• Journal of Plant Biotechnology
• /
• 제42권2호
• /
• pp.93-103
• /
• 2015

The isolation, cloning, and characterization of an R2R3-MYB transcription factor gene (MtMYB70) from the model legume Medicago truncatula is reported. MtMYB70 consists of a 768-bp coding sequence corresponding to 255 amino acids. Sequence alignment revealed that MtMYB70 cDNA contains conserved R2R3-type MYB domains with highly divergent C terminal regions. MtMYB70 was found to have relatively low sequence homology with known R2R3-MYB genes. Phylogenetic analysis placed the R2R3-MYB domain of MtMYB70 closest to PtMYB1, a known activator of lignin biosynthesis. Overexpression of MtMYB70 under the control of the 35S promoter in transgenic poplar did not cause a significant difference in total lignin content relative to the control, but glucan content was significantly increased in transgenic poplar. Therefore, MtMYB70 might have regulatory role in the biosynthesis of cell wall structural carbohydrates.

• 한국버섯학회지
• /
• 제2권3호
• /
• pp.145-148
• /
• 2004

이 연구는 약용버섯 번데기 동충하초로부터 특이적 자실체 형성 유전자를 선별하기 위하여 수행하였다. cDNA는 버섯의 분화 4단계 균사체, 원기, 미성숙 자실체, 성숙한 자실체로부터 분리한 각각의 total RNA를 이용하여 합성하였다. 특이적으로 발현된 유전자의 선별은 cDNA와 랜덤한 primer set을 이용한 DD-PCR에 의해 수행되었고, pGEM 클로닝 벡터를 이용하여 6개의 partial 유전자 서열을 확인하였다. 6개의 DD-PCR product는 보고된 유전자와 유사도를 확인하기 위해 NCBI BLAST search를 사용하여 GenBank에서 비교하였고, 6개의 유전자는 보고되지 않은 유전자임을 확인하였다.

• Applied Biological Chemistry
• /
• 제45권3호
• /
• pp.129-133
• /
• 2002

키토산 분해물을 시금치와 상추에 살포하였을 때, nitrate 함량이 감소되었으며, 이러한 감소는 nitrate reductase 활성의 증가에 기인한 것으로 나타났다. 이에 따라 채소 중 질산염을 가장 많이 축적하는 채소 중 하나인 시금치의 nitrate reductase를 식물체내에 과량 발현시켜 질산염 축적을 줄이기 위한 연구를 수행하였다. 첫 단계로 시금치 mRNA로부터 RT-PCR을 이용하여 cDNA를 분리, 증폭하고 벡터에 클로닝하여 염기서열을 결정하였다. 시금치 nitrate reductase cDNA의 염기서열은 다른 식물체에서 분리된 nitrate reductase 유전자들과 상당히 높은 상동성($71{\sim}82%$)을 보였고, 이미 발표된 시금치 nitrate reductase cDNA의 염기서열과 비교하였을 때 단지 두 염기만이 달랐다.

• Journal of Microbiology and Biotechnology
• /
• 제16권2호
• /
• pp.256-263
• /
• 2006

In the model legume Medicago truncatula, two mutants, sickle and sunn, exhibit morphologically and genetically distinct hypernodulation phenotypes. However, efforts to isolate the single recessive and single semidominant genes for sickle and sunn, respectively, by map-based cloning have so far been unsuccessful, partly due to the absence of clones that enable walks from linked marker positions. To help resolve these difficulties, a new bacterial artificial chromosome (BAC) library was constructed using BamHI-digested genomic fragments. A total of 23,808 clones were collected from ligation mixtures prepared with double-size-selected high-molecular-weight DNA. The average insert size was 116 kb based on an analysis of 88 randomly selected clones using NotI digestion and pulsed-field gel electrophoresis. About 18.5% of the library clones lacked inserts. The frequency of the BAC clones carrying chloroplast or mitochondrial DNA was 0.98% and 0.03%, respectively. The library represented approximately 4.9 haploid M. truncatula genomes. Hybridization of the BAC clone filters with a $C_{0}t-l$ DNA probe revealed that approximately 37% of the clones likely carried repetitive sequence-enriched DNA. An ordered array of pooled BAC DNA was screened by polymerase chain reactions using 13 sequence-characterized molecular markers that belonged to the eight linkage groups. Except for two markers, one to five positive BAC clones were obtained per marker. Accordingly, the sickle- and sunn-linked BAC clones identified herein will be useful for the isolation of these biotechnologically important genes. The new library will also provide clones that fill the gaps between preexisting BAC contigs, facilitating the physical mapping and genome sequencing of M. truncatula.

• Journal of Microbiology and Biotechnology
• /
• 제13권1호
• /
• pp.139-145
• /
• 2003

The gene encoding Thermus caldophilus GK24 polyphosphate kinase (Tca PPK) was cloned and sequenced. The gene contains an open reading frame encoding 608 amino acids with a calculated molecular mass of 69,850 Da. The deduced amino acid sequence of Tca PPK showed a 40% homology to Escherichia coli PPK, and $39\%$ to Klebsiella aerogenes PPK. The Tca ppk gene was expressed under the control of the T7lac promoter on pET-22b(+) in E. coli and its enzyme was purified about 70-fold with $36\%$ yield, following heating and HiTrap chelating HP column chromatography. The native enzyme was found to have an approximate molecular mass of 580,000 Da and consisted of eight subunits. The optimum pH and temperature of the enzyme were 5.5 and $70^{\circ}C$, respectively. A divalent cation was required for the enzyme activity, with $Mg^2+$ being the most effective.

• International Journal of Industrial Entomology and Biomaterials
• /
• 제18권1호
• /
• pp.28-32
• /
• 2009

We describe here the cloning and characterization of a cDNA encoding the glutathione S-transferase (GST) from the bumblebee Bombus ignitus. The Delta-class B. ignitus GST (BiGSTD) gene spans 1668 bp and consists of four introns and five exons that encode 216 amino acid residues with a calculated molecular weight of approximately 24561 Da and a pI of 8.03. The N-terminal domain of BiGSTD has a conserved Ser residue, as well as conserved Lys, Pro, Glu, Ser and Tyr residues that are involved in the GSH-binding site of GST. The BiGSTD showed 60% protein sequence identity to the Bombyx mori GSTT1, 58% to Musca domestica GST, 57% to Drosophila melanogaster GST, and 55% to Anopheles gambiae GST1. BiGSTD was close to the insect-specific Delta class of GSTs in a phylogenetic tree. Northern blot analysis showed that BiGSTD is highly expressed in the fat body and midgut, and less so in the muscles of B. ignitus worker bees.

• 한국작물학회:학술대회논문집
• /
• 한국작물학회 2017년도 9th Asian Crop Science Association conference
• /
• pp.89-89
• /
• 2017

A gene encoding squalene synthase from grain amaranth was cloned and characterized. The full-length cDNA was 1805-bp long and contained a 1248-bp open reading frame encoding a protein of 416 amino acids with a molecular mass of 47.6 kDa. Southern blot analysis revealed that the A. cruentus genome contained a single copy of the gene. Comparison of the cDNA and genomic sequences indicated that the amaranth SQS gene had 12 introns and 13 exons. All of the exons contributed to the coding sequence. The predicted amino acid sequence of the SQS cDNA shared high homology with those of SQSs from several other plants. It contained conserved six domains that are believed to represent crucial regions of the active site. We conducted qRT-PCR analyses to examine the expression pattern of the SQS gene in seeds at different developmental stages and in several tissues. The amaranth SQS gene was low levels of SQS transcripts at the initial stage of seed development, but the levels increased rapidly at the mid-late developmental stages before declining at the late developmental stage. These findings showed that the amaranth SQS is a late-expressed gene that is rapidly expressed at the mid-late stage of seed development. In addition, we observed that the SQS mRNA levels in stems and roots increased rapidly during the four- to six-leaf stage of development. Therefore, our results showed that the expression levels of SQS in stem and root tissues are significantly higher than those in leaf tissues. In present study provides useful information about the molecular characterization of the SQS clone isolated from grain amaranth. Finally, a basic understanding of these characteristics will contribute to further studies on the amaranth SQS.

• Asian-Australasian Journal of Animal Sciences
• /
• 제14권1호
• /
• pp.13-16
• /
• 2001

Bovine ${\beta}1$-adrenergic receptor (AR) cDNA was cloned using degenerative primers. Bovine ${\beta}1$-AR coded for 467 amino acids and the comparison of the deduced amino acid sequence with that of sheep showed 93.4% identity. Northern blot analysis indicated that transcript size for the bovine ${\beta}1$-AR was 3.6 kb in the adipose tissue. The expression level of three $\beta$-ARs (1, 2, and 3) in bovine abdominal, subcutaneous, and perirenal adipose tissues were examined using reverse transcription-polymerase chain reaction (RT-PCR), and the levels of ${\beta}1$- and ${\beta}3$-AR mRNA were found to be lower in the subcutaneous adipose tissue than in the abdominal and perirenal adipose tissues. These results suggest that the expression of $\beta$-ARs mRNA are differentially regulated among the adipose tissues.

• 한국식물병리학회지
• /
• 제11권1호
• /
• pp.73-79
• /
• 1995

Complementary DNAs (cDNAs) to the coat protein gene of an ordinary Korean strain of potato virus Y (PVY-OK) isolated from potato (cv. Superior) were synthesized and cloned into a plasmid pUC119 and sequenced. The RNA of the virus propagated in tobacco (Nicotinaa sylvestris) was extracted by the method of phenol extraction. The first strand of cDNAs to the coat protein penomic RNA of the virus was made by Moloney murine leukemia virus reverse transcriptase. The cDNA were synthesized and amplified by the method of polymerase chain reaction (PCR) using a pair of oligonucleotide primers. PVYCP3P and PVYCP3M. The size of cDNAs inserted in pUC119 plasmid was estimated as about 840 bp upon agarose gel electrophoresis. Double stranded cDNAs were transformed into the competent cell of E. coli JM109. Sequence analysis of cDNAs was conducted by the dideoxynucleotide chain termination method. Homology of cDNAs of the PVY-OK coat protein genomic RNA with those of PVY-O (Japan), PVY-T (Japan), PVY-TH (Japan), PVYN (The Netherlands),and PVYY (France) was represented as 97.3%, 88.9%, 89.3%, 89.6% and 98.5%, respectively. Homology at the amino acid level turned out to the be 97.4%, 92.5%, 92.9%, 92.9% and 98.5%, respectively.

• Journal of Microbiology and Biotechnology
• /
• 제24권4호
• /
• pp.447-452
• /
• 2014

To isolate novel and useful microbial enzymes from uncultured gastrointestinal microorganisms, a fecal microbial metagenomic library of the pygmy loris was constructed. The library was screened for amylolytic activity, and 8 of 50,000 recombinant clones showed amylolytic activity. Subcloning and sequence analysis of a positive clone led to the identification a novel gene (amyPL) coding for ${\alpha}$-amylase. AmyPL was expressed in Escherichia coli BL21 (DE3) and the purified AmyPL was enzymatically characterized. This study is the first to report the molecular and biochemical characterization of a novel ${\alpha}$-amylase from a gastrointestinal metagenomic library.