[보고서]식물의 생체시계 조절과 저온내성간의 상호 작용 연구

김외연

보고서 정보

주관연구기관

경상대학교
GyeongSang National University

연구책임자

김외연

참여연구자

이성신 , 이다연 , 허진성 , 정송이 , 신경임 , 지명근 , 최홍민 , 구성순 , 성현정 , 김진아 , 랍 맥클링 , 데이비드 소머즈 , 김연정 , 정미정 , 이수인

보고서유형

최종보고서

발행국가

대한민국

언어

한국어

발행년월

2018-02

과제시작연도

2017

주관부처

농촌진흥청
Rural Development Administration(RDA)

등록번호

TRKO201800043291

과제고유번호

1395050216

사업명

차세대바이오그린21

DB 구축일자

2018-12-15

키워드

저온 저항성.생체시계.애기장대.배추.Cold tolerance.Circadian clock.Arabidopsis.Brassica.

DOI

https://doi.org/10.23000/TRKO201800043291

표 열충격 단백질에 의한 생체시계 조절
표 식물체의 염스트레스 저항성 기작 및 GI 단백질의 내염성 기작 조절 역할
표 생체시계 유전자를 조절하여 애기장대 냉해 저항성 획득 전략
표 TOC1과 ABAR 사이의 상호작용과 조절과정에서 ABA와 생체시계의 영향
표 추위에 의한 CBF1/2/3의 전사 유도과정에서 생체시계의 조절
표 생체시계와 면역반응간의 crosstalk 모델
표 열충격 스트레스와 빛 신호체계의 공동 작용 모델
표 Model of CCG regulation by PRR9, PRR7 and PRR5
표 애기장대와 배추의 생체리듬 기작 모식도
표 HOS15의 개화 조절 기작 A, 개화 표현형 사진; B, 각 hos15 돌연변이체 라인별 bolting시 잎의 개수
표 hos15 돌연변이체에서의 개화조절 유전자 발현 변화 A, Transcriptome analysis; B-E, 광주기 의존 개화 조절 유전자 CONSTANS (CO, B), SUPPRESSOR OF OVEREXPRESSION OF CO1(SOC1, C), FLOWERING LOCUS T (FT, D), GIGANTEA (GI, E)의 일주기성 발현 변화
표 HOS15 돌연변이체 (hos15-2)의 생체주기 변화 A, pCAB:LUC; B, pCCA1:LUC
표 개화 기작에 HOS15과 GI간 유전적 상관관계 A. 개화 표현형 사진; B, 잎 수를 통한 개화시기
표 저온 저항성 기작에 HOS15과 GI간 유전적 상관관계 A, 저온 저항성 표현형 사진; B, 저온 스트레스 후 생존율
표 HOS1 돌연변이체 (hos1-3)의 생체시계 주기 변화
표 생체주기 조절에서의 HOS15와 GI의 유전적 상관관계
표 HOS15 단백질이 GI 프로모터 상에 결합 후 deacetylation 반응을 유도함 A, Primary genomic structure of GI; B, LUX 항체를 이용한 ChiP; C, HOS15 항체를 이용한 ChiP; D, acetylated Histone H3를 이용한 ChiP
표 HOS15 단백질과 evening complex (ELF3-LUX) 간의 단백질 결합 확인 Yeast two hybrid(A), BiFC(B) 및 Co-IP(C-E)를 통한 HOS15-LUX-EC 복합체 형성
표 개화표현형을 통해 HDA homolog들 중 HOS15 파트너 탐색(A와 B) 및 HOS15과 HDA9간 유전적 상관관계(C와 D)
표 Co-IP(A)와 Chip(B)를 통한 HOS15과 HDA9의 상호작용 검증 A, Co-IP를 통한 HOS15-HDA9 결합 확인; B, ChiP 분석을 통한 각 돌연변이체내 HOS15의 GI 프로모터 결합 확인; C, Co-IP를 통한 HOS15-EC-HDA9 간 단백질 결합 확인
표 HOS15-EC repressor 복합체의 histone modification에 의한 GI 발현 조절
표 저온환경에서의 hos15 mutant의 개화 지연 insensitive 표현형
표 저온-유도 개화 표현형 관찰을 통한 광주기 개화 조절 인자들과 HOS15의 유전적 상관관계
표 저온환경에서 hos15 mutant의 생체시계 주기 변화
표 저온 순화 처리에 따른 hos15-2, gi-2, hos15-2 gi-2 double mutant의 냉해저항성 검증
표 저온에 의해 유도된 GI 단백질 분해
표 저온과 HOS15에 의한 GI 단백질 조절
표 HOS15와 GI의 단백질 결합 확인 A. 면역침강법 2. BiFC
표 배추 생체리듬 유전자 중 저온 저항성 기작 관련 유전자 발현 분석
표 BrGI-RNAi 형질전환체 내 BrGI 유전자 발현 정량 분석
표 BrGI-RNAi 형질전환체의 염 스트레스에 대한 저항성 검정 수경재배에서 형질전환체의 생존율(A), 염저항성 배추의 표현형(B)
표 BrGI-RNAi 형질전환체의 염 스트레스에 대한 저항성 검정 염저항성 결구배추 및 GI 유전자 결핍 유지형 배추의 배지내에서의 발아율(A), 및 광합성율 분석(B)
표 저온 순화 안한 결구 배추 및 형질전환체의 내동성 검정 (좌) 온도 변화에 따른 EC 값 (우) 통계 처리한 EL50
표 5일 저온 순화 후 결구 배추 및 형질전환체의 내동성 검정 (좌) 온도 변화에 따른 EC 값 (우) 통계 처리한 EL50
표 동해 스트레스 실험 조건 및 샘플링 방법
표 배추 RNAseq reads의 통계 분석
표 샘플간의 clustering 결과
표 대조구와 GI 발현조절 배추의 저온 조건 반응 유전자 발현 비교 (MA plots)
표 저온처리 후 시간경과에 따른 유전자 발현 변화 비교 (MA plots) (위) 대조구(야생형)의 저온처리와 비처리간 비교 (아래) GI 발현조절 배추의 저온처리와 비처리간 비교
표 저온 처리 시간대별 대조구와 GK 배추간 발현 차이를 보이는 유전자의 개수
표 저온 처리 시간대별 대조구와 GK 배추간 발현 차이를 보이는 유전자 선발
표 CCA1 과발현 벡터 형질전환 배추 재분화(왼쪽) 및 온실 순환(오른쪽)
표 형질전환 배추의 CCA1 유전자 삽입 분석 및 발현량 분석
표 Luminescence analysis of PRR7pro::PRR7-LUC, PRR7::PRR7-LUC, and 35S::PRR7-LUC in N. benthamiana leaves. Luminescence signals of PRR7pro::PRR7- LUC, PRR7::PRR7-LUC, and 35S::PRR7-LUC were analyzed 3 days after Agrobacterium infiltration
표 Luminescence analysis of PRR7::PRR7-LUC and 35S::PRR7-LUC in T1 transgenic plants in constant red light condition. Luminescence signals of PRR7::PRR7- LUC/Col-0 and 35S::PRR7-LUC/Col-0 were analyzed under continuous red light condition to confirm circadian oscillation of PRR7-LUC. A, Seven PRR7::PRR7-LUC/ Col-0 T1 seedlings selected from first screening. B, Three PRR7::PRR7-LUC/Col-0, six 35S::PRR7-LUC/Col-0, and one PRR7::PRR7-LUC/prr7-11 obtained from second screening. C, Five PRR7::PRR7-LUC/Col-0 T1 seedlings selected from third screening WTP7LUC, PRR7::PRR7-LUC/Col-0; WT35SLUC, 35S::PRR7-LUC/Col-0; P7LUC, PRR7::PRR7-LUC/prr7-11
표 PRR7-LUC expression in 28-days old PRR7::PRR7-LUC/Col-0, 35S::PRR7-LUC/Col-0, and PRR7::PRR7-LUC/prr7-11 T1 transgenic plants grown in 12L:12D condition. Total proteins were extracted from 28-days old T1 transgenic plants at ZT10, and PRR7-LUC proteins were detected by anti-LUC. ADK protein was used as a loading control. Representative luciferase and PRR7-LUC proteins were indicated by red arrows. WTP7LUC, PRR7::PRR7-LUC/Col-0; WT35SLUC, 35S::PRR7-LUC/Col-0 ; P7LUC, PRR7::PRR7-LUC/prr7-11
표 PRR7::PRR7-LUC/Col-0 H2 and H4 T3 progeny test. Individual T3 lines of PRR7::PRR7-LUC/Col-0 H2 and H4 were subjected to luminescence analysis at ZT9 to determine homozygosity. Homozygous lines are indicated by rectangles
표 Cycling pattern of PRR7-LUC protein abundance in PRR7::PRR7-LUC/Col-0 H4. Protein abundances of luciferase and PRR7-LUC were analyzed by anti-LUC immunoblotting after TCA protein precipitation. PRR7-LUC is indicated by a blue arrow showing a cycling pattern consistent with luminescence signals. Red and blue arrows indicate PRR7-LUC and luciferase, respectively. ADK protein was used as a loading control. A, short exposed image of immunoblot; B, long exposed image of immunoblot; C, relative luminescence intensity obtained by image analysis
표 The selected EMS-mutagenized PRR7::PRR7-LUC/Col-0 showing increased PRR7-LUC signal. The luminescence signals of PRR7::PRR7-LUC/Col-0 M2 seedlings were analyzed under constant red and blue light at 22℃
표 Interaction between HOS1 and PRR7 in planta. Transient expression in N. benthamiana of Arabidopsis PRRn-GFP proteins and HOS1-HA and the interaction between PRRn-GFP and HOS1-HA were analyzed co-immunoprecipitation followed by immunoblot. Two trials showed similar results
표 Interaction between HOS15 and PRR7 in planta. Transient expression in N. benthamiana of Arabidopsis PRRn-GFP proteins and HOS15-HA and the interaction between PRRn-GFP and HOS15-HA were analyzed co-immunoprecipitation followed by immunoblot. (그림 7 and 8 data from WY Kim lab)
표 Diurnal cycle of PRR7 expression in Col-0 (WT), hos1 and hos15 at 22℃. The expression of GFP/FLAG-PRR7 in WT, hos1 and hos15 mutant background grown under 12h/12h light/dark cycle at 22 ℃ were examined. ADK protein was used as a loading control for normalization
표 Diurnal expression analysis of PRR7 protein in hos1-3 entrained at 15℃. F7G/WT and F7G/hos1-3 were grown in 12L/12D at 22℃ for 8 days and transferred to growth chamber under 12L/12D 15℃ condition. After 4 days, the 12 days-old seedlings were collected from ZT 1 to ZT21 every 4 hours, and GFP-PRR7 protein levels were analyzed by western blot. Same experiments were performed with different segregated progenies. ADK was used to equally quantify the amount of input proteins
표 Diurnal expression analysis of PRR7 protein in hos15-2 entrained at 15℃. F7G/WT and F7G/hos15-2 were cultivated in 12L/12D at 22℃ for 8 days and transferred to 12L/12D 15℃. After 4 days, the 12 day-old seedlings were harvested from ZT 1 to ZT21 every 4 hours, and GFP-PRR7 protein levels were analyzed by western blot. Same experiments were performed with different segregated progenies. ADK was used to equally quantify the amount of input proteins. Bar graph indicates relative intensities of protein bands
표 Analysis of PRR7 protein stability in hos15-2 entrained at 22℃ in constant white light condition followed by CHX treatment. F7G/WT and F7G/hos15-2 were cultivated in 12L/12D at 22℃ for 8 days and transferred to 12L/12D 22℃. After 4 days, 12 day-old seedlings were transferred to liquid media, and CHX or ethanol was added to the media. The seedlings were collected 0, 1, 4, 7, 10 hours after CHX treatment in constant light condition at 22℃. ADK was used to equally quantify the amount of input proteins. Bar graph indicates relative intensities of protein bands
표 Analysis of PRR7 protein stability in hos15-2 entrained at 15℃ in constant white light followed by CHX treatment. F7G/WT and F7G/hos15-2 were cultivated in 12L/12D at 22℃ for 8 days and transferred to growth chamber under 12L/12D at 15℃ After 4 days, 12 day-old seedlings were transferred to liquid media, and CHX or ethanol was added to the media. The seedlings were collected 0, 1, 4, 7, 10 hours after CHX treatment in constant light condition at 15℃ PRR7 protein levels were analyzed by western blot. ADK was used to equally quantify the amount of input proteins. Same experiments were repeated three times. Bar graph indicates relative intensities of protein bands
표 Transcript level of PRR7 protein stability in hos15-2 entrained at 15℃. F7G/hos15-2 were cultivated under 12L/12D 22℃ for 8 days and transferred to 12L/12D 15℃. After 4 days, the 12 day-old seedlings were collected from ZT 1 to ZT21 every 4 hours, and PRR7 transcript levels were analyzed by quantitative real-time PCR. Experiments were performed twice
표 Analysis of PRR7 protein stability in hos15-2 entrained at 15℃ in darkness followed by CHX treatment. F7G/WT and F7G/hos15-2 were cultivated in 12L/12D at 22℃ for 8 days and transferred to 12L/12D at 15℃. After 4 days, 12 day-old seedlings were transferred to liquid media, and CHX or ethanol was added to the media. The seedlings were collected 0, 1, 4, 7, 10 hours after CHX treatment in constant dark at 15℃. ADK was used to quantify the amount of input proteins. Result represents an average of two biological replicates
표 Analysis of PRR7 protein stability in hos15-2 entrained at 22℃ in darkness followed by CHX treatment. F7G/WT and F7G/hos15-2 were cultivated in 12L/12D at 22℃ for 8 days and transferred to growth chamber under 12L/12D at 1 5℃. After 4 days, 12 day-old seedlings were transferred to liquid media, and CHX or ethanol was added to the media. The seedlings were collected 0, 1, 4, 7, 10 hours after CHX treatment in constant dark at 22℃. ADK was used to quantify the amount of input proteins
표 Expression analysis of PRR7 protein at extended night in hos15-2 entrained at 15℃. F7G/WT and F7G/hos15-2 were grown under 12L/12D 22℃ for 8 days and transferred to 12L/12D 15℃. After 4 days, the 12 day-old seedlings were collected at ZT13, ZT33, ZT37, and ZT41 in darkness
표 Diurnal transcript expression analysis of main clock genes in hos15-2 entrained at 15℃. Col-0 and hos15-2 was cultivated under 12L/12D 22℃ for 8 days and transferred to 12L/12D 15℃. After 4 days, the 12 days-old seedlings were collected from ZT 1 to ZT21 every 4 hours, and PRR7 protein levels were analyzed by western blot. Ubiquitin was used to normalize the amount of input RNA
표 Interaction test between HA-HOS1 and PRRn-GFP by coimmunoprecipitation in N. benthamiana. HA-HOS1 and PRRn-GFP were expressed by bacterial infiltration in N. benthamiana and those protein-protein interactions were analyzed by coimmunoprecipitation. Experiments were performed twice
표 Interaction test between HA-HOS15 and PRRn-GFP by coimmunoprecipitation in N. benthamiana. HA-HOS15 and PRRn-GFP were expressed by bacterial infiltration in N. benthamiana and those protein-protein interactions were analyzed by coimmunoprecipitation. Experiments were performed twice
표 Cold acclimation of B. rapa R500. Freezing tolerance, as determined by electrolyte leakage, increases with duration of exposure to 4°C and saturates after about 14 days
표 Genetic map of B. rapa with QTL for freezing tolerance (blue) and associated candidate gene(s) (red) and other cold related genes of interest (green)
표 Expression of candidate genes in FPsc and R500 in response to cold. Transcript accumulation was analyzed by quantitative RT-PCR. Plants were grown at 22°C for two weeks before transfer to 4°C at dawn (T=0). RNA was isolated from entire shoots in triplicate and three technical replicates were performed for each sample. Data (mean ± SEM) are expressed relative to the average of UBC11 and TFC-A mRNA abundances
표 Loss of GI function confers increased freezing tolerance. EL50 is the temperature at which 50% of electrolytes are lost(Xie et al. 2015)
표 Antisense knockdown of GI expression in B. rapa Chinese cabbage DB. Two individual lines derived from each of 4 independent transgenic insertion events are shown. Actin mRNA is a loading control
표 Freezing tolerance of B. rapa varieties as determined by electrolyte leakage. EL50 is the temperature at which 50% of electrolytes are lost. EL50 was determined following 5 or 14 days acclimation at 4°C
표 Expression analysis of Brassica rapa response to cold. Transcript accumulation was analyzed by quantitative RT-PCR. Plants were grown at 22°C for two weeks before transfer to 4°C at dawn (T=0). RNA was isolated from entire shoots in triplicate and three technical replicates were performed for each sample. Data (mean ± SEM ) are expressed relative to the average of UBC11 and TFC-A mRNA abundances
표 Overexpression of B. rapa CBF1 in Arabidopsis confer constitutive freezing tolerance. Two weeks old plants were transferred to -6ºC for up to 6 hours before being transferred back to 22°C for a week
표 Assembly metrics of the B. rapa R500 genome (de novo assembly) compared to those of the FPsc de novo assembly and the reference B. rapa Chiifu V1.5 genomes
$Circular map of B. rapa R500 de novo genome assembly. Outer ring indicate blocks of synteny with Arabidopsis. Blue indicates Least fractionated and red indicates Most Fractionated genomes. Second ring indicates gene density. Third rings indicate genes that exhibit circadian cycling in transcript abundance. Innermost blue lines indicate LTR density$ 표 Circular map of B. rapa R500 de novo genome assembly. Outer ring indicate blocks of synteny with Arabidopsis. Blue indicates Least fractionated and red indicates Most Fractionated genomes. Second ring indicates gene density. Third rings indicate genes that exhibit circadian cycling in transcript abundance. Innermost blue lines indicate LTR density
표 Experimental design for RNA-seq analysis of circadian transcriptome
표 Weighted Gene Co-Expression Network Analysis of Brassica rapa circadian transcriptome.Six most populous co-expression sets assorted according to circadian phase
표 Differential expression of clock gene paralogs following entrainment to either thermocycles (HC) or photocycles (LD). In each case, the paralog in the Least Fractionated genome is expressed at the highest level
표 Weighted gene co-expression network analysis reveals 10901 genes that can be sorted into multiple modules that differ on the basis of circadian phase of peak expression
표 Application of a second stringent filter, JTK_Cycle (see text), for rhythmicity defines a set of 6131 genes that exhibit robust circadian cycling of transcript abundance following photocycle entrainment
표 Experimental design for evaluation of early effects of mild drought treatment on Brassica rapa R500 plants (A) and soil water potential (B) during the experiment
표 Specific time-of-day differences in (A) Photosynthetic rate, A (mol C O 2 m - 2 s - 1 ) , ( B ) S t o m a t a l c o n d u c t a n c e , g s ( m molH2Om-2s-1,(C)MaximumefficiencyofPSIIinlightadaptedconditions,Fv’/Fm’, and (D) Non-Structural Carbohydrates (NSC) progression in well-watered (green symbols, solid lines) and mild droughted (orange symbols, dotted line) plants. Asterisks represent a significant difference (P < 0.01) between treatments
표 Variation in gene expression over a day is greater than the effects of mild drought. Hierarchical clustering was performed on the log2 FPKM values for the Day 4 time points for well-watered and droughted plants
표 Expansion of gene expression patterns uncovered in the droughted network. The wM5 module from the well-watered network (A) overlaps with dM1, dM5, and dM10 modules from the droughted network (B). The mean log2 FPKM expression values (shading shows SEM) for the genes in common between the wM5 module and each droughted module is shown. White and black bars along the x-axis represent the day and night time points, respectively
표 Dynamic changes in physiology are correlated with co-expression networks. (A and B) Correlations between module eigengenes and physiology measurements for well-watered (A) and droughted (B) plants. The numbers within the heat map represent correlations and P values (in parentheses; red, positively correlated, blue, negatively correlated) for the module – trait associations. (C) Scatterplots of gene significance versus module membership for the photosynthetic rate with dM1 (red), PSII efficiency with dM6 (blue), and stomatal conductance with dM13 (black). Significant correlations imply that hub genes within the module are also highly correlated with the physiology measure
표 Modules positively correlated with physiology are associated with metabolism and light harvesting processes. (A) Mean log2 FPKM expression profiles of genes in the dM1, dM5, and dM10 modules that are positively correlated with stomatal conductance and identified by JTK-CYCLE as having an amplitude change between well-watered and droughted samples. Grey lines are always well-watered. White and black bars along the x-axis represent the day and night time points, respectively. (B) Top 10 associated GO terms for dM1 (top), dM5 (middle), and dM10 (bottom). (C) Genes identified in the dM1, dM5, dM10, and dM19 modules with known Arabidopsis orthologs are shown in the network view. Nodes are colored based on module and size based on module membership. The larger nodes are highly connected within a module and have greater ‘hubness’. The nodes circled in purple were validated by Nanostring
표 Modules negatively correlated with physiology are associated with abiotic stress and light response. (A) Mean log2 FPKM expression profiles and of genes in the dM2, dM6, dM7, dM8, dM11, and dM20 modules that are negatively correlated with stomatal conductance and identified by JTK-CYCLE as having an amplitude change between well-watered and droughted samples. Grey lines are always well-watered. White and black bars along the x-axis represent the day and night time points, respectively. (B) Top 10 associated GO terms for dM2 (top), dM6 (middle), and dM11 (bottom). (C) Genes identified in the dM2, dM6, dM7, dM8, dM11, and dM20 modules with known Arabidopsis orthologs are shown in the network view. Nodes are colored based on module and size based on module membership. The larger nodes are highly connected within a module and have greater ‘hubness’. The nodes circled in magenta were validated by Nanostring

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식물의 생체시계 조절과 저온내성간의 상호 작용 연구
Studies on the relationship between circadian clock and cold tolerance in plant 원문보기

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식물의 생체시계 조절과 저온내성간의 상호 작용 연구 Studies on the relationship between circadian clock and cold tolerance in plant 원문보기

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식물의 생체시계 조절과 저온내성간의 상호 작용 연구
Studies on the relationship between circadian clock and cold tolerance in plant 원문보기

표/그림 (85)

표/그림 (85)