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Kafe 바로가기주관연구기관 | 서울대학교 Seoul National University |
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보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 | 한국어 |
발행년월 | 2016-02 |
과제시작연도 | 2014 |
주관부처 | 농촌진흥청 Rural Development Administration(RDA) |
등록번호 | TRKO201600003400 |
과제고유번호 | 1395039546 |
사업명 | 국책기술개발 |
DB 구축일자 | 2016-06-25 |
DOI | https://doi.org/10.23000/TRKO201600003400 |
Ⅳ. 연구개발결과
선발된 유전자를 대상으로 특이적 발현 프로모터를 이용하여 환경스트레스에 저항성을 가지는 형질 전환체의 제작, 그의 뿌리 생체구조가 대조구에 비해 발달되어 있는 이벤트의 선발 및 농업형질 조사를 통해 생산량의 증가 확인.
근권 공생 신호 전달 유전자 네트워크 해독, 에틸렌 조절을 받는 전사인자와 receptor like kine를 포함하는 신호전달 관련 유전자 후보 선발 및 그의 기능 분석 확인. 밀양23/O. glaberrima 조합 이입계통 및 일품/모로베레칸 조합 이입계통의 농업형질이 증가 확인.
Growth of crop plants in the field is often limited by the effect of atmospheric CO2, temperature and water supply, which can be promoted by restructuring plant architecture to develop low-input and high-yield crops. The root-specific overexpression of OsNACs, plant specific transcription
Growth of crop plants in the field is often limited by the effect of atmospheric CO2, temperature and water supply, which can be promoted by restructuring plant architecture to develop low-input and high-yield crops. The root-specific overexpression of OsNACs, plant specific transcription factors, increases the root diameter due to an enlarged xylem and augmented aerenchyma cell size, which confers enhanced drought resistance to rice plants at the reproduction stage, resulting in grain yield improvements under drought conditions. One major challenge that confronts plant biotechnologists is to engineer crop plants that are able to use fertilizers, especially nitrogen, more efficiently and therefore would require less fertilizers for optimal growth and production of harvestable parts. NAC (NAM,ATAFandCUC) transcription factors are involved in development and stress responses in plants. Here, we report the results of five-year field evaluation (T5-T9) following four years of pre-screening (T1-T4) of OsNAC6 mutants. Root-specific and whole-body OsNAC6-overexpressing transgenic plants showed higher grain yield both under normal and drought stressed conditions. Root analysis revealed vigorous root system architecture with increased root number, diameter and volume in both overexpression lines. Integration of ChIP-seq and RNA-seq data sets revealed that OsNAC6 directly targets genes that are important in drought response and root development. These results suggest that OsNAC6 regulates root system architecture and drought-stress response genes, thus improving grain yield and drought tolerance of rice.Auxin signaling is a fundamental part of many plant growth processes and stress responses and operates through Aux/IAA protein degradation and the transmission of the signal via auxin response factors (ARFs). A total of 31 Aux/IAA genes have been identified in rice (Oryza sativa), some of which are induced by drought stress. However, the mechanistic link between Aux/IAA expression and drought responses is not well understood. In this study we found that the rice Aux/IAA gene OsIAA6 is highly induced by drought stress and that its overexpression in transgenic rice improved drought tolerance, likely via the regulation of auxin biosynthesis genes. We observed that OsIAA6 was specifically expressed in the axillary meristem of the basal stem, which is the tissue that gives rise to tillers. A knock-down mutant of OsIAA6 showed abnormal tiller outgrowth, apparently due to the regulation of the auxin transporter OsPIN1 and the rice tillering inhibitor OsTB1. Our results confirm that the OsIAA6 gene is involved in drought stress responses and the control of tiller outgrowth.
A number of QTL studies reported that one genomic region was associated with several traits, indicating linkage and/or pleiotropic effects. The question of pleiotropy versus tight linkage in these studies should be solved using a large-size population combined with high-density mapping. For example, if each of the 2 parents has a TGW-increasing or SPP-increasing QTL that is tightly linked, complementary combination of the 2 beneficial QTLs by using molecular markers could produce higher yields compared to the 2 parents. However, a pleiotropic QTL with opposite effects on the SPP and 1,000-grain weight (TGW) is complicated and challenging in terms of its application to rice improvement. Brassinosteroids (BRs) are growth-promoting steroid hormones that regulate diverse physiological processes in plants. Most BR biosynthetic enzymes belong to the cytochrome P450 (CYP) family. The gene encoding the ultimate step of BR biosynthesis in Arabidopsis likely evolved by gene duplication followed by functional specialization in a dicotyledonous plant-specific manner. To gain insight into the evolution of BRs, we performed a genomic reconstitution of Arabidopsis BR biosynthetic genes in an ancestral vascular plant, the lycophyte Selaginella moellendorffii. Selaginella contains four members of the CYP90 family that cluster together in the CYP85 clan. Similar to known BR biosynthetic genes, the Selaginella CYP90s exhibit eight or ten exons and Selaginella produces a putative BR biosynthetic intermediate. Therefore, we hypothesized that Selaginella CYP90 genes encode BR biosynthetic enzymes. In contrast to typical CYPs in Arabidopsis, Selaginella CYP90E2 and CYP90F1 do not possess amino-terminal signal peptides, suggesting that they do not localize to the endoplasmic reticulum. In addition, one of the three putative CYP reductases (CPRs) that is required for CYP enzyme function co-localized with CYP90E2 and CYP90F1. Treatments with a BR biosynthetic inhibitor, propiconazole, and epi-brassinolide resulted in greatly retarded and increased growth, respectively. This suggests that BRs promote growth in Selaginella, as they do in Arabidopsis. However, BR signaling occurs through different pathways than in Arabidopsis. A sequence homologous to the Arabidopsis BR receptor BRI1 was absent in Selaginella, but downstream components, including BIN2, BSU1, and BZR1, were present. Thus, the mechanism that initiates BR signaling in Selaginella seems to differ from that in Arabidopsis. Our findings suggest that the basic physiological roles of BRs as growth-promoting hormones are conserved in both lycophytes and Arabidopsis; however, different BR molecules and BRI1-based membrane receptor complexes evolved in these plants.
Legume and rhizobia symbiosis plays an important role in conversion of atmospheric dinitrogen to ammonia. On a global scale, this interaction represents a key entry point for reduced nitrogen into the biosphere, and as a consequence this symbiosis is important in both natural and agricultural systems. Symbiotic development of nodule organ is triggered by chito-oligosaccharide signals (Nod factors) from the bacterium which are perceived by the legume root. Understanding the molecular and cellular processes that underlie Nod factor perception is one focus of legume biology. Although forward genetics has proved to be an important tool to identify key players in Nod factor perception, we still know relatively little regarding the functional networks of genes and proteins that connect the earliest steps of Nod factor perception to immediate downstream outcomes. To identify the genes and proteins that link Nod factor perception to cellular and physiological responses we are taking a discovery-based strategy based on whole transcriptome profiling using RNA-seq analysis. In the roots of barrel medic(Medicago truncatula) after inoculation with Sinorhizobium medicae. We choose mutants with absent or reduced Nod factor sensitivities (nfp and lyk3, respectively) and an ethylene-insensitive, Nod factor hypersensitive mutant(skl). for the clues of signaling regulatory mechanism. Nine points of the dataset were shown remarkable the observation of the symbiotic regulation of diverse biological processes with high temporal resolution. We identified thousands of novel candidate kinase and transcription factor genes related the early Nod factor-induced, ethylene regulated expression of ethylene signaling and biosynthesis. These results suggest that Nod factor signaling activates ethylene production to reduce its own signal. We provide these transcriptome data through a searchable online resource. In addition, we find the interest flowering regulation gene among the Medicago genes. FLOWERING TIME CONTROL PROTEIN, FPA gene encode RNA Recognition Motif (RRM) domain protein and plays important roles in flowering time control in Arabidopsis. Floral transition is significant for reproductive products in all flowering plants. However, little is known about the functions of Medicago autonoumous pathway gene. We had cloned the FPA gene on Medicago based on the sequence similarity of Arabidopsis FPA sequence. The RT-qPCR analysis of MtFPA expression patterns showed that the MtFPA transcripts accumulated ubiquitously in roots, leaves, stems, flowers, and pods. When fused to the green fluorescence protein, MtPFA-GFP was localized in the nucleus as speckle pattern of protoplast from Arabidopsis. To examine the function of MtFPA, 35S::MtFPA transgenic plants were generated in Arabidopsis late flowering mutant background, fpa-2. Overexpression of MtFPA specifically caused early flowering under long day conditions compared with non-transgenic plants. In MtFPA transgenic lines, AtFLC expression were down-regulated whereas the floral ingegrators, AtFT and AtSOC1 were up-regulated as compare with control plant. As these results, MtFPA suggest that is a functional ortholog of the Arabidopsis and may play an important role in the regulation of flowering transition in Medicago.
Lines of Milyang 23 (M23), a Tongil type cultivar, and Ilpum (IP), a japonica type cultivar, respectively, introgressed with chromosome segments, derived from Oryza glaberrima (OG), African agricultural species, and O. sativa ssp. J aponica cv. Moroberekan (MB), a African tropical japonica type cultivar, respectively, were developed to improve agricultural characteristics of Korean rice cultivars, M23 and IP, respectively. These lines were investigated to know their phenotypical responses to salt stress in this study. First, on 2014, fifty five lines of M23/OG combination were examined in the Kyehwa experiment paddy field in the National Institute of Crop Science (NICS), a salty paddy field with 0.2 % salt (the salty field), and in the Iksan experiment paddy field in the NICS (the normal field), respectively. After transplanting, it takes 69 days in the normal field and 83 days for heading of M23 in the salty field, respectively, whereas O. glaberrima did not head in both fields. For heading date, lines of M23/OG combination were grouped into two groups, including a M23-similar heading date group and a delayed heading date group.
Heading date of both two groups was mostly delayed in the salty field. In M23, culm length and panicle length, respectively, decreased by 9.5 % and 15.4 %, respectively, in the salty field, compared to those in the normal field. Most of lines showed much lower culm length in the salty field than those in the normal field, but, in the salty field, panicle length of most lines was similar to those in the normal field. Interestingly, in two lines, OG29 and OG36, panicle length increased by over 10 % in the salty field than those in the normal field. Compared to M23, most lines in the normal field had more panicle numbers, and lots of lines showed much more panicle numbers in the salty field than those in the normal field. Spikelet numbers per a panicle of M23 showed 215 in the normal field, but, in the salty field, M23 exhibited a decrease of 38.1 % in spikelet numbers per a panicle. Most of lines in the normal field showed much less spikelet numbers per a panicle than those of M23, and also exhibited decrease in spikelet numbers per a panicle in the salty field as shown in M23 in the salty field. Grain filling rate of M23 was similar in both the normal and salty fields, but, in most of lines, grain filling rate increased in the salty field. Especially, in 9 lines, including OG5, OG8, OG14, OG18, OG26, OG29, OG30, OG31 and OG50, grain filling rate increased by 20 % in the salty field.
Second, at the same time with lines of M23/OG combination, forty two lines of IP/MB combination were also tested in both the salty and normal fields. Heading date of IP and MB, respectively, was not changed in both the salty and normal fields. Most of lines showed similar patterns as shown in IP and MB in both the salty and normal fields. Culm length of IP and MB, respectively, decreased in the salty field, compared to those in the normal field, and most of lines also showed much lower culm length in the salty field than those in the normal field. Panicle length of IP and MB, respectively, was slightly lower in the salty field than those in the normal field, but panicle length of lines in the salty field was similar to those in the normal field. Specifically, compared to panicle length data in the normal field, three lines, including IM3, IM10 and IM14, exhibited an increase of 10 % in panicle length in the salty field as shown in two lines of M23/OG combination. In the normal field, IP had 11 panicle numbers, which were much higher than 6 of MB, but both parent cultivars showed no significant differences in their panicle numbers in the salty field. Interestingly, most of lines exhibited an increase of panicle numbers in the salty field. Spikelet numbers per a panicle in IP were 130 in the normal field and 111 in the salty field, respectively, and those in MB were 91 in the normal field and 87 in the salty field, respectively. Most of lines had IP-similar spikelet numbers per a panicle and showed a decrease of spikelet numbers per a panicle in the salty field, compared to those in the normal field. Grain filling rate of IP and MB, respectively, was not significantly changed under salt stress.
Symbiosis between legume plants and soil rhizobia culminates in the formation of a novel root organ, the 'nodule', containing bacteria differentiated as facultative nitrogen-fixing organelles. MtNF-YA1 is a Medicago truncatula CCAAT box-binding transcription factor (TF), formerly called HAP2-1, highly expressed in mature nodules and required for nodule meristem function and persistence. Here a role for MtNF-YA1 during early nodule development is demonstrated. Detailed expression analysis based on RNA sequencing, quantitiative real-time PCR (qRT-PCR), as well as promoter-β-glucuronidase (GUS) fusions reveal that MtNF-YA1 is first induced at the onset of symbiotic development during preparation for, and initiation and progression of, symbiotic infection.
Moreover, using a new knock-out mutant, Mtnf-ya1-1, it is shown that MtNF-YA1 controls infection thread (IT) progression from initial root infection through colonization of nodule tissues. Extensive confocal and electronic microscopic observations suggest that the bulbous and erratic IT growth phenotypes observed in Mtnf-ya1-1 could be a consequence of the fact that walls of ITs in this mutant are thinner and less coherent than in the wild type. It is proposed that MtNF-YA1 controls rhizobial infection progression by regulating the formation and the wall of ITs.
The legume-rhizobium symbiosis is initiated through the activation of the Nodulation (Nod) factor-signaling cascade, leading to a rapid reprogramming of host cell developmental pathways. In this work, we combine transcriptome sequencing with molecular genetics and network analysis to quantify and categorize the transcriptional changes occurring in roots of Medicago truncatula from minutes to days after inoculation with Sinorhizobium medicae.
To identify the nature of the inductive and regulatory cues, we employed mutants with absent or decreased Nod factor sensitivities (i.e. Nodulation factor perception and Lysine motif domain-containing receptor-like kinase3, respectively) and an ethylene (ET)-insensitive, Nod factor-hypersensitive mutant (sickle). This unique data set encompasses nine time points, allowing observation of the symbiotic regulation of diverse biological processes with high temporal resolution. Among the many outputs of the study is the early Nod factor-induced, ET-regulated expression of ET signaling and biosynthesis genes. Coupled with the observation of massive transcriptional derepression in the ET-insensitive background, these results suggest that Nod factor signaling activates ET production to attenuate its own signal. Promoter:β-glucuronidase fusions report ET biosynthesis both in root hairs responding to rhizobium as well as in meristematic tissue during nodule organogenesis and growth, indicating that ET signaling functions at multiple developmental stages during symbiosis. In addition, we identified thousands of novel candidate genes undergoing Nod factor-dependent, ET-regulated expression. We leveraged the power of this large data set to model Nod factor- and ET-regulated signaling networks using MERLIN, a regulatory network inference algorithm. These analyses predict key nodes regulating the biological process impacted by Nod factor perception. We have made these results available to the research community through a searchable online resource.
The NITROGEN LIMITATION ADAPTION (NLA) gene was initially shown to function in nitrogen limitation responses; however, recent work shows that the nla mutant hyperaccumulates Pi, phenocopying the Pi signaling mutant pho2. PHO2 encodes a putative E2 conjugase, UBC24. Here, we show that NLA is an E3 ligase that specifically requires UBC24 for polyubiquitination in Arabidopsis thaliana. Among five members of the Pht1 Pi-transporter family tested, NLA associates only with PT2 (Pht1;4). The NLA-UBC24 pair mediates polyubiquitination of PT2 but not PT1. Posttranslational decay of PT2 at high Pi is blocked in pho2 and inhibited by MG132, indicating the requirement of UBC24 and 26S proteasomes. Consistent with NLA/UBC24 function, induced NLA expression causes a UBC24-dependent decrease in PT2 levels. Confocal microscopy of fusion proteins revealed an NLA/PT2 interaction at the plasma membrane. Collectively, these results show that under Pi-replete conditions, NLA and UBC24 target the PT2 transporter for destruction. During the Pi deprivation response, NLA and PHO2 transcripts are cleaved by miR399 and miR827, respectively, and our results suggest that this downregulation relieves the posttranslational repression of PT2, allowing it to accumulate and participate in Pi uptake.
Our work provides additional molecular details describing Pi signaling/homeostasis regulation by identifying NLA and UBC24 as partners and PT2 as one of their downstream targets.
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