Cell-Specific Expression of the rolC Gene of the TL-DNA of Ri Plasmid in Transgenic Tobacco Plants

Transgenic tobacco plants possessing a chimeric gene of thepromoter region of the Ri plasmid ORF12 gene (rolC) and a structuralgene of bacterial ß-glucuronidase were obtained. Histochemicalanalysis showed that the chimeric gene is expressed in phloemcells throughout whole plants. Stable inheritance of such cellspecific expression of ORF12 promoter was also confirmed. ²Present address: Research Institute of Agricultural Resources,Ishikawa Agricultural College, Nonoichi-machi, Ishikawa, 921Japan. (Received September 30, 1988; Accepted March 30, 1989)     

雪花莲凝集素基因（gna）的改造及其抗蚜性

用定点突变方法对编码雪花莲凝集素（Galanthus nivalis agglutinin,GNA)前体蛋白的DNA序列进行了改造和转基因烟草9Nicotana tabacum L.）抗蚜性的研究。结果表明,将GNA编码序列中含有的稀有密码子改造后,GNA的表达水平从占总可溶性蛋白的0.17％增加到0.25％,转基因烟草的抗蚜性也随之增强,从平均抑制桃蚜（Myzus per-sicae(Sulzer））虫口密度63.7％显地提高到71.0％。     

发根农杆菌研究进展

ReviewofStudiesonObtainingTransgenicPlantsbyAgrobacteriumrhizogenesMediatedGeneTransferSystemZhouYanqingZhanggenfaYuanBaojun(DepartmentofBiology,HenanNormalUniversity,Xinxiang453002)②苑保军现在河南省周口地区农业科学技术研究所工作．在植物基因工程中,农杆菌质粒介导的基因转移系统[37]是比较完善与有效的基因转移方法。目前,在根癌农杆菌Ti质粒的结构、功能及其被改造为载体系统与应用等方面均已取得很大进展的情况下,与之同属于根瘤菌科的发根农杆菌及其所携带的Ri质粒开始被广泛研究。本文就发根农杆菌Ri…     

Production and Analysis of Tobacco Transgenic Plants Expressing the Agrobacterial Gene for Tryptophan Monooxygenase

The expression of the agrobacterial iaaM gene for tryptophan monooxygenase, the enzyme catalyzing the first step in the auxin biosynthesis, induced substantial physiological and biochemical changes in transgenic tobacco (Nicotiana tabacum L.) plants. All lines of transgenic plants grown in vitro manifested abnormal phenotypes: enhanced root formation, adventitious roots on stems, and curled leaves. When grown in vivo, plants manifested abnormal, normal, or intermediate phenotype. Under conditions of a greenhouse, the abnormal plants contained the highest amount of auxins in their leaves and manifested an increased number of adventitious roots, poor reproductivity, and the loss in seed germination. Transgenic plants with the normal phenotype did not substantially differ from the wild-type plants in their morphology, and their auxin content was lower than in the abnormal plants. The intermediate-phenotype plants were devoid of some morphological properties characteristic of the abnormal plants. Only the seeds of normal- and intermediate-phenotype transgenic plants germinated at a high rate.     

Transgenic Brassica campestris Plants Expressing the gfp Gene

A protocol has been worked out for efficient regeneration and genetic transformation of summer rape (Brassica campestris L. var. oleifera). Cotyledons of the 5-day-old seedlings were transformed with Agrobacterium tumifaciens strain AGL cells comprising a binary vector with a selectable neomycin phosphotransferase II gene and a reporter gene encoding green fluorescent protein (GFP). Explants were cultured on a regeneration MS medium supplemented with ABA, and transformants were selected on the same medium (minus ABA and plus 70 mM AgNO₃ and 15 mg/l kanamycin). The frequency of shoot regeneration on explant petioles was 30–40%. Transgenic plants were identified by GFP fluorescence and by polymerase chain reaction and Western blotting analysis. The transformation efficiency was as high as 75% of the total number of regenerated shoots.     

Transgenic regal pelargoniums that express the rolC gene from Agrobacterium rhizogenes exhibit a dwarf floral and vegetative phenotype

Summary The regal pelargonium, ev. Dubonnet, was transformed using the disarmed Agrobacterium tumefaciens strains LBA4404 or EHA105 containing the binary vector pLN70. This plasmid carries on its T-DNA the rolC gene from Agrobacterium rhizogenes under control of the CaMV 35S promoter and the npt II selectable marker gene under a NOS promoter. Six independent transformants were produced and grouped according to their phenotypic characteristics. Two transformants showed the same phenotype as the untransformed control plants. Three transformants exhibited a dwarf phenotype and one displayed a super-dwarf phenotype. Southern hybridization analyses of the T-DNA left border region using a npt II probe showed that the six transformants all arose from independent transformation events. Northern hybridization analyses showed that the rolC gene was expressed only in the four transformants that exhibited a dwarf phenotype. Our data show that the phenotypic effects of rolC expression in regal pelargoniums include reductions in plant height, leaf area, petal area, and corolla length. Earlier flowering of the rolC transgenics by up to 22d was also observed.     

Synergistic Function of rolB, rolC, ORF13 and ORF14 of TL-DNA of Agrobacterium rhizogenes in Hairy Root Induction in Nicotiana tabacum

Comparison of the frequency of rooting in the tobacco leaf segmentsinoculated with Agrobacterium tumefaciens harboring variouscombinations of rolB, rolC, ORF13 and ORF14 of TL-DNA of Riplasmid (pRiHRI) revealed that the genes differ in their functionto stimulate adventitious root induction. A single gene rolBinduced roots, while rolC, ORF13 and ORF14 independently promotedthe root induction by the rolB gene. The effects of these geneson the rolB-mediated rooting were in the order of ORF13>rolCORF14. Present address: Laboratory of Phylogenetic Botany, Departmentof Biology, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba,263-8522 Japan. ² Present address: Department of Chemical and Biological Sciences,Faculty of Science, Japan Women's University, 2-8-1 Mejirodai,Bunkyo-ku, Tokyo, 112-8681 Japan.     

Transgenic Tobacco Plants Expressing Bacterial Genes for Thermostable Glucanases

It is shown that bacterial genes for thermostable -glucanases are expressed retaining their activity and substrate specificity. The leader peptide of the carrot extensin exerts effective secretion of the bacterial enzymes into the intercellular space of the plant tissue. Expression of the bacterial gene for -1,3-glucanase in plant tissues alters their morphogenetic potential. Regeneration of shoots from the calli of these plant lines requires a six- to eightfold increase in cytokinin (6-BAP) concentration in comparison with the control lines and the transgenic lines expressing -1,3-1,4-glucanase. Rooting of transgenic plants expressing the bacterial gene for -1,3-glucanase occurs much faster. The transgenic plants obtained in the study are proposed as model objects for investigating the role of glucanases in plants.     

表达barstar基因及bar基因的转基因油菜的研究

从细菌Ｂａｃｉｌｕｓａｍｙｌｏｌｉｑｕｅｆａｃｉｅｎｓ染色体ＤＮＡ中克隆了ｂａｒｎａｓｅ抑制剂ｂａｒｓｔａｒ的基因,构建了带有ＴＡ－２９基因５′调控区（－１３００－＋３）与ｂａｒｓｔａｒ基因编码区、ＣａＭＶ３５Ｓ启动子与除草剂抗性基因ｂａｒ两个表达框架的植物表达质粒ｐＢＢＳ。以“双低”油菜“５－４”的子叶柄为受体,通过农杆菌介导的遗传转化,获得了在含有１０ｍｇ／Ｌ卡那霉素和２０ｍｇ／ＬＰＰＴ的筛选培养基上再生的转基因植株。ＰＣＲ分析结果表明,ｂａｒｓｔａｒ基因已整合到油菜染色体上;Ｎｏｒｔｈｅｒｎｂｌｏｔ检测表明,ｂａｒｓｔａｒ基因及ｂａｒ基因在转基因植物中得到了正确的调控与表达。以转基因油菜“５－４”为父本授粉给表达ｂａｒｎａｓｅ基因的雄性不育植株,不育株能正常结实。     

Deletion Analysis of the 5'-Upstream Region of the Agrobacterium rhizogenes Ri Plasmid rolC Gene Required for Tissue-Specific Expression

The cis-acting elements located in the −848 and +23 region of the 5′-upstream region of the rolC gene of the Agrobacterium rhizogenes Ri plasmid were investigated. The cis-acting DNA region required for phloem-specific expression was found within the −153 region, whereas a minimum region needed for the expression in the seed embryo was located at position −120.     

The ORF8 gene product of Agrobacterium rhizogenes TL-DNA has tryptophan 2-monooxygenase activity

The open reading frame 8 (ORF8) is located on the TL-DNA of the phytopathogenic soil bacterium Agrobacterium rhizogenes strain A4. The predicted ORF8 protein has a particular structure and is possibly a natural fusion protein. The N-terminal domain shows homology to the A. rhizogenes rolB protein and may modulate the auxin responsiveness of host cells. The C terminus has up to 38% homology to tryptophan 2-monooxygenases (t2m). We show that ORF8 overexpressing plants contain a fivefold higher concentration of indole-3-acetamide (IAM) than untransformed plants. Protein extracts from seedlings and Escherichia coli overexpressing ORF8 show significantly higher turnover rates of tryptophan to IAM than negative controls. We conclude that the ORF8 gene product has tryptophan 2-monooxygenase activity.     

Transgenic Tobacco (Nicotiana tabacum SR1) Plants Expressing the Gene Coding for Serratia marcescens Nuclease

The gene coding for the secreted Serratia marcescens endonuclease was fused with the mannopine synthase promoter of Agrobacterium tumefaciens Ti plasmid and transferred to Nicotiana tabacum SR1 plants. The promoter is leaf- and root-specific. The resulting transgenic plants demonstrated elevated nuclease activity. The level of the transgene product was determined in the transgenic lines.     

Physiological and Biochemical Characteristics of Tobacco Transgenic Plants Expressing Bacterial Dioxygenase

Expression of the 1,2-dihydroxynaphthalene dioxygenase (nahC) gene from Pseudomonas putida in tobacco transgenic plants produces notable phenotypic and biochemical changes: retarded growth and rooting and earlier flowering; chlorotic and necrotic spots on leaves; and a threefold increase in the total phenolics in the leaves of 6-week-old plants (94.51 g/g fr wt as compared to 33.18 g/g fr wt in the control) and in the phenylalanine-ammonia lyase activity in 4-week-old plants (0.035 U/g fr wt as compared to 0.014 U/g in the control plants of the same age). The transgenic plants expressing the nahC bacterial gene may serve as a model to study the putative functions of dioxygenases and phenol compounds in plant growth, development, and stress responses.     

表达尾穗苋凝集素基因的转基因烟草对桃蚜(Myzus persicae)繁殖的影响

为研究尾穗苋凝集素(ACA)在植物中可能的抗虫作用,通过RH-PCR克隆了ACA cDNA并通过RACE分析证实了cDNA序列的正确性.构建了ACA基因的韧皮部特异表达载体pBCACAc并通过根癌杜菌介导转化了烟草(Nicotiana tabacum L.).PCR和Southern blot分析结果证明,ACA基因已经整合到转化再生植物的基因组中,其插入插贝数1~4个不等.对转基因烟草叶片蛋白时行行免疫反应的结果表明,ACA基因已被转录和翻译.用桃蚜(Myzuspersicae Sulzer)对转基因烟草离体叶片进行了的接虫试验结果表明,测试过的78%的烟草对桃蚜口密度增长的平均抑制率在75%以上,在抗性植株上观察到有桃蚜若虫死亡的现象.以上结果表明,ACA基因是一个有效的抗蚜基因,在作物抗蚜分子育种具有应具应用价值.     

Effects of the Over-Expression of the rolC Gene on Leaf Development in Transgenic Periclinal Chimeric Plants

Using Agrobacterium tumefaciens harboring a vector that carriedthe rolC gene under the control of the 35S RNA promoter of cauliflowermosaic virus, we produced several transgenic plants. Two ofthem were periclinal chimeras with altered leaves that had wrinkleddark green margins and inner pale green regions. One chimericplant had shortened internodes, reduced apical dominance, smallflowers and exhibited male sterility, but the other had a normalphenotype. Analysis of proteins, RNA and DNA indicated thatthe inner pale green tissues consisted of transformed cellswhile the outer dark green tissues were composed of non-transformedcells. Histological analysis indicated that mesophyll cellswere distorted and larger intercellular spaces were presentin the transformed pale green regions. Furthermore, in youngleaves, transformed mesophyll cells were larger than non-transformedcells. However, the normal parts had larger numbers of cellsper unit area than the transformed parts. These observationssuggest that ths expression of 35S-rolC in leaves caused inhibitionof cell division in developing leaves and that the undulatingmargins, composed of non-transformed cells, were a consequenceof the requirement for accommodating more cells in less spacewithin the region of rolC-transformed cells. (Received January 29, 1993; Accepted May 17, 1993)     

表达尾穗苋凝集素基因的转基因烟草对桃蚜(Myzus persicae)繁殖的影响(英文)

To investigate the possible function of the agglutinin from Amaranthus caudatus L. (ACA) in plant defending against insect pests, ACA cDNA was cloned by RT-PCR and the 5‘ and 3‘ sequences were confirmed by rapid amplification of cDNA ends (RACE). The phloem-specific expression vector of ACA gene, pBCACAc, was constructed based on the plant binary vector pBC438 and transfered into tobacco plants via Agrobacterium-mediated transformation method. Results from PCR and Southern blotting analysis showed that AOA gene was integrated into the genomes of transformed plants and the transgene integration varied from one to four estimated copies per genome. Western blotting analysis indicated that ACA gene was transcribed and translated in the transgenic plants. The bioassay of Myzus persicae Sulzer on detached leaves demonstrated that the 78% transgenic tobacco plants displayed an average aphid-resistant rate of more than 75%. Some apterous progeny of M. persicae were found dead on the resistant plants. These results indicate that ACA gene should be an effective aphid-resistant gene and could be valuable for application in crop breeding for aphid resistance.     

Characterization of Transgenic Rice Plants Expressing an Arabidopsis FAD7

Fatty acid -3 desaturase (FAD) is the key enzyme catalyzing the formation of trienoic fatty acids. We utilized an Arabidopsis FAD7 gene and the seven independent transgenic rice plants harbouring 1 to 3 copies of this gene were generated. The expression of FAD7 mRNA was different among independent transgenic lines regardless of the copy number. The total linolenic acid (18:3) contents reduced by about 7 – 32 % in transgenic rice plants but the linoleic acid (18:2) content increased accordingly. With or without wounding treatments, the jasmonate content was higher in transgenic lines than in wild-type rice plant. The transgenic lines overproducing jasmonate also showed increased expression of PR1b mRNA and allene oxide synthase inresponse to wounding.     

农杆菌介导籼稻优良恢复系bar基因的遗传转化研究

应用农杆菌介导转化体系,成功地将含有CaMv35s启动子启动的bar基因导入籼稻幼胚来源的愈伤组织,获得籼稻优良恢复系T461、R402和752三个品种（系）共47个抗除草剂Basta的转基因株系,Southem分析结果表明,转基因植株基因组中检测到bar基因的整合,转基因植株自交后代Basta除草剂抗性鉴定表现出分离,且大多数为1-2个整合位点的孟德尔方式遗传。结果表明,根癌农杆菌介导法可以有效且可靠地转化籼稻。