• Journal of Microbiology and Biotechnology
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• 제19권2호
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• pp.136-139
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• 2009

Avermectin and its analogs are major commercial antiparasitic agents in the fields of animal health, agriculture, and human infections. Previously, comparative transcriptome analysis between the low-producer S. avermitilis ATCC31267 and the high-producer S. avermitilis ATCC31780 using a S. avermitilis whole genome chip revealed that 50 genes were overexpressed at least two-fold higher in S. avermitilis ATCC31780. To verify the biological significance of some of the transcriptomics-guided targets, five putative regulatory genes were individually cloned under the strong-and-constitutive promoter of the Streptomyces expression vector pSE34, followed by the transformation into the low-producer S. avermitilis ATCC31267. Among the putative genes tested, three regulatory genes including SAV213, SAV3818, and SAV4023 exhibited stimulatory effects on avermectin production in S. avermitilis ATCC31267. Moreover, overexpression of SAV3818 also stimulated actinorhodin production in both S. coelicolor M145 and S. lividans TK21, implying that the SAV3818, a putative TetR-family transcriptional regulator, could be a global upregulator acting in antibiotic production in Streptomyces species.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2017년도 9th Asian Crop Science Association conference
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• pp.164-164
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• 2017

High throughput transcriptome investigations of immunity in plants highlight the complexity of gene networks leading to incompatible interaction. To identify genes crucial to resistance against Xanthomonas oryzae pv oryzae, functional genetic analysis of selected differentially expressed genes from our microarray data set was carried out. A total of 13 overexpression vector constructs were made using 35S CaMV promoter which drive constitutive expression in rice. Most of the genes are developmentally expressed especially during maximum tillering stage and are commonly highly expressed in the leaves. When screened against Xoo strain K2, the transgenic plants displayed shorter lesion length compared with wild type Dongjin which indicates partial resistance. The levels of ROS continuously magnified after inoculation which indicates robust cellular sensing necessary to initiate cell death. Elevated transcripts levels of several defense-related genes at the downstream of defense signal network also corroborate the phenotype reaction of the transgenic plants. Moreover, expression assays revealed regulation of these genes by cross-communicating signal-transductions pathways mediated by salicylic and jasmonic acid. These collective findings revealed the key immune signaling conduits critical to mount full defense against Xoo.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2014년도 추계학술대회 및 정기총회
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• pp.9-9
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• 2014

Null mutants generated by targeted gene replacement are frequently used to reveal function of the genes in fungi. However, targeted gene deletions may be difficult to obtain or it may not be applicable, such as in the case of redundant or lethal genes. Constitutive expression system could be an alternative to avoid these difficulties and to provide new platform in fungal functional genomics research. Here we developed a novel platform for functional analysis genes in Magnaporthe oryzae by constitutive expression under a strong promoter. Employing a binary vector (pGOF1), carrying $EF1{\beta}$ promoter, we generated a total of 4,432 transformants by Agrobacterium tumefaciens-mediated transformation. We have analyzed a subset of 54 transformants that have the vector inserted in the promoter region of individual genes, at distances ranging from 44 to 1,479 bp. These transformants showed increased transcript levels of the genes that are found immediately adjacent to the vector, compared to those of wild type. Ten transformants showed higher levels of expression relative to the wild type not only in mycelial stage but also during infection-related development. Two transformants that T-DNA was inserted in the promotor regions of putative lethal genes, MoRPT4 and MoDBP5, showed decreased conidiation and pathogenicity, respectively. We also characterized two transformants that T-DNA was inserted in functionally redundant genes encoding alpha-glucosidase and alpha-mannosidase. These transformants also showed decreased mycelial growth and pathogenicity, implying successful application of this platform in functional analysis of the genes. Our data also demonstrated that comparative phenotypic analysis under over-expression and suppression of gene expression could prove a highly efficient system for functional analysis of the genes. Our over-expressed transformants library would be a valuable resource for functional characterization of the redundant or lethal genes in M. oryzae and this system may be applicable in other fungi.

• 한국초지조사료학회지
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• 제21권1호
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• pp.21-26
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• 2001

환경 스트레스에 의해 야기되는 활성 산소종에 의한 피해에 내성을 가지는 목초의 개발을 위하여 오차드그래스의 배반 조직 유래의 캘러스에 배추유래의 cytosolic glutathione reductase 유전자(BcGRl)를 Agrobucterium tumefaciens EHA101을 매개로 형질전환시켰다. Hygromcin으로 선발된 캘러스로부터 재분화된 식물체는 야생형과 비교하여 형태적으로 차이를 나타내지 않았다. PCR 및 Southern blot 분석을 통하여 형질전환 식물체의 염색체 내에 BcGRl 유전자가 integration 되었음을 확인하였다. 오차드그래스의 잎으로부터 total RNA를 분리하여 Northern blot 분석을 실시한 결과, 도입된 유전자가 형짙전환 식물체 내에서 지속적으로 발현된다는 것을 확인하였다.

• 한국산림과학회지
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• 제84권1호
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• pp.77-86
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• 1995

임목을 대상으로 삽입된 외래 유전자의 공간적, 시기별 발현 특성을 이해하기 위한 기초연구로서 온실에서 생육 중인 형질전환된 2년생 잡종 포플러 (Populus alba X P. grandidentata) Hansen 클론을 대상으로 삽입된 외래 유전자의 발현정도를 각 기관별로 조사하였다. Agrobacterium binary vector pRT45, pRT102 및 pRT104에 의해서 형질전환된 3계통의 형질전환체 Tr15, Tr345, Tr665 모두는 선발가능한 표식 유전자로서 nos promoter-NPT II 유전자가 대상 식물체의 genome에 삽입되어 있으며, 그외에, pin2 promoter-CAT 유전자(pRT45), nos promoter-PIN2 유전자(pRT102), Cauliflower Mosaic Virus 35s promoter-PIN2 유전자(pRT104)가 3계통의 형질전환체에 제각각 삽입되어 있는 잡종 포플러이다. 이들 3계통의 형질전환 포플러 식물체의 DNA를 PCR 검정 기법을 이용하여 분석해 본 결과 선발 가능한 표식 유전자인 NPT-II가 삽입되어 있음이 입증되었다 발현 정도를 비교 분석하기 위해서 NPT-ELISA 검정을 실시하였다. 삽입된 NPT II 유전자는 형질전환된 포플러의 잎, 엽병, 형성층 조직, 줄기의 목질부, 뿌리에서 발현되었으며, 발현 정도는 형질전환된 식물체의 계통에 따라서, 그리고 형진전환된 식물체의 부위에 따라서 다양하게 나타났다. pRT45에 의해서 형질전환된 Tr15 형질전환체의 경우, 늙은 잎과 엽병에서 NPT II 유전자가 가장 높은 수준으로 발현되었으며, 어린 잎과 뿌리 조직에서 가장 낮게 발현되었다. 삽입된 외래 유전자가 각 식물체간에, 각 기관에 따라서 각각 상이한 발현 정도를 나타내는 이와 같은 결과는 형질전환된 식물체에 대한 효과적인 선발과정이 요구됨을 의미함은 물론이고, 형질전환 식물체의 발달 과정에 따라서 삽입된 외래 유전자가 공간적, 시기적으로 각각 다르게 발현할 수 있다는 것을 나타낸다.

• Journal of Microbiology and Biotechnology
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• 제25권12호
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• pp.2100-2105
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• 2015

Probiotic bacteria must have not only tolerance against bile salt but also no genes for antibiotic resistance. Leuconostoc citreum is a dominant lactic acid bacterium in various fermented foods, but it is not regarded as a probiotic because it lacks bile salt resistance. Therefore, we aimed to construct a bile salt-resistant L. citreum strain by transforming it with a bile salt hydrolase gene (bsh). We obtained the 1,001 bp bsh gene from the chromosomal DNA of Lactobacillus plantarum and subcloned it into the pCB4170 vector under a constitutive P710 promoter. The resulting vector, pCB4170BSH was transformed into L. citreum CB2567 by electroporation, and bile salt-resistant transformants were selected. Upon incubation with glycodeoxycholic acid sodium salt (GDCA), the L. citreum transformants grew and formed colonies, successfully transcribed the bsh gene, and expressed the BSH enzyme. The recombinant strain grew in up to 0.3% (w/v) GDCA, conditions unsuitable for the host strain. In in vitro digestion conditions of 10 mM bile salt, the transformant was over 67.6% viable, whereas only 0.8% of the host strain survived.

• 한국미생물·생명공학회지
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• 제48권3호
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• pp.296-302
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• 2020

Rapamycin, derived from Streptomyces rapamycinicus, is an important bioactive compound having a therapeutic value in managing Parkinson's disease, rheumatoid arthritis, cancer, and AIDS. Because of its pharmaceutical activity, studies over the past decade have focused on the biosynthesis of rapamycin to enhance its yield. In this study, the effect of rapG on rapamycin production was investigated. The rapG expression vector was constructed by utilizing the integration vector pSET152 under the control of the erythromycin resistance gene (ermE^⁎), a strong constitutive promoter. The rapamycin yield of wild type (WT) and WT/rapG overexpression mutant strains, under fermentation conditions, was analyzed by high-performance liquid chromatography (HPLC). Our results revealed that overexpression of rapG increased rapamycin production by approximately 4.9-fold (211.4 mg/l) in MD1 containing 15 g/l of glycerol, compared to that of the WT strain. It was also found that Illicium verum powder (10 g/l), containing shikimic acid, enhanced rapamycin production in both WT and WT/rapG strains. Moreover, the amount of rapamycin produced by the WT/rapG strain was statistically higher than that produced by the WT strain. In conclusion, the addition 15 g/l glycerol and 15 g/l I. verum powder produced the optimal conditions for rapamycin production by WT and WT/rapG strains.

• 대한수의학회지
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• 제39권2호
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• pp.327-337
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• 1999

The transposon TnphoA was used to generate avirulent mutants from a type A Pasteurella multocida. A suicide vector plasmid pRT733 carrying TnphoA, having the kanamycin resistant gene and harbored in Escherichia coli K-12 strain SM10(${\lambda}pir$), was mated with streptomycin resistant P. multocida P-1059 strain as recipient. This resulted in the generation of two TnphoA insertion mutants (transconjugants, tc95-a and tc95-b) which were resistant both to kanamycin ($Km^{R}$) and streptomycin ($Sm^{R}$), secreted alkaline phosphatase, and were avirulent to turkeys. Southern blot hybridization using two probes derived from internal fragments of TnphoA, confirmed the insertion of TnphoA into 12.9kb or 13.7kb DNA fragment from the EcoRV digested genomic fragments of transconjugants. The two transconjugants, tc95-a and tc95-b, were distinguishable from their parent strains by differences in ribotypes, and outer membrane protein profiles. TnphoA insertion in both transconjugants also resulted in constitutive expression of a 33Kd iron regulated outer membrane protein (IROMP). The gene encoding $Sm^{R}$ was also located within the same 12.9kb EcoRV genomic fragment from both transconjugants. Furthermore, our finding that the recipient P. multocida P-1059 $Sm^{R}$ strain and both transconjugants were avirulent to turkeys suggest that the either 12.9kb or 13.7kb genomic DNA contains the virulence gene and speculate that the presence of $Sm^{R}$ gene or TnphoA insertion may be responsible for regulating and inactivating the gene(s) encoding virulence in P. multocida.

• Journal of Microbiology and Biotechnology
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• 제20권11호
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• pp.1491-1499
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• 2010

Rhizosphere microorganisms possessing phytase activity are considered important for rendering phytate-phosphorus (P) available to plants. In the present study, the Citrobacter braakii phytase gene (appA) was overexpressed in rhizobacteria possessing plant growth promoting (PGP) traits, for increasing their potential as bioinoculants. AppA was cloned under the lac promoter in the broadhost-range expression vector pBBR1MCS-2. Transformation of the recombinant construct pCBappA resulted in high constitutive phytase activity in all of the eight rhizobacterial strains belonging to genera Pantoea, Citrobacter, Enterobacter, Pseudomonas (two strains), Rhizobium (two strains), and Ensifer that were studied. Transgenic rhizobacterial strains were found to display varying levels of phytase activity, ranging from 10-folds to 538-folds higher than the corresponding control strains. The transgenic derivative of Pseudomonas fluorescens CHA0, a well-characterized plant growth promoting rhizobacterium, showed the highest expression of phytase (~8 U/mg) activity in crude extracts. Although all transformants showed high phytase activity, rhizobacteria having the ability to secrete organic acid showed significantly higher release of P from Ca-phytate in buffered minimal media. AppA overexpressing rhizobacteria showed increased P content, and dry weight (shoot) or shoot/ root ratio of mung bean (Vigna radiata) plants, to different extents, when grown in semisolid agar (SSA) medium containing Na-phytate or Ca-phytate as the P sources. This is the first report of the overexpression of phytase in rhizobacterial strains and its exploitation for plant growth enhancement.

• Journal of Microbiology and Biotechnology
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• 제31권4호
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• pp.550-558
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• 2021

Lactobacillus kefiranofaciens contains two types of β-galactosidase, LacLM and LacZ, belonging to different glycoside hydrolase families. The difference in function between them has been unclear so far for practical application. In this study, LacLM and LacZ from L. kefiranofaciens ATCC51647 were cloned into constitutive lactobacillal expression vector pMG36e, respectively. Furtherly, pMG36n-lacs was constructed from pMG36e-lacs by replacing erythromycin with nisin as selective marker for food-grade expressing systems in Lactobacillus plantarum WCFS1, designated recombinant LacLM and LacZ respectively. The results from hydrolysis of o-nitrophenyl-β-galactopyranoside (ONPG) showed that the β-galactosidases activity of the recombinant LacLM and LacZ was 1460% and 670% higher than that of the original L. kefiranofaciens. Moreover, the lactose hydrolytic activity of recombinant LacLM was higher than that of LacZ in milk. Nevertheless, compare to LacZ, in 25% lactose solution the galacto-oligosaccharides (GOS) production of recombinant LacLM was lower. Therefore, two β-galactopyranosides could play different roles in carbohydrate metabolism of L. kefiranofaciens. In addition, the maximal growth rate of two recombinant strains were evaluated with different temperature level and nisin concentration in fermentation assay for practical purpose. The results displayed that 37℃ and 20-40 U/ml nisin were the optimal fermentation conditions for the growth of recombinant β-galactosidase strains. Altogether the food-grade Expression system of recombinant β-galactosidase was feasible for applications in the food and dairy industry.