Kyung-Hwan Kim

Increased α-tocopherol content in soybean seed overexpressing the Perilla frutescens γ-tocopherol methyltransferase gene

Tocopherols, with antioxidant properties, are synthesized by photosynthetic organisms and play important roles in human and animal nutrition. In soybean, γ-tocopherol, the biosynthetic precursor to α-tocopherol, is the predominant form found in the seed, whereas α-tocopherol is the most bioactive component. This suggests that the final step of the α-tocopherol biosynthetic pathway catalyzed by γ-tocopherol methyltransferase (γ-TMT) is limiting in soybean seed. Soybean oil is the major edible vegetable oil consumed, so manipulating the tocopherol biosynthetic pathway in soybean seed to convert tocopherols into more active α-tocopherol form could have significant health benefits. In order to increase the soybean seed α-tocopherol content, the γ-TMT gene isolated from Perilla frutescens was overexpressed in soybean using a seed-specific promoter. One transgenic plant was recovered and the progeny was analyzed for two generations. Our results demonstrated that the seed-specific expression of the P. frutescens γ-TMT gene resulted in a 10.4-fold increase in the α-tocopherol content and a 14.9-fold increase in the β-tocopherol content in T2 seed. Given the relative contributions of different tocopherols to vitamin E activity, the activity in T2 seed was calculated to be 4.8-fold higher than in wild-type seed. In addition, the data obtained on lipid peroxidation indicates that α-tocopherol may have a role in preventing oxidative damage to lipid components during seed storage and seed germination. The increase in the α-tocopherol content in the soybean seed could have a potential to significantly increase the dietary intake of vitamin E.

Ectopic expression of AtPAD4 broadens resistance of soybean to soybean cyst and root-knot nematodes

BackgroundThe gene encoding PAD4 (PHYTOALEXIN-DEFICIENT4) is required in Arabidopsis for expression of several genes involved in the defense response to Pseudomonas syringae pv. maculicola. AtPAD4 (Arabidopsis thaliana PAD4) encodes a lipase-like protein that plays a regulatory role mediating salicylic acid signaling.ResultsWe expressed the gene encoding AtPAD4 in soybean roots of composite plants to test the ability of AtPAD4 to deter plant parasitic nematode development. The transformed roots were challenged with two different plant parasitic nematode genera represented by soybean cyst nematode (SCN; Heterodera glycines) and root-knot nematode (RKN; Meloidogyne incognita). Expression of AtPAD4 in soybean roots decreased the number of mature SCN females 35 days after inoculation by 68 percent. Similarly, soybean roots expressing AtPAD4 exhibited 77 percent fewer galls when challenged with RKN.ConclusionsOur experiments show that AtPAD4 can be used in an economically important crop, soybean, to provide a measure of resistance to two different genera of nematodes.

MiniMax, a new diminutive Glycine max genotype with a rapid life cycle, embryogenic potential and transformation capabilities

We developed Glycine max cv MiniMax (PI643148) that has a rapid life cycle, short stature and characteristic simple sequence repeat (SSR) markers that could make it useful for mutant screening, functional genomics, genetic mapping and other studies involving soybeans. We demonstrate that MiniMax is able to make somatic embryos (SEs) that rapidly develop into plantlets. Thus, the rapid cycling habit carries over into aspects of plant regeneration. Chimaeras (having transformed roots with untransformed aerial stocks) have been produced rapidly under non-axenic conditions using Agrobacterium rhizogenes-mediated transformation. Part of these experiments involved the engineering an enhanced green fluorescent protein (eGFP) reporter cassette outside the multi-cloning site of a plant expression vector, permitting non-invasive visual screening of the transformed roots. The rapid cycling growth habit of MiniMax, its ability to efficiently generate SEs and ability to be transformed should prove useful for basic aspects of G. max molecular and genetic research.

Expression of hemagglutinin-neuraminidase protein of Newcastle disease virus in transgenic tobacco

Newcastle disease is one of the most important pathogens of domestic poultry including chickens. The hemagglutinin-neuraminidase (HN) of Newcastle disease virus is the principal target of neutralizing and protective antibodies against Newcastle disease. In this paper, we transformed tobacco plants with Agrobacterium tumefaciens EHA105 to generate the plants expressing HN of Newcastle disease virus (NDV). The insertion and copy numbers of HN gene in the genomic DNA of phosphinothricin-resistant plants were confirmed by PCR and Southern blot, respectively. The presence of the HN-specific transcript in the total RNAs of the leaves of transgenic plants was verified by Northern analysis. The recombinant HN proteins were detected by Western blot analysis using a polyclonal antibody against GST–HN fusion proteins. The highest level of expression of HN in leaves of transgenic plants was approximately 0.069% of the total soluble protein. ELISA assay showed that the recombinant protein extracted from transformants has normal immunoactivity. Transgenic tobacco expressing HN of NDV with sterilized PBS was fed to 6-week-old chickens. Immunized chickens developed slightly high titers of anti-HN serum IgG compared with those of the wild type plant. These results suggest that oral immunization with HN-transgenic tobacco provides a potential means of protecting chickens from NDV. Further modified animal experiments would be needed to increase the immunity of HN by co-administration of classical adjuvants or other trials.

Seed specific expression of perilla γ-tocopherol methyltransferase gene increases α-tocopherol content in transgenic perilla (Perilla frutescens)

Increasing vitamin E activity in economically important oil crops such as perilla will enhance the nutritional value of these crops. Perilla (Perilla frutescens Britt) represents an important oil crop in Asian countries, including Korea. Using Agrobacterium-mediated transformation, we have engineered perilla with the γ-tocopherol methyltransferase (γ-TMT) gene under the control of seed-specific vicillin promoter. Molecular characterization including PCR, Southern and Northern blots confirmed that the γ-TMT transgene was successfully inherited to and expressed in the progeny plants. The γ -TMT transgene was specifically expressed in immature seeds of transgenic plants, leading to efficient conversion of γ-tocopherol to α-tocopherol and dramatic increase in seed α-tocopherol content, as detected by high performance liquid chromatography analysis. We also showed that such a high α-tocopherol content phenotype was transmitted to the progeny plants. In addition, there was no significant change in fatty acid composition in transgenic seeds as compared with untransformed control Yeupsil cultivar, suggesting the lack of interplay between the fatty acid and tocopherol biosynthesis pathways. This was the first report on over expression of the γ-TMT gene in transgenic perilla displaying desirable high α-tocopherol content phenotype. Since α-tocopherol has the highest vitamin E activity, the transgenic perilla with high α-tocopherol content in seeds developed in this study will benefit both human and animal health.

Post-transcriptional gene silencing of the gene encoding aldolase from soybean cyst nematode by transformed soybean roots

Plant parasitic nematodes cause approximately 157 billion US dollars in losses worldwide annually. The soybean cyst nematode (SCN), Heterodera glycines, is responsible for an estimated one billion dollars in losses to the US farmer each year. A promising new approach for control of plant parasitic nematode control is gene silencing. We tested this approach by silencing the SCN gene HgALD, encoding fructose-1,6-diphosphate aldolase. This enzyme is important in the conversion of glucose into energy and may be especially important in actin-based motility during parasite invasion of its host. An RNAi construct targeted to silence HgALD was transformed into soybean roots of composite plants to examine its efficacy to reduce the development of females formed by SCN. The number of mature females on roots transformed with the RNAi construct designed to silence the HgALD gene was reduced by 58%. These results indicate that silencing the aldolase gene of SCN +can greatly decrease the number of female SCN reaching maturity, and it is a promising step towards broadening resistance of plants against plant-parasitic nematodes.

Carotenoids Biosynthesis and Their Metabolic Engineering in Plants

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.

Screening of GLA (γ-Linolenic Acid) from Fungi by Gas Chromatography and Mass Spectroscopy

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).

Perilla transformation using selection markers containing antibiotics and basta

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.】

Effects of Transgenic Soybean Cultivation on Soil Microbial Community in the Rhizosphere

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.