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Confirmation of Green Fluorescent Protein Transformants using PCR
전선영 ( Sunyoung Jeon ),( Denver Walitang ),( Shamim Amed ),( Aritra Roy Choudhury ),사동민 ( Tongmin Sa ) 한국환경농학회 2017 한국환경농학회 학술대회집 Vol.2017 No.-
Plant growth promoting bacteria (PGPB) have beneficial effects on plant growth via an enhancement of the nutrient status of their host plant. The means by PGPB enhance the nutrient status of host plants can be categorized into five areas: biological N2 fixation, increasing the availability of nutrients in the rhizosphere, inducing increases in root surface area, enhancing other beneficial symbioses of the host, and combination of modes of action. These bacteria have plant growth promoting traits such as 1-aminocyclopropane-1-carboxylate (ACC) deaminase, indole acetic acid (IAA) production, salicyclic acid, phosphate solublization, siderophore production. In endophytic relationships, PGPB actually reside inside the host plant. PGPB maybe found in the surface of the root, stems or leaves. To find the location of PGPB in plant, Green Fluorescent Protein from jellyfish Aequoria victoria used as a valuable molecular marker which can be expressed in many organisms. GFP transformants emitting fluorescence were suitable for studying plant colonization under environmental condition. This study aims to assess the process of transformation of GFP insert into bacteria by PCR amplification. For the development of gfp mutant, triparental mating was carried out in Pseudomonas frederiksbergensis OB145, Pseudomonas umsongensis OB243, Pseudomonas umsongensis OB133, Pseudomonas moorei OR108, Pantoea sp CBMB55, Brevibacterium iodium RS16 and Burkholderia pyrrocinia CBPB-HOD using donor E.coli strain pFAJ1820 and helper E.coli strain pRK2013. Antibiotic resistance is used as a selection marker to ensure that bacteria in a culture contain a particular plasmid. Transfer of plasmid pFAJ1820 into these strains was confirmed through PCR amplification with specific primer. Band had been confirmed by using Gel-doc. In this study, plant growth promoting bacteria were successfully checked for GFP transformation. Confocal microscopy will be used to confirm effective colonization and intercellular localization of bacteria strains in plants.
( Sandipan Samaddar ),김기윤 ( Kiyoon Kim ),( Poulami Chatterjee ),전선영 ( Sunyoung Jeon ),사동민 ( Tongmin Sa ) 한국환경농학회 2017 한국환경농학회 학술대회집 Vol.2017 No.-
Ammonia oxidation is the first and rate-limiting step in nitrification, the oxidation of ammonia to nitrate via nitrite. However, we know little about archaeal population under field conditions, or how their relative contributions to soil nitrification respond to the combination of cropping treatment and seasonal conditions. Archaea especially Ammonia oxidizing archaea are responsible for the rate limiting step in nitrification; a key nitrogen (N) loss pathway in agricultural systems. Among the factors reported to influence the abundance and diversity of archaea are fertilizers (ammonium addition).The main objective of this study is to investigate the abundance and diversity of archaeal community especially nitrifying archaea in paddy fields under five different treatments (No fertilizer (CON), Chemical Fertilizer (CF), Compost (CO), Chemical fertilizer along with Compost (FCO), P & K [N free] (NFR) and how it is correlated to physicochemical parameters and nitrification potential in soil. Soil samples were collected from paddy fields of Department of Southern Area Crop Science, Paddy Crop research Division, Miryang, Republic of Korea and were used to characterize the abundance and diversity of ammonia oxidizing archaeal community based on ammonia monooxygenase gene (amoA). Soil enzyme activities, chemical parameters and nitrification potential were determined in laboratory using standard procedures. Soil nucleic acid was extracted from 0.25 grams of soil using PowerSoil DNA isolation kit (MoBIO, Carlsbad, CA, USA) following the manufacturer`s protocol. Quantitative PCR (qPCR) reactions were performed on a Rotor-Gene 6000 real-time rotary qPCR analyzer (Corbett Research) using SYBR Green PCR master mix (Applied Biosystems). Pyrosequencng of amoA gene was performed on a 454 GS FLX Platform by Macrogen, South Korea. Results revealed the treatments lead to considerable changes in community structure of nitrifying archaea and lead to separate taxonomical placement in a reference tree. The soil factor organic matter was found to contribute significantly for shaping this change in community structure.