K proteins in embryonic development of silkworm Bombyx mori

The silkworm Bombyx mori possesses a 30K protein family of 3×10~4 Da,the biologicalfunctions of which have not been fully identified.The relationship between the 30K protein family and theembryonic development of temperature sensitive sex-linked mutant strain of silkworm was investigated bytwo dimensional polyacrylamide gel electrophoresis(2D-PAGE)and Matrix assisted laser desorption ionization-time of flight mass spectrometry(MALDI-TOF MS).The results show that protein spots 1-5 of the 30Kprotein family,mainly existing in normal strain,are possibly related to embryonic development.The earlyconsumption of a 30K protein named 6G1-30K-1 and the accumulation of 30K proteins named 6G1-30K-3and 6G1-30K-4 are likely caused by the destruction of physiological balance in normal embryonic development,which may lead to lower hatchability of the temperature sensitive strain.The results suggest that reasonablemetabolism of 30K proteins is a prerequisite for the embryo's normal development.     

New and highly efficient method for silkworm transgenesis using Autographa californica nucleopolyhedrovirus and piggyBac transposable elements

We have developed a new method for the transgenesis of the silkworm, Bombyx mori. This method couples the use of recombinant baculoviruses with the use of the piggyBac transposable element. One recombinant AcNPV, designated the helper virus, is designed to express the piggyBac transposase under the control of the Drosophila hsp70 promoter. Another recombinant AcNPV encoded the gene to be incorporated into the silkworm genome, in this case a green fluorescent protein (GFP) gene, under the control of B. mori actin A3 promoter and franked by the piggyBac inverted terminal repeats. Preblastoderm eggs were inoculated with a fine needle coated with a mixture of these two recombinant baculoviruses. Most of the inoculated larvae hatched and a high proportion of the newly hatched G0 larvae expressed the GFP marker. Transgenesis was confirmed by Southern blot analysis of G1 insects, sequencing the insertion site junctions isolated by inverse PCR, and the marker segregated in Mendelian fashion, as evidenced by the appearance of green fluorescence in G2 insects. Thus, transgenic silkworms were easily and efficiently obtained using this new method.     

Report on construction of gene-targeting vector for homologous recombination and transformation in silkworm, Bombyx mori L.

Abstract:  This paper reports the methods of construction of gene-targeting vector for transformation of silkworm, Bombyx mori L. The genomic DNA was isolated from the posterior silk gland of the fifth-instar silkworm larvae. The short fragment (0.5 kb) and long fragment (5 kb) of the fibroin light-chain gene were obtained by polymerase chain reaction (PCR) analysis with special primers and genome DNA as templates and then recombined with pBlueselect vector into pBs-FS-FL. The target green flourescent protein (GFP) gene, was derived from pGEP-1 vector and recombined with pUC19 vector into pUCG vector. GFP was recovered after cutting with restriction endonucleases, Pst I and Bam HI. Finally, GFP was recombined with pBs-FS-FL into gene-targeting vector, pBs-FS-GFP-FL.     

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。     

Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector

We have developed a system for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni. The transformation constructs consist of the piggyBac inverted terminal repeats flanking a fusion of the B. mori cytoplasmic actin gene BmA3 promoter and the green fluorescent protein (GFP). A nonautonomous helper plasmid encodes the piggyBac transposase. The reporter gene construct was coinjected into preblastoderm eggs of two strains of B. mori. Approximately 2% of the individuals in the G1 broods expressed GFP. DNA analyses of GFP-positive G1 silkworms revealed that multiple independent insertions occurred frequently. The transgene was stably transferred to the next generation through normal Mendelian inheritance. The presence of the inverted terminal repeats of piggyBac and the characteristic TTAA sequence at the borders of all the analyzed inserts confirmed that transformation resulted from precise transposition events. This efficient method of stable gene transfer in a lepidopteran insect opens the way for promising basic research and biotechnological applications.     

逆转录病毒载体介导胸苷磷酸化酶在胰腺癌细胞表达

人胸腺嘧啶核苷磷酸化酶（ＴＰ）在一些肿瘤组织中活性增高,但其功能目前了解尚少。构建了表达ＴＰ的重组逆转录病毒载体,直接导入人胰腺癌ＰＣ－２细胞,ｍＰＣＲ扩增、Ｓｏｕｔｈｅｒｎ及Ｎｏｒｔｈｅｒｎ印迹和原位杂交证实转染细胞有外源ＴＰ的整合及表达,酶活性检测发现含外源ＴＰ细胞ＴＰ活性比ＰＣ－２细胞的内源性ＴＰ活性高ＴＰ活性高４０－７０倍,生长曲线和＾３Ｈ－ＴｄＲ参入率检测未发现含外源ＴＰ细胞生物学行为的     

绿色荧光蛋白基因在昆虫细胞中的克隆与表达

将绿色荧光蛋白(GFP)基因亚克隆到转移载体pVLneo的多角体蛋白基因(ocu)启动子下游,与杆状病素AcNPV DNA共转染昆虫细胞,通过同源重组和G418筛选,构建了整合有GFP基因的重组病毒。在昆虫细胞中表达的GFP,MW为30kDa,在荧光显微镜下呈现美丽的绿色,荧光光谱表明其激发波长395nm,发射波长509nm。Southern blot杂交证明,重组病毒的1kb EcoRI片段与GFP cDNA探针有很强的杂交信号,这是GFP基因在杆状病毒基因组中整合的直接证据。     

Targeted gene expression using the GAL4/UAS system in the silkworm Bombyx mori

The silkworm Bombyx mori is one of the most well-studied insects in terms of both genetics and physiology and is recognized as the model lepidopteran insect. To develop an efficient system for analyzing gene function in the silkworm, we investigated the feasibility of using the GAL4/UAS system in conjunction with piggyBac vector-mediated germ-line transformation for targeted gene expression. To drive the GAL4 gene, we used two endogenous promoters that originated from the B. mori actin A3 (BmA3) and fibroin light-chain (FiL) genes and the artificial promoter 3xP3. GFP was used as the reporter. In initial tests of the function of the GAL4/UAS system, we generated transgenic animals that carried the UAS-GFP construct plus either BmA3-GAL4 or 3xP3-GAL4. GFP fluorescence was observed in the tissues of GFP-positive animals, in which both promoters drove GAL4 gene expression. Animals that possessed only the GAL4 gene or UAS-GFP construct did not show GFP fluorescence. In addition, as a further test of the ability of the GAL4/UAS system to drive tissue-specific expression we constructed FiL-GAL4 lines with 3xP3-CFP as the transformation marker. FiL-GAL4 x UAS-GFP crosses showed GFP expression in the posterior silk gland, in which the endogenous FiL gene is normally expressed. These results show that the GAL4/UAS system is applicable to B. mori and emphasize the potential of this system for controlled analyses of B. mori gene function.     

Effects of egt gene transfer on the development of Bombyx mori

This study investigated the effects of gain of ecdysteroid UDP-glucosyltransferase (EGT) gene function mutation on the development of the silkworm, Bombyx mori. A novel piggyBac-derived plasmid containing the egt gene from B. mori nucleopolyhedrovirus (BmNPV) driven by a heat-shock protein (hsp) 23.7 promoter, with a neomycin-resistance gene (neo) controlled by the BmNPV ie-1 promoter and a green fluorescent protein gene (gfp) under the control of the B. mori actin 3 (A3) promoter was constructed. The vector was transferred into silkworm eggs by sperm-mediated gene transfer. Transgenic silkworms were produced after screening for neo and gfp genes and gene transfer was verified by polymerase chain reaction, dot-blot hybridization and western blotting. The hatching rate of G1 generation silkworm eggs was about 60% lower than that of normal silkworm eggs. The duration of the G1 generation larval period was extended, and the G2 generation pupal stage lasted four days longer than that in non-transgenic silkworms. The ecdysone blood level in G2 silkworms in the third instar molting stage was reduced by up to 90%. These results show that EGT suppressed transgenic silkworm molting, and that egt expression in egt-transgenic silkworms resulted in arrest of metamorphosis from pupae to moths.     

拯救细胞系恢复重组家蚕核型多角体病毒包涵体实现外源蛋白在家蚕中产业化生产

构建家蚕Bombyx mori肌动蛋白（BmA3）启动子驱动的家蚕核型多角体病毒（BmNPV）多角体基因（ph）和OpNPV极早期启动子（IE1）驱动的zeocin抗性筛选基因转座供体载体,与鳞翅目辅助转座质粒pie2piggyBac共转染家蚕卵巢细胞BmN,经200μg/ml zeocin抗生素筛选一个月,成功获得持续表达BmNPV多角体蛋白的稳定细胞系BmN-A3ph。多角体缺陷型重组病毒BmBac-GF P感染拯救细胞系BmN- A3ph, 细胞成功装配出病毒包涵体颗粒,其包装效率约为野生型病毒感染正常BmN细胞的8%。用拯救型包涵体病毒颗粒喂食家蚕幼虫进行复感染,结果表明稳定细胞系所包装的包涵体病毒与野生型病毒一样能够通过口服途径感染宿主,却并不在宿主体内形成包涵体,从而保证外源基因高效表达。拯救型包涵体病毒可望解决传统注射感染效率较低问题,通过喂食感染可促进杆状病毒介导的家蚕生物反应器产业化进程。