Search

Most oilseeds contain polyunsaturated fatty acids, such as linoleic acid (LA) and α-linolenic acid (ALA) that are important essential fatty acids for human health. γ-Linolenic acid (GLA) and stearidonic acid (SDA), synthesized from LA and ALA by Δ6 desaturase, respectively, are mainly obtained from algae and fish. Given the benefits of GLA and SDA to human health, many researchers have investigated ways to produce these fatty acids in oilseed crops. Canola-type oilseed rape (Brassica napus), widely cultivated in temperate and microthermal zones, accumulates oleic acid to a relatively greater extent (60–70% oleic acid content) than do other oilseed crops. We transformed the Phytophthora citrophthora Δ6 desaturase (PcD6DES) gene under the control of seed-specific vicilin into canola-type oilseed rape ‘Youngsan’. D6DES products (GLA, SDA, and 18:2Δ6,9) accumulated in mature PcD6DES-transformed rapeseeds and leaves. D6DES products of PcD6DES rapeseeds were over 20% in T1 and more than 25% in T2. Expression levels of the PcD6DES gene and the content of D6DES products coincided with each other and were related to expression levels and fatty acid composition in leaves and developing seeds 15, 25, 35, and 45 days after flowering. Seed weights of PcD6DES rapeseeds were not lower than those of Youngsan. In this study, PcD6DES oilseed rapes accumulated GLA, SDA, and putative 18:2Δ6,9 content to a maximum of 25% in the seed oil. Results show that PcD6DES rapeseed oil can potentially be used as a health food to improve human health.

Vegetable oils, which are stored in seeds as triacylglycerol (TAG), represent a valuable source of food, fuel, and industrial raw materials. Given the increasing demand for vegetable oils, it is essential to increase vegetable oil production in a sustainable manner. To boost the supply of vegetable oil, strategies to produce oils in the vegetative tissues of biomass crops are being investigated. Producing oils in leaves is challenging, due to the major role of leaves in producing sugars, via photosynthesis, that are then transported to storage tissues. Several strategies have been developed to overcome this limitation, such as increasing fatty acid and TAG biosynthesis, stabilizing lipid droplets, and reducing carbon flux to starch biosynthesis in the leaves. For example, overexpressing the WRINKLED 1 (WRI1) transcription factor, which upregulates fatty acid biosynthesis; DIACYLGLYCEROL ACYLTRANSFERASE 1 (DGAT1) enzymes, which participate in the last step of TAG biosynthesis; and oleosin, which stabilizes oil droplets and simultaneously suppressing the expression of genes encoding enzymes involved in lipogenesis and lipases led to the accumulation of significant levels of oil in leaves in tobacco, Arabidopsis, and sugarcane. To facilitate the sustainable production of biofuels and industrial raw materials, efforts are underway to modulate metabolic flux between the carbohydrate and lipid biosynthesis pathways in non-edible biomass plants that can grow on barren or reclaimed lands.

Dry mouth is a common complaint among the elderly population. The aim of this study was to investigate the effect of Ixeris dentata (IXD) extract on aging-induced dry mouth. We used young (two months) and aged (20 months) SD rats in our study. Using water as the vehicle, IXD extract (25, 50, and 100 mg/kg) was given via oral gavage to the young and aged rats for eight weeks. We found that the salivary flow rate relative to the submandibular gland weight was differently influenced by IXD extract treatment. IXD extract augmented the submandibular gland acinar cells, which are depleted during aging. In addition, the decreased salivary alpha-amylase, inositol triphosphate receptor, and aquaporin-5 in the aging rats were upregulated by IXD treatment. Free radical-induced oxidative stress in the aging rats was also alleviated in the IXD-treated group. The formation of high molecular weight complexes of protein disulfide isomerase, decreased expression of an ER chaperone (GRP78), and increased ER stress response (ATF-4, CHOP and p-JNK) in aging rats was regulated with IXD treatment, and eventually increased salivary secretions from the aging submandibular glands. These are the first data to suggest that IXD extract might ameliorate aging-associated oral dryness by regulating the ER environment.

Rapeseed (Brassica napus) is now the second largest oilseed crop after soybean. Cold temperature tolerance is an important agronomic trait in winter rapeseed that determines the plant's ability to control below freezing temperatures. To improve cold tolerance of rapeseed plants, an expression vector containing an Barley Ice recrystallization inhibition protein (HvIRIP) cDNA driven by a cauliflower mosaic virus 35S promoter was transferred into rapeseed plants. Transgenic expression of HvIRIP was proved by southern- and northern-blot analyses. The level of freezing tolerance of transgenic plants was found to be significantly greater than that of wild-type rapeseed plants by freezing assay. Proline accumulation during cold stress was also highly induced in the transgenic rapeseed plants. The transgenic plants exhibited considerable tolerance against oxidative damage induced by cold stress. Our results indicated that heterologous HvIRIP expression in transgenic rapeseed plants may induce several oxidative-stress responsive genes to protect from cold stress.

Stearidonic acid (STA; 18:4n-3) and γ-linolenic acid (GLA; 18:3n-6) are significant intermediates in the biosynthetic pathway for the very-long-chain polyunsaturated fatty acids of eicosapentaenoic acid (EPA; 20:5n-3) and arachidonic acid (ARA; 20:4n-6), respectively. To develop a sustainable system for the production of dietary polyunsaturated fatty acids, we focused on the action of the enzyme delta 6-desaturase (D6DES) on the essential acids, linoleic acid (LA; 18:2n-6) and α-linolenic acid (ALA; 18:3n-3). A 1,335-bp full-length cDNA encoding D6DES (McD6DES) was cloned from Muraenesox cinereus using degenerate PCR and RACE-PCR methods. To investigate the enzymatic activity of McD6DES in the production of n-6 and n-3 fatty acids, a recombinant plasmid expressing McD6DES (pYES-McD6DES) was transformed into and expressed in Saccharomyces cerevisiae. The exogenously expressed McD6DES produced GLA and STA at conversion rates of 14.2% and 45.9%, respectively, from the exogenous LA and ALA substrates. These results indicate that McD6DES is essentially a delta 6-desaturase involved in very-long-chain polyunsaturated fatty acid synthesis.

The cDNA coding for a polyunsaturated fatty acid elongase (McELOVL5) was isolated from the brain of the pike eel (Muraenesox cinereus) being based on available sequences in 23 types of fish. Four sequence variants were identified with different amino acid substitutions as compared with two clones of McELOVL5 gene (McELOVL5 11.7 and McELOVL5 12.4). When the two variants of McELOVL5 were expressed in Saccharomyces cerevisiae, the two recombinant yeasts elongated γ-linolenic acid (GLA, 18:3n-6) to di-homo-γ-linolenic acid (DGLA, 20:3n-6) but differed in the rate of GLA conversion to DGLA. Cells transformed with McELOVL5 12.4 also converted arachidonic acid (20:4n-6) and eicosapentaenoic acid (20:5n-3) to docosatetraenoic acid (22:4n-6) and docosapentaenoic acid (22:5n-3), respectively. However McELOVL5 11.7 lost its function for the elongation of C20 fatty acids. The four sequence variants have changed substrate specificities. Three-dimensional models of the McELOVL5 proteins are suggested.

BACKGROUND: Genetically modified(GM) trigonal cactus(Hylocereus trigonus Saff.) contained a coat protein gene of cactus virus X (CVX), which conferred resistance to the virus, phosphinothricin acetyltransferase (bar) gene, which conferred herbicide resistance, and a cauliflower mosaic virus 35S promoter (CaMV 35S). This study was conducted to evaluate the possible impact of GM trigonal cactus cultivation on the soil microbial community. METHODS AND RESULTS: Microorganisms were isolated from the rhizosphere of GM and non-GM trigonal cactus cultivation soils. The total numbers of bacteria, and actinomycete in the rhizosphere soils cultivated GM and non-GM trigonal cactus were similar to each other, and there was no significant difference. Dominant bacterial phyla in the rhizosphere soils cultivated with GM and non-GM trigonal cactus were Proteobacteria, Uncultured archaeon, and Uncultured bacterium. The denaturing gradient gel electrophoresis (DGGE) profiles show a similar patterns, significant difference was not observed in each other. DNA was isolated from soil cultivated GM and non-GM trigonal cactus, we analyzed the persistence of the inserted gene by PCR. Amplification of the inserted genes was not observed in the soil DNA, which was collected after harvest. CONCLUSION(S): This result suggests that the GM trigonal cactus cultivation does not change significantly the microbial community.

We observed that oil began to accumulate at 25 seed days after flowering (DAF) and reached the maximum potential at 35 seed DAF of oilseed rape, and the greatest weight of 100 seeds was obtained at 35 seed DAF. To survey a broad analysis of gene expression in developing embryos of Brassica napus, the Bn 300k microarray have been constructed. The Bn 300k Microarrary was designed from 80,696 unigenes clustered from 543,448 ESTs and 780 cDNA at NCBI. These arrays have been hybridized in a series of experiments with probes derived from seeds and leaf of B. napus. Approximately 8.5% of the 7,000 genes were expressed as ratios 2-fold higher in seed (25 DAF) than leaves and 0.4% at ratios 10. Also we observed that storage and cell differentiation- related genes were highly expressed at 10 DAF, whereas energy- related genes including fatty acid metabolism were increased up depending on seed maturation using Microarray, which was confirmed by reverse transcriptase polymerase chain reaction. These results suggest that B. napus arrays provide a very useful data set of seed-specific expression that can be further analyzed by examination of the promoter regions of these genes and help our understanding of the complex regulatory network in developing seeds.

BACKGROUND: The carotenoid-biofortified (PAC) rice was generated by inserting phytoene synthase (Psy) and carotene desaturase (Crtl) genes isolated from Capsicum annuum cv. Nockwang and Pantoea ananatis into the genome of a conventional variety of rice (Nakdongbyeo). In our present study, we studied the effects on insecticide susceptibility of Rice Water Weevil (Lissorhoptrus oryzophilus). METHODS AND RESULTS: The L. oryzophilus were fed on carotenoid-biofortified (PAC) rice and its near non-genetically modified (GM) counterparts (Nakdongbyeo) under , humidity of , and photoperiod 16L:8D for more than 60 days. Ten adults were soaked in the Clothianidin SC solution for 5 second in different concentrations, and were detected the mortalities after 24, 48 and 72 hours respectively. Every experiment was conducted with three replications. The cumulative mortalities of L. oryzophilus exposed to Clothianidin SC were similar between two types of feed administration. CONCLUSION: The results of this study suggested that carotenoid-biofortified rice might not affect the insecticide susceptibilities of Lissorhoptrus oryzophilus.

BACKGROUND: Genetically modified (GM) crops must receive relevant regulator’s authorization before they can be sold as seed or used food, feed and processing. Before approving any GM crop, the relevant government ministries are required to examine environmental risk assessment to make scientifically sound and socially acceptable decisions. But one of the least studied and understood areas in the environmental risk assessment of GM crops are their impact on soil microbial community. METHODS AND RESULTS: Recently, advanced methods have been developed to characterize the soil microbial community in various environments. In this study, the culture-dependent and culture -independent technical appr-oaches for profiling soil microbial communities are summa-rized and their applicability to assess GM crops are discussed. CONCLUSION(S): We concluded that the effect of GM crops on soil microbial community need to be assessed on a case by case basis. The combination of culture-dependent and culture-independent method was necessary for reliable and detailed assessment of effect of GM crops on soil microbial community.

BACKGROUND: Cultivation of genetically modified(GM) crops rapidly has increased in the global agricultural area. Among those, herbicide resistant GM crops are reported to have occupied 89.3 million hectares in 2010. However, cultivation of GM crops in the field evoked the concern of the possibility of gene transfer from transgenic plant into soil microorganisms. In our present study, we have assessed the effects of herbicide-resistant GM Chinese cabbage on the surrounding soil microbial community. METHODS AND RESULTS: The effects of a herbicideresistant genetically modified (GM) Chinese cabbage on the soil microbial community in its field of growth were assessed using a conventional culture technique and also culture-independent molecular methods. Three replicate field plots were planted with a single GM and four non-GM Chinese cabbages (these included a non-GM counterpart). The soils around these plants were compared using colony counting, denaturing gradient gel electrophoresis and a species diversity index assessment during the growing periods. The bacterial, fungal and actinomycetes population densities of the GM Chinese cabbage soils were found to be within the range of those of the non-GM Chinese cabbage soils. The DGGE banding patterns of the GM and non-GM soils were also similar, suggesting that the bacterial community structures were stable within a given month and were unaffected by the presence of a GM plant. The similarities of the bacterial species diversity indices were consistent with this finding. CONCLUSION: These results indicate that soil microbial communities are unaffected by the cultivation of herbicide-resistant GM Chinese cabbage within the experimental time frame.

BACKGROUND: Soybean [Glycine max (L.) Merrill] is a legume and an important oil crop worldwide. This study was conducted to evaluate the possible impact of transgenic soybean cultivation on the soil microbial community. METHODS AND RESULTS: Microorganisms were isolated from the rhizosphere soils . Microbial community was identified based on the culture-dependent and molecular biology methods. The total numbers of bacteria, fungi, and actinomycete in the rhizosphere soils cultivated with transgenic and non-transgenic soybeans were similar to each other, and there was no significant difference between transgenic and non-transgenic soybeans. Dominant bacterial phyla in the rhizosphere soils cultivated with transgenic or non-transgenic soybeans were Actinobacteria, Firmicutes, and Proteobacteria. The microbial communities in transgenic and non-transgenic soybean soils were characterized using the denaturing gradient gel electrophoresis (DGGE). The DGGE profiles showed the different patterns, but didn’t show significant difference to each other at 0.05 significance level. DNAs were isolated from soils cultivating transgenic or non-transgenic soybeans and analyzed for persistence of transgenes in the soil by using PCR. PCR analysis revealed that there were no amplified γ-tmt and bar gene in soil DNA. CONCLUSION(S): The results of this study suggested that microbial community of soybean field were not significantly affected by cultivation of the transgenic soybeans.

Insect-resistant transgenic rice was developed by inserting the mCry1Ac1 a modified gene fromthe soil bacterium Bacillus thuringiensis (Bt). For biosafety assessment, we studied the effects onsurvival of cantor Daphnia magna, a commonly used as a model organism in ecotoxicologicalstudies. D. magna fed on Bt rice and its near non-genetically modified (GM) counterparts(Nakdong) grown in the same environment (100% ground rice suspension). The Bt rice wascomfirmed to have the insertion of T-DNA and protein expression by the polymerase chainreaction and ELISA analysis. Feeding study showed similar cumulative immobility and abnormalresponse of D. magna between Bt rice and non-GM counterparts. 48 h-EC

The purposes of this study were to analyze the weediness potential associated with 'Vitamin A enforced rice' and to evaluate the relationships between growth characteristics and environmental adaption related to survival ability in the natural environment. 'Vitamin A enforced rice', 'Nagdong', donor variety of 'Vitamin A enforced rice' and three other varieties were used for this study. The 'Vitamin A enforced rice' was experimented according to ratio of ripened grain, shattering of grain, germination traits, germination ratio of low temperature, emergence ratio after wintering, ratio of viviparity, and growth ratio after ratooning ability. 'Vitamin A enforced rice' was not significant between 'Nagdong' and other varieties in seven characters. Germination ratio was similar of Vitamin A enforced rice' and 'Nagdong', but average germination ability of was different at low temperature. Also, shattering of grain and germination ratio of alternating temperature were significant between 'Nagdong' and other varieties. These results suggested that the relationships between growth characteristics and environmental adaption in 'Vitamin A enforced rice' could be applied to the stability of weediness p otential and biosafety guide in GM rice.

Tension wood is a specialized tissue that develops in the upper side of leaning stem and branches in angiosperm. In yellow poplar, tension wood does not form a G-layer, which is one of the most characteristic features of typical tension wood. In order to determine whether the chemical modification associated with tension wood formation in yellow poplar is consistent with those of G-layer-forming angiosperm species, tension wood was induced via mechanical bending treatment for 7 and 14 days in the stem of 2-year-old yellow poplars, and its major cell wall components were analyzed. Whereas the cellulose contents of the tension wood were higher than those in opposite wood, its lignin contents were lower; the result is consistent with that seen in other angiosperm tension wood. Interestingly, the lignin contents differed between tension wood and opposite wood sides at 7 days in the bent sample, but became similar levels to each other at later stages of tension wood formation. The S/G ratio of tension wood was...

Delta 6-fatty acid desaturase (D6DES) is used in the synthesis of polyunsaturated fatty acids (PUFAs) from microorganisms to higher animals, including arachidonic acid (ARA) and eicosapentaenoic acid (EPA). A 1,338 bp full-length cDNA encoding D6DES was cloned from Acanthopagrus schlegeli (AsD6DES) through degenerate- and RACE-PCR methods. A recombinant vector expressing AsD6DES (pYES-AsD6DES) was subsequently constructed and transformed into Saccharomyces cerevisiae to test the enzymatic activity of AsD6DES towards the production of n-6 and n-3 fatty acids. The exogenously expressed AsD6DES produced γ-linolenic acid (18:3 n-6) and stearidonic acid (18:4n-3) at 26 and 36% from exogenous linoleic acid (18:2n-6) and α-linolenic acid (18:3n-3), respectively, indicating that it is essentially a delta 6-fatty acid desaturase.

식물 종자오일의 지방산은 인간에 필수 지방산을 공급하는 식용 및 생필품 생산에 필요한 다양한 산업원료로 사용된다. 식물 오일의 식용 및 산업용 적합성과 경제성을 극대화하기 위해 유전공학에 의한 종자오일의 양과 지방산 조성 변형을 위한 대사조절연구가 계속 진행되고 있다. 하지만 식물에 일반적으로 존재하지 않는, 산업적으로 유용한 특이지방산의 합성과 종자오일로의 축적은 한계가 있음이 알려져 있다. 그 이유는 재배가 용이하며 생산성이 높은 오일식물의 acyltransferase가 특이지방산에 대한 기질특이성이 떨어지며 또한 특이지방산에 대한 세포막지질에서 종자오일로 전환시키는 편집기작 (editing mechanism)이 없기 때문으로 사료된다. 최근에 모델식물의 종자오일의 축적에 관여하는 acyltransferase에 관한 유전자들이 클로닝되었고, 특이지방산이 합성되는 인지질에서의 편집기작에 관여하는 acyltransferase 유전자들이 밝혀져 이들 유전자들의 정보를 이용하여 특이지방산을 효과적으로 생산.증진할 수 있는 기술이 개발될 수 있을 것으로 기대한다. 피마자오일의 주성분인 산업용 특이지방산인 리시놀레인 지방산을 오일식물에서 생산하기 위해 이에 관여할 것으로 추정되는 11개의 acyltransferase 유전자를 피마자 유전체 데이터베이스에서 존재함을 확인하였다. 이들 유전자들의 도입에 의해 형질전환 식물이 갖고 있지 않은 리시놀레인산에 대한 기질 특이성을 부여하여 종자오일 내의 특이지방산의 생산을 증가시킬 것으로 기대된다. 【Fatty acids in seed oil from plants are essential for human nutrients and have been used for industrial purpose. The growing demands of seed oil as food resources and feedstocks for industrial uses have attempted to modify fatty acid composition and to increase oil content in transgenic plants. However, production of unusual fatty acids in transgenic plants are limited, which is not synthesized the level same as original plants. This bottleneck was common for production of several unusual fatty acids in transgenic plants and suggests that there is different for substrate preference in oil metabolic pathway enzymes between host oil plants and original wild plants. Review of acyltransferases involved in acyl-editing and seed oil accumulation of oil plant and wild-plant producing unusual fatty acids will design strategies to maximize the production of unusual fatty acids in transgenic plants. In here, we identified eleven acyltransferase genes in castor based on sequence homology, which will be useful to increase hydroxy unusual fatty acids in transgenic plants.】

Medium-chain fatty acids (MCFA) are composed of 8-12 carbon atoms, and are found in coconut, cuphea, and palm kernel oil. MCFA were introduced into clinical nutrition in the 1950s for dietary treatment of malabsorption syndromes because of their rapid absorption and solubility. Recently, MCFA have been applied to Gastrointestinal Permeation Enhancement Technology (GIPET), which is one of the most important parts in drug delivery system in therapeutics. Therefore, to accumulate the MCFA in seed oil of rapeseed, much effort has been conducted by classical or molecular breeding. Laurate can be successfully accumulated up to 60 mol% in the seed oil of rapeseed by the expression of bay thioesterase (Uc FatB1) alone or crossed with a line over-expressing the coconut lysophosphatidic acid acyltransferase (LPAAT) under the control of a napin seed-storage protein promoter. Also, caprylate and caprate were obtained 7 mol% and 29 mol%, respectively, from plants over-expressing of the medium-chain specific thioesterase (Ch FatB2) alone or together with the chain-length-specific condensing enzyme (Ch KASIV). Despite the success of some research in utilizing parallel classical and molecular breeding to produce MCFA, commercially available seed oils have for the most part, not been realized. Recent research in the field of developing MCFA-enriched transgenic plants has established that there is no single rate-limiting step in the production of the target fatty acids. The purpose of this article is to review some of the recent progress in understanding the mechanism and regulation of MCFA production in seed oil of rapeseed.

【Polyunsaturated fatty acids (PUFA), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have significantly beneficial effects on health in relation to cardiovascular, immune, and inflammatory conditions and they are involved in determining the biophysical properties of membranes as well as being precursors for signaling molecules. PUFA biosynthesis is catalyzed by sequential desaturation and fatty acyl elongation reactions. This aerobic biosynthetic pathway was thought to be taxonomically conserved, but an alternative anaerobic pathway for the biosynthesis of PUFA is now known to contain analogous polyketide synthases (PKS). Certain fish oil can be a rich source of PUFA although processed marine oil is generally undesirable as food ingredients because of the associated objectionable flavors that are difficult and cost-prohibitive to remove. Oil-seed plants contain only the 18-carbon polyunsaturated fatty acid alpha-linolenic acid, which is not converted in the human body to EPA and DHA. It is now possible to engineer common oilseeds which can produce EPA and DHA and this has been the focus of a number of academic and industrial research groups. Recent advances and future prospects in the production of EPA and DHA in oilseed crops are discussed here.】

The proteins that are responsive to toxic volatile organic compounds (VOC) such as formaldehyde and toluene were analyzed with proteome analysis using two-dimensional difference image gel electrophoresis (DIGE) technology. Twenty-one days after germination (DAG) seedlings of Arabidopsis thaliana were exposed either to the gaseous formaldehyde or toluene in an airtight box installed in a plant growth chamber maintained at 24 degrees C under the long day condition with relatively low light condition. Comparative expression analysis revealed 14 and 22 protein spots as proteins displaying at least 1.5-fold differences in expression upon formaldehyde and toluene treatment, respectively, compared to those of untreated control. Most of the isolated spots were successfully identified by peptide analysis using LC-MS-MS. The VOC-responsive proteins contain ATP synthase CF1, ribulose-1,5-bisphosphate carboxylase/oxygenase, photosystem II light harvesting complex, and enolase, which are components of photosynthesis and carbohydrate metabolism. Despite the relatively low light intensity was applied, many identified VOC-induced proteins were previously known to be up-regulated upon high light stimulus. In addition, proteins involved in the toxin catabolic process and stress hormone-related proteins were identified as toluene-induced proteins. Although the exact function of most of the VOC-responsive proteins identified in these experiments had not been characterized, the protein expression analysis using DIGE was clearly demonstrated that plants are capable of responding actively to VOCs at translational level, and identified proteins may provide valuable tools to account for the effects of abiotic stress caused by air pollutants such as VOCs in plant.

선발마커로 두 종류의 항생제 (hpt와 nptII)와 제초제(bar) 유전자를 사용하여 아그로박테리움을 이용한 수정된 들깨 형질전환방법을 개발하였다. 들깨 배축 절편을 pMOG6-Bar 운반체 혹은 pCK-Bar 운반체를 가진 아그로박테리움 EHA 105와 각각 3일간 공동배양 하였다. 1차로 형성된 신초는 하이그로마이신(15mg/L)이나 카나마이신(125 mg/L)을 사용하여 선발하였고 재분화 된 신초들은 확실한 형질전환체를 얻기 위해서 포스피노트리신(1.2 mg/L)에서 한번 더 선발하였다. 뿌리는 호르몬이 없는 MS배지에서 재분화 신초로 부터 유도하였으며 80개의 재분화개체를 획득하였다. 들깨 지놈으로 형질전환유전자의 삽입은 서던 블럿으로 확인하였고 유전자의 발현은 노던 블럿으로 분석하였다. To 식물체로부터 유도된 T1들깨 종자들은 후대로의 안정된 유전자 전이를 확인하기 위하여 0.3% 바스타 제초제 살포로 확인하였다. 【A modified method of Agrobacterium-mediated perilla transformation was developed using two selection markers of an antibiotics (either hpt or nptll) and an herbicidal (bar) gene. Perilla hypocotyl explants were cocultured with Agrobacterium tumefaciens EHA 105 strain harboring plasmid vector (either pMOG6-Bar or pCK-Bar) for three days, respectively. Primary shoots were selected with antibiotics of hygromycin (15 mg/L) or kanamycin (125 mg/L) and regenerated shoots were further selected with herbicide phosphinothricin (ppt,1.2 mg/L) to obtain authentic transformants. Roots were induced for the regenerated shoots on the MS medium without hormone and 80 putative transgenic plants were obtained. Transgene integration into perilla genome was confirmed by Southern blot and their expression was analyzed by Northern blot. T1 perilla seeds drived from To plants were tested 0.3% basta spray for identification of stable gene delivery to next generation.】

The MYB transcription factors play important roles in the regulation of many secondary metabolites at the transcriptional level. We evaluated the possible roles of the Arabidopsis R2R3-MYB transcription factors in flavonoid biosynthesis because they are induced by UV-B irradiation but their associated phenotypes are largely unexplored. We isolated their genes by RACE-PCR, and performed transgenic approach and metabolite analyses in lettuce (Lactuca sativa). We found that one member of this protein family, AtMYB60, inhibits anthocyanin biosynthesis in the lettuce plant. Wild-type lettuce normally accumulates anthocyanin, predominantly cyanidin and traces of delphinidin, and develops a red pigmentation. However, the production and accumulation of anthocyanin pigments in AtMYB60-overexpressing lettuce was inhibited. Using RT-PCR analysis, we also identified the complete absence or reduction of dihydroflavonol 4-reductase (DFR) transcripts in AtMYB60- overexpressing lettuce (AtMYB60-117 and AtMYB60-112 lines). The correlation between the overexpression of AtMYB60 and the inhibition of anthocyanin accumulation suggests that the transcription factorAtMYB60 controls anthocyanin biosynthesis in the lettuce leaf. Clarification of the roles of the AtMYB60 transcription factor will facilitate further studies and provide genetic tools to better understand the regulation in plants of the genes controlled by the MYB-type transcription factors. Furthermore, the characterization of AtMYB60 has implications for the development of new varieties of lettuce and other commercially important plants with metabolic engineering approaches.

In order to select acid (GLA)-producing fungi, a total of forty-four strains of 4 genera such as Phytophthora, Pythium, Mucor and Rhizopus were obtained from Koran Agricultural Culture Collection (KACC) and then analysed by using GC-FID and GC-MS. GLA was detected on 39 fungal strains, and the highest rate of GLA was found as 24.8% of total fatty acids on Mucor hiemalis f. sp. hiemalis KACC 40264. Total GLA content of Zygomycota was comparatively high - Mucor (14.2%) and Rhizopus (14.3%), whereas that of Oomycetes was low - Phytophthora (3.3%) and Pythium (3.0%). Moreover, total fatty acids of the Zygomycota fungi such as Mucor (15.4 mg/100 ml) and Rhizopus (7.1 mg/100 ml) were higher compared with the Oomycetes such as Phytophthora (2.6 mg/100 ml) and Pythium (4.5 mg/100 ml). Thus, two genera such as Mucor and Rhizopus have higher potential as an useful microbial resource. The total fatty acid content varies even within the strains of the same genus e.g. Mucor. M. blumbeus KACC 40935 showed the highest values on productivity (18.2%) of GLA and total fatty acid contents (50.8 mg/100 ml liquid medium).

Quercetin is a potent free radical–scavenging antioxidant exerting chemopreventive activity by reducing oxidative stresses. However, oxidized quercetin behaves as a prooxidant, which can lead to paradoxical effects. This study was conducted to investigate the effects of quercetin supplementation on 1,2-dimethylhydrazine (DMH)–induced oxidative DNA damage and precancerous lesion formation in the colon. Male Sprague-Dawley rats were randomized into 5 groups. The rats in groups 3, 4, and 5 were treated with DMH (30 mg/kg body weight IP), twice on weeks 2 and 3. The experimental diets were as follows: control diet (group 1), 2% quercetin diet (group 2), control diet (group 3), 0.2% quercetin diet (group 4), and 2% quercetin diet (group 5). The DMH-induced oxidative tissue damage was examined 24 hours after the first DMH injection, and the aberrant crypt foci formation was measured at week 12. The results show the following: (1) that DMH significantly increased the level of 8-hydroxyguanine (8-OH-Gua) in the liver, where the metabolic conversion of DMH occurs, but did not significantly change the level of 8-OH-Gua in the colon; (2) that both the 0.2% and 2% quercetin supplementation suppressed the extent of 8-OH-Gua formation in the liver; (3) that dietary quercetin did not affect the formation of aberrant crypt foci; and (4) that 2% quercetin supplementation only slightly increased the liver oxidative damage without statistical significance. These results indicate that quercetin may not be an effective anticarcinogen against DMH-induced colon cancer, where oxidative tissue damage is not a primary event in the formation of a precancerous region. The context of quercetin as a hepatoprotective agent, however, must be emphasized.

Reconstitution of nonnative, very-long-chain polyunsaturated fatty acid (VLC-PUFA) biosynthetic pathways inArabidopsis thalianawas undertaken. The introduction of three primary biosynthetic activities to cells requires the stable coexpression of multiple proteins within the same cell. Herein, we report that C22VLC-PUFAs were synthesized from C18precursors by reactions catalyzed byΔ6-desaturase, an ELOVL5-like enzyme involved in VLC-PUFA elongation, andΔ5-desaturase. Coexpression of the corresponding genes (McD6DES, AsELOVL5, andPtD5DES) under the control of the seed-specific vicilin promoter resulted in production of docosapentaenoic acid (22:5 n-3) and docosatetraenoic acid (22:4 n-6) as well as eicosapentaenoic acid (20:5 n-3) and arachidonic acid (20:4 n-6) inArabidopsisseeds. The contributions of the transgenic enzymes and endogenous fatty acid metabolism were determined. Specifically, the reasonable synthesis of omega-3 stearidonic acid (18:4 n-3) could be a useful tool to obtain a sustainable system for the production of omega-3 fatty acids in seeds of a transgenic T3 line 63-1. The results indicated that coexpression of the three proteins was stable. Therefore, this study suggests that metabolic engineering of oilseed crops to produce VLC-PUFAs is feasible.

Carrot seeds normally have surface spines. The availability of a spineless mutant would be agronomically beneficial, eliminating the current efforts to remove those spines pre-sowing. Furthermore, the identification of spine-specific genes would provide insights into spine development in wild-type carrot seed. This effort could be facilitated through the use of an annealing control primer (ACP) system. Here, we employed a new and accurate reverse transcriptionpolymerase chain reaction (RT-PCR) that involves ACPs for identifying genes of interest. With these techniques, 11 expressed sequence tags (ESTs) were obtained for cloning and sequencing the genes that are differentially expressed in wild-type spiny seeds, but not in the spineless mutant. In all, 7 cDNAs exhibited significant sequence similarity with known genes from other species. These included cell wall-associated hydrolase, tail fiber assembly protein, transcriptional regulatory protein, berberine bridge enzyme, S-adenosyl methionine synthase, transketolase, and phenylalanyl t-RNA synthetase beta chain. Four other cDNA sequences had no significant identities with known genes. As revealed by RT-PCR, these genes regulate spine formation during the developmental stage. Our results suggest that PCR-based differential display RT-PCR techniques are a very useful tool for identifying spine-specific genes from carrot seeds.

Camellia sinensis (tea) is a commercially important crop that is valued for its secondary metabolites. Higher levels of catechins are accumulated in young leaves than in mature leaves. To understand the molecular regulation of secondary metabolism in tea leaves, differentially expressed genes of interest should be identified, cloned, and studied in detail. A total of 588 cDNA clones from a subtractive cDNA library were randomly picked, sequenced, and analyzed. High-quality sequences were isolated for 508 of these clones. BlastX comparisons indicated that about 8.7% of the clones encoded enzymes involved in secondary metabolism, with a particularly high abundance of flavonoid-metabolism proteins (5.1%). These ESTs facilitated the isolation and characterization of genes involved in the flavonoid pathway in tea: chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), flavonoid 3 � 5 � -hydroxylase (F3 � 5 � H), flavonol synthase (FLS), dihydroflavonol 4-reductase (DFR), and leucoanthocyanidin reductase (LCR). The three genes encoding F3H, DFR, and LCR were more highly expressed in young leaves than in mature leaves. These results indicate that flavonoid biosynthesis genes from tea are differentially regulated in the developmental stages. The subtractive cDNA library and EST database described in this study represent a valuable resource for future research aimed at improving economically important secondary metabolic characteristics in crops. © 2003 Elsevier Ireland Ltd. All rights reserved.

Carotenoids are synthesized from the plastidic glyceraldehyde-3-phosphate (GAP)/pyruvate pathway in isoprenoids biosynthetic system of plants. They play a crucial role in light harvesting, work as photoprotective agents in photosynthesis of nature, and are also responsible for the red, orange and yellow colors of fruits and flowers in plants. In addition to biological actions of carotenoids as antioxidants and natural pigments, they are essential components of human diet as a source of vitamin A. It has been also suggested that some kinds of carotenoids might provide protection against cancer and heart disease as human medicines. In this article, we review the commercial applications on the basis of biological functions of carotenoids, summarize the studies of genes involved in the carotenoid biosynthetic pathway, and introduce recent results achieved in metabolic engineering of carotenoids. This effort for understanding the carotenoids metabolism will make us to increase the total carotenoid contents of crop plants, direct the carotenoid biosynthetic machinery towards other useful carotenoids, and produce a new array of carotenoids by further metabolizing the new precursors that are created when one or two key enzymes in carotenoid biosynthetic pathway are exchanged through gene manipulation in the near future.

Flavonoid biosynthesis is one of the most extensively studied areas in the secondary metabolism. Due to the study of flavonoid metabolism in diverse plant system, the pathways become the best characterized secondary metabolites and can be excellent targets for metabolic engineering. These flavonoid-derived secondary metabolites have been considerably divergent functional roles: floral pigment, anticancer, antiviral, antitoxin, and hepatoprotective. Three species have been significant for elucidating the flavonoid metabolism and isolating the genes controlling the flavonoid genes: maize (Zea mays), snapdragon (Antirrhinum majus) and petunia (Prtunia hybrida). Recently, many genes involved in biosynthesis of flavonoid have been isolated and characterized using mutation and recombinant DNA technologies including transposon tagging and T-DNA tagging which are novel approaches for the discovery of uncharacterized genes. Metabolic engineering of flavonoid biosynthesis was approached by sense or antisense manipulation of the genes related with flavonoid pathway, or by modified expression of regulatory genes. So, the use of a variety of experimental tools and metabolic engineering facilitated the characterization of the flavonoid metabolism. Here we review recent progresses in flavonoid metabolism: confirmation of genes, metabolic engineering, and applications in the industrial use.

Background The perturbation of the steady state of reactive oxygen species (ROS) due to biotic and abiotic stresses in a plant could lead to protein denaturation through the modification of amino acid residues, including the oxidation of methionine residues. Methionine sulfoxide reductases (MSRs) catalyze the reduction of methionine sulfoxide back to the methionine residue. To assess the role of this enzyme, we generated transgenic rice using a pepper CaMSRB2 gene under the control of the rice Rab21 (responsive to ABA protein 21) promoter with/without a selection marker, the bar gene. Results A drought resistance test on transgenic plants showed that CaMSRB2 confers drought tolerance to rice, as evidenced by less oxidative stress symptoms and a strengthened PSII quantum yield under stress conditions, and increased survival rate and chlorophyll index after the re-watering. The results from immunoblotting using a methionine sulfoxide antibody and nano-LC-MS/MS spectrometry suggest that porphobilinogen deaminase (PBGD), which is involved in chlorophyll synthesis, is a putative target of CaMSRB2. The oxidized methionine content of PBGD expressed in E. coli increased in the presence of H2O2, and the Met-95 and Met-227 residues of PBGD were reduced by CaMSRB2 in the presence of dithiothreitol (DTT). An expression profiling analysis of the overexpression lines also suggested that photosystems are less severely affected by drought stress. Conclusions Our results indicate that CaMSRB2 might play an important functional role in chloroplasts for conferring drought stress tolerance in rice.

Fatty acid delta 6-desaturase (D6DES) and elongases are key enzymes in the synthesis of polyunsaturated fatty acids (PUFAs) including arachidonic acid (ARA) and eicosapentaenoic acid (EPA) from microorganisms to higher animals. To identify the genes encoding D6DES and elongases for PUFAs, we isolated each cDNA with a high similarity to the D6DES and ELOVL5-like elongases of mammals and fishes via degenerate PCR and RACE-PCR from Acanthopagrus schlegelii. A recombinant vector expressing AsD6DES was subsequently constructed and transformed into Saccharomyces cerevisiae to test the enzymatic activity toward n-6 and n-3 fatty acids in the PUFA biosynthesis. The heterologously expressed AsD6DES produced γ-linolenic acid (GLA, C18:3 n-6) and stearidonic acid (STA, C18:4 n-3) at conversion rates of 26.3–35.6 % from exogenous linoleic acid (LA, C18:2 n-6) and α-linolenic acid (ALA, C18:3 n-3) substrates, respectively. When AsELOVL5 was expressed in yeast, it conferred an ability to elongate GLA to di-homo-γ-linolenic acid (DGLA, C20:3 n-6). In addition, AsELOVL5 showed an ability to convert ARA (C20:4 n-6) and EPA (C20:5 n-3) to dodecylthioacetic acid (DTA, C22:4 n-6) and docosapentaenoic acid (DPA, C22:5 n-3), respectively. In these results, the AsD6DES encodes a delta 6-fatty acid desaturase and the AsELOVL5 encoding a long-chain fatty acid elongase shows activity to enlongate C18Δ6/C20Δ5, but not C22.

To identify the genes encoding fatty acid elongases for the biosynthesis of polyunsaturated fatty acids (PUFAs), we isolated a cDNA via degenerate PCR and RACE-PCR from Acanthopagrus schlegelii with a high similarity to the ELOVL5-like elongases of mammals and fishes. This gene is termed AsELOVL5 and encodes a 294 amino acid protein. When AsELOVL5 was expressed in Saccharomyces cerevisiae, it conferred an ability to elongate γ-linolenic acid (18:3 n-6) to di-homo-γ-linolenic acid (20:3 n-6). In addition, the transformed cells converted arachidonic acid (20:4 n-6) and eicosapentaenpic acid (20:5 n-3) to docosatetraenoic acid (22:4 n-6) and docosapentaenoic acid (22:5 n-3), respectively. These results indicate that the AsELOVL5 gene encodes a long-chain fatty acid elongase capable of elongating C(18)Δ6/C(20)Δ5 but not C(22) PUFA substrates.

Improvements in plant productivity (biomass) and yield have centered on increasing the efficiency of leaf CO(2) fixation and utilization of products by non-photosynthetic sink organs. We had previously demonstrated a correlation between photosynthetic capacity, plant growth, and the extent of leaf starch synthesis utilizing starch-deficient mutants. This finding suggested that leaf starch is used as a transient photosynthetic sink to recycle inorganic phosphate and, in turn, maximize photosynthesis. To test this hypothesis, Arabidopsis thaliana and rice (Oryza sativa L.) lines were generated with enhanced capacity to make leaf starch with minimal impact on carbon partitioning to sucrose. The Arabidopsis engineered plants exhibited enhanced photosynthetic capacity; this translated into increased growth and biomass. These enhanced phenotypes were displayed by similarly engineered rice lines. Manipulation of leaf starch is a viable alternative strategy to increase photosynthesis and, in turn, the growth and yields of crop and bioenergy plants.

Flavonols, a class of bioactive polyphenols present in plants, are the products of flavonol desaturation catalyzed by flavonol synthase (FLS). We cloned the cDNA coding for the enzyme FLS from Camellia sinensis (CsFLS) by end-to-end PCR followed by 5'- and 3'-RACE. The putative CsFLS had 333 amino acid residues, displayed identities to the FLSs of Arabidopsis and Ginkgo of 53% and 52.5%, respectively, and contained several conserved elements found in the 2-oxoglutarate-Fe(II)-dioxygenase superfamily. The cDNA of CsFLS was subcloned into pET28a(+) and introduced into Escherichia coli (BL21-CodonPlus-RIL). Induction with 0.1mM IPTG at low temperature (20 degrees C) led to higher amounts of CsFLS in the soluble fraction than induction at 30 degrees C. The enzyme aggregated into inclusion bodies could be rescued by denaturation with 6M urea and purification with a His. Bind purification kit. The purified protein was desalted by Amicon Ultra-15 centrifugal filter unit, and the His-tag was removed with thrombin. The finally purified protein was assayed with dihydroquercetin as substrate and the products were analyzed by HPLC. The addition of FeSO(4) to the buffers used in the CsFLS purification significantly increased the recovery of active enzyme. The CsFLS obtained in this study was found to have higher specific activity and lower K(m) than previously reported FLSs.

Polyphenols have been reported to possess anti-inflammatory activity, thereby act as chemopreventive agents. The objective of this study was to evaluate quercetin and keampferol as anti-inflammator...

Molecular analysis of gene expression differences between green and red lettuce leaves was performed using the SSH method. BlastX comparisons of subtractive expressed sequence tags (ESTs) indicated that 7.6% of clones encoded enzymes involved in secondary metabolism. Such clones had a particularly high abundance of flavonoid-metabolism proteins (6.5%). Following SSH, 566 clones were rescreened for differential gene expression using dot-blot hybridization. Of these, 53 were found to overexpressed during red coloration. The up-regulated expression of six genes was confirmed by Northern blot analyses. The expression of chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), and dihydroflavonol 4-reductase (DFR) genes showed a positive correlation with anthocyanin accumulation in UV-B-irradiated lettuce leaves; flavonoid 3′,5′-hydroxylase (F3′,5′H) and anthocyanidin synthase (ANS) were expressed continuously in both samples. These results indicated that the genes CHS, F3H, and DFR coincided with increases in anthocyanin accumulation during the red coloration of lettuce leaves. This study show a relationship between red coloration and the expression of up-regulated genes in lettuce. The subtractive cDNA library and EST database described in this study represent a valuable resource for further research for secondary metabolism in the vegetable crops.

Nodules are formed on legume roots as a result of signaling between symbiotic partners and in response to the activities of numerous genes. We cloned fragments of differentially expressed genes in spot-inoculated soybean (Glycine max) roots. Many of the induced clones were similar to known genes related to oxidative stress, such as thioredoxin and β-carotene hydroxylase. The deduced amino acid sequences of full-length soybean cDNAs for thioredoxin and β-carotene hydroxylase were similar to those in other species. In situ RNA hybridization revealed that the thioredoxin gene is expressed on the pericycle of 2-d-old nodules and in the infected cells of mature nodules, suggesting that thioredoxin is involved in nodule development. The thioredoxin promoter was found to contain a sequence resembling an antioxidant responsive element. When a thioredoxin mutant of yeast was transformed with the soybean thioredoxin gene it became hydrogen peroxide tolerant. These observations prompted us to measure reactive oxygen species levels. These were decreased by 3- to 5-fold in 7-d-old and 27-d-old nodules, coincident with increases in the expression of thioredoxin and β-carotene hydroxylase genes. Hydrogen peroxide-producing regions identified with cerium chloride were found in uninoculated roots and 2-d-old nodules, but not in 7-d-old and 27-d-old nodules. RNA interference-mediated repression of the thioredoxin gene severely impaired nodule development. These data indicate that antioxidants such as thioredoxin are essential to lower reactive oxygen species levels during nodule development.

We have characterized a novel type I ribosome-inactivating protein (CAP30) from the leaves of Chenopodium album. Purified native CAP30 depurinated the ribosomes of Chenopodium, tomato, and tobacco leaves in vitro. To further characterize this protein, cDNA clones were isolated from a leaf cDNA library using a DNA probe derived from the N-terminal amino acid sequence. Two full-length cDNA clones, CAP30A and CAP30B, were isolated. The two clones were highly homologous (91.4% identity over 280 amino acids) at the deduced amino acid level. Both contain a putative signal peptide of 25 amino acid and a conserved domain commonly found in ribosome-inactivating proteins. This suggests that CAP30 is a single-chain ribosome-inactivating protein. Expression of CAP30 mRNA peaked twice, at 12 and 72 h, after tobacco mosaic virus (TMV) infection or wounding. Transformed Escherichia coli cells expressing pre- or mature CAP had greatly reduced growth rates. These results suggest that CAP30 functions as a broad-spectrum defense-related protein with both antiviral and anti-microbial activity.

ADP-glucose pyrophosphorylase (AGPase) is a key regulatory enzyme of bacterial glycogen and plant starch synthesis as it controls carbon flux via its allosteric regulatory behavior. Unlike the bacterial enzyme that is composed of a single subunit type, the plant AGPase is a heterotetrameric enzyme (alpha2beta2) with distinct roles for each subunit type. The large subunit (LS) is involved mainly in allosteric regulation through its interaction with the catalytic small subunit (SS). The LS modulates the catalytic activity of the SS by increasing the allosteric regulatory response of the hetero-oligomeric enzyme. To identify regions of the LS involved in binding of effector molecules, a reverse genetics approach was employed. A potato (Solanum tuberosum L.) AGPase LS down-regulatory mutant (E38A) was subjected to random mutagenesis using error-prone polymerase chain reaction and screened for the capacity to form an enzyme capable of restoring glycogen production in glgC(-) Escherichia coli. Dominant mutations were identified by their capacity to restore glycogen production when the LS containing only the second site mutations was co-expressed with the wild-type SS. Sequence analysis showed that most of the mutations were decidedly nonrandom and were clustered at conserved N- and C-terminal regions. Kinetic analysis of the dominant mutant enzymes indicated that the K(m) values for cofactor and substrates were comparable with the wild-type AGPase, whereas the affinities for activator and inhibitor were altered appreciably. These AGPase variants displayed increased resistance to P(i) inhibition and/or greater sensitivity toward 3-phosphoglyceric acid activation. Further studies of Lys-197, Pro-261, and Lys-420, residues conserved in AGPase sequences, by site-directed mutagenesis suggested that the effectors 3-phosphoglyceric acid and P(i) interact at two closely located binding sites.

Background Foot-and-mouth disease virus (FMDV) is a small single-stranded RNA virus which belongs to the family Picornaviridae, genus Apthovirus. It is a principal cause of FMD which is highly contagious in livestock. In a wild type virus infection, infected animals usually elicit antibodies against structural and non-structural protein of FMDV. A structural protein, VP1, is involved in neutralization of virus particle, and has both B and T cell epitopes. A RNA-dependent RNA polymerase, 3D, is highly conserved among other serotypes and strongly immunogenic, therefore, we selected VP1 and 3D as vaccine targets. Methods VP1 and 3D genes were codon-optimized to enhance protein expression level and cloned into mammalian expression vector. To produce recombinant protein, VP1 and 3D genes were also cloned into pET vector. The VP1 and 3D DNA or proteins were co-immunized into 5 weeks old BALB/C mice. Results Antigen-specific serum antibody (Ab) responses were detected by Ab ELISA. Cellular immune response against VP1 and 3D was confirmed by ELISpot assay. Conclusion The results showed that all DNA- and protein-immunized groups induced cellular immune responses, suggesting that both DNA and recombinant protein vaccine administration efficiently induced Ag-specific humoral and cellular immune responses.