Generation and DNA Characterization of High-lysine Mutants by Biochemical Selection from Callus Culture of 'Hwayeongbyeo'

Yi Gi-Hwan,Choi Jun-Ho,Kim Kyung-Min,Jeong Eung-Gi,Park Hyang-Mi,Kim Doh-Hoon,Ku Yeon Chung,Eun Moo-Young,Kim Ho-Yeong,Nam Min-Hee The Plant Resources Society of Korea 2005 Plant Resources Vol.8 No.1

Lysine is the first essential amino acid for optimal nutrient quality in rice grain. For the narrow genetic diversities of lysine contents in rice, somaclonal variation was the source of mutation in our breeding program. Biochemical selection was conducted using 1 mM S-(2-aminoethyl) cysteine followed by two passages of 5 mM lysine plus threonine in the callus subculture medium. The lysine contents in endosperm of all progenies recovered from the biochemical selection were higher than those of their donor cultivar 'Hwayeongbyeo'. These elevated lysine levels of mutants were successfully transmitted to $M_4$ generation. The lysine contents in endosperm varied 3.85 to $4.80\%$ compare to their donor cultivar 'Hwayeongbyeo' was $3.85\%$. Three of high-lysine germplasms, Lys-l, Lys-2 and Lys-7 were selected by biochemical selection and rapid screening methods. DNA analysis showed that a new insertion of Tos 17 which mapped to rice chromosome 11 on the high-lysine mutant, Lys-2.

고-Lysine 보리 돌연변이 계통 M98 종실의 아미노산 조성 및 Proteome Profile 특성

김대욱,김홍식,박형호,황종진,김선림,이재은,정건호,황태영,김정태,김시주,권영업 한국작물학회 2012 한국작물학회지 Vol.57 No.2

본 연구는 찰쌀보리와 M98의 종실에 함유된 아미노산 조성 및 단백질 profile의 특성 차이를 분석하여 M98의 고-Lysine 및 함몰배유 형질에 관련된 생화학적 정보를 얻고자 수행하였으며, 그 결과를 요약하면 다음과 같다. 1. 찰쌀보리와 M98 종실의 총 16개 아미노산 조성을 분석한 결과, lysine을 비롯하여 총 6개 아미노산의 조성비가 M98에서 찰쌀보리에 비해 1.2~1.8배 높았다. Proline과 그것의 전구물질인 glutamic acid의 조성비는 M98에서 찰쌀보리에 비해 절반수준으로 낮았는데, glutamic acid를 전구물질로 하여 합성되는 arginine의 조성비는 M98에서 찰쌀보리보다 1.8배 높았다. 2. 찰쌀보리와 M98간의 종실 단백질을 이차원전기영동으로 분리한 결과, 총 70개 단백질 spot에서 발현양상의 차이가 확인되었다. 찰쌀보리에 비해 M98에서 발현양이 높은 단백질 spot은 44개였으며, 발현양이 낮은 단백질 spot은 18개였다. 나머지 8개 단백질 spot에서 7개의 발현은 찰쌀보리에서만 확인되었고, 1개의 발현은 M98에서만 확인되었다. 3. 찰쌀보리와 M98의 종실에서 발현양상 차이를 나타낸 총 53개의 단백질 spot을 선택하여 nESI-LC-MS/MS 방식으로 동정하였으며, 대부분의 단백질 spot은 종실의 다양한 생물학적 과정에 관여하는 단백질이었다. 특히, 28개 단백질 spot은 M98의 함몰배유 및 고-Lysine 형질과 연관된 단백질이었다. 4. 본 연구에서 밝혀진 M98 종실의 아미노산 조성 및 단백질 발현특성 정보는 향후 M98 유래 계통을 이용한 유전학적 검토 등을 통하여 고-Lysine 형질 연관 바이오마커 및 보리 신품종 개발에 유용한 정보로 활용될 수 있을 것으로 판단되었다. Lysine is the first limiting essential amino acid in cereals for humans and monogastric animals, although its content is generally low. A chemically induced high-lysine barley mutant, M98, has an agronomically undesirable shrunken endosperm trait. In order to obtain detailed insight into the atypical traits of M98 grains, we characterized amino acid composition and protein profiles of M98 and its parent cultivar Chalssalbori. Among a total of 16 amino acids, the percentage of each of the 7 amino acids, including lysine, was 1.2~1.8 times higher in M98, comparing to Chalssalbori. The percentage of proline and its precursor, glutamic acid, in M98 was about the half of that of the amino acids in Chalssalbori, but arginine synthesized from glutamic acid was 1.8 times higher in M98, compared that in the parent cultivar. Theses results indicated that the mutation in M98 grains might alter the proportion of amino acids linked to each other in a biosynthetic pathway. A comparison of grain proteome profiles between Chalssalbori and M98 revealed 70 differentially expressed protein spots, where 45 protein spots were up-regulated and 25 protein spots down-regulated in M98 compared to those in Chalssalbori. Of these changed protein spots, 53 were identified using nano-electrospray ionization liquid chromatography mass spectrometry. Most of these identified proteins were involved in various biological processes. In particular, 28 protein spots such as β -amylase, serpins and B3-hordein were identified as proteins associated with the atypical traits of M98. It was thought that a genetic study on the unique protein profile of M98 would be needed to develop an agronomically feasible barley cultivar with high-lysine trait.

A Liquid-Based Colorimetric Assay of Lysine Decarboxylase and Its Application to Enzymatic Assay

( Yong Hyun Kim ),( Ganesan Sathiyanarayanan ),( Hyun Joong Kim ),( Shashi Kant Bhatia ),( Hyung Min Seo ),( Jung Ho Kim ),( Hun Seok Song ),( Yun Gon Kim ),( Kyungmoon Park ),( Yung Hun Yang ) 한국미생물 · 생명공학회 2015 Journal of microbiology and biotechnology Vol.25 No.12

A liquid-based colorimetric assay using a pH indicator was introduced for high-throughput monitoring of lysine decarboxylase activity. The assay is based on the color change of bromocresol purple, measured at 595 nm in liquid reaction mixture, due to an increase of pH by the production of cadaverine. Bromocresol purple was selected as the indicator because it has higher sensitivity than bromothymol blue and pheonol red within a broad range and shows good linearity within the applied pH. We applied this for simple determination of lysine decarboxylase reusability using 96-well plates, and optimization of conditions for enzyme overexpression with different concentrations of IPTG on lysine decarboxylase. This assay is expected to be applied for monitoring and quantifying the liquid-based enzyme reaction in biotransformation of decarboxylase in a high-throughput way.

A New High Lysine with Disease Resistance Rice Variety "Yeonganbyeo"

You Chun Song,No Bong Park,Sang Jong Lim,Do Yeon Kwak,Sae Jun Yang,Ho Yeong Kim,Min Hee Nam,Hung Goo Hwang,Un Goo Ha,Byeong Gen Oh,Un Sang Yeo,Jong Rae Kang,Gi Hwan YiJae Ki 한국육종학회 2007 한국육종학회지 Vol.39 No.3

“Yeonganbyeo”, a new japonica rice variety (Oryza sativa L.), is a mid- maturing ecotype with high lysine content in kernels that was developed by the rice breeding team of National Yeongnam Agricultural Experiment Station (NYAES) in 2001 and released in 2002. This variety was originated from the single cross of Milyang 122/YR13616 Acp1 (in 1992/1993 winter) and was selected by means of a mixed method of bulk and pedigree breeding. The pedigree of “Yeonganbyeo” was YR15815-B-B-B-30 and designated in 1998 as “Milyang 164”. It has about 83cm in culm length with lodging tolerance. This variety is resistant to bacterial leaf blight (K1, K2, K3), stripe virus, and moderately resistant to leaf blast disease. Milled rice kernels of “Yeonganbyeo” has high lysine content of 4.31% (ratio of amino acid components in total protein), a clear translucent with non-glutinous endosperm and clear in chalkness and good at eating quality by pannel test. The yield potential of “Yeonganbyeo” in milled rice is about 5.45 MT/ha at ordinary fertilizer level of local adaptability test. This cultivar would be adaptable to the southern plain of Korea.

Identification of causative mutations in patients with Leigh syndrome and MERRF by mitochondrial DNA-targeted next-generation sequencing

Hong, Hyun Dae,Kim, Eunja,Nam, Soo Hyun,Yoo, Da Hye,Suh, Bum Chun,Choi, Byung-Ok,Chung, Ki Wha Korean Society of Medical Genetics and Genomics 2015 대한의학유전학회지 Vol.12 No.2

Purpose: Mitochondrial diseases are clinically and genetically heterogeneous disorders, which make their exact diagnosis and classification difficult. The purpose of this study was to identify pathogenic mitochondrial DNA (mtDNA) mutations in 2 Korean families with myoclonic epilepsy with ragged-red fibers (MERRF) and Leigh syndrome, respectively. Materials and Methods: Whole mtDNAs were sequenced by the method of mtDNA-targeted next-generation sequencing (NGS). Results: Two causative mtDNA mutations were identified from the NGS data. An m.8344A>G mutation in the tRNA-Lys gene (MT-TK) was detected in a MERRF patient (family ID: MT132), and an m.9176T>C (p.Leu217Pro) mutation in the mitochondrial ATP6 gene (MT-ATP6) was detected in a Leigh syndrome patient (family ID: MT130). Both mutations, which have been reported several times before in affected individuals, were not found in the control samples. Conclusion: This study suggests that mtDNA-targeted NGS will be helpful for the molecular diagnosis of genetically heterogeneous mitochondrial diseases with complex phenotypes.

Identification of causative mutations in patients with Leigh syndrome and MERRF by mitochondrial DNAtargeted next-generation sequencing

Hyun Dae Hong,Eunja Kim,Soo Hyun Nam,Da Hye Yoo,Bum Chun Suh,Byung-Ok Choi,Ki Wha Chung 대한의학유전학회 2015 대한의학유전학회지 Vol.12 No.2

Purpose: Mitochondrial diseases are clinically and genetically heterogeneous disorders, which make their exact diagnosis and classification difficult. The purpose of this study was to identify pathogenic mitochondrial DNA (mtDNA) mutations in 2 Korean families with myoclonic epilepsy with ragged-red fibers (MERRF) and Leigh syndrome, respectively. Materials and Methods: Whole mtDNAs were sequenced by the method of mtDNA-targeted next-generation sequencing(NGS). Results: Two causative mtDNA mutations were identified from the NGS data. An m.8344A>G mutation in the tRNA-Lys gene(MT-TK ) was detected in a MERRF patient (family ID: MT132), and an m.9176T>C (p.Leu217Pro) mutation in the mitochondrial ATP6 gene (MT-ATP6) was detected in a Leigh syndrome patient (family ID: MT130). Both mutations, which have been reported several times before in affected individuals, were not found in the control samples. Conclusion: This study suggests that mtDNA-targeted NGS will be helpful for the molecular diagnosis of genetically heterogeneous mitochondrial diseases with complex phenotypes.

Identiἀcation of causative mutations in patients with Leigh syndrome and MERRF by mitochondrial DNA-targeted next-generation sequencing

Hyun Dae Hong,Eunja Kim,Soo Hyun Nam,Da Hye Yoo,Bum-ChunSuh,Byung-Ok Choi,정기화 대한의학유전학회 2015 대한의학유전학회지 Vol.12 No.2

Purpose: Mitochondrial diseases are clinically and genetically heterogeneous disorders, which make their exact diagnosis and classiἀcation difἀcult. The purpose of this study was to identify pathogenic mitochondrial DNA (mtDNA) mutations in 2 Korean families with myoclonic epilepsy with ragged-red ἀbers (MERRF) and Leigh syndrome, respectively. Materials and Methods: Whole mtDNAs were sequenced by the method of mtDNA-targeted next-generation sequencing (NGS). Results: Two causative mtDNA mutations were identiἀed from the NGS data. An m.8344A>G mutation in the tRNA-Lys gene (MT-TK) was detected in a MERRF patient (family ID: MT132), and an m.9176T>C (p.Leu217Pro) mutation in the mitochondrial ATP6 gene (MT-ATP6) was detected in a Leigh syndrome patient (family ID: MT130). Both mutations, which have been reported several times before in affected individuals, were not found in the control samples. Conclusion: This study suggests that mtDNA-targeted NGS will be helpful for the molecular diagnosis of genetically heterogeneous mitochondrial diseases with complex phenotypes.