Investigation of Agrobacterium-mediated Transient dsRNA Expression in Tobacco

최원균,임혜송,서한규,김동욱 한국하천호수학회 2019 생태와 환경 Vol.52 No.4

The Agrobacterium tumefaciens mediated gene transfer is widely used to generate genetic transformation of plants and transient assay of temporal exogenous gene expression. Syringe infiltration system into tobacco (Nicotiana benthamiana) leaves is a powerful tool for transient expression of target protein to study protein localization, protein-protein binding and protein production. However, the protocol and technical information of transient gene expression, especially double strand RNA (dsRNA), in tobacco using Agrobacterium is not well known. Recently, dsRNA is crucial for insecticidal effect on destructive agronomic pest such as Corn rootworm. In this study, we investigated the factor influencing the dsRNA expression efficiency of syringe agro-infiltration in tobacco. To search the best combination for dsRNA transient expression in tobacco, applied two Agrobacterium cell lines and three plant vector systems. The efficiency of dsRNA expression has estimated by real-time PCR and digital PCR. As a result, pHellsgate12 vector constructs showed the most effective accumulation of dsRNA in the cell. These results indicated that the efficiency of dsRNA expression was depending on the kind of vector rather than Agrobacterium cells. In summary, the optimized combination of transient dsRNA expression system in tobacco might be useful to in vivo dsRNA expression for functional study and risk assessment of dsRNA.

Agrobacterium을 이용한 고추의 Transient Expression 시스템

성은수,정영희,최도일,Seong, Eun-Soo,Joung, Young-Hee,Choi, Doil 한국식물생명공학회 2004 식물생명공학회지 Vol.31 No.3

Agrobacterium을 이용한 GUS 유전자를 효과적으로 발현시키기 위하여 수행되어진 실험 결과를 요약하면 다음과 같다. 박테이라 농도별 실험을 수행한 결과 균의 전처리 배양 농도 O $D_{600nm}$ 0.3일때 원심분리한 후 얻어진 균을 희석한 후의 최종 농도는 O $D_{600nm}$ 0.8로 맞춘 실험 처리구에서 GUS 유전자 발현율이55%로 가장 높게 나타났다. 병원성 유도 배지 내에 Acetosyringone (AS)이 첨가되지 않은 경우 GUS 유전자가 발현된 고추 잎을 얻을 수 없었으나, 200$\mu$M을 첨가했을 때 90%의 가장 높은 GUS 유전자 발현율을 나타내어 많은 수의 GUS spots을 관찰할 수 있었다. Agrobacterium에 의한 고추 잎의 감염 정도를 조사한 바 Agrobacterium으로 감염시킨지 3일째부터는 박테리아 에한 감염 정도가 심해져서 GUS 유전자 발현 정도가 약해지므로 Agrobacterium으로 감염시킨지 2일째 되었을 때 GUS 유전자 발현이 가장 강하게 나타난 것을 확인하였다. 이 같은 결과는 박테리아에 의한 감염이 일어난지 3일째 부터는 식물체의 감염부위 고사를 일으키는 것과 관련된 것으로 보인다. We established a transient gene expression system in chili pepper leaves based on Agrobacterium-mediated transformation of GUS gene. For the best GUS transient expression, two step culture system was adopted. When the Agrobacterium tumefaciens cell density of pre-culture was $A_{600nm}$ 0.3, the cells were harvested and diluted to $A_{600nm}$ 0.8 with virulence induction medium after cell harvested. The addition of acetosyringone (200 $\mu$M) in virulence induction step was a key factor for successful transient expression. Additionally, Younger leaves showed more effective transient expression than older leaves. Temporally, the strongest intensity of GUS expression was detected at 2 days after infiltration. These results demonstrate that Agrobacterium-mediated transient expression can be used for a simple in vivo assays of plant promoters, transcription factors and furthermore provide efficient protocol for chili pepper transformation.

Transient Expression of β-gulucuronidase (GUS) gene in Immature Ovules and Calli Derived from Cottonwood Species (Populus deltoides) by Microprojectile Bombardment

강호덕,강상구,배한홍,박교수,Kang, Hoduck,Kang, Sang-Gu,Bae, Hanhong,Park, Kyo-Soo,Hall, Richard B. Korean Society of Forest Science 1997 한국산림과학회지 Vol.86 No.3

미류나무의 미성숙 ovule과 줄기로부터 유기된 캘러스에 plasmid pBI221 유전자를 유전자총을 이용하여 인위적으로 삽입하였다. Plasmid pBI221은 CaMV-35S 유전자에 의하여 발현되는 ${\beta}$-glucuronidase(GUS) reporter 유전자를 포함하고 있다. Plasmid pBI221이 물리적으로 삽입된 후 GUS 유전자의 발현정도는 5-bromo-4-chloro-3-indolyl-${\beta}$-gluconide(X-glue)의 반응에 의해 분석되었고, 유전자의 일시적 발현현상은 ovule, 캘러스 시료에 X-glue substrate의 반응에 의하여 나타나는 뚜렷한 점(spot)의 수에 따라서 조사하였다. 본 실험에서 particle bombardment후 GUS유전자의 발현검정에 있어 가장 중요한 요인은 bombardment 횟수와 X-glue substrate에 노출된 시간이었다. X-glue substrate와의 반응결과, 캘러스와 ovule에서 각각 56.8%, 75.9%의 반응 점들을 나타냈다. 여러 처리중 두번의 연속적인 shot와 bombardment 이후, X-glue과 sample을 48시간 반응시킨 후 24시간 동안 alcohol로의 침지가 가장 많은 수의 점을 유기시켰고, 이들 반응으로부터 평균 $25.75{\pm}2.77$(ovule), $11.43{\pm}1.22$(calli)개의 반응 점을 보였다. 유전자총에 의한 외래 유전자 도입에 관한 본 연구는 두종류의 시료로부터 빠른 시간 내에 유전자 발현을 볼 수 있을 뿐만 아니라, 지금까지 Agrobacterium을 이용한 형질전환이 보고되지 않은 미류나무(Populus deltoides)의 형질전환 연구에 대체 방법을 제공하리라 생각된다. Excised immature ovules and calli derived from the stems of cottonwood were bombarded with microprojectiles carrying plasmid DNA containing CaMV-35S promoter and ${\beta}$-glucuronidase(GUS) gene. After bombarded, the expression of GUS gene was detected by the assay of 5-bromo-4-chloro-3-indolyl-${\beta}$-gluconide(X-gluc). Transient gene expression was measured by counting the number of distinct regions of GUS activity per explant. As major parameters, the number of shots and the period of exposure to X-gluc after the bombardment were investigated for detecting GUS gene expression. In this experiment, the percents of GUS gene expression showing spots were 56.8 from immature ovules and 75.9 from micro-calli of cottonwood species. Among the treatments, two consecutive shots and 48 hour exposure produced about $25.75{\pm}2.77$(per ovule), $11.43{\pm}1.22$(per mini petridish) spots, respectively, Microprojectile particle bombardment provides a useful method to assay transient expression in both types of explants. Furthermore, our results represent that the excised ovule and/or the calli might be stably transformed by the biolistics.

Large-scale production of foreign proteins via the novel plant transient expression system in Pisum sativum L

Liang Li,Xiufeng Wang,Liping Yang,Yajun Fan,Xiaojuan Zhu,Xingzhi Wang 한국식물생명공학회 2016 Plant biotechnology reports Vol.10 No.4

Transient expression of foreign genes by Agrobacterium infiltration is a versatile technique that can be used as a rapid tool for functional protein production in plants. A reproducible protocol of large-scale production of foreign proteins via the novel plant transient expression system in Pisum sativum L. was established in our study. Non-detached plants from soil-independent culture were used as the target organ, and vacuum infiltrating mediated by Agrobacterium tumefaciens harboring green fluorescent protein (GFP) gene was performed. Step-by-step optimization was performed and showed that the quality of plant material as well as agro-infiltration conditions were the major factors influencing the gene expression. Monitoring the transient GFP expression daily, the highest expression level was achieved on the 8th day post-infiltration. Evidence of anti-acidic fibroblast growth factorsingle chain variable fragment (anti-aFGF-scFv) gene expression in pea seedling was also achieved using agromediated vacuum infiltration system. Our work proves that the system is suitable for the largescale production of pharmaceutical proteins. The in planta infiltration system described here provides a powerful tool to explore easily gene expression in Pisum sativum L. avoiding tissue culture steps and the labor-intensive generation of transgenic plants.

Agrobacterium을 이용한 토마토 떡잎에서 CRISPR-Cas9 시스템의 임시발현 시 토마토 떡잎 발달 단계에 따른 유전자교정 효율 변화

김의연 ( Euyeon Kim ),양소희 ( So Hee Yang ),박효선 ( Hyosun Park ),구연종 ( Yeonjong Koo ) 한국환경농학회 2021 한국환경농학회지 Vol.40 No.3

BACKGROUND: Before generating transgenic plant using the CRISPR-Cas9 system, the efficiency test of sgRNAs is recommended to reduce the time and effort for plant transformation and regeneration process. The efficiency of the sgRNA can be measured through the transient expression of sgRNA and Cas9 gene in tomato cotyledon; however, we found that the calculated efficiency showed a large variation. It is necessary to increase the precision of the experiment to obtain reliable sgRNA efficiency data from transient expression. METHODS AND RESULTS: The cotyledon of 11^th, 15^th, 19^th, and 23^rd-day-old tomato (Solanum lycopersicum cv. Micro-Tom) were used for expressing CRISPR-Cas9 transiently. The agrobacterium harboring sgRNA for targeting ALS2 gene of tomato was injected through the stomata of leaf adaxial side and the genomic DNA was extracted in 5 days after injection. The target gene edition was identified by amplifying DNA fragment of target region and analyzing with Illumina sequencing method. The target gene editing efficiency was calculated by counting base deletion and insertion events from total target sequence read. CONCLUSION: The CRISPR-Cas9 editing efficiency varied with tomato cotyledon age. The highest efficiency was observed at the 19-day-old cotyledons. Both the median and mean were the highest at this stage and the sample variability was also minimized. We found that the transgene of CRISPR-Cas9 system was strongly correlated with plant leaf development and suggested the optimum cotyledon leaf age for Agrobacterium-mediated transfection in tomato.

Transformation of Wheat Immature Embryos by Particle Bombardment

Wu Li-Min,Wei Yu-Ming,Zheng You-Liang The Korean Society of Plant Biotechnology 2005 Plant molecular biology and biotechnology research Vol.7 No.2

The objective of this study was to identify the major parameters controlling DNA delivery by particle bombardment to immature embryos of Chinese spring wheat (Triticum aestivum L.). Efficiency of DNA (uidA gene) delivery was assessed by transient GUS (${\beta}$-glucuronidase) expression in bombarded tissues. Of the parameters analyzed, acceleration pressure, bombardment distance, chamber vacuum pressure, bombardment times, osmotic conditioning of culture had a remarkable influence on transient gene expression. A bombardment procedure suitable for Chinese spring wheat cultivars was developed which allowed high-efficiency DNA delivery combined with reduced damage to target tissues. The high efficiency made the system practical for wheat genetic transformation research and accelerating wheat breeding programs.

Hijacking Tobacco Hairy Roots and Leaves in Order to Produce IpaD Antigen by Means of Different Signal Peptides

Shahram Shokrian Hajibehzad,Fariba Abooei Mehrizi,Hossein Honari,Houshang Alizadeh 한국작물학회 2017 Journal of crop science and biotechnology Vol.20 No.5

IpaA, IpaB, IpaC, and IpaD are Shigella dysenteriae Ipa operon genes which collectively contribute in invasion to the epithelial cells of the human gut. Among them, IpaD has been demonstrated to play the most crucial role in shigellosis. Noteworthy, due to the more efficient, cost-effective and no need for advanced equipment in comparison with traditional systems, plant-based expression systems are considered as a novel strategy for production of recombinant proteins. As an aim of this research, attempts were carried out to examine and compare IpaD antigen production in three different plant-based platforms, including transgenic tobacco hairy roots and leaves as well as a transient based expression. Furthermore, different signal peptides (i.e. Zera and Extensin) were also employed in order to improve the production level. Based on TAS-ELISA result, the highest yield of IpaD acquired by ER-derived protein bodies (Zera ) which was more than 1.29-fold higher as compared with apoplastic space based on TSP% in both transgenic tobacco hairy roots and leaves. Furthermore, transgenic tobacco hairy roots were more abundant than transgenic leaves averaging 0.52 ng of IpaD per μg TSP and with a maximum of 0.94 ng IpaD per μg TSP in the best-performing construct of pBI-ZeCIpaD. Totally, the results of quantitative RT-PCR and TAS-ELISA indicated that the best time point for the production of IpaD using agroinfiltration was 72 h post infiltration and during 72 to 96 hpi, expression levels descended rapidly. To the best of our knowledge, this is the first report representing and combining the potential effects of signal peptides and plant-based expression platforms on stably production of IpaD antigen in transgenic tobacco leaves and hairy roots.

누에 핵다각체병 바이러스 gp64 유전자의 특성조사 및 transient 발현 벡터 개발

김미향,최재영,우수동,이해광,제연호 한국산업미생물학회 2001 한국미생물·생명공학회지 Vol.29 No.1

누에 핵다각체병 바이러스의 gp64 프로모터를 이용한 transient 발현 벡터를 제작하기 위해서 gp64 유전자의 구조를 분석하였다. Southern blotting 분석을 통해 genome DNA에서 gp64 유전자를 탐색하고 gp64 구조유전자를 포함하는 2,277 nucleotide의 염기를 분석하였으며 gp64의 early, late 프로모터 발현을 조절하는 인자들을 확인하였다. gp64프로모터를 이용한 transient 발현 벡터를 제작하고 외래유전자로써 lacZ 유전자를 Bm5 세포주에서 transient 발현시켰다. gp64 프로모터의 외래유전자 발현성 여부를 조사하기 위하여 gp64 프로모터 하에 lacZ 유전자를 가지는 재조합 바이러스를 제작하고 β-galactosidase in situ staining을 수행한 결과 전체적인 발현량은 매우 약한 것으로 판단되었다. BmNPV-K1의 gp64 프로모터를 이용한 벡터는 더욱 민감한 표지 유전자를 발현시켜 재조합 바이러스의 분리에 이용하거나 숙주세포에 독성을 보이는 유전자 산물의 소량 발현에 더욱 유용할 것으로 판단된다. Expression of the baculovirus major envelope glycoprotein gene (gp64) is regulated by transcription from both early and late promoters. To develop a transient expression vector under the control of gp64 gene promoter, the gp64 gene of Bombyx mori nucleopolyhedrovirus-K1 (BmNPV-K1) was characterized. The gp64 gene was localized at EcoR I-Pst I 7.38-kb fragment of the BmNPV-K1 genome. The EcoR I-Pst I 7.38-kb fragment was cloned and the nucleotide sequence of 2,277 bases including the coding region of gp64 gene was determined. Based on these results, transient expression vector using gp64 gene promoter was constructed and named as pBm64. E. coli lacZ gene was introduced into pBm64 as a reporter gene and expressed transiently in B. mori 5 (Bm5) cells. The expression vector transfected into the cells was maintained stably for 1 to 5 days. In order to confirm the expression of the reporter gene by gp64 promoter, recombinant virus was constructed. The recombinant virus has two independent transcription units in opposite orientations with two promoters; gp64 and polyhedrin gene promoters each initiating transcription of β-galactosidase and polyhedrin, respectively. Polyhedra formation and expression of β-galactosidase in Bm5 cells infected with the recombinant virus were observed with phase contrast microscope and in situ staining.

Preparation of Leaf Mesophyll Protoplasts for Transient Gene Expression in Brachypodium distachyon

Shin-Young Hong,서필준,Shin-Hae Cho,박충모 한국식물학회 2012 Journal of Plant Biology Vol.55 No.5

Transient gene expression systems using protoplasts have been widely used for rapid functional characterization of genes in many plant species. Brachypodium distachyon has recently been employed as a model plant for studies on biofuel grass species and grass crops because of its small genome size, short life-span, and availability of efficient transformation systems. Here, we report the an efficient protocol for the preparation of leaf mesophyll protoplasts from Brachypodium seedlings. We also modified the polyethylene glycol (PEG)-mediated transformation procedure to optimize experimental conditions, such as duration of enzyme digestion, PEG incubation time, and plasmid DNA concentration and size. The green fluorescence protein (GFP)- and β-glucuronidase (GUS)-coding genes were used as reporters to evaluate the feasibility of this transient expression system. We found that the yield of viable protoplasts was highest after 3 h of enzymatic digestion. Viability of enzyme-digested protoplasts was moderately maintained up to 24 h in Mmg preincubation solution. In addition, the highest transient expression of reporter genes was obtained when protoplasts were transformed with 20 μg of plasmid DNA and incubated for 16 h. Together with the recent completion of the Brachypodium genome sequence,the Brachypodium transient expression system using leaf mesophyll protoplasts can be widely used for cellular,molecular, and biochemical studies of genes involved in carbon metabolism and signaling pathways mediating intrinsic and environmental cues.

Transient gene expression system in zoysiagrass leaf mesophyll protoplasts

Kim Jin Hee,Doan Phan Phuong Thao,이효연,김정식 한국식물생명공학회 2022 Plant biotechnology reports Vol.16 No.1

Transient expression of genes in protoplasts is a versatile technique for rapid functional characterization of genes by assessing protein localization or effector–reporter responses. In addition, protoplasts have been widely used for generating transgenic or gene-edited plants in model or crop plants. Zoysiagrass (Zoysia japonica Steud.) is one of the most economically important turf plants used in many living places or natural fields, but its management is labor-intensive. Therefore, genetic manipulation using valuable genes is highly demanded in zoysiagrass. Although transient expression systems in zoysiagrass facilitates the identification of valuable zoysiagrass genes, transient expression use in the zoysiagrass protoplasts is yet to be reported. Here we describe the methodology and feasibility for transient expression of genes in the zoysiagrass protoplasts isolated from green leaves. We obtained more than 70% of transfection efficiency in the zoysiagrass protoplasts using polyethylene glycol- mediated transfection. Additionally, we showed the feasibility of cellular, biochemical, and molecular approaches using the zoysiagrass protoplasts transiently expressing genes of interest through fluorescent microscopic observation, immunoblot analysis, and luciferase-based promoter activity assay. Along with the genome draft of the Zoysia family, transient expres- sion will be a valuable tool to rapidly investigate gene functions. An initial assessment of gene function using protoplasts facilitate the identification of valuable genes that can improve the economic value of the Z. japonica or its closely related species by gene manipulation.