가뭄 스트레스 특이적인 cis-regulatory element의 특성을 기반으로 한 신규 프로모터 구축 = Construction of novel promoters based on the characteristics of drought stress specific cis-regulatory element

다국어 초록 (Multilingual Abstract)

Droughts are one of the abiotic stresses that hinders the growth and productivity of crop plants. Coping with abiotic stress is necessary to understand the molecular regulatory networks that makes plants respond to adverse environmental conditions. In our experiment to find a combination that can cope with abiotic stress (respond to drought), we screened 5 stressinducible promoters that are expressed only under stress conditions. This founded 36 cis-elements in stress-inducible promoters. With the result we designed 2 synthetic promoters (BL1, BL2) for fine-controlled regulation by assembling ciselements from the native promoters, which are expressed only under stress caused by droughts. Analysis of the transgenic plant (BL1-GUS, BL2-GUS) showed that the synthetic promoters increased the expression of β-glucuronidase (GUS) in transgenic plants under desiccation. Also in the transient activation assay demonstrated that synthetic promoters induced the cotransformation of effector DREB1A and DREB2C. These results expect that the synthetic promoter with a combination of droughtspecific elements can be used to respond to various abiotic stress and is resistant to stress without causing growth retardation.

참고문헌 (Reference)

1 Bhaskara GB, "Unique drought resistance functions of the highly ABA-induced clade A protein phosphatase 2Cs" 160 (160): 379-395, 2012

2 Ramakrishna C, "The membrane tethered transcription factor EcbZIP17 from finger millet promotes plant growth and enhances tolerance to abiotic stresses" 8 (8): 1-14, 2018

3 Gong W, "The development of protein microarrays and their applications in DNA-protein and protein-protein interaction analyses of Arabidopsis transcription factors" 1 (1): 27-41, 2008

4 Söderman E, "The Arabidopsis homeobox gene ATHB-7 is induced by water deficit and by abscisic acid" 10 (10): 375-381, 1996

5 Baker SS, "The 5′-region of Arabidopsis thaliana cor15a has cis-acting elements that confer cold-, drought-and ABA-regulated gene expression" 24 (24): 701-713, 1994

6 Abe H, "Role of Arabidopsis MYC and MYB homologs in drought-and abscisic acid-regulated gene expression" 9 (9): 1859-1868, 1997

7 Vitha S, "Quantitative βglucuronidase assay in transgenic plants" 35 (35): 151-155, 1993

8 Chang WC, "PlantPAN : Plant promoter analysis navigator, for identifying combinatorial cisregulatory elements with distance constraint in plant gene groups" 9 (9): 561-, 2008

9 Yang L, "Overexpression of the maize E3 ubiquitin ligase gene ZmAIRP4enhances drought stress tolerance in Arabidopsis" 123 : 34-42, 2018

10 Hu H, "Overexpressing a NAM, ATAF, and CUC(NAC)transcription factor enhances drought resistance and salt tolerance in rice" 103 (103): 12987-12992, 2006

11 Yamaguchi-Shinozaki K, "Organization of cis-acting regulatory elements in osmotic-and cold-stress-responsive promoters" 10 (10): 88-94, 2005

12 Kirby J, "NAN fusions: a synthetic sialidase reporter gene as a sensitive and versatile partner for GUS" 32 (32): 391-400, 2002

13 Maruyama K, "Metabolic pathways involved in cold acclimation identified by integrated analysis of metabolites and transcripts regulated by DREB1A and DREB2A" 150 (150): 1972-1980, 2009

14 Kim WC, "MYB 46directly regulates the gene expression of secondary wall-associated cellulose synthases in Arabidopsis" 73 (73): 26-36, 2013

15 Shi H, "Low temperature-induced 30(LTI30)positively regulates drought stress resistance in Arabidopsis : effect on abscisic acid sensitivity and hydrogen peroxide accumulation" 6 : 893-, 2015

16 Kim HJ, "Light signalling mediated by phytochrome plays an important role in cold-induced gene expression through the C-repeat/dehydration responsive element (C/DRE) in Arabidopsis thaliana" 29 (29): 693-704, 2002

17 Hundertmark M, "LEA(late embryogenesis abundant)proteins and their encoding genes in Arabidopsis thaliana" 9 (9): 118-, 2008

18 Mubeen H, "In Silico approach to identify transcription factor binding sites and Cis-regulatory elements in tubulin gene promoter" 6 (6): 31-33, 2010

19 Kasuga M, "Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor" 17 (17): 287-291, 1999

20 Taji T, "Important roles of droughtand cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana" 29 (29): 417-426, 2002

21 Hernandez-Garcia CM, "Identification and validation of promoters and cis-acting regulatory elements" 217 : 109-119, 2014

22 Van der Weele CM, "Growth of Arabidopsis thaliana seedlings under water deficit studied by control of water potential in nutrient-agar media" 51 (51): 1555-1562, 2000

23 Daryanto S, "Global synthesis of drought effects on maize and wheat production" 11 (11): e0156362-, 2016

24 Jefferson RA, "Genetic engineering" Springer 247-263, 1988

25 Zhou Y, "GNS4, a novel allele of DWARF11, regulates grain number and grain size in a high-yield rice variety" 10 (10): 1-11, 2017

26 Hsieh EJ, "Functional characterization of an abiotic stress-inducible transcription factor AtERF53 in Arabidopsis thaliana" 82 (82): 223-237, 2013

27 Clough SJ, "Floral dip : a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana" 16 (16): 735-743, 1998

28 Ko JH, "Ectopic expression of MYB46identifies transcriptional regulatory genes involved in secondary wall biosynthesis in Arabidopsis" 60 (60): 649-665, 2009

29 Leavitt JM, "Coordinated transcription factor and promoter engineering to establish strong expression elements in Saccharomyces cerevisiae" 11 (11): 866-876, 2016

30 Yamaguchi-Shinozaki K, "Characterization of the expression of a desiccation-responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants" 236 (236): 331-334, 1993

31 Msanne J, "Characterization of abiotic stress-responsive Arabidopsis thaliana RD29A and RD29B genes and evaluation of transgenes" 234 (234): 97-107, 2011

32 Cheng MC, "Arabidopsis RGLG2, functioning as a RING E3 ligase, interacts with AtERF53 and negatively regulates the plant drought stress response" 158 (158): 363-375, 2012

33 Chen H, "Arabidopsis DREB2C functions as a transcriptional activator of HsfA3during the heat stress response" 401 (401): 238-244, 2010

34 Yoo SD, "Arabidops is mesophyll protoplasts : a versatile cell system for transient gene expression analysis" 2 (2): 1565-, 2007

35 Kim JS, "An ABRE promoter sequence is involved in osmotic stressresponsive expression of the DREB2A gene, which encodes a transcription factor regulating drought-inducible genes in Arabidopsis" 52 (52): 2136-2146, 2011

36 Savouré A, "Abscisic acid-independent and abscisic acid-dependent regulation of proline biosynthesis following cold and osmotic stresses in Arabidopsis thaliana" 254 (254): 104-109, 1997

37 De Bruxelles GL, "Abscisic acid induces the alcohol dehydrogenase gene in Arabidopsis" 111 (111): 381-391, 1996

38 Bradford MM, "A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding" 72 (72): 248-225, 1976

39 Yamaguchi-Shinozaki K, "A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, lowtemperature, or high-salt stress" 6 (6): 251-264, 1994

40 Xiang C, "A mini binary vector series for plant transformation" 40 (40): 711-717, 1999

41 Kasuga M, "A combination of the Arabidopsis DREB1A gene and stress-inducible rd29A promoter improved drought-and low-temperature stress tolerance in tobacco by gene transfer" 45 (45): 346-350, 2004