• Horticultural Science & Technology
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• v.28 no.3
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• pp.442-448
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• 2010

The objectives of this study were to analyze mutant lines of Chinese cabbage ($Brassica$ $rapa$ ssp. $pekinensis$) using gene tagging system (plasmid rescue and inverse polymerase chain reaction) and to observe the phenotypic characteristics. Insertional mutants were derived by transferring DNA (T-DNA) of $Agrobacterium$ for functional genomics study in Chinese cabbage. The hypocotyls of Chinese cabbage 'Seoul' were used to obtain transgenic plants with $Agrobacterium$ $tumefaciens$ harboring pRCV2 vector. To tag T-DNA from the Chinese cabbage genomic DNA, plasmid rescue and inverse PCR were applied for multiple copies and single copy insertional mutants. These techniques were successfully conducted to Chinese cabbage plant with high efficiency, and as a result, T-DNA of pRCV2 vector showed distinct various integration patterns in the transgenic plant genome. The polyploidy level analysis showed the change in phenotypic characteristics of 13 mutant lines was not due to variation in somatic chromosome number. Compared with wild type, the $T_1$ progenies showed varied phenotypes, such as decreased stamen numbers, larger or smaller flowers, upright growth habit, hairless leaves, chlorosis symptoms, narrow leaves, and deeply serrated leaves. The polyploidy level analysis showed the change in phenotypic characteristics of 13 mutant lines was not due to variation in somatic chromosome number. To tag T-DNA from the Chinese cabbage genomic DNA, plasmid rescue and inverse PCR were applied for multiple copies and single copy insertional mutants. Mutants that showed distinct phenotypic difference compared to wild type with 1 copy of T-DNA by Southern blot analysis, and with 2n = 20 of chromosome number were selected. These selected mutant lines were sequenced flanking DNA, mapped genomic loci, and the genome information of the lines is being recorded in specially developed database.

• Plant Biotechnology Reports
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• v.4 no.3
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• pp.201-211
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• 2010

We present a highly effective T-DNA inserted gene screening method as part of a reverse genetics model system using the Chinese cabbage (Brassica rapa L. spp. pekinensis). Three-step two-dimensional (2D) matrix strategies are potentially accurate and useful for the identification of specific T-DNA inserted mutants from a large population. To construct our Chinese cabbage model, we utilized a forward genetics screening approach for the abnormal phenotypes that were obtained from transgenic plants of Brassica rapa generated with Agrobacteria tumefaciens containing the pRCV2 vector. From one transgenic plant with an abnormal phenotype, we observed that the st1 gene (which is related to senescence-associated process proteins) contained a T-DNA fragment, and that its expression level was decreased. This T-DNA insert was then used as a control to construct an effective screening pool. As a result, the optimum template concentration was found to be 0.1-1 ng in our PCR strategy. For other conditions, positive changes to the Gibbs free energy prevented the formation of oligo dimers and hairpin loop structures, and autosegment extension gave better results for long fragment amplification. Using this effective reverse genetics screening method, only 23 PCR reactions were necessary to select a target gene from a pool of 100 individual DNAs. Finally, we also confirmed that the sequence we obtained from the above method was identical to the flanking sequence isolated by rescue cloning.

• Journal of Life Science
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• v.11 no.5
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• pp.423-431
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• 2001

Genomic and cDNA clones containing the RAT3 gene involving in Agobacterium-mediated plant transformation were identified using plant DNA flanking the righ border of a T-DNA rescued from the rat3 mutant as hy-bridization probe. Two highly homologous cDNA clones were identified; one (RAT3-1) weakly hybridized with the probe whereas another (RAT3-2) strongly hybridized with the probe. Both Rat3-1 and Rat3-2 proteins contain a putative signal peptide for secretion. The deduced molecular weights of encoded proteins are 15 kDa. The results of genomic DNA blot analysis and DNA sequencing indicated that RAT3-1 and RAT3-2 exist as single copy genes and they were arranged side by side with just 600 bp distance between them. RAT3-1 was disrupted by the integration of T-DNA into the 3 untranslated region in rat3 mutant. A BLAST search showed that both RAT3-1 and RAT3-2 proteins have homology with only the C-terminal region of $\beta$-1,3-glucanase homologues from Triticum aestivum and Arabidopsis thaliana. Thses $\beta$-1,3-glucanase homologues contain an unusually long C-terminal region with no sig-nificant homology to other $\beta$-1,3-glucanase.

• Microbiology and Biotechnology Letters
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• v.47 no.4
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• pp.667-672
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• 2019

We compared two integration systems for stable expression of heterologous genes in Saccharomyces cerevisiae. A Candida glabrata-derived gene was used as the selective marker for the Cre/loxP system, and XYLP, XYLB, GRE3, and XYL2 genes were used as model heterologous genes and ligated into the universal pRS-CMT vector. The resulting pRS-XylP, pRS-XylB, pRS-Gre3, and pRS-Xyl2 plasmids were sequentially integrated into yeast chromosome VII by four integration processes (marker rescue and gene integration). The four introduced genes were successfully expressed. Further, the pRS-PBG2 plasmid harboring expression cassettes for the four genes was constructed for one-step integration. The four genes that were introduced were stably maintained as a gene cluster and were simultaneously expressed. The one-step integration was more effective for the simultaneous integration and expression of the four genes related to xylan/xylose metabolism. This method will enable the generation of a useful biosystem through appropriate use of gene integration methods.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.30 no.1
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• pp.46-54
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• 1997

Sporulation in the fission yeast Schizosaccharomyces pombe has been regarded as an important model of cellular development and differentiation. S. pombe cells proliferate by mitosis and binary fission on growth medium. Deprivation of nutrients especially nitrogen sources, causes the cessation of mitosis and initiates sexual reproduction by malting between two sexually compatible cell types. Meiosis is then followed in a diploid cell in the absence of nitrogen source. DNA fragment complemented with the mutations of sporulation gene was isolated from the S. pombe gene library constructed in the vector, pDB 248' and designated as pDB (spo 5)1. We futher analyzed six recombinant plasmids, pDB (spo 5)2, pDB(spo 5)3, pDB(spo 5)4, pDB(spo 5)5, pDB(spo 5)6, pDB(spo 5)7, and found each plasmids is able to rescue the spo 5-2, spo 5-3, spo 5-4, spo 5-5, spo 5-6, spo 5-7, mutations, respectively. Mapping of the integrated plasmid into the homologous site of the S. pombe chromosomes demonstrated that pDB (spo 5)1, and pDB (spo 5)R1 contained the spo 5 gene. Transcipts of spo 5 gene were analyzed by Northern hybridization. Two transcripts of 3.2 kb and 25 kb were detected with 5 kb Hind III fragment containing a part of the spo 5 gene as a probe. The small mRNA (2.5 kb) appeared only when a wild-type strain was cultured in the absence of nitrogen source in which condition the large mRNA (3.2 kb) was produced constitutively. Appearance of a 2.5 kb spo 5-mRNA depends upon the function of the mei1, mei2 and mei3 genes.

• The Korean Journal of Food And Nutrition
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• v.15 no.2
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• pp.112-118
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• 2002

Sporulation in the fission yeast Schizosaccharomyces pombe has been regarded as an important model of cellular development and differentiation. S. pombe cells proliferate by mitosis and binary fission on growth medium. Deprivation of nutrients especially nitrogen sources, causes the cessation of mitosis and initiates sexual reproduction by matting between two sexually compatible cell types. Meiosis is then followed in a diploid cell in the absence of nitrogen source. DNA fragment complemented with the mutations of sporulation gene was isolated from the S. pombe gene library constructed in the vector, pDB 248' and designated as pDB(spo5)1. We futher analyzed six recombinant plasmids, pDB(spo5)2, pDB(spo5)3, pDB(spo5)4, pDB(spo5)5, pDB (spo5)6, pDB(spo5)7 and found each of these plasmids is able to rescue the spo5-2, spo5-3, spo5-4, spo5-5, spo5-6, spo5-7 mutations, respectively. Mapping of the integrated plasmid into the homologous site of the S. pombe chromosomes demonstrated that pDB(spo5)1, and pDB(spu5)Rl contained the spo5 gene. Transcripts of spo5 gene were analyzed by Northern hybridization. Two transcripts of 3.2 kb and 2.5kb were detected with 5kb Hind Ⅲ fragment containing a part of the spo5 gene as a probe. The small mRNA(2.5kb) appeared only when a wild-type strain was cultured in the absence of nitrogen source in which condition the large mRNA (3.2kb) was produced constitutively. Appearance of a 2.5kb spo5-mRNA depends upon the function of the meil, mei2 and mei3 genes.