Introduction of a citrus blight-associated gene into Carrizo citrange [<Emphasis Type="Italic">Citrus sinensis</Emphasis> (L.) Osbc. × <Emphasis Type="Italic">Poncirus trifoliata</Emphasis> (L.) Raf.] by <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation

The protein p12 accumulates in leaves of trees with citrus blight (CB), a serious decline of unknown cause. The function of p12 is not known, but sequence analysis indicates it may be related to expansins. In studies to determine the function of p12, sense and antisense constructs were used to make transgenic Carrizo citrange using an Agrobacterium-mediated transformation system. Homogeneous -glucuronidase⁺ (GUS⁺) sense and antisense transgenic shoots were regenerated using kanamycin as a selective agent. Twenty-five sense and 45 antisense transgenic shoots were in vivo grafted onto Carrizo citrange for further analyses. In addition, 20 sense and 18 antisense shoots were rooted. The homogeneous GUS⁺ plants contained either the p12 sense or antisense gene (without the intron associated with the gene in untransformed citrus) as shown by PCR and Southern blotting. Northern blots showed the expected RNA in the sense and antisense plants. A protein of identical size and immunoreactivity was observed in seven of nine sense plants but not in nine antisense or non-transgenic plants. At the current stage of growth, there are no visual phenotypic differences between the transgenic and non-transgenic plants. Selected plants will be budded with sweet orange for field evaluation for resistance or susceptibility to CB and general rootstock performance.Abbreviations AS Acetosyringone - CaMV 35S P Cauliflower mosaic virus 35S promoter - CaMV 35S poly A Cauliflower mosaic virus 35S poly A terminator - CB Citrus blight - 2,4-D 2,4-Dichlorophenoxyacetic acid - FMV Figwort mosaic virus - GUS -Glucuronidase - GUS gene uidA - IBA Indole-3-butyric acid - MES 2-(N-Morpholino) ethane sulfonic acid - MSI Inoculation medium - MSP-10M Plasmolysis solution with 10% maltose - MSP-8S Plasmolysis solution with 8% sucrose - NAA -Naphthaleneacetic acid - NOS Nopaline synthase - NP Nopaline synthase promoter - NT Nopaline synthase terminator - NPTII Neomycin phosphotransferase II - p12 Blight-associated protein p12     

Promoter cassettes, antibiotic-resistance genes, and vectors for plant transformation

We have constructed a set of plant transformation vectors, promoter cassettes, and chimeric antibiotic-resistance genes for the transformation and expression of foreign genes in plants sensitive to Agrobacterium infection. The different vectors allow for either concurrent or consecutive selection for kanamycin and hygromycin resistance and have a number of unique restriction sites for the insertion of additional DNA. The promoter cassettes utilize the CaMV 19S and CaMV 35S promoters and are constructed to allow for the easy insertion of foreign genes. The cloned gene can then easily be inserted into the transformation vectors. We have utilized the promoter cassettes to express the hygromycin-resistance gene either from the CaMV 35S or the CaMV 19S promoters, with both chimeric resistance genes allowing for the selection of hygromycin-resistant tobacco plants.     

Comparison of promoters and selectable marker genes for use in Indica rice transformation

The effectiveness of different promoters for use in Indica rice transformation was compared. Plasmids encoding the Escherichia coli uidA (gus) gene under the control of CaMV 35S, Emu, Act1 or Ubi1 promoters were delivered into cell suspension cultures by particle bombardment. Transient gene expression, 48 h after delivery, was greatest from plasmids utilising the constitutive promoters, Act1 and Ubi1. Gene expression in stably transformed tissue was examined by bombarding embryogenic Indica rice calli with a pUbi1-gas plasmid and a plasmid containing either the selectable marker gene, hph, which confers hygromycin resistance, or bar, which confers resistance to the herbicide phosphinothricin (BASTA) each under the control of the CaMV 35S, Emu, Act1 or the Ubi1 promoters. The bombarded calli were placed on the appropriate selection media and stained for GUS activity at 1 day, 3 weeks and 5 weeks after shooting. Callus bombarded with the pUbi1-hph or the pEmu-hph constructs gave a dramatic increase in the size of the GUS staining areas with time. No such increase in the size of GUS staining areas was observed in calli co-bombarded with pUbi1-gus and any of the bar containing constructs.Co-bombardment of calli with either the pEmu-hph or pUbi1-hph construct and a virus minor coat protein (cp) gene construct resulted in many fertile transgenic Indica rice plants, containing one to eight copies of both the hph and cp genes. These genes were stably inherited by the T1 generation.     

35S驱动Pib基因启动区和编码区的转基因初步分析

将包含Pib基因启动区及下游完整编码区的9.9 kb DNA片段克隆到双元载体pPZP2Ha3(+)中, 构建了35S驱动的正义表达载体pNAR701(20.3 kb); 同时将Pib基因编码区6 986~9 392 bp之间的DNA片段, 克隆到双元载体pPZP2Ha3(-)中, 构建了35S驱动的反义表达载体pNAR703(12.8 kb); 用农杆菌介导法转入中感稻瘟病水稻品种R109中。PCR、Southern blot鉴定以及转基因T0代种子的潮霉素抗性鉴定证明, 目的基因已经整合到R109基因组中, 并能在后代稳定遗传。Northern blot分析表明含有启动区及下游完整编码的Pib基因片段在35S驱动下能够在转基因后代中表达。对T1代苗期转基因植株和分蘖期离体叶片进行抗稻瘟病初步分析, 结果显示pNAR701转基因植株对稻瘟病生理小种ZD1和ZG1的抗性较对照增强, 而转反义片段的pNAR703转基因植株对稻瘟病的抗性较对照减弱。     

胰蛋白酶抑制剂KSTI3基因新型真核表达载体的构建及其在盐藻中的表达

应用PCR技术扩增Nos基因、 CaMV35S 启动子片段和氯霉素抗性基因 Cat ,并与胰蛋白酶抑制剂 KSTI3 基因连接,构建真核表达载体pMDCKN-Cat。DNA序列分析结果表明:表达载体中的胰蛋白酶抑制剂 KSTI3 基因、启动子 CaMV35S 、终止子 Nos 和氯霉素抗性基因 Cat 与已知序列完全一致。采用LiAc/PEG介导法将质粒pMDCKN-Cat转化至盐藻细胞中,通过氯霉素抗性基因筛选和PCR鉴定获得转基因盐藻细胞。经Western blotting检测,在硝酸纤维素膜上出现清晰的条带,分子量为20.1kDa,证明胰蛋白酶抑制剂 KSTI3 基因在盐藻中得到成功表达。     

Expression of 10 kD Sulfur-Rice Prolamin Gene of Rice in Potato

Using 10 kD sulfur-rich prolamin gene of rice (PLG) as target gene, the authers constructed the expression vectors pBinLG and pBinLGP, which contained CaMV 35S promoter/PLG/NOS terminator, and Patafin Class Ⅰ promoter/PLG/NOS terminator respectively. They were transformed into Agrobacterium tumefaciens strain LBA4404 (pAL4404) by direct transformation method with incubating the leaf and tuber explants of potato (Solanum tuberosum L. ) with LBA4404 (pAL4404) and selecting in the medium containing 100 mg/L kanamycin, regenerated resistant plants were obtained. The NPT Ⅱ enzyme activity analysis, polymerase chain reaction (PCR), Southern blotting, Northern dot blotting and Western blotting demonstrated that the target gene was integrated into the genome of potato ceils and well expressed in the plant.     

Analysis of Arabidopsis thaliana transgenic plants transformed with CER2 and CER3 genes in sense and antisense orientations

 A number of genes involved in the biosynthesis of the epicuticular wax (EW) of Arabidopsis thaliana have recently been isolated through genetic approaches. In view of the evidence in favor of the importance of EW compounds in the adaptation of higher plants to a number of physiological and ecological stresses, we have used clones of some of these genes to genetically engineer constructs with which to manipulate EW biosynthesis in transgenic A. thaliana plants. All our constructs were placed under the control of the near constitutive CaMV 35S promoter. We were able to complement mutant plants with the construction in the sense orientation as well as induce phenocopies of the eceriferum phenotype by transforming wild-type plants with both the sense and antisense constructs. We observed reduced fertility in the wild-type plants transformed with the 35SCER3sense or 35SCER3antisense constructs but not in those transformed with the 35SCER2sense or 35SCER2antisense constructs. Received: 28 December 1997 / Accepted: 31 March 1998     

Herbicide-resistant Indica rice plants from IRRI breeding line IR72 after PEG-mediated transformation of protoplasts

The commercially important Indica rice cultivar Oryza sativa cv. IR72 has been transformed using direct gene transfer to protoplasts. PEG-mediated transformation was done with two plasmid constructs containing either a CaMV 35S promoter/HPH chimaeric gene conferring resistance to hygromycin (Hg) or a CaMV 35S promoter/BAR chimaeric gene conferring resistance to a commercial herbicide (Basta) containing phosphinothricin (PPT). We have obtained so far 92 Hg^r and 170 PPT^r IR72 plants from protoplasts through selection. 31 Hg^r and 70 PPT^r plants are being grown in the greenhouse to maturity. Data from Southern analysis and enzyme assays proved that the transgene was stably integrated into the host genome and expressed. Transgenic plants showed complete resistance to high doses of the commercial formulations of PPT.