共查询到10条相似文献,搜索用时 140 毫秒
1.
2.
T-DNA insertional mutagenesis for functional genomics in rice 总被引:56,自引:0,他引:56
Jeon JS Lee S Jung KH Jun SH Jeong DH Lee J Kim C Jang S Yang K Nam J An K Han MJ Sung RJ Choi HS Yu JH Choi JH Cho SY Cha SS Kim SI An G 《The Plant journal : for cell and molecular biology》2000,22(6):561-570
We have produced 22 090 primary transgenic rice plants that carry a T-DNA insertion, which has resulted in 18 358 fertile lines. Genomic DNA gel-blot and PCR analyses have shown that approximately 65% of the population contains more than one copy of the inserted T-DNA. Hygromycin resistance tests revealed that transgenic plants contain an average of 1.4 loci of T-DNA inserts. Therefore, it can be estimated that approximately 25 700 taggings have been generated. The binary vector used in the insertion contained the promoterless beta-glucuronidase (GUS) reporter gene with an intron and multiple splicing donors and acceptors immediately next to the right border. Therefore, this gene trap vector is able to detect a gene fusion between GUS and an endogenous gene, which is tagged by T-DNA. Histochemical GUS assays were carried out in the leaves and roots from 5353 lines, mature flowers from 7026 lines, and developing seeds from 1948 lines. The data revealed that 1.6-2.1% of tested organs were GUS-positive in the tested organs, and that their GUS expression patterns were organ- or tissue-specific or ubiquitous in all parts of the plant. The large population of T-DNA-tagged lines will be useful for identifying insertional mutants in various genes and for discovering new genes in rice. 相似文献
3.
4.
Transgenic papaya plants from Agrobacterium-mediated transformation of somatic embryos 总被引:1,自引:0,他引:1
Maureen M. M. Fitch Richard M. Manshardt Dennis Gonsalves Jerry L. Slightom 《Plant cell reports》1993,12(5):245-249
Summary Transgenic papaya (Carica papaya L.) plants were regenerated from embryogenic cultures that were cocultivated with a disarmed C58 strain of Agrobacterium tumefaciens containing one of the following binary cosmid vectors: pGA482GG or pGA482GG/cpPRV-4. The T-DNA region of both binary vectors includes the chimeric genes for neomycin phosphotransferase II (NPTII) and ß-glucuronidase (GUS). In addition, the plant expressible coat protein (cp) gene of papaya ringspot virus (PRV) is flanked by the NPTII and GUS genes in pGA482GG/cpPRV-4. Putative transformed embryogenic papaya tissues were obtained by selection on 150 g·ml–1 kanamycin. Four putative transgenic plant lines were obtained from the cp gene– vector and two from the cp gene+ vector. GUS and NPTII expression were detected in leaves of all putative transformed plants tested, while PRV coat protein expression was detected in leaves of the PRV cp gene+ plant. The transformed status of these papaya plants was analyzed using both polymerase chain reaction amplification and genomic blot hybridization of the NPTII and PRV cp genes. Integration of these genes into the papaya genome was demonstrated by genomic blot hybridizations. Thus, like numerous other dicotyledonous plant species, papayas can be transformed with A. tumefaciens and regenerated into phenotypically normal-appearing plants that express foreign genes.Journal Series no. 3757 of the Hawaii Institute of Tropical Agriculture and Human Resources 相似文献
5.
6.
Radhamony Resminath Anand Mohan Prasad Dhiraj R. Thakare C. Sivanandan S. R. Bhat Srinivasan 《Journal of plant biochemistry and biotechnology.》2005,14(1):1-8
A new promoter trap vector was constructed based on the juxtaposition of T-DNA right border to coding sequence of GUS. The new vector pRN-1 carried an intron in the GUS coding region. Promoter trap vectors pGKB5 and pRN-1 vectors were used to transform Arabidopsis ecotype Columbia using the floral dip transformation system. The transformants were selected on appropriate selection media and the primary transformants were confirmed by PCR using gene specific primers. Approximately 50 % of the T2 lines segregated for a 3:1 ratio indicating presence of T-DNA at single locus. Approximately 15% of the transformed lines showed expression of GUS. Morphological mutants for male sterility and dwarfism were also identified in the T2 population. A T-DNA tagged line was identified in T2 with GUS expression specifically in the floral parts. The number of T-DNA loci in this line was confirmed by Southern blot hybridization. T-DNA flanking region isolated from this line suggested insertions into chromosome 2 at two closely linked loci. The results demonstrate that the population generated can be used effectively to identify and characterize gene regulatory elements. 相似文献
7.
8.
The experimental control of gene expression in specific tissues or cells at defined time points is a useful tool for the analysis of gene function. GAL4/VP16-UAS enhancer trap lines can be used to selectively express genes in specific tissues or cells, and an ethanol-inducible system can help to control the time of expression. In this study, the combination of the two methods allowed the successful regulation of gene expression in both time and space. For this purpose, a binary vector, 962-UAS::GUS, was constructed in which the ALCR activator and β-glucuronidase (GUS) reporter gene were placed under the control of upstream activator sequence (UAS) elements and the alcA response element, respectively. Three different GAL4/VP16-UAS enhancer trap lines of Arabidopsis were transformed, resulting in transgenic plants in which GUS activity was detected only on ethanol induction and exclusively in the predicted tissues of the enhancer trap lines. As a library of different enhancer trap lines with distinct green fluorescent protein (GFP) patterns exist, transformation with a similar vector, in which GUS is replaced by another gene, would enable the control of the time and place of transgene expression. We have constructed two vectors for easy cloning of the gene of interest, one with a polylinker site and one that is compatible with the GATEWAY™ vector conversion system. The method can be extended to other species when enhancer trap lines become available. 相似文献
9.
Hairy Root-activation Tagging: a High-throughput System for Activation Tagging in Transformed Hairy Roots 总被引:2,自引:0,他引:2
Activation tagging is a powerful technique for generating gain-of-function mutants in plants. We developed a new vector system
for activation tagging of genes in “transformed hairy roots”. The binary vector pHR-AT (Hairy Root-Activation Tagging) and
its derivative pHR-AT-GFP contain a cluster of rol (rooting locus) genes together with the right border facing four tandem repeats of the cauliflower mosaic virus (CaMV) 35S
enhancer element on the same T-DNA. Transformation experiments using Arabidopsis, potato, and tobacco as model plants revealed that upon inoculating plants with Agrobacterium tumefaciens harboring these vectors, a large number of independently transformed roots could be induced from explants within a short
period of time, and root culture lines were subsequently established. Molecular analyses of the pHR-AT-GFP-transformed Arabidopsis lines showed that expression of the genes adjacent to the T-DNA insertion site was significantly increased. This system may
facilitate application of the activation-tagging approach to plant species that are recalcitrant to the regeneration of transgenic
plants. High-throughput metabolic profiling of activation-tagged root culture lines will offer opportunities for identifying
regulatory or biosynthetic genes for the production of valuable secondary metabolites of interest. 相似文献
10.
Sung-Ryul Kim Dong-Yeon Lee Jung-Il Yang Sunok Moon Gynheung An 《Journal of Plant Biology》2009,52(1):73-78
We developed various binary vectors that can be used for expressing a foreign gene in rice. Vectors pGA3426, pGA3436, and
pGA3626 are intended for overexpression of a gene using the maize Ubiquitin promoter, whereas pGA3780 is for rather mild expression of a gene using the rice Actin1 promoter. Vector pGA3777 is for expressing two genes simultaneously. We also developed binary vectors for expressing a fusion
protein with a tag. Four vectors (pGA3427, pGA3428, pGA3429, and pGA3438) are for protein tags with sGFP, HA, His, and Myc,
respectively. Vector pGA3383 is for analyzing promoter activity using the GUS reporter. In this vector, multiple cloning sites in front of GUS can be utilized for accepting a promoter fragment. We also generated transient expression vectors for studying the subcellular
localization of a protein. Vectors pGA3452, pGA3651, and pGA3652 are for GFP fusion; pGA3574 for RFP fusion; pGA3697 for Myc
tag; and pGA3698 for HA tag. In addition, we generated pGA3506, pGA3516, pGA3592, and pGA3593 for facilitating the subcloning
of full-length cDNA clones into our binary vectors. 相似文献