Dr. Thavrak Huon from South Korea: 2004

December 20, 2004

Over-expression of the bacterial nhaA gene in rice enhances salt and drought tolerance

The Escherichia coli nhaA gene encodes a Na+/H+ antiporter, which plays a critical role in ion homeostasis. We transferred a bacterial nhaA gene into rice (Oryza sativa L. ssp. japonica) and detected high expression in the transgenic rice. The germination rate, growth, and average yield per plant of the transgenic lines were better than those of control lines under salt or drought stress. Moreover, the sodium and proline content of the transgenic lines under salt or drought stress was also higher than in control lines, implying that nhaA over-expression enhances osmoregulation by activating the biosynthesis of proline. Tolerance to both salt and drought was compared between transgenic rice over-expressing nhaA and that over-expressing Arabidopsis δ-OAT encoding ornithine-8-aminotransferase. The transgenic plants with nhaA grew better than those with δ-OAT at high salinity, while the opposite was true for drought stress.

Source: Plant science (2005) vol. 168, p. 297-302

September 18, 2004

Heat-tolerant basmati rice engineered by over-expression of hsp101

Rice is sensitive to high-temperature stress at almost all the stages of its growth and development. Considering the crucial role of heat shock protein 101 (Hsp 101) in imparting thermotolerance to cells, we introduced Arabidopsis thaliana hsp101 (Athsp101) cDNA into the Pusa basmati 1 cultivar of rice (Oryza sativa L.) by Agrobacterium-mediated transformation. Stable integration and expression of the transgene into the rice genome was demonstrated by Southern, northern and western blot analyses. There appeared no adverse effect of over-expression of the transgene on overall growth and development of transformants. The genetic analysis of tested T1 lines showed that the transgene segregated in a Mendelian fashion. We compared the survival of T2 transgenic lines after exposure to different levels of high-temperature stress with the untransformed control plants. The transgenic rice lines showed significantly better growth performance in the recovery phase following the stress. This thermotolerance advantage appeared to be solely due to over-expression of Hsp101 as neither the expression of low-molecular-weight heat shock proteins (HSPs) nor of other members of Clp family proteins was altered in the transgenic rice. The production of high temperature tolerant transgenic rice cultivars would provide a stability advantage under supra-optimal temperature regime thereby improving its overall performance.

Source: Plant Mol Biol. (2003) vol 51, p. 677-686

August 28, 2004

Transgenics of an elite indica rice variety Pusa Basmati 1 harbouring the codA gene are highly tolerant to salt stress

Transgenic lines of indica rice were generated by Agrobacterium-mediated transformation with the choline oxidase ( codA) gene from Arthrobacter globiformis. Choline oxidase catalyses conversion of choline to glycine betaine. Glycine betaine is known to provide tolerance against a variety of stresses. Molecular analyses of seven independent transgenic lines as performed by Southern, Northern and Western hybridization revealed integration and expression of the transgene as well as inheritance in the progeny plants. A good correlation was observed between levels of mRNA and protein accumulation, and a significant amount of choline oxidase product, i.e. glycine betaine, accumulated in R0 as well as R1 plants. Mendelian as well as non-Mendelian segregation patterns were obtained in the progeny plants. Challenge studies performed with R1 plants by exposure to salt stress (0.15 M NaCl) for 1 week, followed by a recovery period, revealed that in some cases more than 50% of the transgenic plants could survive salt stress and set seed whereas wild-type plants failed to recover.

Source: Theor Appl Genet. (2002) vol 106, p. 51-57

July 11, 2004

Highly efficient production and characterization of T-DNA plants for rice ( Oryza sativa L.) functional genomics

We investigated the potential of an improved Agrobacterium tumefaciens-mediated transformation procedure of japonica rice ( Oryza sativa L.) for generating large numbers of T-DNA plants that are required for functional analysis of this model genome. Using a T-DNA construct bearing the hygromycin resistance ( hpt), green fluorescent protein ( gfp) and beta-glucuronidase ( gusA) genes, each individually driven by a CaMV 35S promoter, we established a highly efficient seed-embryo callus transformation procedure that results both in a high frequency (75-95%) of co-cultured calli yielding resistant cell lines and the generation of multiple (10 to more than 20) resistant cell lines per co-cultured callus. Efficiencies ranged from four to ten independent transformants per co-cultivated callus in various japonica cultivars. We further analysed the T-DNA integration patterns within a population of more than 200 transgenic plants. In the three cultivars studied, 30-40% of the T(0) plants were found to have integrated a single T-DNA copy. Analyses of segregation for hygromycin resistance in T(1) progenies showed that 30-50% of the lines harbouring multiple T-DNA insertions exhibited hpt gene silencing, whereas only 10% of lines harbouring a single T-DNA insertion was prone to silencing. Most of the lines silenced for hpt also exhibited apparent silencing of the gus and gfp genes borne by the T-DNA. The genomic regions flanking the left border of T-DNA insertion points were recovered in 477 plants and sequenced. Adapter-ligation Polymerase chain reaction analysis proved to be an efficient and reliable method to identify these sequences. By homology search, 77 T-DNA insertion sites were localized on BAC/PAC rice Nipponbare sequences. The influence of the organization of T-DNA integration on subsequent identification of T-DNA insertion sites and gene expression detection systems is discussed.

Source: Theor Appl Genet. (2003) vol 106, p. 1396-1408

July 10, 2004

Highly efficient production and characterization of T-DNA plants for rice ( Oryza sativa L.) functional genomics

May 29, 2004

Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses

Trehalose is a nonreducing disaccharide of glucose that functions as a compatible solute in the stabilization of biological structures under abiotic stress in bacteria, fungi, and invertebrates. With the notable exception of the desiccation-tolerant “resurrection plants,” trehalose is not thought to accumulate to detectable levels in most plants. We report here the regulated overexpression of Escherichia coli trehalose biosynthetic genes (otsA and otsB) as a fusion gene for manipulating abiotic stress tolerance in rice. The fusion gene has the advantages of necessitating only a single transformation event and a higher net catalytic efficiency for trehalose formation. The expression of the transgene was under the control of either tissue-specific or stress-dependent promoters. Compared with nontransgenic rice, several independent transgenic lines exhibited sustained plant growth, less photo-oxidative damage, and more favorable mineral balance under salt, drought, and low-temperature stress conditions. Depending on growth conditions, the transgenic rice plants accumulate trehalose at levels 3–10 times that of the nontransgenic controls. The observation that peak trehalose levels remain well below 1 mg/g fresh weight indicates that the primary effect of trehalose is not as a compatible solute. Rather, increased trehalose accumulation correlates with higher soluble carbohydrate levels and an elevated capacity for photosynthesis under both stress and nonstress conditions, consistent with a suggested role in modulating sugar sensing and carbohydrate metabolism. These findings demonstrate the feasibility of engineering rice for increased tolerance of abiotic stress and enhanced productivity through tissue-specific or stress-dependent overproduction of trehalose.

Source: Proc Natl Acad Sci U S A. (2002) vol 99, p. 15898–15903

April 24, 2004

Research

Doctoral Degree (2004 - Now):
* Part I:
1. Production of transgenic rice overexpressing AOS-LOX fusion gene (T0, T1, T2)

2. Genetic Analysis of transgenic rice
- Analysis of DNA (by PCR and/or Southern), RNA (by Northern), Protein (Western)
- Selection of homozygous lines of transgenic rice

3. Biochemical Analysis of transgenic rice
- Lipid analysis
- JA analysis
- Dihydro JA analysis

4. Stress response of overexpressed transgenic rice
- Ion leakage experiments in cold, salt stress
- Wound and HR (local, systemic) response
- Blast fungi resistance

5. Metabolomics of overexpressed transgenic rice
- Lipid peroxidation experiments
- Product analysis by HPLC, GC, GC-MS
- Metabolic changes by wound and other stresses

* Part II:
1. Characterization of Fusion Genes (AOS-LOX, AOS-AOC and AOC-AOS)

Master Degree (2000 - 2002):

1. Production of transgenic rice overexpressing allene oxide synthase gene

2. Kinetic properties of V311M mutant Bacillus subtilis protoporphyrinogene oxidase
- Protein expression
- Protein purification
- Enzyme kinetics

April 23, 2004

Publications

Updated on December 22, 2009

Thavrak Huon, Sungkuk Jang, Kyoungwon Cho, Randeep Rakwal, Je Chang Woo, Ilchul Kim, Seung Wook Chi, Oksoo Han (2009) A Substrate Serves as a Hydrogen Atom Donor in the Enzyme-Initiated Catalytic Mechanism of Dual Positional Specific Maize Lipoxygenase-1. Bulletin of the Korean Chemical Society. 30: 719-723 [Abstract] [PDF Full Text]

Sungkuk Jang, Thavrak Huon, Keumhwa Kim, Eunji Um, and Oksoo Han (2007) Regiochemical and stereochemical evidence for enzyme-initiated catalysis in dual positional specific maize lipoxygenase-1. Organic letters 16: 3113-3116 [Abstract] [PDF Full Text]

Kyoungwon Cho, Sungkuk Jang, Thavrak Huon, Sangwook Park and Oksoo Han (2007) Biochemical Characterization of the Dual Positional Specific Maize Lipoxygenase and the Dependence of Lagging and Initial Burst Phenomenon on pH, Substrate, and Detergent during Pre-steady State Kinetics. Journal of Biochemistry and Molecular Biology 40: 100-106 [Abstract] [PDF Full Text]

Eunjoo Jeong, Thavrak Huon, Yongin Kuk, Eun-Seon Kim, Hema Kumar Chandru, Myunggi Baik, Kyoungwhan Back, Ja-Ock Guh, and Oksoo Han (2003) A point mutation of valine-311 to methionine in Bacillus subtilis protoporphyrinogen oxidase does not greatly increase resistance to the diphenyl ether herbicide oxyfluorfen. Bioorganic Chemistry 31: 389-397 [Abstract] [PDF Full Text]

Thavrak Huon (2002) Kinetic properties of V311M mutant Bacillus subtilis protoporphyrinogen oxidase. Thesis of Master degree, Department of Genetic Engineering, Chonnam National University. Gwangju, Republic of Korea

Thavrak Huon (1997) The effect of organic and inorganic fertilization on the growth of Tilapia fingerling. Thesis of Bachelor degree, Faculty of Fisheries, Royal University of Agriculture. Phnom Penh, Cambodia