FOX hunting system을 이용한 배추 기능유전자 탐색
Systematic approaches to identify functional genes using the FOXhunting system in Chinese cabbage
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Full-length cDNAs are essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. To elucidate the functions of a large population of Chinese cabbage (Brassica rapa) genes and to search efficiently for agriculturally useful genes, we have been taking advantage of the full-length cDNA Over-eXpresser (FOX) gene hunting system. With oligo dT column it purify the each mRNA from the flower organs, leaf and stem tissue. And about 120,000 cDNAs from the library were transformed into λ-pFLCIII-F vector. Of which 115,000 cDNAs from the library were transformed into T-DNA binary vector, pBigs for transformation study. We used normalized full-length cDNA and introduced each cDNA into Arabidopsis by in planta transformation. Fulllength Chinese cabbage cDNAs were expressed independently under the CaMV 35S promoter in Arabidopsis. Selfed seeds were harvested from transgenic Arabidopsis. We had selected 2,500 transgenic plants by hygromycin antibiotic tolerant test, and obtained a number of transgenic mutants. Each transgenic Arabidopsis was investigated in morphological changes, fertility and leaf colour. As a result, 285 possible morphological mutants were identified. Introduced cDNA was isolated by PCR amplification of the genomic DNA from the transgenic mutants. Sequencing result and BLAST analysis showed that most of the introduced cDNA were complete cDNAs and functional genes. Also, we examined the effect of Bromelain on enhancing resistance to soft rot in transgenic Chinese cabbage ‘Osome’. The bromelain gene identified from FOX hunting system was transformed into Chinese cabbage using Agrobacterium methods. Transformants were screened by PCR, then RT-PCR and real time PCR were performed to analyze gene expression of cysteine protease in the T1 and T2 generations. The anti-bacterial activity of bromelain was tested in Chinese cabbages infected with soft rot bacteria. The results showed that the over-expressed bromelain gene from pineapple conferred enhanced resistance to soft rot in Chinese cabbage.
Abstract
Full-length cDNAs are essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. To elucidate the functions of a large population of Chinese cabbage (Brassica rapa) genes and to search efficiently for agriculturally useful genes, we have been taking advantage of the full-length cDNA Over-eXpresser (FOX) gene hunting system. With oligo dT column it purify the each mRNA from the flower organs, leaf and stem tissue. And about 120,000 cDNAs from the library were transformed into λ-pFLCIII-F vector. Of which 115,000 cDNAs from the library were transformed into T-DNA binary vector, pBigs for transformation study. We used normalized full-length cDNA and introduced each cDNA into Arabidopsis by in planta transformation. Fulllength Chinese cabbage cDNAs were expressed independently under the CaMV 35S promoter in Arabidopsis. Selfed seeds were harvested from transgenic Arabidopsis. We had selected 2,500 transgenic plants by hygromycin antibiotic tolerant test, and obtained a number of transgenic mutants. Each transgenic Arabidopsis was investigated in morphological changes, fertility and leaf colour. As a result, 285 possible morphological mutants were identified. Introduced cDNA was isolated by PCR amplification of the genomic DNA from the transgenic mutants. Sequencing result and BLAST analysis showed that most of the introduced cDNA were complete cDNAs and functional genes. Also, we examined the effect of Bromelain on enhancing resistance to soft rot in transgenic Chinese cabbage ‘Osome’. The bromelain gene identified from FOX hunting system was transformed into Chinese cabbage using Agrobacterium methods. Transformants were screened by PCR, then RT-PCR and real time PCR were performed to analyze gene expression of cysteine protease in the T1 and T2 generations. The anti-bacterial activity of bromelain was tested in Chinese cabbages infected with soft rot bacteria. The results showed that the over-expressed bromelain gene from pineapple conferred enhanced resistance to soft rot in Chinese cabbage.
참고문헌 (32)
- An S, "Generation and analysis of end sequence database for T‐DNA tagging lines in rice", Plant Physiol, 133, pp.2040~2047 (2003)
- Chaudhury AM, "Non-destructive transformation of Arabidopsis", Plant Mol. Bio. Rep., 7, pp.258~265 (1989)
- Chen S, "Distribution and characterization of over 1,000 T‐DNA tags in rice genome", Plant J., 36, pp.105~113 (2003)
- Engwerda CR, "Bromelain modulates T cell and B cell immune responses in vitor and in vivo", Cell Immunol., 25, pp.66~75 (2001)
- Fujita M, "Identification of stress‐tolerance‐related transcription‐factor genes via mini‐scale full‐length cDNA over‐expressor (FOX) gene hunting system", Bioche. Biophy. Res. Communi., 364, pp.250~257 (2007)
- Futamura N, "Characterization of expressed sequence tags from a fulllength enriched cDNA library of Cryptomerica japonica male strobili", BMC Genomics, 9, pp.383~ (2008)
- Gaspani L, "In vivo and in vitro effects of bromelain on PEG2 and SP concentrations in the inflammatory exudate in rats", Pharmacology, 65, pp.83~86 (2002)
- Hirochika H, "Rice mutant resources for gene discovery", Plant Mol. Biol., 54, pp.325~334 (2004)
- Ichikawa T, "Sequence database of 1,172 T‐DNA insertion sites in Arabidopsis activation tagging lines that showed phenotypes in T1 generation", Plant J., 36, pp.421~429 (2003)
- Ichikawa T, "The FOX hunting system: an alternative gain‐of‐function gene hunting technique", Plant J., 48, pp.974~985 (2006)
- Jeong DH, "T‐DNA insertional mutagenesis for activation tagging in rice", Plant Physiol., 130, pp.1636~1644 (2002)
- Manhart N, "Administration of proteolytic enzymes bromelain and trypsin diminish the number of CD4+ cells and the interferon-gamma response in Peyer’s patches and spleen in endotoxemic balb", c mice, 2002, pp.215, 113~119
- Maurer HR, "Bromelain: biochemistry, pharmacology and medical use", Cell Mol. Life Sci., 58, pp.1234~1245 (2001)
- Meyerowitx EM, "Arabidopsis thaliana and Plant Molecular Genetics", Science, 229, pp.1214~1218 (1985)
- Miyao A, "A large‐scale collection of phenotypic data describing an insertional mutant population to facilitate functional analysis of rice genes", Plant Mol. Biol., 63, pp.625~635 (2007)
- Miyao A, "Target site specificity of the Tos17 retrotransposon shows a preference for insertion within genes and against insertion in retrotransposon ‐rich regions of the genome", Plant Cell, 15, pp.1771~1780 (2003)
- Mynott TL, "Proteolytic inhibition of Salmonella enterica serovar typhimuriuminduced activation of the mitogen-activated protein kinases ERK and JNK in cultured human intestinal cells", Infect Immun., 70, pp.86~95 (2002)
- Nakamura H, "A genome‐wide gain-of -function analysis of rice genes using the FOX‐hunting system", Plant Mol. Biol., 65, pp.357~371 (2007)
- Nanjo, T., "Functional annotation of 19,841 Populus nigra fulllength enriched cDNA clones", BMC Genomics, 8, pp.448~ (2007)
- Park SH, "Analysis of gene‐trap Ds rice populations in Korea", Plant Mol. Biol., 65, pp.373~384 (2007)
- Patton DA, "High-frequency plant regeneration from culture cotyledons of Arabidopsis thaliana", Plant Cell Rep., 7, pp.233~237 (1988)
- Pruitt RE, "Characterization of the genome of Arabidopsis thaliana", J. Mol. Biology, 187, pp.169~183 (1986)
- Ryu CH, "Generation of T‐DNA tagging lines using a bi‐directional gene trap vector and the establishment of an insertion‐site database", Plant Mol. Biol., 54, pp.489~502 (2004)
- Sallaud C, "High throughput T‐DNA insertion mutagenesis in rice: a first step towards in silico reverse genetics", Plant J., 39, pp.450~464 (2004)
- Seki M, "RIKEN arabidopsis full‐lenth (RAFL) cDNA and its applications for expression profiling under abiotic stress conditions", J. Experimental Botany, 55, pp.213~223 (2004)
- Taji T, "Large-scale collection and annotation of full-length enriched cDNAs from a model halophyte, Thellungiella halophila", BMC Plant Biol., 8, pp.115~ (2008)
- Takasaki T, "Factors influencing Agrobacteriummediated transformation of Brassica rapa L", Breeding Science, 47, pp.127~134 (1997)
- Anai T, "Sequencing and analysis of approximately 40,000 soybean cDNA clones from a full-length-enriched cDNA library", DNA Res., 15, pp.333~346 (2008)
- Upadhyaya NM, "An iAc", Ds gene and enhancer trapping system for insertional mutagenesis in rice, 2002, pp.29, 547~559
- Weigel D, "Activation Tagging in Arabidopsis", Plant Physiol, 122, pp.1003~1013 (2000)
- Yokotani N, "Tolerance to various environmental stresses conferred by the salt-responsive rice gene ONAC063 in transgenic Arabidopsis", Planta, 229, pp.1065~1075 (2009)
- Yokotani N, "Expression of rice heat stress transcription factor OsHsfA2e enhances tolerance to environmental stresses in transgenic Arabidopsis", Planta, 227, pp.957~967 (2008)