The sequence of carnation etched ring virus DNA: comparison with cauliflower mosaic virus and retroviruses

Carnation etched ring virus (CERV) DNA comprises 7932 bp. CERV primer binding sites and overall genome organization are similar to those of the related cauliflower mosaic virus (CaMV). The six open reading frames of CERV showed amino acid homology (50-80%) with CaMV ORFs I-VI; no homologues of CaMV ORFs VII or VIII were found. CERV ORFs 1-5 interface each other with the sequence ATGA. The comparison of CERV ORF5 with CaMV ORFV highlighted regions which show homologies to retrovirus gag/pol protease, RNase H and DNA polymerase domains; the possibility that the DNA polymerase domain comprises two subdomains, operating off different templates, is discussed. Both CERV and CaMV ORFs I have sequence homology to tobacco mosaic virus P30 and plastocyanin.     

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     

Evidence for a Dual Strategy in the Expression of Cauliflower Mosaic Virus Open Reading Frames I and IV

Studies have indicated that cauliflower mosaic virus (CaMV) gene expression is mediated by the translation of polycistronic 35S pregenomic RNA, but the involvement of some minor subgenomic RNA species is also suspected. We examined the involvement of the 35S promoter in the expression of CaMV open reading frames (ORFs) I and IV using both 35S RNA-driven and promoter-less ORF I- and ORF IV-β-glucuronidase (GUS) fusion constructs. In addition to the 35S promoter-dependent expression of both ORF I- and IV-GUS fusions, we detected the 35S promoter-independent expression of both fusion genes via subgenomic mRNAs, which were detected by Northern blotting in the protoplasts transfected with the 35S promoter-driven constructs as well as in those transfected with the promoter-less constructs. These results suggest the involvement of subgenomic RNAs in the expression of CaMV ORFs I and IV, and the operation of a dual strategy in the expression of two viral genes.     

The Commelina yellow mottle virus promoter is a strong promoter in vascular and reproductive tissues.

Commelina yellow mottle virus (CoYMV) is a double-stranded DNA virus that infects the monocot Commelina diffusa. Although CoYMV and cauliflower mosaic virus (CaMV; another double-stranded DNA virus) probably replicate by a similar mechanism, the particle morphology and host range of CoYMV place it in a distinct group. We present evidence that a prompter fragment isolated from CoYMV confers a tissue-specific pattern of expression that is different from that conferred by the CaMV 35S promoter. When the CoYMV promoter is used to drive expression of the beta-glucuronidase reporter gene in stably transformed tobacco plants, beta-glucuronidase activity occurs primarily in the phloem, the phloem-associated cells, and the axial parenchyma of roots, stems, leaves, and flowers. Activity is also detected throughout the anther, with highest activity in the tapetum. In contrast, the CaMV 35S promoter is active in most cell types. The CoYMV promoter is a strong promoter, and when the activity of the CoYMV promoter is compared with that of a duplicated CaMV 35S promoter, it is 30% as active in tobacco suspension cells and up to 25% as active in maize suspension cells. These properties of the CoYMV promoter make it potentially useful for high-level expression of engineered genes in vascular cells.     

A promoter from sugarcane bacilliform badnavirus drives transgene expression in banana and other monocot and dicot plants

A 1369 bp DNA fragment (Sc) was isolated from a full-length clone of sugarcane bacilliform badnavirus (ScBV) and was shown to have promoter activity in transient expression assays using monocot (banana, maize, millet and sorghum) and dicot plant species (tobacco, sunflower, canola and Nicotiana benthamiana). This promoter was also tested for stable expression in transgenic banana and tobacco plants. These experiments showed that this promoter could drive high-level expression of the -glucuronidase (GUS) reporter gene in most plant cells. The expression level was comparable to the maize ubiquitin promoter in standardised transient assays in maize. In transgenic banana plants the expression levels were variable for different transgenic lines but was generally comparable with the activities of both the maize ubiquitin promoter and the enhanced cauliflower mosaic virus (CaMV) 35S promoter. The Sc promoter appears to express in a near-constitutive manner in transgenic banana and tobacco plants. The promoter from sugarcane bacilliform virus represents a useful tool for the high-level expression of foreign genes in both monocot and dicot transgenic plants that could be used similarly to the CaMV 35S or maize polyubiquitin promoter.     

Mutation of either G box or I box sequences profoundly affects expression from the Arabidopsis rbcS-1A promoter.

A deletion analysis of the Arabidopsis thaliana rbcS-1A promoter defined a 196 bp region (-320 to -125) sufficient to confer light-regulated expression on a heterologous Arabidopsis alcohol dehydrogenase (Adh) reporter gene in transgenic Nicotiana tabacum (tobacco) leaves. This region, which contains DNA sequences I, G and GT boxes, with homology to other ribulose-1,5-bisphosphate carboxylase small subunit (RBCS) gene promoter sequences, directed expression independent of orientation and relative position in the Adh promoter. Site-specific mutagenesis of these conserved sequences and subsequent expression analysis in transgenic tobacco showed that both G box and I box mutations in the context of the full (-1700 to +21) rbcS-1A promoter substantially reduced the expression of Adh and beta-glucuronidase (GUS) reporter genes. The G box has previously been shown to specifically bind in vitro a factor isolated from nuclear extracts of tomato and Arabidopsis. This factor (GBF) is distinct from the factor GT-1 which binds to adjacent GT boxes in the pea rbcS-3A promoter. Multiple mutations in putative Arabidopsis rbcS-1A promoter GT boxes had no pronounced affect on expression, possibly due to a redundancy of these sites. Experiments in which rbcS-1A promoter fragments were fused to truncated 35S CaMV (cauliflower mosaic virus) promoter--GUS reporter constructs showed that cis-acting CaMV promoter elements could partially restore expression to G-box-mutated rbcS-1A sequences.     

麻疯树逆境蛋白(curcin 2)基因在烟草中的表达

麻疯树（Jatropha curcas）幼苗在干旱、高低温胁迫和真菌浸染下,其叶片中诱导产生了一种新的毒蛋白curcin 2。这意味着curcin 2在其它植物中的异源表达可能会增强植物对外界胁迫的抵抗。curcin 2 cDNA的两个片断：cur2p片断（编码前成熟蛋白）和cur2m片断（编码成熟蛋白）,通过农杆菌的介导分别转化烟草并获得转基因植株。但是,只有在插入了cur2p片断的烟草中检测到了curcin 2蛋白的表达。同时,curcin 2在烟草中的表达增强了植株对烟草花叶病毒（TMV）的抗性。     

萼脊兰ACC氧化酶基因片段的克隆及其反义基因表达载体的构建

根据已报道的几种不同属的兰科植物ACO基因序列,设计并合成了一对特异引物,以萼脊兰的基因组DNA为模板,用PCR扩增方法克隆出萼脊兰ACO基因的部分片段,将其连接到pMD19-T质粒载体上进行测序.结果显示,该片段的长度为333 bp,序列和蝴蝶兰(Phalaenopsis hybrid)、卡特兰(Cattleya intermedia)、两棱蕾丽兰(Laelia anceps)的相应ACO片段的同源性分别达到达到99.7%、92.49%和92.19%;和大花蕙兰(Cymbidium hybrid)、石斛兰(Dendrobium crumenatum)的同源性分别是89.49%和89.19%.将此片段反向插入植物表达载体pBI221的CaMV35S启动子和NOS终止子之间,成功构建了萼脊兰ACO的反义基因植物表达载体pBI-antiACO.     

Expression of active hBMP2 in transgenic tobacco plants

Bone morphogenetic protein 2 (BMP2) is important for bone tissue repair. The goal of this research is to construct a high level human BMP2 (hBMP2) expression system using transgenic tobacco plants as a bioreactor. Cauliflower mosaic virus (CaMV) 35S promoter, alfalfa mosaic virus (AMV) enhancer, tobacco mosaic virus (TMV) enhancer, matrix attachment regions (MARs) sequence, and “Kozak” sequence were used to construct recombinant expression vectors and the high-expression vectors were screened out through GUS-fusions assay. The promoter is the most important factor; double-CaMV 35S promoter is more effective than single promoter. The AMV or TMV enhancer is able to promote the foreign protein expression. After four-step purification, the activated hBMP2 (0.02% total soluble protein) was obtained. Our results suggested that the transgenic tobacco has great potential to be used as a bioreactor to produce hBMP2.     

Construction of hybrid promoters of caulimoviruses and analysis of their activity in transgenic plants

By the techniques of DNA shuffling, PCR, and restriction-ligation, chimeric forms of cauliflower (Brassica oleracea) mosaic virus (CaMV), dahlia (Dahlia pinnata) mosaic virus (DMV), and carnation (Dianthus caryophillus) etching ring virus (CERV) promoters were obtained at various combinations. Twelve chimeric promoters were cloned into pCambia binary vectors comprising the reporter GUS gene, and their activities in transgenic tobacco (Nicotiana tabacum) plants were determined fluorimetrically. 35S promoter and those of DMV (442 bp) and CERV (371 and 501 bp) were used as controls. Seven of analyzed promoters displayed higher and seven promoters lower activity in transgenic tobacco plants than 35S promoter. The highest activity was characteristic of natural DMV promoter, and the least one — natural CERV promoter 501 bp in size. The CERV promoter 371 bp in size was approximately similar in strength to 35S promoter.     

花椰菜花叶病毒(CaMV)的基因表达调控

花椰菜花叶病毒（CaMV）是一种具有重要经济价值和生物学意义的植物双链DNA病毒,它有7个开放阅读框（ORF）,其中6个呆各自编码一种蛋白产物。35S启动子区域含有3个转录因子专一的结合位点;RNA多腺苷化位点具有AAUAAA特征序列,它和其上游序列对35SRNA的加工和翻译有影响作用;下游ORFI在转录激活因子存在时可被翻译。由此表明,CaMV的表达调控表现在不同调控机制工作的基础之上。     

二氢黄酮还原酶基因的克隆与转基因烟草的获得

以蒺藜苜蓿(Medicagotrunctulacv.5160)幼果总RNA为模板,采用RT-PCR技术克隆到二氢黄酮还原酶(DFR)基因的cDNA序列,所获得的cDNA序列全长1018 bp,具有完整的ORF,编码337个氨基酸。Blast分析表明,该片段与GenBank中注册的DFR基因同源性为99.80%。以植物表达载体pBI121为基础,构建了Ca MV35S启动子驱动的DFR基因植物表达载体pBIDFR;采用直接转化法将pBIDFR导入根癌农杆菌EHA105,用该菌株对普通烟草进行遗传转化获得6株转基因植株。     

Preliminary Functional Studies on AC2, a Novel Trans-acting Factor from Cotton Leaf Curl Virus

Studies on tomato golden mosaic virus and African cassava mosaic virus suggested that virion sense promoter was trans -activated in transient expression by A C2 encoded by geminivirus. The AC2 gene fragment of cott on leaf curl virus (CLCuV) was obtained from total DNA of CLCuV infected tobacco leaves by polymerase chain reaction, and the amplified DNA fragment was cloned into vector. Transient expres sion vectors were constructed by fusing the AC2 gene fra gment with CaMV 35S prom oter and nopaline terminator. These constructs were delivered into tobacco [ WT(Nicotiana tabacum L.) and cotton ( Gossypium hirsutum L.) leaf cells for transient expression by particle bombardment. Results indicated that activity of virion sense promoter was activated by AC2 and increased remarkably. However, the activity of trans-activated virion sense promoter was still lower than that of complementary sense promoter. Expression pattern of transactivated virion sense promoter was similar to that of complementary sense promoter with the high activity in both mesophyll and vascular of leaf vein. In this paper, the expression behavior of AC2 in Agrobacterium -mediated transgenic plants was also discussed. 