[보고서]식물의 뿌리체계 발달 조절 호르몬 시그널링 및 상호작용 네트워크의 시스템 분석

김정묵

식물의 뿌리체계 발달 조절 호르몬 시그널링 및 상호작용 네트워크의 시스템 분석
Systems analysis of hormone signaling networks regulating plant root architecture 원문보기

보고서 정보
주관연구기관	전남대학교 Chonnam National University
연구책임자	김정묵
참여연구자	김민정 , 강나영 , 김진선 , 전병욱 , 하세훈 , Nguyen Thu Hien
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2021-02
과제시작연도	2020
주관부처	농촌진흥청 Rural Development Administration(RDA)
등록번호	TRKO202100009749
과제고유번호	1395062942
사업명	차세대바이오그린21(R&D)
DB 구축일자	2021-09-18
키워드	식물뿌리체계.식물발달.식물호르몬.세포신호전달.유전자 조절네트워크.Plant root architecture.Plant development.Plant hormones.Cell signaling.Gene regulatory network.

초록 ▼

측근형성을 촉진하는 전사인자 LBD18의 1 차 반응 표적 유전자로 brassinosteroid hormone receptor BRI1의 homolog인 BRI1-LIKE1(BRL1) 및 BRI1-LIKE3(BRL3) 유전자 분리, RiD-BRL1/BAK1 기능획득 형질전환 식물체확보, BRL1 및 BRL3 유전자의 이중돌연변이체 제작 및 뿌리 표현형 분석 수행.결과를 종합하면 BRL1 및 BRL3 유전자는 측근의 형성과정에 있어서 양성적으로 발달을 촉진하는 역할 수행 확인. BRL1과 BRL3 유전자는 옥신 신호전달 과정 중 ARF7/ARF19 전사인자 하부에서 발현조절을 받는 유전자임. 옥신신호전달 과정 중 LBD18 단백질은 BRL1 및 BRL3 유전자 발현을 온전히 조절하는 유일한 인자는 아니지만 ARF7/ARF19-LBD18 전사인자 모듈을 경유하여 측근 형성에 관여하는 인자임. 또한, 각종 BR 신호전달 관련 유전자 돌연변이체 및 형질전환체의 측근의 표현형 분석결과 측근발달과정에서 BR 신호전달 경로 주요 구성성분들이 기능하는 것으로 추정됨. 그리고 뿌리조직에서 ABA에 의해 발현이 증가하는 HB 유전자군 발굴, HB 유전자의 기능상실 및 기능획득 돌연변이체 제작 및 ABA 반응에서의 뿌리 표현형 및 종자 발달 및 유묘생장 표현형 분석 수행. 결과 HB7과 HB21 유전자는 뿌리발달 중 측근형성의 양성조절자로, HB7, HB21, HB40 유전자는 종자발달 중 ABA 반응에서 발아억제과정의 음성조절자로 기능하는 것으로 확인하였음.

(출처 : 요약서 3p)

Abstract ▼

□ Purpose & Contents
The present research is aimed at understanding plant hormone signaling pathways involved in the control of plant root architecture by molecular systems analysis. The present study is focused on molecular and functional characterization of gene regulatory network of the ARF7/ARF19-LBD18 transcriptional module during lateral root formation in auxin response, signaling interactions between auxin/brassinosteroids, and gene regulatory network of ABA-mediated control of lateral root formation in Arabidopsis thaliana.

□ Results
BRI1-LIKE1 (BRL1) and BRI1-LIKE3 (BRL3), the homologs of BRASSINOSTEROID (BR) INSENSITIVE1 (BRI1), that act as BR receptors, were identified as positive regulators of lateral root formation. BRL1 and BRL3 were shown to be regulated downstream of ARF7/ARF19-LBD18 transcriptional module. Analyses of BR signaling mutants and gain-of-function transgenic plants indicated that BR signaling components play roles in lateral root formation. ABA-inducible HB genes in Arabidopsis roots were found to play roles in lateral root formation and ABA-mediated inhibition of seed germination.

□ Expected Contribution
• This research contributes to molecular systems understanding of plant hormone signaling involved in the control of plant root architecture.
• Genes identified in this study can be used as useful genetic resources for promoting root development and for crop control to improve plant yield.
• Further studies are necessary for filing patents and for more publications in high-profile SCI journals.

(출처 : SUMMARY 5p)

목차 Contents

표지 ... 1
제 출 문 ... 2
보고서 요약서 ... 3
국 문 요 약 문 ... 4
Summary ... 5
목차 ... 6
제 1 장 연구 개발 과제의 개요 ... 7
제1절 연구 개발 목적 ... 7
제2절 연구 개발의 필요성 ... 8
제3절 연구 개발 범위 ... 12
제 2 장 연구 수행 내용 및 결과 ... 12
제 3 장 목표달성도 및 관련분야 기여도 ... 64
제1절 목표대비 달성도 ... 64
제2절 정량적 성과(논문게재, 특허출원, 기타)를 기술 ... 65
제 4 장 연구 결과의 활용 계획 ... 67
제 5 장 연구 개발 결과의 보안 등급 ... 67
제 6 장 연구시설·장비종합정보시스템에등록한 연구시설·장비현황 ... 69
제 7 장 연구개발과제의 대표적 연구실적 ... 70
제 8 장 기타사항 ... 70
제 9 장 참고문헌 ... 71
끝페이지 ... 73

표/그림 (46)

표 A Schematic diagram for genome-wide analysis of genes regulated by LBD18 using microarray with Pro35S:HA:LBD18:GR/ lbd18 transgenic Arabidopsis
표 Summary of downstream genes regulated by LBD18 in Arabidopsis roots. Initiation indicates number of genes involved in lateral root initiation. Emergence indicates number of genes involved in lateral root emergence
표 Analysis of LR densities of wild-type and transgenic Arabidopsis expressing RiD-BRL1KD/BAK1KD with or without rapamycin treatment. Plants were grown vertically for 7 DAG, and primary root length and LR number were measured. LR densities were calculated by the numbers of LR divided by primary root length (#/cm). Bars indicate ±SE of three biological replicates
표 Genotyping of brl1, brl3, and bak1 mutants
표 Expression level of BRL1 and BRL3 genes in various loss-of-function mutants. (A) Gene structure of BRL1 and BRL3. Total RNA was isolated from Arabidopsis seedlings. Each RNA sample was subjected to RT-PCR (B) or RT-qPCR (C) analysis. ACTIN7 was used as a normalization control
표 Analysis of LR densities of wild-type and brl1-1 brl3-3 double mutants. Seven days-old plants were used in this assay. LR densities were calculated by the numbers of LR divided by primary root length (#/cm). Mean ±SD values were determined
표 Analysis of the LR phenotype of wild-type and transgenic Arabidopsis expressing RiD-BRL3KD/BAK1KD with or without rapamycin treatment. Bars indicate ±SE of three biological replicates
표 Generation of the brl1, brl3 single and double mutants. (A) Gene structure of BRL1 and BRL3. T-DNA tagging positions are indicated by triangle. (B) Genotyping of brl1, brl3 single mutants. (C) Genotyping of various brl1 brl3 double mutants. (D-E) Expression level of BRL1 and BRL3 genes in various loss-of-function mutants. Total RNA was isolated from Arabidopsis seedlings. Each RNA sample was subjected to RT-PCR analysis in single mutants (D) and double mutants (E). ACTIN7 was used as a normalization control
표 Genotyping of brl1, brl3, bak1 double and triple mutants
표 Analysis of LR densities of wild-type and brl1, brl3 single and double mutants. Plants were grown vertically for 8 DAG. Then primary root length and LR number were measured. LR densities were calculated by the numbers of LR divided by primary root length (#/cm). Bars indicate ±SE of three biological replicates
표 Dark-grown hypocotyl elongation of brl1, brl3 single and double mutants
표 Time-course expression of BRL1 and BRL3 genes in WT and arf7 arf19 double mutants in response to auxin. Seven 9-day-old seedlings were incubated with 20uM IAA, and total RNA was isolated. Each RNA sample was subjected to RTq-PCR analysis. Bars indicate ±SE of three biological replicates
표 Expression analysis of BRL1 and BRL3 in WT and lbd18 in response to auxin. 9-day-old seedlings were incubated with or without 20 uM IAA for 8 or 24 hours, respectively. Then total RNA was isolated. Each RNA sample was subjected to RT-qPCR analysis. Bars indicate ±SE of three biological replicates
표 GUS staining of ProLBD16:GUS and ProARF19:GUS transgenic Arabidopsis with GUS staining of ProLBD16:GUS and ProARF19:GUS transgenic Arabidopsis with the mock, BL(brassinolide), and IAA(Indole-3-acetic acid) treatments. Seedlings of 7 DAG were incubated with each concentration of phytohormone for 24 h and subjected to GUS staining
표 Analysis of LR phenotypes of wild-type, bri1-116, bzr1-1D, and bzr1-1D bri1-116 mutants. (A) Plants were grown vertically for 7 DAG and primary root length and LR number were measured. LR densities were calculated from the numbers of LR divided by primary root length (#/cm). Bars indicate ±SE of three biological replicates. (B) LR primordia densities of wild-type, bri1-116, bzr1-1D, and bzr1-1D bri1-116 mutants. LR primordia per root length (#/cm) were measured at given developmental stage before the emergence of LRs. Mean ± SD values were determined from 20 seedlings
표 Analysis of LR phenotype for BL treatment. (A) Analysis of the LR phenotype for wild-type Arabidopsis with varying concentrations of BL. Plants were grown vertically for 7 DAG and primary root length and LR number was measured. LR densities were calculated by the numbers of LR divided by primary root length (#/cm). Mean ± SD values were determined from 20 seedlings. (B) LR densities of wild-type, bri1-116, bzr1-1D, and bzr1-1D bri1-116 mutants in the absence or presence of 1nM BL. Plants were grown vertically for 7 DAG and measured the primary root length and the numbers of LR. LR densities were measured by the numbers of LR per root length (#/cm). Bars indicate ±SE of three biological replicates
표 Analysis of LR densities of wild-type, bri1-116, bzr1-1D, and bzr1-1D bri1-116 mutants with 2 μM PPZ treatment. (A) Seven DAG seedlings of wild-type, bri1-116, bzr1-1D, and bzr1-1D bri1-116 mutants are shown to be treated with or without PPZ. (B) LR densities of wild-type, bri1-116, bzr1-1D, and bzr1-1D bri1-116 mutants in the absence or presence of 2 μM PPZ. Plants were grown vertically for 7 DAG and primary root length and LR number were measured. LR densities were calculated by the numbers of LR divided by primary root length (#/cm). Bars indicate ±SE of three biological replicates
표 Expression analysis of LBD16 and ARF19 in Col-0, bri1-116, bzr1-1d, bzr1-1d bri1-116 mutants and ProBZR1:CFP plants. Plants were grown vertically for 7 DAG, total RNAs were isolated from roots, and each RNA sample was subjected to RT-qPCR analysis. Bars indicate ±SD of three technical replicates
표 mRNA expression analysis of the wild-type, bzr1, and bzr2 T-DNA insertion mutants. Plants were grown for 7 days, total RNAs were isolated from seedlings, and each RNA sample was subjected to RT-PCR analysis
표 Expression analysis of LBD16 and ARF19 in Col-0, bzr1, and bzr2 T-DNA insertion mutants. Plants were grown vertically for 7 days, total RNAs were isolated from root sample, and each RNA sample was subjected to RT-qPCR analysis
표 Analysis of LR densities of wild-type, bzr1d-OX, bzr1DΔEAR, ProBZR1-bzr1D:CFP1, ProBZR1-bzr1D:CFP2, bzr1-1d, bzr1, bzr2-1, and bzr2-2 mutants. (A) Plants were grown on 1/2MS medium (mock) and 1/2MS medium with 2uM PPZ vertically for 7 days after germination (DAG) and measured the primary root length and the numbers of LR. LR densities were measured by the numbers of LR per root length (#/cm). Mean ± SD values were determined from 24 seedlings. (B) Plants were grown on 1/2MS medium (mock) and 1/2MS medium with 1nM BL vertically for 7 days after germination (DAG) and measured the primary root length and the numbers of LR. LR densities were measured by the numbers of LR per root length (#/cm). Mean ± SD values were determined from 24 seedlings
표 Genotyping of pin mutants
표 Genotyping of pin mutants
표 Expression analysis of LBD16 in Col-0 plant of various BL concentration(A) and various treatment time(B). Plant were grown vertically for 7 days, seedlings were treated by various BL concentrations and various time, total RNA were isolated from root sample, and each RNA sample was subjected to RT-qPCR analysis
표 Expression analysis of LBD16 (A) and ARF19 (B) in Col-0 and various bzr mutant plants with or without BL treatment for 2 h. Plant were grown vertically for 7 days, seedlings were treated by 10 nM BL for 2 h, total RNAs were isolated from root sample, and each RNA sample was subjected to RT-qPCR analysis
표 Genotyping of taa1 and wei8 mutants
표 Genotyping of taa1 bzr1 and wei8 bzr1 F1 mutants
표 Genotyping of pin bzr1-1d F2 mutants
표 Sequencing analysis of bzr1-1d mutation in pin bzr1-1d F2 mutants
표 Time-course expression of HB7 and HB12 genes in response to ABA. Seven-day-old wild type seedlings were incubated with 5 uM ABA, and total RNA was isolated from root sample. Each RNA sample was subjected to RT-qPCR analysis. RD29A was used as a marker gene for ABA response. Bars indicate ±SE of three biological replicates
표 GUS expression of ProHB12:GUS transgenic Arabidopsis. GUS expression of seedling, primary root tip, lateral root tip and the lateral root primordium at each stage of development
표 Expression analysis of LBD14, LBD37, LBD38, LBD39 in Pro35S:HB12 and Pro35S:HB12:SRDX transgenic Arabidopsis plants. Plants were grown vertically for 7 days, total RNAs were isolated from root sample, and each RNA sample was subjected to RT-qPCR analysis. Bars indicate ±SD of three technical replicates
표 Transient gene expression assay using ProLBD14(-1526):LUC reporter in Arabidopsis mesophyll protoplasts (A) or root protoplasts (B)
표 List of Arabidopsis athb mutants isolated and confirmed for T-DNA tagging. The athb mutants were selected based on the expression of HB genes in the root in response to ABA
표 Genotyping of athb mutants
표 Genotyping of athb mutants
표 Genotyping of athb21 mutants
표 Genotyping of athb40 mutants
표 Expression levels of HB6, HB7, HB21, and HB40 in loss-of-function mutants. (A) Scheme of gene structures of HB6, HB7, HB21, and HB40. Total RNA was isolated from Arabidopsis roots. Each RNA sample was subjected to RT-PCR. ACTIN7 was used as a normalization control
표 Analysis of LR densities of wild-type and hb mutants in the presence or absence of ABA. Arabidopsis plants were grown on 1/2MS vertical plate for 3 days, then transferred to 1/2MS agar plate containing DMSO or different concentrations of ABA. LR densities were calculated by the numbers of LR divided by primary root length (#/cm). Mean ± SE values were determined at three biological replicates
표 Dose-dependent effects of ABA on seed germination of wild-type and various hb mutants. Bars indicate ±SE of three biological replicates
표 Analysis of LR densities of hb double mutants in response to ABA. Arabidopsis plants were grown on 1/2MS vertical plate for 3 days, then transferred to 1/2MS containing DMSO or different concentrations of ABA. Bars indicate ± SD
표 Seed dormancy analysis of the wild-type and hb7, hb21, and hb40 single mutants. Germination efficiency were examined in wild-type and hb single mutants with stratification for 4 days (A) and 0 days (B). Error bars indicate ±SE of three biological replicates
표 Post-germination growth assays of the wild-type and hb7, hb21, and hb40 mutants. The rate of green cotyledon areas were calculated after 7 days growing on 1/2MS supplemented with or without ABA. Bars indicate ±SE of three biological replicates
표 PCR analysis of three transgenic Arabidopsis expressing HB7, HB21, and HB40 driven by the CaMV 35S promoter
표 Seed phenotypes of Pro35S:HB40 transgenic Arabidopsis T2 generation. Picture showing testa rupture after imbibition in Pro35S:HB40 seeds (upper panel) and young seedlings grown on 1/2MS for 3 days (lower panel)

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연합인증

식물의 뿌리체계 발달 조절 호르몬 시그널링 및 상호작용 네트워크의 시스템 분석
Systems analysis of hormone signaling networks regulating plant root architecture 원문보기