등검은말벌과 꿀벌의 장내 세균 군집 비교

김의연 ( Euyeon Kim ),서정원 ( Jeongwon Seo ),양소희 ( So Hee Yang ),김인선 ( In-seon Kim ),구연종 ( Yeonjong Koo ) 한국환경농학회 2018 한국환경농학회지 Vol.37 No.2

한국에서 말벌은 양봉 산업에 큰 영향을 미치는 종이다. 특히 비교적 최근에 전국적으로 번식하는 말벌 종인 등검은 말벌 (Vespa velutina nigrithorax)은 작은 체구에도 불구하고 높은 사냥 능력으로 꿀벌을 공격하여 양봉 업계에 큰 피해를 주고 있다. 그러나 이 새로운 침입 종에 대한 연구는 아직 시작단계에 있으며, 본 연구에서는 등검은말벌의 장내 박테리아 군집을 조사하고 이 결과를 꿀벌의 장내 박테리아 군집과 비교하여 등검은말벌의 방제를 위한 자료로 이용하고자 했다. 아시아 말벌과 꿀벌 장의 박테리아 게놈 DNA를 수집하여 16S rDNA를 증폭시켜 가변 부위인 V3, V4 염기서열을 판독 하였다. 계통별 분석결과 목 (order) 수준에서 Flavobacteriales는 등검은말벌에서 가장 우점인 박테리아였고, Aeromonadales와 Pseudomonadales가 그 뒤를 이었다. Flavobacteriaes는 꿀벌의 박테리아 군집과 비교했을 때 등검은말벌에서 증가하였으며, Aeromonadales와 Pseudomonadales는 우점종이지만 꿀벌에 비해 낮은 점유율을 보이는 등 두 개체 간의 차이를 확인할 수 있었다. 한편 이번 연구를 통해 이전에 동정되지 않은 등검은말벌 장 박테리아의 16S rDNA염기 서열을 분석하였고, 이들 박테리아는 Thalassomonas, Caedobacter, Vampirovibrio, Alkaliphilus 및 Calothrix 속으로 분류되었다. BACKGROUND: The Asian hornet (Vespa velutina nigrithorax), a wasp species, has attacked honey bee populations and affected the beekeeping industry in Korea over the past 15 years. However, little research has been done with this invasive species. In this study, we investigated the intestine bacterial microbiota of Asian hornets and honey bees to design an attractive trap for Asian hornets. METHODS AND RESULTS: Genomic DNAs isolated from the intestine microorganisms of Asian hornets and honey bees were utilized to amplify bacterial 16S rDNA for the comparative sequence analysis. The next generation sequencing analysis identified that the orders Flavobacteriales as the most abundant intestinal microorganisms in Asian hornets, showing a clear difference compared to honey bees in which Aeromonadales are dominant. We also report five newly identified 16S rDNA sequences of Asian hornet intestinal bacteria. According to the sequence blast search, these five bacteria belong to the genera Thalassomonas, Caedobacter, Vampirovibrio, Alkaliphilus and Calothrix. CONCLUSION: While Asian hornets and honey bees show similar intestine bacterial diversity, the relative ratio of bacterial populations is different. providing useful information to design pest control agents specifically targeting Asian hornets.

CRISPR-Cas9 시스템을 활용한 제초제 저항성 토마토 개발 전략

김의연 ( Euyeon Kim ),박효선 ( Hyosun Park ),양소희 ( Sohee Yang ),구연종 ( Yeonjong Koo ) 한국환경농학회 2020 한국환경농학회 학술대회집 Vol.2020 No.-

The development of herbicide-tolerant plants provided a convenient farming method. The five most popular herbicides are divided into glyphosate,sulfonylureas/imidazolinones, glufosinate, norflurasone and oxyfluorfen depending on the reaction target by their mode of action and these herbicides inhibit 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), acetohydroxyacid synthase (AHAS), glutamine synthetase (GS), Phytoene desaturase (PDS) and protoporphyrinogen oxidase (PPO), respectively. Inhibition of EPSPS and AHAS reduces the biosynthesis of phenolic and branched amino acids, respectively, and inhibition of GS and PDS enhances the production of reactive oxygen species and induces plant necrosis. Inhibition of PPO decreases chlorophyll biosynthesis and inhibits plant photosynthesis. These herbicides induce plant death by interacting with their target proteins and the development of herbicide resistant plants is based on the discovery of mutant proteins insensitive to these herbicides. We reviewed the development of herbicide-tolerant plants and derived target amino acids for the production of herbicide-tolerant proteins using the CRISPR/Cas9 system in tomatoes.

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.

군유전체학을 활용한 후코이단 반응 토양 미생물 분리 및 식물생장촉진 활성 규명

양소희 ( Sohee Yang ),박효선 ( Hyosun Park ),김의연 ( Euyeon Kim ),구연종 ( Yeonjong Ko ) 한국환경농학회 2020 한국환경농학회 학술대회집 Vol.2020 No.-

Seaweed and seaweed extract have been studied and developed as a crop fertilizer because they have various plant growth promoting compounds and antibacterial reagents. To use seaweed as fertilizer, the major carbohydrates of seaweed, fucoidan and alginate, should be digested efficiently in the soil. In this paper, we looked at changes in the bacterial community by adding fucoidan and alginate. With 16s ribosomal DNA analysis, bacteria that was either fucoidan and alginate resistant (increasing their number) or susceptible (decreasing their number) were selected successively from paddy soil. Most of the bacteria that increased their number with fucoidan and alginate had specificity to each carbohydrate, while the decreasing bacteria showed susceptibility to both carbohydrates. This report found some useful bacteria to apply as a biofertilizer with seaweed and also demonstrated that a high throughput approach is an efficient method to find bacteria with specific properties

후숙 조절 유전자 Pectate lyase와 Phytoene Synthase 편집용 CRISPR-Cas9 sgRNA의 유전자 편집 효율 측정

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

BACKGROUND: Tomato genome editing using CRISPR-Cas9 is being actively conducted in recent days, and lots of plant researches have been aiming to develop high valued crops by editing target genes without inserting foreign genes. Many researchers have been involved in the manipulation of the crop ripening process because fruit ripening is an important fruit phenotype for increasing fruit shelf life, taste, and texture of crops. This paper intends to evaluate target sgRNA to edit the two ripening-related genes encoding pectate lyase (PL) and phytoene synthase (Psy) with the CRISPR-Cas9 system. METHODS AND RESULTS: The CRISPR-Cas9 expression vector was cloned to target the PL (Solyc03g111690), Psy1 (Solyc03g031860), and Psy2 (Solyc02g081330) genes, which are the ripening genes of tomatoes. Tomatoes injected with Agrobacterium containing the CRISPR-Cas9 expression vector were further cultured for 5 days and used to check gene editing efficiency. As a result of the target gene sequence analysis by the next generation sequencing method, gene editing efficiency was calculated, and the efficient target location was selected for the PL and Psy genes. CONCLUSION: Therefore, this study was aimed to establish target sgRNA data that could have higher efficiency of the CRISPR-Cas9 system to obtain the delayed ripening phenotype of tomato. The developed method and sgRNA information is expected to be utilized in the development of various crops to manage its ripening processes.