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방사선 유도 내염성 증진 사료용 옥수수 돌연변이체 특성 분석

조철오1, 김경화1, 최만수1, 전재범1, 서미숙1, 정남희1, 진민아1, 손범영2, 김둘이1

Characterization of a Gamma Radiation-Induced Salt-Tolerant Silage Maize Mutant

Chuloh Cho1, Kyung Hwa Kim1, Man-Soo Choi1, Jaebuhm Chun1, Mi-Suk Seo1, Namhee Jeong1, Mina Jin1, Beom-Young Son2, Dool-Yi Kim1
Korean Journal of Breeding Science 2019;51(4):318-325.
Published online: December 1, 2019

1농촌진흥청 국립식량과학원 작물기초기반과

2중부작물과

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* Corresponding Author (E-mail: dykim22@korea.kr, Tel: +82-63-238-5323, Fax: +82-63-238-5305)
• Received: July 16, 2019   • Revised: September 16, 2019   • Accepted: September 16, 2019

Copyright © 2019 by the Korean Society of Breeding Science

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  • Salt stress is a significant factor limiting growth and productivity in crops. However, little is known about the response and resistance mechanism to salt stress in maize. The objective of this research was to develop an enhanced salt-tolerant silage maize by mutagenesis with gamma radiation. To generate gamma radiation-induced salt-tolerant silage maize, we irradiated a KS140 inbred line with 100 Gy gamma rays. Salt tolerance was determined by evaluating plant growth, morphological changes, and gene expression under NaCl stress. We screened 10 salt-tolerant maize inbred lines from 2,248 M2 mutant populations and selected a line showing better growth under salt stress conditions. The selected 140RS516 mutant exhibited improved seed germination and plant growth when compared with the wild-type under salt stress conditions. Enhanced salt tolerance of the 140RS516 mutant was attributed to higher stomatal conductance and proline content. Using whole-genome re-sequencing analysis, a total of 328 single nucleotide polymorphisms and insertions or deletions were identified in the 140RS516 mutant. We found that the expression of the genes involved in salt stress tolerance, ABP9, CIPK21, and CIPK31, was increased by salt stress in the 140RS516 mutant. Our results suggest that the 140RS516 mutant induced by gamma rays could be a good material for developing cultivars with salt tolerance in maize.
염분은 농업 생산성을 저해하는 가장 주요한 환경 스트레스 중 하나이다(Zhu 2001). 경작지에서 과도한 화학 비료의 사용과 환경 변화 등 다양한 요인에 의해 토양의 염화가 나타나며, 이는 지속 가능한 식량 생산을 위협하고 있다(Munns & Tester 2008, Ondrasek et al. 2011). 토양의 염화로 인해 전 세계적으로 전체 경작지의 약 20%와 관개 농지의 33%가 고농도의 염에 영향을 받고 있으며, 염화토양은 다양한 요인들에 의해 연간 10%의 비율로 증가되는 것으로 보인다(FAO 2015, Shrivastava & Kumar 2015). 또한 2050년경 경작할 수 있는 토지의 약 50% 이상이 염화토양이 될 것으로 추정된다(Jamil et al. 2011).
옥수수(Zea mays L.)는 전 세계적으로 가장 중요한 식량 및 사료작물 중 하나로써 전세계 식량 생산의 36%를 차지하고 있다(Yadav et al. 2019). 또한 벼, 밀과 함께 다양한 용도를 가지고 있으며, 환경 적응 범위가 넓어 다양한 곳에서 재배가 가능하다. 최근에는 바이오연료의 원료로써 옥수수의 중요성이 부각됨에 따라 재배면적이 더욱 증가할 것으로 예상된다(Edgerton 2009). 옥수수는 다양한 곳에서 재배가 가능하지만 염 스트레스에 민감하여 뿌리와 신초 생장이 각각 20%와 50% 이상 저해된다(Geilfus et al. 2010). 지속적으로 증가하고 있는 토양의 염화는 이러한 옥수수의 생산성에 심각한 위협이 될 수 있다(Ouda et al. 2008, Sugirtharan et al. 2008, Carpici et al. 2009). 따라서 염화토양에서 옥수수의 생산성을 증진시키기 위해서는 옥수수의 염 스트레스 반응에 대한 생리⋅생화학적 그리고 분자생물학적 메커니즘에 대한 연구가 필요하다.
염 스트레스는 식물의 수분 포텐셜 감소, 이온의 불균형, 기공의 상태 변화, 영양분의 흡수 감소, 광합성 효율 및 탄소 이용 변화를 포함하는 다양한 생리학적인 반응을 초래하여 작물의 생육 및 생산성을 감소시킨다(Brugnoil & Lauteri 1991, Carpici et al. 2010). 광합성에 의한 이산화탄소 동화 작용은 염분에 의해 감소되며, 이러한 동화 작용의 감소는 기공전도도의 감소로 인해 초래된다(Downton et al. 1985, Seemann & Critchley 1985). 식물체는 세포 내, 세포 및 기관 수준에서 기공 조절, 호르몬 균형의 변화, 항산화 방어 시스템의 활성화, 삼투압 조절, 조직 수분 함량의 유지 및 독성 이온 배제 메커니즘과 같은 다양한 적응과정을 거쳐 염 스트레스에 반응한다고 알려져 있다(Hichema et al. 2009, Schubert et al. 2009, Kaya et al. 2010, Farooq et al. 2015). 또한 식물은 염 스트레스에 반응하여 아미노산의 일종인 proline을 축적 시킴으로써 삼투압 조절, 세포 구조의 안정화 및 활성 산소의 제거를 통하여 염 스트레스에 대해 내성을 부여하며(Schobert & Tschesche 1978, Nanjo et al. 1999, Munns & Tester 2008), 스트레스 저항성 유전자의 발현 조절 및 세포 내 대사 물질들을 변화시킴으로써 염, 건조 및 저온을 포함하는 다양한 환경 스트레스에 반응한다(Gupta & Huang 2014). 애기장대에서 옥수수의 ABP9 과발현 식물체는 식물 호르몬 ABA 신호 전달과 세포 내 활성 산소를 조절함으로써 다양한 스트레스 내성을 보이며(Zhang et al. 2011), 목화에서 식물 호르몬 ABA 신호 전달 조절을 통해 염과 건조 스트레스에 내성을 증가시킨다고 알려져 있다(Wang et al. 2017). 또한 애기장대에서 칼슘 신호 전달과정에서 기능하는 CIPK21은 삼투와 염 스트레스에 반응하여 내성을 부여하며(Pandey et al. 2015), 야생잔디의 BdCIPK31은 기공 세포의 개폐, 이온의 항상성, 활성 산소 제거와 염 스트레스 관련 유전자들의 발현을 조절함으로써 건조와 염 스트레스에 내성을 부여하였다(Luo et al. 2017). 하지만 옥수수에서 염 스트레스 내성을 증가시키기 위한 연구 및 내염성 작물의 개발은 많이 보고되지 않고 있어 다양한 스크리닝을 통한 효과적인 내염성 육종소재 개발이 요구되고 있는 실정이다.
감마선은 실험의 용이성과 함께 식물에 높은 침투성을 가지고 있어 다른 이온화 방사선과 비교할 때 경제적이고 효과적으로 돌연변이를 유발할 수 있으며, 따라서 다양한 식물 육종에 사용되고 있다(Moussa 2006). 감마선 돌연변이 육종을 통해 참깨의 생육과 수확량에 영향을 미치는 역병 저항성 참깨 품종이 개발되었으며(Kumari et al. 2018), 스리랑카에서 320 Gy 감마선 조사를 통해 풋마름병 저항성 토마토 품종이 개발되었다(Peiris et al. 2008). 또한 감마선 돌연변이 육종을 통해 녹두, 밀, 콩 및 벼와 같은 다양한 작물에서 내병성 연구가 진행되어 있다(IAEA 2015). 하지만 옥수수에서 감마선을 이용한 환경 스트레스 내성에 관한 연구는 아직까지 보고된 바가 없다.
본 연구는 사료용 옥수수 종자에 감마선을 조사하여 내염성 옥수수 계통을 개발하고, 염 스트레스에 대한 내염성 옥수수의 형태학적, 생리학적 반응과 유전변이를 분석함으로써 옥수수의 염 스트레스 관련 기작을 규명하고자 하였다.
식물 재료 및 감마선 조사
본 연구에서는 사료용 옥수수 품종인 강다옥의 모본 KS140을 이용하였다. 감마선 조사는 한국원자력연구원의 60Co gamma irradiator (IR222; MDS Nordion Inc., Ottawa, CA) 시설을 이용하여 조사하였다. 내염성 옥수수 개발을 위한 돌연변이 집단을 육성하기 위해 약 3,000립의 KS140 종자를 petridish에 200립씩 나눠 담은 후 0, 100, 200, 300 Gy를 4시간 동안 실온에서 건조 종자에 직접 조사하였다.
감마선 유도 내염성 계통 선발
감마선 유도 내염성 계통 선발을 위해 감마선 0, 100, 200, 300 Gy를 조사한 종자를 파종하여 100 Gy를 조사한 집단은 자가 교배를 통해 세대를 진전하여 2,248개의 M2 집단을 확보한 반면 생육 상태가 불량한 200과 300 Gy가 조사된 집단은 종자를 확보하지 못하였다. 확보된 M2 집단을 자가 교배하여 M3 종자를 수확하였다. 내염성 계통 선발을 위해 50공 포트에 파종 후 0.7% NaCl을 처리하여 3주간 재배하였고 식물의 생육상태를 확인하여 내염성 증진 계통을 선발하였다. 내염성이 약한 계통들은 잎이 노랗게 변하는 반면 내염성이 강한 계통들은 3주 후에도 대조군과 비교하여 양호한 생육상태를 보였다.
종자 발아 및 간장 길이 측정
내염성 계통으로 선발된 140RS516 돌연변이체의 염 스트레스에 대한 발아 및 생육 정도를 조사하기 위해 대조군인 KS140과 내염성 계통 140RS516 종자를 하루 동안 물에 불린 후, 0, 0.1, 0.2, 0.3, 0.4, 0.5% NaCl이 첨가된 배지에 파종하였다. 종자 발아율 및 간장 길이 조사는 기내 파종 10일 후 측정하였다.
기공전도도 측정
광합성 시 기공세포에서 대기로의 수분 확산 속도를 측정하기 위해 대조군인 KS140과 내염성 계통 140RS516 종자를 온실에서 두 달간 육성하여 0.5% NaCl을 7일간 처리한 후 6~8엽을 이용하여 기공전도도를 측정하였다. 기공전도도는 휴대용 장치 SC-1 Leaf Porometer (ICT, New South Wales, AU)를 사용하여 잎의 뒷면에서 확산되는 수분을 측정하였다.
Proline 함량 측정
염 스트레스에 반응하여 식물에 축적되는 proline 함량을 조사하기 위해 대조군 KS140과 140RS516을 0.5% NaCl이 첨가된 배지(Plant agar 0.3%, NaCl 0.5%)에 파종 후 10일간 배양하였다. 염 스트레스 처리 후 식물 전체를 액체질소를 이용해 분쇄하였고, 분쇄된 시료 100 mg을 20% 에탄올 1ml로 균질화하여 14,000×g로 5분간 원심분리 하였다. 이 후 proline과 반응하여 파란색을 띄는 isatin을 도포한 여과지(Isatin 1 g, 100 ml methanol, 2.5 ml glacial acetic acid)에 상층액 10 ㎕를 떨어뜨린 후 상온에서 30분간 건조시켰다(Boctor 1971). 이 후 90°C 오븐에서 20분간 반응하여 proline의 염색 정도를 확인하였다. 염색의 정도를 확인하기 위해 L-Proline을 이용하여 표준 곡선을 작성하였고, ImageJ (NIH, Maryland, USA) 프로그램을 이용하여 염색의 강도를 정량하였다(Ábrahám et al. 2010).
염기서열재분석(Whole-genome re-sequencing)과 SNP 분석
유전변이 분석을 위해 전체 게놈 염기서열재분석(Whole-genome re-sequencing)과 단일염기다형성(SNP) 분석을 수행하였다. 대조군 KS140과 140RS516으로부터 DNeasy Plant Mini Kit (Qiagen, Hilden, DE)를 이용하여 DNA를 추출하였다. 추출된 DNA를 이용하여 Illumina HiSeq X 장비를 사용하여 염기서열을 분석하였고, 생산된 염기서열 정보로부터 low quality read와 중복 read를 Trimmomatic (version 0.38)을 이용하여 제거하였다. High-quality read들을 옥수수 inbred line B73의 표준 유전체 서열(http://plants.ensembl.org/Zea_mays/Info/Index, B73_RefGen_v4, database version 90.7)에 Burrows-Wheeler Aligner (BWA, version 0.7.17)을 이용하여 mapping하였다. 이후 mapping 결과를 SAMtools (version 1.9)을 이용하여 BAM 형식으로 변환하고, Picard package (version 1.112, http://broadinstitute.github.io/picard/)를 이용하여 중복된 read들을 제거하였다. Genome Analysis Toolkit (GATK, version 3.5)의 HaplotypeCaller module를 이용하여 변이를 선발하고 VCF 파일을 작성하였다. 이 파일로부터 유의미한 SNP를 min coverage, 5; max coverage, 250; min quality, 20; SNP ratio, >=0.8-0.9 parameter를 적용하여 선발하였다. KS140과 140RS516를 각각 표준 유전체(B73)에 맵핑하여 발굴한 유전형 결과를 최종 비교하여 KS140으로부터 발생한 140RS516의 유전변이를 탐색하였다.
내염성 반응 유전자 발현 분석
염 스트레스 처리 시 내염성과 관련된 유전자들의 발현 양상을 확인하기 위해 Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) 분석을 수행하였다. 염 스트레스 처리를 위해 KS140과 140RS516 돌연변이 식물체를 0.5% NaCl이 첨가된 배지에서 1일간 배양하였다. 이후 액체질소를 이용해 식물을 곱게 분쇄하였고, RNeasy Mini Kit (Qiagen, Hilden, DE)를 이용해 RNA를 추출하였다. 추출한 RNA는 NanoVue™ Plus (GE healthcare, Illinois, USA)를 이용하여 정량하였고, RNA to cDNA EcoDry™ Premix (Oligo dT) kit (Takara, Moriyama, JP)를 사용하여cDNA를 합성하였다. 유전자 발현 분석은 Supplementary Table 1에 표기된 프라이머를 사용하여 수행하였다.
Table 1
Summary of genetic variations in 140RS516 mutant line.
Table 1
Variant category Number of variants
5'-UTRz 3
Coding (Exonic) 4
Intronic 18
3'-UTR 1
Intergenic 302
Total 328
Nonsynonymous 3
Synonymous 1
Total Coding 4

zUTR, Untranslated region.

데이터 분석
모든 시험은 3반복 수행하여 결과를 획득하였으며, 각각의 결과에 대한 통계 분석은 SPSS 통계 패키지 22.0 (SPSS Inc., Illinois, USA)를 이용하였다 통계적 검증은 t-test를 수행하여 p값이 0.05이하인 경우를 유의한 차이가 있는 것으로 판단하였다.
내염성 옥수수 돌연변이체 선발
감마선을 조사한 종자는 포장에 파종하여 발아 및 생육 상태를 관찰하였고, 100 Gy 감마선을 처리한 종자의 경우 일부 형태학적 변이는 관찰되었지만 생육에는 큰 문제가 발견되지 않았다. 반면, 200과 300 Gy 감마선을 처리한 종자는 발아가 거의 되지 않았으며 발아된 일부 개체 또한 정상적인 생육 상태를 보이지 않았다. 후세대 종자가 확보된 100 Gy 조사 집단에 대해 파종 2~3개월 후 자가 교배를 통하여 2,248 계통의 M2 종자를 확보하였다. Inbred 계통을 육성하기 위해 확보된 M2 종자를 세대 진전하고 자가교배하여 M3 종자를 확보하였다. 수확한 M3 종자를 온실내에서 계통 당 20립을 파종하여 3주간 0.7% NaCl 농도에서 내염성이 증진된 10계통을 선발하고 그 중에서 가장 강한 생육상태를 보이는 140RS516 계통을 최종 선발하여 M6 세대까지 진전하였다(Fig. 1).
Fig. 1
Schematic representation of procedures for the selection of gamma radiation-induced salt-tolerant lines. Total 3,000 of M0 seeds were irradiated with 100 Gy gamma rays for 4 hours at room temperature. For selection of salt-tolerant lines, M3 plants were grown for 3 weeks on soil containing 0.7% NaCl in the greenhouse.
KJBS-51-318-f1.jpg
선발 내염성 계통의 발아 및 생육
내염성 증진 10계통 중 염 스트레스 조건에서 가장 강한 생육을 보이는 140RS516를 이용하여 기내에서 염 스트레스에 대한 발아율과 식물체 간장을 조사하였다(Fig. 2). 염 농도가 증가함에 따라 KS140은 생장이 강하게 저해 받는 반면 140RS516는 대조군과 비교하여 양호한 생육 상태를 보였다(Fig. 2A). 염 농도별 발아 상태를 조사한 결과 NaCl 0.1% 농도에서는 대조군과 큰 차이를 보이지 않았지만, 0.2% 이상의 농도에서는 대조군이 87.5%의 발아율을 보이는 반면 140RS516은 100%의 발아율을 보였다(Fig. 2B). 염 조건에서 140RS516의 생육 상태를 조사하기 위해 다양한 농도의 NaCl이 첨가된 배지에 종자를 파종 후 간장 길이를 측정하였다. 정상 조건과 저농도 염 조건에서는 생육에 큰 차이를 보이지 않았으나, 0.3% 이상의 농도에서는 대조군 2.7 cm의 간장 길이와 비교하여 140RS516의 간장 길이는 3.1 cm로 돌연변이체의 생육이 보다 양호함을 확인할 수 있었다(Fig. 2C). 본 실험의 결과 140RS516이 KS140 대조군과 비교하여 염 스트레스에 대한 내성을 가지고 있는 것으로 판단된다.
Fig. 2
Phenotype analysis of KS140 and 140RS516 after salt stress. (A) Morphological phenotype of KS140 and 140RS516 in response to various concentration of NaCl. Germination rate (B) and stem length (C) of KS140 and 140RS516 in response to NaCl. Plants were grown for 10 days on medium containing each concentration of NaCl. Data are means ±SD determined from three independent biological replicates. Asterisks denote statistical significance at p<0.05 (*) by Student’s t-test.
KJBS-51-318-f2.jpg
140RS516의 내염성 조사
140RS516 돌연변이체의 내염성을 조사하기 위해 KS140과 140RS516을 온실에 파종하여 두 달간 키운 후 0.5% NaCl이 첨가된 물을 7일간 처리하였다. 대조군과 140RS516의 표현형은 정상 조건에서 큰 차이를 보이지 않았으나, 염 처리 후 대조군의 잎은 위조 현상을 보이며 말리는 반면 140RS516은 정상 조건과 큰 차이를 보이지 않았다. 이 후 물을 주어 2일간 회복시킨 결과 대조군과 140RS516 모두 정상적인 생육 상태를 보였다(Fig. 3A).
Fig. 3
Effect of salt stress on morphological phenotype, stomatal conductance, and proline contents of KS140 and 140RS516. (A) Phenotypes were analyzed after 0.5% NaCl treatment. Plants were grown for 2 months on soil then treated 0.5% NaCl for 7 days and recovered for 2 days. Magnified images of the regions boxed in (A) are shown in a, b, c and d. Stomatal conductance (B) and proline contents (C) of KS140 and 140RS516. Data are means ±SD determined from three independent biological replicates. Asterisks denote statistical significance at p<0.05 (*) and p<0.01 (**) by Student’s t-test. Scale Bars: 10 cm.
KJBS-51-318-f3.jpg
KS140과 140RS516의 염 스트레스에 대한 광합성 능력을 확인하기 위해 기공전도도를 측정하였다(Fig. 3B). 염 스트레스 처리 후 KS140과 140RS516 모두 기공전도도가 감소하였으나, 대조군이 0.05 mol/m2⋅s의 기공전도도를 보인 반면 140RS516 돌연변이체는0.1 mol/m2⋅s으로 두배 이상 높은 기공전도도를 보였다. 또한 2일간 회복한 식물체에서 기공전도도를 측정한 결과, 표현형에서는 큰 차이를 보이지 않았음에도 불구하고 140RS516의 0.23 mol/m2⋅s에 비해 KS140은 0.16 mol/m2⋅s의 기공전도도를 보였다. 이러한 결과는 염 스트레스에 반응하여 140RS516이 대조군인 KS140과 비교하여 높은 기공전도도를 보이며, 이는 높은 광합성 효율을 가짐으로써 염 스트레스에 대한 내성이 증가된 것으로 사료된다. 내염성 반응의 척도인 proline 함량을 알아보기 위해 본 실험에서는 KS140과 140RS516을 0.5% NaCl이 첨가된 배지에서 10일간 키운 후 isatin 분석법을 이용하여 proline 함량을 측정하였다. 무처리구에서는 대조군과 140RS516은 큰 차이를 보이지 않았지만 염 스트레스 처리 후 140RS516은 대조군과 비교하여 0.55 μg/mg에서 0.72 μg/mg으로 증가된 proline 함량을 보였다(Fig. 3C). 이상의 결과를 종합해볼 때 140RS516은 대조군과 비교하여 염 스트레스에 반응하여 높은 기공전도도를 나타내고 proline의 함량을 증가시켜 수분이용효율을 향상시킴으로써 염 스트레스에 대한 저항성을 획득한 것으로 판단된다.
140RS516의 유전변이 분석
자연계에서 유전적 변이로 일어나는 돌연변이는 식물을 포함한 모든 유기체에서 다양한 스트레스에 대한 적응 및 내성 능력을 강화시켜 왔다(FAO 2011, Mickelbart et al. 2015). 이러한 사실에 착안하여 감마선 유도 내염성 140RS516에서 유전변이를 탐색하기 위해 전체 게놈 염기서열재분석(Whole genome re-sequencing)을 수행하였다. 염기서열재분석 결과를 통해 140RS516에서 단일염기다형성(SNP)과 염기삽입-결실(InDels)을 포함하는 총 328개의 유전변이가 감지되었다. 이러한 유전변이는 UTR 영역에 4개, exon 영역에 4개, intron 영역에 18개 그리고 intergenic 영역에서 302개가 관찰되었다(Table 1). Exon 영역에서 관찰된 4개의 변이 중 아미노산 서열을 변화시키는 non-synonymous 변이는 1번 염색체에 2개 그리고 3번 염색체에서 1개가 확인되었다(Table 2). ATP synthase subunit gamma mitochondrial은 alanine (A)이 glycine (G)으로 변이가 발생했으며, nucleotide binding protein는 aspartic acid (D)가 glutamic acid (E)로 변이가 일어났다. 또한 serine/threonine-protein kinase AGC1-5는 leucine (L)이 glutamine (Q)으로 변이가 발생하였다(Table 2). 염 스트레스에 의해 발현이 증가한다고 알려진 COP1-interacting protein (Moazzam-Jazi et al. 2018), Protein YABBY (Hou et al. 2019) 그리고 Aldo-keto reductase (Simpson et al. 2009) 유전자의 경우 모두 intron 영역에서 변이가 관찰되었다(Table 2). 지금까지 옥수수에서 내염성에 관여하는 유전자 변이는 명확하게 밝혀져 있지 않다. 본 실험에서 내염성 옥수수 유전변이 결과를 토대로 추가적인 연구를 통해 옥수수의 염 스트레스 내성에 관여하는 유전자를 탐색할 필요가 있을 것으로 사료된다.
Table 2
List of genetic variations on genic region of 140RS516 mutant line.
Table 2
zChr. ReferenceAllele MutationAllele AnnotationType Gene ID Gene Description
1 TTTACTC T Intron Zm00001d027392 Callose synthase 9
1 G C Nonsynonymous Zm00001d028207 ATP synthase subunit gamma mitochondrial
1 T C Intron Zm00001d033237 Uncharacterised conserved protein
1 G T Nonsynonymous Zm00001d033304 Nucleotide binding protein
1 T G Intron Zm00001d034931 COP1-interacting protein-related
2 A G Intron Zm00001d003284 SWI/SNF complex subunit SWI3A
2 T C 5'-UTR Zm00001d005100 RNA binding (RRM/RBD/RNP motifs) family protein
2 A G 5'-UTR Zm00001d007444 Unknown
3 G C Synonymous Zm00001d039282 Serine/threonine-protein kinase AGC1-5
3 A T Nonsynonymous Zm00001d039282 Serine/threonine-protein kinase AGC1-5
3 C T Intron Zm00001d040104 Unknown
3 A G Intron Zm00001d042303 Beclin 1 protein 253B Beclin-1-like protein
4 C G Intron Zm00001d053561 Pentatricopeptide repeat-containing protein mitochondrial
5 C T Intron Zm00001d013070 Transcription factor MYB98
5 C T 3'-UTR Zm00001d013073 Transcription factor bHLH7
6 A G Intron Zm00001d038365 Aldo-keto reductase family 4 member C9
6 T G Intron Zm00001d038650 Xylem bark cysteine peptidase 3
7 T TAC Intron Zm00001d018829 Protein YABBY
7 A T Intron Zm00001d019627 ABC transporter B family member 28
7 AC ACC Intron Zm00001d019915 CASP-like protein
7 C T Intron Zm00001d021070 Pyruvate dehydrogenase4
7 TGGGACG T 5'-UTR Zm00001d021810 Protein strubbelig-receptor family 1
8 G T Intron Zm00001d012363 Protein strictosidine synthase-like 11
10 T A Intron Zm00001d024234 Terpene synthase 23
10 T A Intron Zm00001d025679 Putative MYB DNA-binding domain superfamily protein
10 A C Intron Zm00001d025679 Putative MYB DNA-binding domain superfamily protein

zChr., Chromosome.

140RS516에서 내염성 관련 유전자 발현 양상 분석
식물은 환경 스트레스에 반응하여 다양한 유전자의 발현을 조절함으로써 환경 스트레스에 반응한다(Gupta & Huang 2014). 140RS516에서 내염성 관련 유전자들의 발현 양상을 관찰하기 위해 qRT-PCR 분석을 수행하였다(Fig. 4). 무처리구에서 ABP9과 CIPK31은 대조군에 비해 140RS516에서 높은 발현을 보였으며, 염 스트레스 처리 조건에서도 높은 발현을 보였다. 반면 CIPK21은 무처리구에서는 큰 차이를 보이지 않았으나, 염 스트레스 처리 후 대조군에서 발현이 감소하는 것에 비해 140RS516은 무처리구와 유사한 발현양을 유지하였다. 이러한 결과로 미루어 볼 때, 140RS516에서 관찰되는 내염성이 본 실험에서 발현양을 측정한 유전자와 관련이 있는 것으로 사료된다.
본 연구에서는 대조군인 KS140과 감마선 유도 140RS516 간 내염성 차이를 비교하였다. 내염성이 증진된 140RS516 옥수수는 염 스트레스에서 높은 발아율과 생장, 호흡률 및 삼투물질 함량을 보였으며, 내염성과 관련된 유전자들이 높은 발현을 보였다. 이와 같이 내염성이 증진된 돌연변이체 사료용 옥수수는 고농도의 염에 영향을 받고 있는 우리나라 간척지에 재배될 수 있으며 염 스트레스에 대한 식물의 반응 기작을 규명하기 위한 유용한 재료로 활용될 수 있을 것이다.
Fig. 4
qRT-PCR analysis of salt stress responsive genes in KS140 and 140RS516. For salt stress treatment, each seeds were sown in medium with or without 0.5% NaCl. Plants were grown for 10 days with or without 0.5% NaCl. Data are means ±SE determined from three independent biological replicates. Different letters indicate a significant difference determined by one-way ANOVA followed by Duncan’s post hoc test (p<0.05). ABP, ABA-responsive-elementbinding protein 9; CIPK21, CBL-interacting protein kinase 21; CIPK31, CBL-interacting protein kinase 31.
KJBS-51-318-f4.jpg
식물은 다양한 환경 스트레스에 적응하기 위해 스트레스 내성 유전자의 발현과 자연 돌연변이를 통해 외부 환경 및 자극에 대한 반응 특성을 강화시켜 왔다 본 연구는 사료용 옥수수를 대상으로 감마선을 이용하여 돌연변이 집단을 구축하고 내염성이 증대된 계통을 개발하고자 수행되었다. 140RS516은 NaCl 처리 조건에서 대조군인 KS140과 비교하여 증가된 염 스트레스 내성을 보였다. 감마선에 의한 다양한 유전변이를 보인 140RS516 식물체는 염 스트레스 조건에서 대조군보다 높은 발아율과 생장, 기공전도도 그리고 proline함량을 나타냈으며, 내염성에 관여하는 유전자들의 발현이 증가하였다. 따라서 본 연구를 통해 개발된 140RS516 옥수수는 간척지 염화토양과 같이 불량한 환경에서 작물 재배 및 생산이 가능한 내염성 품종을 개발하기 위한 육종 소재로 활용될 수 있을 것이다.
KJBS-51-318_Supple.pdf
본 연구는 농촌진흥청 기관 과제(과제 번호: PJ013514012019)와 차세대 바이오그린21 시스템합성농생명공학사업단(과제 번호: PJ01382042019)의 지원에 의해 이루어졌습니다. 또한 본 연구는 2019년도 농촌진흥청 국립식량과학원 전문연구원 과정 지원사업으로 이루어졌습니다.

보충자료

본문의 Supplementary Table 1은 한국육종학회지 홈페이지에서 확인할 수 있습니다.

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Characterization of a Gamma Radiation-Induced Salt-Tolerant Silage Maize Mutant
Korean. J. Breed. Sci.. 2019;51(4):318-325.   Published online December 1, 2019
DOI: https://doi.org/10.9787/KJBS.2019.51.4.318
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Characterization of a Gamma Radiation-Induced Salt-Tolerant Silage Maize Mutant
Korean. J. Breed. Sci.. 2019;51(4):318-325.   Published online December 1, 2019
DOI: https://doi.org/10.9787/KJBS.2019.51.4.318
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Characterization of a Gamma Radiation-Induced Salt-Tolerant Silage Maize Mutant
Image Image Image Image
Fig. 1 Schematic representation of procedures for the selection of gamma radiation-induced salt-tolerant lines. Total 3,000 of M0 seeds were irradiated with 100 Gy gamma rays for 4 hours at room temperature. For selection of salt-tolerant lines, M3 plants were grown for 3 weeks on soil containing 0.7% NaCl in the greenhouse.
Fig. 2 Phenotype analysis of KS140 and 140RS516 after salt stress. (A) Morphological phenotype of KS140 and 140RS516 in response to various concentration of NaCl. Germination rate (B) and stem length (C) of KS140 and 140RS516 in response to NaCl. Plants were grown for 10 days on medium containing each concentration of NaCl. Data are means ±SD determined from three independent biological replicates. Asterisks denote statistical significance at p<0.05 (*) by Student’s t-test.
Fig. 3 Effect of salt stress on morphological phenotype, stomatal conductance, and proline contents of KS140 and 140RS516. (A) Phenotypes were analyzed after 0.5% NaCl treatment. Plants were grown for 2 months on soil then treated 0.5% NaCl for 7 days and recovered for 2 days. Magnified images of the regions boxed in (A) are shown in a, b, c and d. Stomatal conductance (B) and proline contents (C) of KS140 and 140RS516. Data are means ±SD determined from three independent biological replicates. Asterisks denote statistical significance at p<0.05 (*) and p<0.01 (**) by Student’s t-test. Scale Bars: 10 cm.
Fig. 4 qRT-PCR analysis of salt stress responsive genes in KS140 and 140RS516. For salt stress treatment, each seeds were sown in medium with or without 0.5% NaCl. Plants were grown for 10 days with or without 0.5% NaCl. Data are means ±SE determined from three independent biological replicates. Different letters indicate a significant difference determined by one-way ANOVA followed by Duncan’s post hoc test (p<0.05). ABP, ABA-responsive-elementbinding protein 9; CIPK21, CBL-interacting protein kinase 21; CIPK31, CBL-interacting protein kinase 31.

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Characterization of a Gamma Radiation-Induced Salt-Tolerant Silage Maize Mutant

Summary of genetic variations in 140RS516 mutant line.

Variant category Number of variants
5'-UTRz 3
Coding (Exonic) 4
Intronic 18
3'-UTR 1
Intergenic 302
Total 328
Nonsynonymous 3
Synonymous 1
Total Coding 4

List of genetic variations on genic region of 140RS516 mutant line.

zChr. ReferenceAllele MutationAllele AnnotationType Gene ID Gene Description
1 TTTACTC T Intron Zm00001d027392 Callose synthase 9
1 G C Nonsynonymous Zm00001d028207 ATP synthase subunit gamma mitochondrial
1 T C Intron Zm00001d033237 Uncharacterised conserved protein
1 G T Nonsynonymous Zm00001d033304 Nucleotide binding protein
1 T G Intron Zm00001d034931 COP1-interacting protein-related
2 A G Intron Zm00001d003284 SWI/SNF complex subunit SWI3A
2 T C 5'-UTR Zm00001d005100 RNA binding (RRM/RBD/RNP motifs) family protein
2 A G 5'-UTR Zm00001d007444 Unknown
3 G C Synonymous Zm00001d039282 Serine/threonine-protein kinase AGC1-5
3 A T Nonsynonymous Zm00001d039282 Serine/threonine-protein kinase AGC1-5
3 C T Intron Zm00001d040104 Unknown
3 A G Intron Zm00001d042303 Beclin 1 protein 253B Beclin-1-like protein
4 C G Intron Zm00001d053561 Pentatricopeptide repeat-containing protein mitochondrial
5 C T Intron Zm00001d013070 Transcription factor MYB98
5 C T 3'-UTR Zm00001d013073 Transcription factor bHLH7
6 A G Intron Zm00001d038365 Aldo-keto reductase family 4 member C9
6 T G Intron Zm00001d038650 Xylem bark cysteine peptidase 3
7 T TAC Intron Zm00001d018829 Protein YABBY
7 A T Intron Zm00001d019627 ABC transporter B family member 28
7 AC ACC Intron Zm00001d019915 CASP-like protein
7 C T Intron Zm00001d021070 Pyruvate dehydrogenase4
7 TGGGACG T 5'-UTR Zm00001d021810 Protein strubbelig-receptor family 1
8 G T Intron Zm00001d012363 Protein strictosidine synthase-like 11
10 T A Intron Zm00001d024234 Terpene synthase 23
10 T A Intron Zm00001d025679 Putative MYB DNA-binding domain superfamily protein
10 A C Intron Zm00001d025679 Putative MYB DNA-binding domain superfamily protein
Table 1 Summary of genetic variations in 140RS516 mutant line.

UTR, Untranslated region.

Table 2 List of genetic variations on genic region of 140RS516 mutant line.

zChr., Chromosome.

Table 1

Table 2

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