Detection of CaMV gene I and gene VI protein products in vivo using antisera raised to COOH-terminal β-galactosidase fusion proteins

Specific antisera were prepared to the inclusion body protein (gene VI product) and the gene I product of cauliflower mosaic virus (CaMV). Translational fusions between the lacZ gene and gene VI or gene I were constructed by cloning the relevant DNA fragments into the expression vectors pUR290, pUR291 or pUR292. Large amounts of fusion protein were synthesized when the inserted DNA fragment was in frame with the lacZ gene of the expression vector. These fusion proteins were used to raise specific antisera to gene VI and gene I proteins of CaMV. Antiserum to the gene VI product detected a range of proteins in crude extracts and in a subcellular fraction enriched for virus inclusion bodies. This range of proteins was further shown to be related to gene VI by Staphylococcus aureus V8 partial proteolysis. Antiserum to the gene I product detected viral specific proteins of 46, 42 and 38 K in preparations of CaMV replication complexes from infected plants but not in any other subcellular fraction.     

伪狂犬病毒gD基因在转基因烟草中的表达

将猪伪狂犬病毒 (pseudorabiesvirus ,PRV)最主要的保护性抗原基因gD完整编码区亚克隆到修饰的植物双元表达载体pBI 35SL中 ,使其置于强启动子CaMV 35S doubleenhancer TEV 5′UTR下游 ,构建的转基因植物双元表达质粒经农杆菌介导转化烟草 .PCR检测叶片筛选阳性植株 ,Southern杂交进一步证实gD已整合到转基因烟草基因组中 .固相酶联斑点试验和Western印迹表明 ,gD在烟草获得正确表达并具有抗原性     

Expression of cauliflower mosaic virus gene I in Saccharomyces cerevisiae

Cauliflower mosaic virus (CaMV) gene I encodes a 40-kDa protein, P1, which is thought to be involved in the cell-to-cell movement of the virus. In order to investigate its functioning, P1 was expressed in Saccharomyces cerevisiae transformed by an expression vector containing CaMV gene I. When produced in yeast, P1 was 40 kDa in size and not N-glycosylated.     

Effect of an enhanced CaMV 35S promoter and a fruit-specific promoter on uida gene expression in transgenic tomato plants

Summary Two different promoters, a cauliflower mosaic virus (CaMV) 35S promoter with a 5′-untranslated leader sequence from alfalfa mosaic virus RNA4 (designated as CaMV 35S/AMV) and an E-8 fruit-ripening-specific promoter, were compared to evaluate their effects on expression of the uidA reporter gene in transgenic tomato plants. In order to generate sufficient numbers of transgenic tomato plants, both a reliable regeneration system and an efficient Agrobacterium transformation protocol were developed using 8-d-old cotyledons of tomato (Lycopersicon ecsulentum Mill. cv. Swifty Belle). Two sets of constructs, both derivatives of the binary vector pBI121, were used in transformation of tomato whereby the uidA gene was driven either by the CaMV 35S/AMV or the E-8 fruit-ripening-specific promoter. Southern blot hybridization confirmed the stable integration of the chimeric uidA gene into the tomato genome. Fruit and leaf tissues were collected from T₀ and T₁ plants, and assayed for β-glucuronidase (GUS) enzyme activity. As expected, both vegetative and fruit tissues of transgenic plants carrying the uidA gene under the control of CaMV 35S/AMV showed varying levels of GUS activity, while no expression was observed in vegetative tissues of transgenic plants carrying the uidA gene driven by the E-8 promoter. All fruits from transgenic plants produced with both sets of constructs displayed expression of the uidA gene. However, when this reporter gene was driven by the CaMV 35S/AMV, GUS activity levels were significantly higher than when it was driven by the E-8 fruit-specific promoter. The presence/absence of the uidA gene in T₁ plants segregated in a 3∶1 Mendelian ratio.     

Generation and immunogenicity of Japanese encephalitis virus envelope protein expressed in transgenic rice

Transgenic plants have become attractive as bioreactors to produce heterologous proteins that can be developed as edible vaccines. In the present study, transgenic rice expressing the envelope protein (E) of Japanese encephalitis virus (JEV), under the control of a dual cauliflower mosaic virus (CaMV 35S) promoter, was generated by Agrobacterium-mediated transformation. Southern blot, Northern blot, Western blot and ELISA analyses confirmed that the E gene was integrated into transgenic rice and was expressed in the leaves at levels of 1.1-1.9 μg/mg of total soluble protein. After intraperitoneal immunization of mice with crude protein extracts from transgenic rice plants, JEV-specific neutralizing antibody could be detected. Moreover, E-specific mucosal immune responses could be detected in mice after oral immunization with protein extracts from transgenic rice plants. These results show the potential of using a transgenic rice-based expression system as an alternative bioreactor for JEV subunit vaccine.     

Altered patterns of gene expression in Arabidopsis elicited by cauliflower mosaic virus (CaMV) infection and by a CaMV gene VI transgene

Cauliflower mosaic virus (CaMV) gene VI protein (P6) is an important determinant of symptom expression. Differential display polymerase chain reaction (PCR) was used to identify changes in gene expression in Arabidopsis elicited by a P6 transgene that causes a symptomatic phenotype. We used slot blot hybridization to measure the abundance of mRNAs complementary to 66 candidate PCR products in transgenic, CaMV-infected, and uninfected Arabidopsis plants. CaMV-infected and P6 transgenic plants showed broadly similar changes in abundance of mRNA species. In P6 transgenic plants we detected 18 PCR products that showed unambiguous changes in abundance plus another 15 that showed more limited changes (approximately twofold). CaMV-infected plants showed 17 unambiguous and 13 limited changes. Down-regulated species include those encoding a novel, phenol-like sulfotransferase, and a glycine-rich, RNA-binding protein. Up-regulated species included ones encoding an myb protein, glycine-rich and stress-inducible proteins, and a member of a previously unreported gene family. CaMV infection causes alterations in expression of many Arabidopsis genes. Transgene-mediated expression of P6 mimics virus infection in its effect on host gene expression, providing a potential mechanism for this process.     

ggpps 5'-侧翼序列的克隆及其启动子功能验证

目的:研究牻牛儿基牻牛儿基焦磷酸合成酶(geranylgeranyl diphosphate synthase,GGPPs)基因启动子的活性;方法:从曼地亚红豆杉细胞中克隆ggpps基因5'-侧翼序列,并将该侧翼序列代替pBI121质粒上的CaMV35S启动子,以Gus基因作为报告基因构建植物表达载体,并进一步导入农杆菌LBA4404中获得阳性转化子,然后用叶盘转化法验证该侧翼序列的启动子活性;结果:本研究从曼地亚红豆杉细胞中成功克隆了ggpps基因的5'-侧翼序列,并且验证了该侧翼序列具有启动子活性;结论:ggpps基因的5'-侧翼序列的测序结果表明本实验成功克隆了该侧翼序列,启动子功能验证结果表明ggpps 5'-侧翼序列具有启动子活性,这些结果为进一步的通过缺失法进行ggpps基因启动子功能研究奠定了基础.     

香蕉凝集素基因启动子的分离、序列分析及鉴定

香蕉是世界上最重要的水果之一,由于香蕉果实是利用转基因方法生产重组药用蛋白或有价值的化合物的理想器官,构建能在香蕉果实中高水平表达异源蛋白质的表达载体是非常有意义的。而一个高效表达的载体,启动子则是其最重要元件之一,因此,果实特异性表达启动子的获得是香蕉作为生物反应器的前提。香蕉凝集素是一种在香蕉果实中大量存在的蛋白质,其基因被证明在果肉组织中大量表达。利用染色体步移法克隆到香蕉凝集素基因5′端上游的一段长702bp的序列,经序列测定及软件分析表明,该序列具有典型的启动子结构。此序列置换植物表达载体pBI121的CaMV35S启动子,构建植物表达载体,命名为pBIL2,该启动子下游为gus基因。利用基因枪法转化香蕉的根、叶和果实薄片,对gus基因的瞬时表达进行测定,结果表明所获得的凝集素基因启动子,只在香蕉果肉中瞬时表达,该启动子的表达具有果实特异性,并且表达量较高。     

Applications of an optimized monocot expression vector in studying transient gene expression and stable transformation of barley

Protoplasts of a barley ( Hordeum vulgare L. cv. Golden Promise) suspension cell line were used for PEG-mediated gene transfer. Transient gene expression in barley protoplasts was studied using a chimeric CaMV 35S cat construct, which was only poorly expressed in barley cells. However, insertion of exon 1 and intron 1 of the maize Shrunken-1 (Sh1) gene in the 5'-untranslated leader of the construct strongly stimulated gene expression. By using the optimized chimeric cat construction the amount of CAT protein that was reached 19 hours after DNA uptake was 0.5% of total protein, which was calculated from western blot data.
As an alternative marker gene for expression studies, we also tested the firefly luciferase gene in barley protoplasts. Low level expression of chimeric CaMV 35S luciferase genes could be highly stimulated when Sh1 exon1 and intron1 were inserted in the 5'-untranslated leader of the constructs. Enhanced luciferase gene expression by Shrunken-1 intronic sequences enabled us to monitor gene integration events early after DNA uptake using a promoterless luciferase marker gene, which could only be expressed after integration behind an endogenous promoter.     

The cauliflower mosaic virus open reading frame VII product can be expressed in Saccharomyces cerevisiae but is not detected in infected plants.

Antiserum was prepared against a synthetic peptide corresponding to the N-terminal 20 amino acids of the protein encoded by cauliflower mosaic virus (CaMV) open reading frame VII (ORF VII). This antiserum was used to detect the expression of CaMV ORF VII either in Saccharomyces cerevisiae transformed by an expression vector containing CaMV ORF VII or in CaMV-infected plants. Only in S. cerevisiae has a 14-kilodalton protein been detected.