|
Abstract To enrich studies of NAC transcription factor in upland cotton (Gossypium hirsutum) and find related NAC genes involved in stresses induced senescence, we cloned and analyzed GhNAC63, a homologous gene of JUNGBRUNNEN1, and its promoter. By qRT-PCR analysis, we explored the expression patterns of GhNAC63 in different cotton tissues, different cotyledon developmental stages, and in responses to different abiotic stresses in various culture conditions. To explore the function of GhNAC63, we constructed 35S::GhNAC63 expression vector, and transferred it into Arabidopsis through Agrobacterium LBA4404 by flower dipping method, and observed its phenotype in homozygous T4 stage. In addition, pYL156::GhNAC63 vector was constructed with pYL156-pYL192 system, and transferred into cotton to knock down GhNAC63 expression by VIGS. Gene sequence analysis showed that GhNAC63 harbored the conserved NAM domain, belonging to the NAC transcription factors, and it was conserved in the N-terminal, but variant in the C-terminal, which was consist with most of the NAC transcription factors. The full length of GhNAC63 was 870 bp, and its predicted protein contained 289 amino acid residues, with a molecular weight about 33.12 kD. Based on the genome sequence of upland cotton, GhNAC63 was located in scaffold246.1, and about 2.0 kb of its 5' UTR was cloned and analyzed. Besides basal elements, TATA-Box and CAAT- Box, a lot of stresses-related Cis-elements were contained in its promoter, such as drought-related, hot-related, phytohormone-related, and light responsive-related elements. Expression patterns analysis showed that GhNAC63 dominantly expressed in fiber, flower, stem, leaf and cotyledon, and it had the highest expression level in the middle stage of cotyledon development. Under closed culture condition, GhNAC63 was upregulated under treatments of MeJA, SA, drought and ABA in leaf, while the expression level of GhNAC63 was down-regulated under other treatments both in leaf and root, and GhNAC63 showed the most sensitive to ethylene treatment. Under open culture condition, GhNAC63 showed different expression patterns under drought, salt, abscisic acid (ABA), methyl jasmonate (MeJA) or ethylene treatments, and its expression level was highly induced by ethylene treatment. Overexpression of GhNAC63 in Arabidopsis made plants more susceptive to ethylene treatment as leaves of transgenic Arabidopsis became yellow after ethylene treatment, in addition, leaves of the transgenic plants also became wilting after drought treatment, which might indicate the function of GhNAC63 in response to ethylene or drought. On the contrary, VIGS induced gene silence led to a low expression level of GhNAC63 in cotton, with about 0.4 times decrease compared with wild type or empty vector control plants. GhNAC63 silenced seedlings can flowering just as wild type seedlings, while empty vector control plants did not bloom, thus low expression level of GhNAC63 could make cotton seedlings being stronger. In conclusion, GhNAC63 was sensitive to ethylene treatment, and it might negatively regulate ethylene or drought treatments in Arabidopsis, and it negatively regulated plant development in cotton. Promoter of GhNAC63 contained various stresses-related Cis-elements, indicating GhNAC63 might response to different stimulates. This study helps to create new breeding material which was related with stresses induced senescence, and enriches the functional studies of NAC transcription factor in upland cotton.
|
Received: 15 June 2016
Published: 13 January 2017
|
Fund:The National High Technology Research and Development Program of China |
|
|
|
[1]宋国立 崔荣霞, 王坤波, 等.改良CTAB法快速提取棉花[J].棉花学报, 1998, 10(5):273-275[2]Chai M, Bellizzi M, Wan C, et al.The NAC transcription factor OsSWN1 regulates secondary cell wall development in Oryza sativa[J].Journal of Plant Biology, 2015, 58(1):44-51[3]Chen X, Wang Y, Lv B, et al.The NAC family transcription factor OsNAP confers abiotic stress response through the ABA pathway[J].Plant and Cell Physiology, 2014, 55(3):604-619[4]Clough S J, Bent A F.Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[J].The Plant Journal, 1998, 16(6):735-743[5]Souer E, van Houwelingen A, Kloos D, et al.The no apical meristem gene of Petunia is required for pattern formation in embryos and flowers and is expressed at meristem and primordia boundaries[J].Cell, 1996, 85(2):159-170[6]Fujita M, Fujita Y, Maruyama K, et al.A dehydration-induced NAC protein,RD26,is involved in a novel ABA-dependent stress-signaling pathway[J].The Plant Journal, 2004, 39(6):863-876[7]Guo Y, Gan S.AtNAP,a NAC family transcription factor,has an important role in leaf senescence[J].The Plant Journal, 2006, 46(4):601-612[8]Han Y J, Kim Y M, Hwang O J, et al.Characterization of a small constitutive promoter from Arabidopsis translationally controlled tumor protein (AtTCTP) gene for plant transformation[J].Plant Cell Reports, 2015, 34(2):265-275[9]He X J, Mu R L, Cao W H, et al.AtNAC2,a transcription factor downstream of ethylene and auxin signaling pathways,is involved in salt stress response and lateral root development[J].The Plant Journal, 2005, 44(6):903-916[10]Ishida T, Aida M, Takada S, et al.Involvement of CUP-SHAPED COTYLEDON genes in gynoecium and ovule development in Arabidopsis thaliana[J].Plant and Cell Physiology, 2000, 41(1):60-67[11]Jiang X, Zhang C, Lü P, et al.RhNAC3,a stress-associated NAC transcription factor,has a role in dehydration tolerance through regulating osmotic stress-related genes in rose petals[J].Plant Biotechnology Journal, 2014, 12(1):38-48[12]Kawaura K, Mochida K, Ogihara Y.Genome-wide analysis for identification of salt-responsive genes in common wheat[J].Functional & Integrative Genomics, 2008, 8(3):277-286[13]Kim J H, Chung K M, Woo H R.Three positive regulators of leaf senescence in Arabidopsis,ORE1,ORE3 and ORE9,play roles in crosstalk among multiple hormone-mediated senescence pathways[J].Genes & Genomics, 2011, 3(4):373-381[14]Li F, Fan G, Wang K, et al.Genome sequence of the cultivated cotton Gossypium arboreum[J].Nature Genetics, 2014, 46(6):567-572[15]Li W, Huang G Q, Zhou W, et al.A cotton (Gossypium hirsutum) gene encoding a NAC transcription factor is involved in negative regulation of plant xylem development[J].Plant Physiology and Biochemistry, 2014, 83:134-141[16]Lin M, Pang C, Fan S, et al.Global analysis of the Gossypium hirsutum LTranscriptome during leaf senescence by RNA-Seq[J].BMC Plant Biology, 2015, 15(1):1-[17]Liu G, Li X, Jin S, et al.Overexpression of rice NAC gene SNAC1 improves drought and salt tolerance by enhancing root development and reducing transpiration rate in transgenic cotton[J].PLoS One, 2014, 9(1):e86895-[18]Livak K J, Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2? ΔΔCT method[J].Methods, 2001, 25(4):402-408[19]Lu P L, Chen N Z, An R, et al.A novel drought-inducible gene,ATAF1,encodes a NAC family protein that negatively regulates the expression of stress-responsive genes in Arabidopsis[J].Plant Molecular Biology, 2007, 63(2):289-305[20]Osakabe Y, Yamaguchi-Shinozaki K, Shinozaki K, et al.ABA control of plant macroelement membrane transport systems in response to water deficit and high salinity[J].New Phytologist, 2014, 202(1):35-49[21]Paterson A H, Wendel J F, Gundlach H, et al.Repeated polyploidization of Gossypium genomes and the evolution of spinnable cotton fibres[J].Nature, 2012, 492(7429):423-427[22]Sakuma Y, Maruyama K, Osakabe Y, et al.Functional analysis of an Arabidopsis transcription factor,DREB2A,involved in drought-responsive gene expression[J].The Plant Cell, 2006, 18(5):1292-1309[23]Shah S T, Pang C, Fan S, et al.Isolation and expression profiling of GhNAC transcription factor genes in cotton (Gossypium hirsutum L) during leaf senescence and in response to stresses[J].Gene, 2013, 531(2):220-234[24]Shah S T, Pang C, Hussain A, et al.Molecular cloning and functional analysis of NAC family genes associated with leaf senescence and stresses in Gossypium hirsutum L[J].Plant Cell, Tissue and Organ Culture (PCTOC), 2014, 117(2):167-186[25]Shang H, Li W, Zou C, et al.Analyses of the NAC transcription factor gene family in Gossypium raimondii Ulbr: chromosomal location,structure,phylogeny,and expression patterns[J].Journal of Integrative Plant Biology, 2013, 55(7):663-676[26]Shang H, Wang Z, Zou C, et al.Comprehensive analysis of NAC transcription factors in diploid Gossypium: sequence conservation and expression analysis uncover their roles during fiber development[J].Science China Life Sciences, 2016, 59(2):142-153[27]Singh B, Avci U, Inwood S E E, et al.A specialized outer layer of the primary cell wall joins elongating cotton fibers into tissue-like bundles[J].Plant Physiology, 2009, 150(2):684-699[28]Song M, Fan S, Pang C, et al.Genetic analysis of the antioxidant enzymes,methane dicarboxylic aldehyde (MDA) and chlorophyll content in leaves of the short season cotton (Gossypium hirsutum L)[J].Euphytica, 2014, 198(1):153-162[29]Sun X L, Sun M, Luo X, et al.A Glycine soja ABA-responsive receptor-like cytoplasmic kinase,GsRLCK,positively controls plant tolerance to salt and drought stresses[J].Planta, 2013, 237(6):1527-1545[30]Zhirnov IV, Trifonova EA, Kochetov AV et al.Virus-induced silencing as a method for studying gene functions in higher plants[J].Russian Journal of Genetics, 2015, 51(5):467-475[31]Wang K, Wang Z, Li F, et al.The draft genome of a diploid cotton Gossypium raimondii[J].Nature Genetics, 2012, 44(10):1098-1103[32]Wright P R.Premature senescence of cotton (Gossypium hirsutum L)–Predominantly a potassium disorder caused by an imbalance of source and sink[J].Plant and Soil, 1999, 211(2):231-239[33]Wu A, Allu A D, Garapati P, et al.JUNGBRUNNEN1,a reactive oxygen species–responsive NAC transcription factor,regulates longevity in Arabidopsis[J].The Plant Cell, 2012, 24(2):482-506[34]Yu X, Peng H, Liu Y, et al.CarNAC2,a novel NAC transcription factor in chickpea (Cicer arietinum L),is associated with drought-response and various developmental processes in transgenic Arabidopsis[J].Journal of Plant Biology, 2014, 57(1):55-66[35]Zhang D, Zhang T, Guo W.Effect of H2O2 on fiber initiation using fiber retardation initiation mutants in cotton (Gossypium hirsutum)[J].Journal of Plant Physiology, 2010, 167(5):393-399[36]Zhao F, Ma J, Li L, et al.GhNAC12,a neutral candidate gene,leads to early aging in cotton (Gossypium hirsutum L)[J].Gene, 2016, 576(1):268-274[37]Zhao JQ, Jiang TF, Liu Z, et al.Dominant Gene cplsr1 Corresponding to Premature Leaf Senescence Resistance in Cotton (Gossypium hirsutum L)[J].Journal of Integrative Plant Biology, 2012, 54(8):577-583[38]Zhu M, Chen G, Zhou S, et al.A new tomato NAC (NAMATAF12CUC2) transcription factor,SlNAC4,functions as a positive regulator of fruit ripening and carotenoid accumulation[J].Plant and Cell Physiology, 2014, 55(1):119-135 |
|
|