Search

Sweet proteins are the natural alternative to the artificial sweeteners as well as flavor enhancers. Among other sweet protein, thaumatin protein was isolated from Thaumatococcus daniellii Benth plant fruit. In this study, pinII Ti plasmid vector was constructed with thaumatin gene, where thaumatin was placed under the control of the duel cauliflower mosaic virus 35S promoter into rice (Oryza sativa L. var. japonica cv. ‘Dongjinbyeo’) by Agrobacterium-mediated transformation to generate transgenic plants. Thirteen plant lines were regenerated and the transgenic rice lines were confirmed by different molecular analysis. The genomic PCR result revealed that all of the plant lines were transgenic. The single copy and intergenic plant lines were selected by Taqman PCR analysis and FST analysis, respectively. Expression of thaumatin gene in transgenic rice resulted in the accumulation of thaumatin protein in the leave. Thaumatin protein was also accumulated in leave of T1 generation. Sensory analysis result suggested that the thaumatin protein expressing transgenic lines exerted sweet tasting activity. These results demonstrated that thaumatin was expressed in transgenic rice plants.

Transgenic rice overexpressing PLCP attenuated the virulence of Xanthomonas oryzae pv. oryzae through extensive activation of transduction signal and transcription activities that orchestrate downstream responses including the biosynthesis of secondary metabolites and up-regulation of several pathogenesis-related proteins. High-throughput transcriptome investigations of plant immunity highlight the complexity of gene networks leading to incompatible interaction with the pathogen. Accumulating findings implicate papain-like cysteine proteases (PLCPs) as a central hub in plant defense. While diverse roles of PLCPs in different pathosystems have become more evident, information on gene networks and signaling pathways necessary to orchestrate downstream responses are lacking. To understand the biological significance of cysteine protease against Xanthomonas oryzae pv. oryzae, PLCP overexpression and knockout rice lines were generated. The pathogenicity test revealed the attenuation of Xanthomonas oryzae pv. oryzae race K3a virulence in transgenic lines which is ascribed to high hydrogen peroxide and free salicylic acid accumulation. Next-generation sequencing of RNA from transgenic and wild-type plants identified 1597 combined differentially expressed genes, 1269 of which were exclusively regulated in the transgenic libraries. It was found that PLCP aids rice to circumvent infection through the extensive activation of transduction signal and transcription factors that orchestrate downstream responses, including up-regulation of multiple pathogenesis-related proteins and biosynthesis of secondary metabolites.

The altered rice leaf color based on the knockout of CAO1 gene generated using CRISPR/Cas9 technology plays important roles in chlorophyll degradation and ROS scavenging to regulate both natural and induced senescence in rice. Rice chlorophyllide a oxygenase (OsCAO1), identified as the chlorophyll b synthesis under light condition, plays a critical role in regulating rice plant photosynthesis. In this study, the development of edited lines with pale green leaves by knockout of OsCAO1 gene known as a chlorophyll synthesis process is reported. Eighty-one genetically edited lines out of 181 T0 plants were generated through CRISPR/Cas9 system. The edited lines have short narrow flag leaves and pale green leaves compared with wild-type ‘Dongjin’ plants (WT). Additionally, edited lines have lower chlorophyll b and carotenoid contents both at seedling and mature stages. A transcriptome analysis identified 580 up-regulated and 206 downregulated genes in the edited lines. The differentially expressed genes (DEGs) involved in chlorophyll biosynthesis, magnesium chelatase subunit (CHLH), and glutamate-1-semialdehyde2, 1-aminomutase (GSA) metabolism decreased significantly. Meanwhile, the gel consistency (GC) levels of rice grains, chalkiness ratios and chalkiness degrees (CD) decreased in the edited lines. Thus, knockout of OsCAO1 influenced growth period, leaf development and grain quality characters of rice. Overall, the result suggests that OsCAO1 also plays important roles in chlorophyll degradation and ROS scavenging to regulate both natural and induced rice senescence.

Meiosis is a specialized cell division, essential in most reproducing organisms to halve the number of chromosomes, thereby enabling the restoration of ploidy levels during fertilization. A key step in meiosis is homologous recombination, which promotes homologous pairing and generates crossovers (COs) to connect homologous chromosomes until their separation at anaphase I. These CO sites, seen cytologically as chiasmata, represent a reciprocal exchange of genetic information between two homologous non-sister chromatids. RAD51, the eukaryotic homolog of the bacterial RecA recombinase, plays a central role in homologous recombination (HR) in yeast and animals. Loss of RAD51 function causes lethality in the flowering plant, Arabidopsis thaliana, suggesting that RAD51 has a meiotic stage-specific function that is different from homologous pairing activity.

Brazzein은 열대식물인 P. brazzeana Baillon의 과실에서 분리된 가장 작은 감미단백질로 토착민들의 단맛원료로 사용되어 왔다. Brazzein은 sucrose보다 분자량 기준으로 500 ~ 2000배, 몰 기준으로 9500배 당도가 높아 감미료로써 매우 높은 평가를 받고 있다. 그러나 이 감미단백질은 재배가 어렵고 생산 비용이 높아서 brazzein 단백질의 이용 가능성을 높이기 위한 대체 생산 시스템으로 형질전환 식물체 육성 하고자 하였다. 본 연구에서는 brazzein 관련 유전자를 벼에 도입하기 위하여 식물형질전환용 Ti-plasmid에 2 x CaMV 35S 프로모터에 의해 지배되어 발현하도록 하고, 선발 마커로 bar 유전자가 삽입된 식물발현 벡터를 구축하여 A. tumefaciens EHA105에 형질전환시켜 17개의 재분화 식물체를 육성하였다. 17개 재분화 식물체는 PCR 및 RT-PCR 분석을 통하여 유전자 도입 및 발현을 확인하였으며, TaqMan PCR을 통해 single copy로 도입된 T0 세대 9개체를 선발하였다. 또한 FST 분석을 통하여 도입 유전자가 intergenic으로 삽입된 개체 5개를 선발하였다. 이들 5개체를 이용하여 western blot 분석에 의해 단백질 발현량을 분석한 결과 선발된 모든 개체에서 발현 밴드를 확인하였다. 그 중 brazzein 단백질의 발현량이 높은 개체를 TG11으로 계통화하여 후대 종자를 육성하였다. TG11 계통은 천연 감미료 brazzein을 생산하는 새로운 벼 품종을 개발하기 위한 육종 소재로 활용 가능하다고 시사된다.

본 연구에서는 국내외에서 수집한 벼 294개 유전자원 핵심집단을 대상으로 벼의 지엽각 특성에 대한 조사를 수행하였고, GWAS를 이용하여 지엽각 연관 유전자를 추출 및 분석하였다. 표현형 데이터를 이용한 GWAS의 Manhattan plot 결과 분석을 통해, 각 집단에서 염색체를 대상으로 표현형과 통계적 유의성을 나타내 연관성을 보이는 SNP를 발굴하였다. 지엽각 관련 특성에 대하여 선행 연구된 QTL region과의 비교를 통하여 본 연구에서 발굴된 SNP간의 유의성을 조사한 결과, 지엽각과 유의성이 있는 SNP (S8-19815442)가 이미 확인된 QTL region에 위치하는 것으로 나타났으며, 후보유전자 Os08g31950 대해 연관 유전자 변이를 관찰하기 위해서 형질 특이적 품종군 간의 염기서열을 비교한 결과 1개의 지역에서 단일염기변이가 검출되었다. Os08g31950의 조직별 RNA의 상대적 발현량 수준을 비교한 결과, Os08g31950 유전자는 모든 조직에서 높은 발현량을 확인할 수 있었으며 조직별로 다양한 발현 양상을 관찰할 수 있었다. 또한, 모두 직립형 품종군에서 상대적으로 발현량이 높게 나타났으며 뿌리보다 잎에서의 발현율이 높게 나타났다. 본 연구를 통해 동정된 지엽각 연관 후보유전자 Os08g31950는 벼 생육 및 수량 증대에 이용할 수 있는 마커제작 및 육종의 기초자료가 될 것으로 기대된다.

Nowadays, rice production faces significant challenges due to population pressure, global climate change, and outbreak of various pests and diseases. Breeding techniques used to improve rice traits include mutant breeding, cross breeding, heterogeneity, transformation, molecular markers, genome-wide association study (GWAS), and so on. Since the recently developed CRISPR/Cas9 technology can directly target a specific part of a desired gene to induce mutation, it can be used as a powerful means to expand genetic diversity of crops and develop new varieties. So far, CRISPR/Cas9 technology has been used for improving rice characteristics such as high yield, good quality, abundant nutrition, pest and disease resistance, herbicide resistance, and biotic and abiotic stress resistance. This review highlights the mechanisms and optimization of the CRISPR system and its application to rice crop, including resistance to biotic and abiotic stresses, and improved rice quality and yield.

벼 품종의 DNA profiling을 위하여 SSR 마커를 활용하여 한국 벼의 품종판별 기술 확립을 위하여 국내에서 육성된 벼 243개 품종에 대하여 최종 선발된 7개의 SSR 마커(RM21, RM257, HsSSR01-52, RM333, RM580, RM1306, RM157)를 이용하여 판별하였다. 판별용으로 이용된 SSR 마커 7개의 총 대립인자 수는 130개였으며, 대립인자 수의 범위는 10 ~ 32개이었고, 평균 대립인자 수는 18.57이었다. PIC 값은 0.679(HsSSR01-52) ~ 0.895 (RM333)의 범위이었으며, 평균 PIC 값은 0.774이었다. SSR 마커 7개의 조합으로 6단계의 판별을 통해 총 243개 품종 중 243개 모든 품종의 구별이 가능하였다. 이와 같은 결과, 본 연구에 이용된 SSR 7개 마커는 한국 벼 품종의 판별과 순도유지에 매우 효율적으로 이용할 수 있을 것으로 여겨진다.

식물의 유일한 활성 스테로이드 호르몬인 Brassinosteroid (BR)는 다양한 내재적 또는 외부 신호 전달 경로와의 통합적인 결합을 통해 식물의 생장 및 발달 과정에서 중요한 기능을 하는 것으로 알려져 있다. 최근 식물학 연구들은 종자의 발아와 초기 발달과정에서 BR과 ABA 사이의 필수적인 상호 작용 메커니즘이 존재하고 있음을 보고하고 있다. 하지만 이들 두 호르몬의 중요한 신호전달 상호작용에 대한 분자 메커니즘은 거의 알려지지 않았다. 식물의 초기 발달과정에서 BR에 의해 매개되는 ABA 신호전달과의 기능학적, 생물학적 상호작용 네트워크를 이해하기 위해 Agilent Arabidopsis 4 x 44K 올리고 칩을 사용하여 비교 전사체 분석을 수행하였다. ABA에 반응하지 않는 bes1-D 돌연변이체에서의 ABA 처리에 따른 다양한 유전자의 발현 패턴을 야생형 식물과 비교 분석하였다. 그 결과 발현의 변화가 발생하는 유전자(DEGs) 2,353 개를 확인하였다. GO 분석을 통해 ABA 신호전달 및 대사에 관여하는 유전자들이 BR 신호전달 경로에 의해 하향 조절되는 것으로 확인되었다. 뿐만 아니라, BR 신호전달 경로는 다양한 비생물학적/생물학적 스트레스, 오옥신 및 ROS 등 다양한 신호전달 체계와 밀접하게 연관되어 있음을 확인하였다. 본 연구를 통해 BR 신호전달의 활성화는 ABA 신호전달에 관여하는 다양한 유전자들의 발현을 억제함을 확인하였다. 또한 본 연구는 다양한 신호 경로 사이의 상호 작용이 다양한 환경요인에 대한 식물의 적응 반응에 중요하게 작용할 수 있음을 보여주고 있다.

Implementation of crop improvement programs relies on genetic diversity. To overcome the limited occurrence of natural mutations, researchers and breeders applied diverse methods, ranging from conventional crossing to classical bio-technologies. Earlier generations of knockout and gain-of-function technologies often result in incomplete gene disruption or random insertions of transgenes into plant genomes. The newly developed editing tool, CRISPR/Cas9 system, not only provides a powerful platform to efficiently modify target traits, but also broadens the scope and prospects of genome editing. Customized Cas9/guide RNA (gRNA) systems suitable for efficient genomic modification of mammalian cells or plants have been reported. Following successful demonstration of this technology in mammalian cells, CRISPR/Cas9 was suc-cessfully adapted in plants, and accumulating evidence of its feasibility has been reported in model plants and major crops. Recently, a modified version of CRISPR/Cas9 with added novel functions has been developed that enables programmable direct irreversible conversion of a target DNA base. In this review, we summarized the milestone applications of CRISPR/ Cas9 in plants with a focus on major crops. We also present the implications of an improved version of this technology in the current plant breeding programs.

To study the transcript levels of epigenetically regulated genes in tobacco, we have developed a transgenic line OX1 overexpressing NtROS2a gene encoding cytosine DNA demethylation and a RNAi plant line RNAi13. It has been reported that salt- and -stress tolerance of these transgenic lines are enhanced with various phenotypic characters (Lee et al. 2015). In this paper, we conducted microarray analysis with Agilent Tobacco 4 x 44K oligo chip by using overexpression line OX1, RNAi plant line RNAi 13, and wild type plant WT. Differentially expressed genes (DEGs) related to metabolism, nutrient supply, and various stressed were up-regulated by approximately 1.5- to 80- fold. DEGs related to co-enzymes, metabolism, and methylation functional genes were down-regulated by approximately 0.03- to 0.7- fold. qRT-PCR analysis showed that the transcript levels of several candidate genes in OX1 and RNAi lines were significantly (p

Single nucleotide polymorphism (SNP) is an abundant form of genetic variation within individuals of species. DNA polymorphism can arise throughout the whole genome at different frequencies in different species. SNP may cause phenotypic diversity among individuals, such as individuals with different color of plants or fruits, fruit size, ripening, flowering time adaptation, quality of crops, grain yields, or tolerance to various abiotic and biotic factors. SNP may result in changes in amino acids in the exon of a gene (asynonymous). SNP can also be silent (present in coding region but synonymous). It may simply occur in the noncoding regions without having any effect. SNP may influence the promoter activity for gene expression and finally produce functional protein through transcription. Therefore, the identification of functional SNP in genes and analysis of their effects on phenotype may lead to better understanding of their impact on gene function for varietal improvement. In this mini-review, we focused on evidences revealing the role of functional SNPs in genes and their phenotypic effects for the purpose of crop improvements.

UDP-glucose 4-epimerase (UGE) catalyzes the reversible conversion of UDP-glucose to UDP-galactose. To understand the biological function of UGE from Brassica rapa, the gene BrUGE1 was cloned and introduced into the genome of wild type rice 'Gopum' using the Agrobacterium-mediated transformation method. Four lines which carried a single copy gene were selected and forwarded to T3 generation. Agronomic traits evaluation of the transgenic T3 lines (CB01, CB03, and CB06) under optimal field conditions revealed enriched biomass production particularly in panicle length, number of productive tillers, number of spikelets per panicle, and filled spikelets. These remarkably improved agronomic traits were ascribed to a higher photosynthetic rate complemented with higher CO2 assimilation. Transcripts of BrUGE1 in transgenic lines continuously accumulated at higher levels after the 20% PEG6000 treatment, implying its probable role in drought stress regulation. This was paralleled by rapid accumulation of soluble sugars which act as osmoprotectants, leading to delayed leaf rolling and drying. Our findings suggest the potential of BrUGE1 in improving rice growth performance under optimal and water deficit conditions.

Anthranilate synthase (AS) is a key enzyme in the biosynthesis of tryptophan (Trp), which is the precursor of bioactive metabolites like indole-3-acetic acid and other indole alkaloids. Alpha anthranilate synthase 2 (OsASA2) plays a critical role in the feedback inhibition of tryptophan biosynthesis. In this study, two vectors with single (F124V) and double (S126F/L530D) point mutations of the OsASA2 gene for feedback-insensitive subunit of rice anthranilate synthase were constructed and transformed into wildtype Dongjinbyeo by Agrobacterium-mediated transformation. Transgenic single and double mutant lines were selected as a single copy using TaqMan PCR utilized nos gene probe. To select intergenic lines, the flanking sequence of RB or LB was digested with a BfaI enzyme. Four intergenic lines were selected using a flanking sequence tagged (FST) analysis. Expression in rice (Oryza sativa L.) of the transgenes resulted in the accumulation of tryptophan (Trp), indole-3-acetonitrile (IAN), and indole-3-acetic acid (IAA) in leaves and tryptophan content as a free amino acid in seeds also increased up to 30 times relative to the wildtype. Two homozygous event lines, S-TG1 and D-TG1, were selected for characterization of agronomic traits and metabolite profiling of seeds. Differentially expressed genes (DEGs), related to ion transfer and nutrient supply, were upregulated and DEGs related to co-enzymes that work as functional genes were down regulated. These results suggest that two homozygous event lines may prove effective for the breeding of crops with an increased level of free tryptophan content.

본 연구는 다양한 LED광 처리가 상추의 생장상, 플라보노이드 생합성 관련 유전자들의 발현양상 및 플라보노이드 함량에 미치는 영향을 분석하였다. 상추의 생장상 분석에서 혼합광 처리가 단일 처리구보다 잎생장이 월등히 우수하였으며, 단일광내에서 청색광이 적색광보다 생체중이 높게 나타났다. LED광질에 따른 플라보노이드 생합성 관련 8개 유전자들의 발현을 분석한 결과 LsF3H와 LsDFR 유전자에서 혼합광 처리한 적상추에서 청상추보다 높게 나타났다. 또한 혼합광 처리가 단일광 처리보다 Gallic acid, Chlorogenic acidn, Quercetin 및 안토시아닌의 함량이 가장 높게 나타났다. 이상의 결과로부터 LED 광질에 따라 상추 생장과 발육차이를 보이며, 유전자의 발현 및 기능성 물질의 축적에 중요한 요인으로 작용하며, 기능성 물질의 축적을 유도하기 위해서는 적색과 청색의 혼합처리가 유용하리라 생각된다.

The novel BrRZFPs genes encoding C3HC4- type RING zinc finger protein were identified from FOX (full length cDNA over-expressing) library of Brassica rapa . Ten full-length cDNAs obtained from the library encode zinc-finger protein containing 346 amino acids, designated BrRZFPs. These genes were classified into four groups by phylogenic analysis showing conserved protein sequences at both termini. The tissue distribution of BrRZFPs trans- cription was examined by qRT-PCR revealing ubiquitous expression pattern. However, each gene was strongly ex- pressed in the specific tissue. Transcriptional analysis showed that those acquired 10 genes were inducible under abiotic stresses. Likewise, the transcript of BrRZFP3 was strongly induced (~12-folds) by exogenous abscisic acid, whereas the transcripts of BrRZFP1, BrRZFP2 and BrRZFP3 were (> 9-folds) induced by cold. We suggest that these BrRZFPs that function as signal or response to abiotic stress are useful for crop improvement.

This study demonstrates the isolation and characterization of cDNA encoding a phosphoinositide phosphatase (PIP) from a stem cell cDNA library of Chinese cabbage ( Brassica rapa ) seedling. The full length gene ( BrSAC1 ; GenBank accession no., GU434275) contained 1999 base pairs (bp), with an open reading frame of 1785 bp, encoding a polypeptide of 594 amino acids with a predicted molecular weight of 65 kDa, including a putative N-terminal signal peptide (the signal peptide counted within the 594 residues). Other regions found within the sequence include a conserved KXKXX COPI-binding motif and a consensus Cx5R (T/S) catalytic motif. BrSAC1 protein shares 92% identity with AtSac1B, and 86% identity with AtRHD4 at the amino acid level. Gene expression analyses revealed that BrsSAC1 was constitutively expressed at high levels in the pistil, stamen and flower bud, whereas it was expressed at low levels in the leaf and stem. In addition to injury, BrSAC1 expression was also induced in response to different types of stress condition, namely cold, desiccation, salt, submergence, abscisic acid and heavy metals. Overexpression of BrSAC1 in transgenic tobacco conferred tolerance to cold, dehydration, and salt stress at the seed germination/seedling stage as reflected by the percentage of germination/green seedlings, the fresh weight of seedlings and their development pattern. Our data suggest that BrSAC1 is an important stress response determinant in plants. Keywords: Abiotic stress, Brassica rapa , phosphoinositide phosphatase, transgenic plant African Journal of Biotechnology Vol. 12(15), pp. 1782-1792

C3H4-type RING zinc finger protein (BrRZP1) from Brassica rapa under abiotic stresses. To elucidate the role of the BrRFP1 transcription factor in gene regulation, we transformed tobacco plants with the BrRZFP1 gone. Plants were regenerated from 82 independently transformed callus lines of tobacco and analysed for transgene expression. Trans-gene integration and expression was confirmed by Southern and RT-PCR analyses, respectively. T2 plants displayed more tolerance to the bacterial pathogens Pectobacterium carotovorum and Ralstonia solanacearum, and the tolerance levels were correlated with BrRZFP1 expression levels. These results suggest that the transcription factor BrRZFP1 is an important determinant of stress response in plants and its overexpression in plants could increase biotic stress resistance.

Brassica rapa (chinese cabbage) is one of the main vegetable crops grown in Asian countries. Pectobacterium carotovorum subsp. carotovorum causes severe economic loss in this crop as well as in other Brassica crops through soft rot disease. Cysteine proteases like bromelain, papain or ficin show toxic effects to herbivorous insects and pathogenic bacteria. They have been known to be critical factors in plant defence mechanisms. The current study investigated the effect of bromelain gene (BL1) of pineapple (Ananas comosus L. Merrill) on enhancing resistance to soft rot in transgenic Brassica rapa ‘Seoulbaechu’. Three homozygous T2 lines were inoculated with Pectobacterium carotovorum subsp. carotovorum and BL8-2 line showed the lowest rate of infected leaves (RIL) in both wound inoculation and non-wound inoculation, when the non-infected line showed 100 % RIL in both cases. The highest expression of BL1 gene was also observed in BL8-2 homozygous line. Thus, the over-expressed BL1 gene conferred enhanced resistance to soft rot in Brassica rapa.

Glycosyltransferases are enzymes (EC 2.4) that catalyze the transfer of monosaccharide moieties from activated nucleotide sugar to a glycosyl acceptor molecule which can be a carbohydrate, glycoside, oligosaccharide, or a poly- saccharide. In this study, a UDP-glucosyltransferase cDNA was isolated from Brassica rapa using a rapid amplification of cDNA ends (RACE) and subsequently named BrUGT. It has a full-length cDNA of 1,236 bp with 119 bp 5'-un- translated region (UTR), a complete ORF of 834 bp encoding a polypeptide of 277 amino acids (31.19 kDa) and a 3'-UTR of 283 bp. BLASTX analysis hits a catalytic domain of Glycos_transf_1 super family (cl12012) that belongs to the Glycosyltransferases group 1 with tetratricopeptide (TPR) regions located between 165 to 350 bp. Expression analysis showed high mRNA transcripts in pistil, followed by petal, seed and calyx of flower. Moreover, expression analysis of BrUGT in Chinese cabbage seedlings under stresses of cold, salt, PEG, H2O2, drought and ABA showed elevated mRNA transcript. Furthermore, when BrUGT gene was transformed into rice using pUbi-1 promoter, overexpression was evident among the T1 plants. This study provides insights into the function of BrUGT in plants.

In plants, the fifth step of the plastidial 2-C- methyl-D-erythritol 4-phosphate (MEP) pathway is catalyzed by 2-C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase (MECP; EC: 4. 6. 1. 12), an enzyme proposed to play a key role in the regulation of isoprenoid biosynthesis. Here we report the isolation and functional characterization of a 823 bp Brassica rapa MECP ( Brmecp ) cDNA encoding a deduced polypeptide of 230 amino acid residues. Tran-scription levels of Brmecp were two-fold higher in petal compared to leaves. In addition, Brmecp expression in cabbage seedlings treated with ABA, H 2 O 2 and drought was higher than control seedlings. These results were consistent with changes in chlorophyll contents in transgenic Arabidopsis . Thus, the Brmecp may contribute to the production of primary (chlorophylls and carotenoids) isoprenoid end-products in chloroplasts. Keywords Brassica rapa , 2-C-methyl-D-erythritol 2,4-cyclo-diphosphate synthase, isoprenoid biosynthesis, 2-C- methyl-D- erythritol 4-phosphate pathway, transgenic

【Knock-out of a gene by insertional mutagenesis is a direct way to address its function through the mutant phenotype. Among ca. 15,000 gene-trapped Ds insertion lines of rice, we identified one line from selected sensitive lines in highly salt stress. We conducted gene tagging by TAIL-PCR, and DNA gel blot analysis from salt sensitive mutant. A gene encoding an oligopeptide transporter (OPT family) homologue was disrupted by the insertion of a Ds transposon into the OsOPT10 gene that was located shot arm of chromosome 8. The OsOPT10 gene (NP_001062118.) has 6 exons and encodes a protein (752 aa) containing the OPT family domain. RT-PCR analysis showed that the expression of OsOPT10 gene was rapidly and strongly induced by stresses such as high-salinity (250 mM), osmotic, drought, $100\;{\mu}M$ ABA. The subcellular localization assay indicated that OsOPT10 was localized specifically in the plasma membrane. Overexpression of OsOPT10 in Arabidopsis thaliana and rice conferred tolerance of transgenic plants to salt stress. Further we found expression levels of some stress related genes were inhibited in OsOPT10 transgenic plants. These results suggested that OsOPT10 might play crucial but differential roles in plant responses to various abiotic stresses.】

Cysteine proteases have been known as a critical factor in plant defense mechanisms in pineapple, papaya, or wild fig. Papain or ficin is one kind of cysteine proteases that shows toxic effects to herbivorous insects and pathogenic bacteria. However, resistance to bacterial soft rot of plants genetically engineered with cysteine protease has been little examined thus far. We cloned a cysteine protease cDNA from Ananas comosus and introduced the gene into Chinese cabbage ( Brassica rapa ) under the control of the cauliflower mosaic virus 35S promoter. The transgene was stably in-tegrated and actively transcribed in transgenic plants. In comparisons with wild-type plants, the T 2 and T 3 transgenic plants exhibited a significant increase in endo-protease activity in leaves and enhanced resistance to bacterial soft rot. A cDNA microarray analysis revealed that several genes were more abundantly transcribed in the transgenic than in the wild type. These genes encode a glyoxal oxidase, PR-1 protein, PDF1, protein kinase, LTP protein, UBA protein and protease inhibitor. These results suggest an important role for cysteine protease as a signaling regulator in biotic stress signaling pathways, leading to the build-up of defense mechanism to pathogenic bacteria in plants.

Full-length cDNAs are essential for the correct annotation of genomic sequences and for the functional an- alysis 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. Full- length Chinese cabbage cDNAs were expressed indepen- dently 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 morpho- logical 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 bro- melain 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 ex- pression of cysteine protease in the T1 and T2 generations. The anti-bacterial activity of bromelain was tested in Chi- nese cabbages infected with soft rot bacteria. The results showed that the over-expressed bromelain gene from pine- apple conferred enhanced resistance to soft rot in Chinese cabbage.

Fructan has been found to accumulate in various tissues during periods when light levels increased carbon fixation where low temperatures reduced growth rates while photosynthesis continued. In this study, we have cloned 1-sucrose:sucrose fructosyl transferase(1-sst) and 1-fructan: fructan fructosyl transferase (1-fft, a key enzyme for the synthesis of fuctan) from Jerusalem Artichoke ( Helianthus tuberosus L.). The recombinant vector with 1-sst and 1-fft has been constructed under the control of 35S promoter of KJGV-B2 vector and transgenic plants obtained by Agro-bacterium tumefaciens LBA4404. PCR analysis carried out on the putative transgenic plants for amplification of the coding region of specific gene (1-sst, 1-fft), and HPT genes. Transgenic lines carrying of 1-sst and 1-fft were confirmed for integration into the rice genome using Southern blot hybridization and RT-PCR. The transgenic plants in T 2 generation were selected and expression pattern analysis revealed that 1-sst and 1-fft were stable. This analysis confirmed the presence of low-molecular-weight fructan in the seedling of the transgenic rices. Therefore, cold tolerance and carbohydrate metabolism will be possible to develop resistant plants using the transgenic rice.

A cDNA clone encoding phytocystatin was isola- ted from Brassica rapa seedlings, through rapid amplification of cDNA ends (RACE). This gene (name as Brcpi1; GenBank accession no.: EF079953) had a total length of 881 bp with an open reading frame of 609 bp, and encoded predicted poly- peptide of 203 amino acid (aa) residues including a putative N-terminal signal peptide. Other relevant regions found its sequence included the G and PW conserved aa motifs, and the consensus LARFAV sequence for phytocystatins and the reactive site QVVAG. The BrCPI1 protein shared 95, 94, 81, 80 and 78% identity with other CPI proterins isolated from Brassica oleracea (BoCPI-1), Arabidopsis thaliana (AtCY SB), Glycine max (GmCPI), Oryza sativa (OsCYS-2) and Zea may (ZmCPI) at amino acid level, respectively. Southern blot analysis showed that Brcpi1 was a low copy gene. Expression pattern analysis revealed that Brcpi1 was a tissue-specific expressing gene during reproductive growth and strongly expressed at mature seedling stages. Furthermore, overexpre- ssion of Brcpi1 in transgenic Arabidopsis was enhanced tolerance to salt and cold stresses. Meanwhile the juvenile seedling of Brcpi1 transgenic plants was not affected by vari- ous concentrations ABA in MS medium. Taken together, the results showed that Brcpi1 functioned as a cysteine protease inhibitor and it exhibited a protective agent against diverse types of abiotic stress, which induced this gene in a tissue- and stress-specific manner.

The recombinant DNAs, pGBF, pGTF, and pZ4F, using soybean ferritin gene have constructed with the promoters derived from seed proteins, glutelin, globulin, and zein. The recombinant ferritin genes were transformed into rice plant by Agrobacterium-mediated transformation. Iron contents and agronomic traits have been evaluated in the transgenic progenies. The embryogenic calli survived from second selection medium were regenerated at the rates of 19.2% with pGBF, 15.0% with pGTF, and 18.4% with pZ4F in Donganbyeo and 6.7% with pGBF, 11.7% with pGTF, and 3.4% with pZ4F in Hwashinbyeo. The introduction of ferritin gene in putative transgenic rice plants was confirmed by PCR and Southern blot analysis and also the expression of ferritin gene was identified by Northern blot and Western blot analysis. The iron accumulation in transgenic rice grains of the transgenic rice plant, T1-2, with zein promoter and ferritin gene contained 171.4 ppm showing 6.4 times higher than 26.7 ppm of Hwashinbyeo seed as wild type rice, but the transgenic plants with globulin and glutelin showed a bit higher iron contents with a range from 2.1 to 3.0 times compare to wild type grain. The growth responses of transgenic plants showed the large variances in plant height and number of tillers. However, there were some transgenic plants having similar phenotype to wild type plants. In the T1 generation of transgenic plants, plant height, culm length, panicle length, and number of tillers were similar to those of wild type plants, but ripened grain ratio ranged from 53.3% to 82.2% with relatively high variation. The transgenic rice plants would be useful for developing rice varieties with high iron content in rice grains.

A repeated DNA fragment (pKRD) was isolated from the genomic library of weedy rice in Korea. The pKRD showed significant homology to Em/Spm CACTA-like transposon in whole genome sequences of rice released in the Blast rice sequence database of NCBI and was closely related to the TNP2 transposase group, including a TNP-like transposable element of rice. A Southern hybridization experiment demonstrated that the pKRD sequence is unique to the Oryza genome. The 126 sequences homologous to pKRD were evenly distributed in all 12 different chromosomes in rice genomes with multiple copy numbers. Different copy numbers ranging from 1,500 to 4,500 corresponding to rice species were detected by slot blot hybridization. In a DNA fingerprinting experiment, a pKRD probe was assessed to be the potential molecular marker for studying evolution and divergence, biodiversity and phylogenic analysis of rice species.

To develop rice (Oryza sativa L.) cultivars to be planted on salt-affected sites, cell lines with enhanced proline content and resistance to growth inhibition by Azetidine-2-carboxylic acid (AZCA), a proline analogue, were screened out among calli irradiated with gamma ray of 50, 70, 90, and 120 Gy. The calli had been derived from embryo culture of the cultivar Donganbyeo. Selected AZCA resistant lines that had high proline accumulation were used as sources for selection of NaCl resistant lines. To determine an optimum concentration for selection of NaCl resistant lines, Donganbyeo seeds were initially cultured on the media containing various NaCl concentrations (0 to 2.5%) for 40 days, and 1.5% NaCl concentration was determined as the optimum concentration. One hundred sixteen salt-tolerant (ST) lines were selected from bulked 20,000 seeds of the AZCA resistant seeds in the medium containing 1.5% NaCl. The putative 33 lines ( generation) considered with salt-tolerance were further analyzed for salt tolerance, amino acid and ion contents, and expression patterns of the salt tolerance-related genes. Out of the 33 lines, 7 lines were confirmed to have superior salt tolerance. Based on growth comparison of the entries, the selected mutant lines exhibited greater shoot length with average 1.5 times, root length with 1.3 times, root numbers with 1.1 times, and fresh weight with 1.5 times than control. Proline contents were increased maximum 20%, 100% and 20% in the leaf, seed and callus, respectively, of the selected lines. Compared to control, amino acid contents of the mutants were 24 to 29%, 49 to 143%, 32 to 60% higher in the leaf, seed and callus, respectively. The ratio of for most of the ST-lines were lower than that of control, ranging from 1.0 to 3.8 for the leaf and 11.5 to 28.5 for the root, while the control had 3.5 and 32.9 in the leaf and root, respectively. The transcription patterns for the P5CS and NHXI genes observed by RT-PCR analysis indicated that these genes were actively expressed under salt stress. The selected mutants will be useful for the development of rice cultivar resistant to salt stress.

Transgenic chrysanthemum plants were constructed to simultaneously express three N-methyltransferases involved in caffeine biosynthetic pathways. Resulting plants produced caffeine at approximately ...

Overexpression of OsGS gene modulates oxidative stress response in rice after exposure to cadmium stress. Our results describe the features of transformants with enhanced tolerance to Cd and abiotic stresses. Glutamine synthetase (GS) (EC 6.3.1.2) is an enzyme that plays an essential role in the metabolism of nitrogen by catalyzing the condensation of glutamate and ammonia to form glutamine. Exposure of plants to cadmium (Cd) has been reported to decrease GS activity in maize, pea, bean, and rice. To better understand the function of the GS gene under Cd stress in rice, we constructed a recombinant pART vector carrying the GS gene under the control of the CaMV 35S promoter and OCS terminator and transformed using Agrobacterium tumefaciens. We then investigated GS overexpressing rice lines at the physiological and molecular levels under Cd toxicity and abiotic stress conditions. We observed a decrease in GS enzyme activity and mRNA expression among transgenic and wild-type plants subjected to Cd stress. The decrease, however, was significantly lower in the wild type than in the transgenic plants. This was further validated by the high GS mRNA expression and enzyme activity in most of the transgenic lines. Moreover, after 10 days of exposure to Cd stress, increase in the glutamine reductase activity and low or no malondialdehyde contents were observed. These results showed that overexpression of the GS gene in rice modulated the expression of enzymes responsible for membrane peroxidation that may result in plant death.

Brassica is a very important vegetable group worldwide and different stresses are a major concern for these crops. Enhancement of resistance against biotic and abiotic stresses by exploiting stress resistance related genes offers the most efficient approach to address this concern. In this study, a stress resistance related gene was identified from the full-length cDNA library of Brassica rapa cv. Osome, which was determined to be Brassica longevity assurance protein ( BrLAP ) after sequence analysis. A comparison study of this gene showed a high degree of homology with other stress resistance related longevity assurance genes and was shown to be expressed in all organs during all of the developmental growth stages. In addition, this gene significantly responded after cold, drought and ABA stress treatments in Chinese cabbage. All these data revealed that this gene may be involved in plant resistance against stresses. Keywords: Brassica rapa , longevity assurance gene, gene expression, biotic and abiotic stress

Asymmetric hybrid plants were obtained from fused protoplasts of a monocotyledon (Oryza sativa L.) and a dicotyledon (Daucus carota L.). X-ray-irradiated protoplasts isolated from a cytoplasmic malesterile (cms) carrot suspension culture were fused with iodoacetoamide-treated protoplasts isolated from a 5-methyltryptophan (5MT)-resistant rice suspension culture by electrofusion. The complementary recovered cells divided and formed colonies, which were then cultivated on regeneration medium supplemented with 25mg/l 5MT to eliminate any escaped carrot cells. Somatic hybrids were regenerated from 5 of the 5MT-resistant colonies. The morphologies of most of the regenerated plants closely resembled that of the parental carrot plants. A cytological analysis of callus cultures induced from these plants indicated that most of the cells possessed 20-22 chromosomes and were resistant to 5MT. An isozyme analysis revealed that several regenerated plants had the peroxidase isozyme patterns of both parents. A Southern hybridization analysis with non-radioactively labelled DNA fragments of the rgp1 gene showed that regenerated plants had hybridizing bands from both rice and carrot. Chloroplast (cp) and mitochondrial (mt) DNAs were also analyzed by Southern hybridization by using several probes. CpDNA patterns of the regenerated plants were indistinguishable from those of the carrot parent. However 1 of the regenerated plants had a novel band pattern of mtDNA that was not detected in either of the parents, indicating a possible recombination of mitochondrial genomes.

A maize plant resistant to 5-methyltrytophan (5MT) was selected from M2 seeds (Zea mays L. Danggin, inbred line) originating from ears treated with ethylmethane sulfonate (0.2%) at 6 hr after self-pollination. Genetic analysis of the progeny of plants selected from a medium containing 50 ppm 5MT showed that 5MT resistance was inherited as a single dominant nuclear gene. This resistance was also expressed in callus and seedling. Analysis of the free amino acids in kernels and calli showed that homozygous resistant plants (MR1) contained higher levels of total free amino acids than sensitive plants and calli. In particular, the their kernels the levels of tryptophan, threonine and serine were, respectively, 4.5, 5.9 and 6.3 times higher than those of the sensitive plants. From the results, it may be expected that mutants resistant to amino acid anologs will be useful not only for studying amino acid biosynthesis but also for improving the nutritional quality of maize.