花椰菜BraERF023a的克隆及在响应盐和干旱胁迫中的功能

doi:10.3864/j.issn.0578-1752.2021.01.011

摘要/Abstract

摘要：

【目的】在前期从花椰菜中筛选、鉴定出多个逆境胁迫应答相关ERF（Ethylene-responsive factors）转录因子的基础上,对其中一个未知功能的转录因子BraERF023a进行克隆,并阐释其在盐及干旱胁迫条件下的表达特征及功能。【方法】对花椰菜中的BraERF023a进行克隆,采用MEGA 6等生物学软件对其序列特征进行分析,采用qRT-PCR方法分析BraERF023a在盐及干旱胁迫条件下的表达特征,进而构建过表达载体并转化拟南芥,对BraERF023a过表达转基因拟南芥株系在盐及干旱胁迫下的表型、生存率进行观察、分析。【结果】序列分析证实,花椰菜BraERF023a编码区长度为597 bp,编码含198个氨基酸的蛋白质,其内含有一个AP2保守结构域。BraERF023a在十字花科芸薹属植物中具有很高保守性。转录表达模式分析显示,盐及干旱胁迫条件均可显著诱导花椰菜BraERF023a的表达。进一步的功能分析显示,在盐及干旱胁迫处理条件下,过表达BraERF023a的转基因拟南芥株系比野生型对照生长势更好,植株生存率更高,盐及干旱耐受能力明显增强。【结论】BraERF023a在花椰菜响应盐及干旱胁迫中发挥重要的正向调控作用,过表达BraERF023a可显著提高转化株对盐及干旱的胁迫耐受。

关键词: 花椰菜, ERF转录因子, BraERF023a, 盐胁迫, 干旱胁迫

Abstract:

【Objective】In our previous study, Ethylene-responsive factors (ERFs) involved in abiotic stress responses in cauliflower were identified. In the present study, one of these ERF transcription factors with unknown function, named BraERF023a, was cloned. The expression profiles and function of BraERF023a under salt and drought stresses were further explored. 【Method】BraERF023a was cloned by RT-PCR in cauliflower. The sequence characteristics of BraERF023a were analyzed by biosoftwares such as MEGA 6. The expression profiles of BraERF023a under salt and drought stresses were explored by qRT-PCR. BraERF023a overexpression vector was then constructed and genetically transformed into Arabidopsis. The phenotypes and survival rate of overexpression BraERF023a transgenic Arabidopsis under salt and drought stresses were observed and analyzed.【Result】Sequence analysis confirmed that BraERF023a coding region was 597 bp in length and encoded a protein containing 198 amino acids with an AP2 conserved domain. BraERF023a was highly conserved in Brassica. The expression level of BraERF023a significantly increased under both salt and drought stresses in cauliflower. Functional analysis indicated that comparative with the wild-type controls, overexpression BraERF023a transgenic Arabidopsis lines exhibited better growth vigor and higher plant survival rate under salt or drought stress.【Conclusion】 BraERF023a played important roles in positive response to salt and drought stresses in cauliflower. Overexpression of BraERF023a in Arabidopsis could significantly improve the tolerance of transgenic lines to salt and drought stresses.

Key words: cauliflower, ERF transcription factor, BraERF023a, salt stress, drought stress

李慧,韩占品,贺丽霞,杨亚苓,尤书燕,邓琳,王春国. 花椰菜BraERF023a的克隆及在响应盐和干旱胁迫中的功能[J]. 中国农业科学, 2021, 54(1): 152-163.

LI Hui,HAN ZhanPin,HE LiXia,YANG YaLing,YOU ShuYan,DENG Lin,WANG ChunGuo. Cloning and Functional Analysis of BraERF023a Under Salt and Drought Stresses in Cauliflower (Brassica oleracea L. var. botrytis)[J]. Scientia Agricultura Sinica, 2021, 54(1): 152-163.

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参考文献 36

[1]	FEDOROFF N V, BATTISTI D S, BEACHY R N, COOPER P J M, FISCHHOFF D A, HODGES C N, KNAUF V C, LOBELL D, MAZUR B J, MOLDEN D, REYNOLDS M P, RONALD P C, ROSEGRANT M W, SANCHEZ P A, VONSHAK A, ZHU J K. Radically rethinking agriculture for the 21st century. Science, 2010,327(5967):833-834. doi: 10.1126/science.1186834 pmid: 20150494
[2]	邵文靖, 敖特根白音, 郎明林. AP2/ERF转录因子对植物非生物胁迫的应答机制研究进展. 分子植物育种, 2020. http://kns.cnki.net/kcms/detail/46.1068.S.20200226.2306.014.html. doi: 10.1111/pbr.12645 pmid: 31598026
	SHAO W J, AO T G B Y, LANG M L. Mechanism of AP2/ERF transcription factor response to plant abiotic stress. Molecular Plant Breeding, 2020. http://kns.cnki.net/kcms/detail/46.1068.S.20200226.2306.014.html.(in Chinese) doi: 10.1111/pbr.12645 pmid: 31598026
[3]	SINGH K B, FOLEY R C, LUIS OATE-FÁNCHEZ. Transcription factors in plant defense and stress responses. Current Opinion in Plant Biology, 2002,5(5):430-436. doi: 10.1016/S1369-5266(02)00289-3
[4]	JOFUKU K D, DEN BOER B G, VAN MONTAGU M, OKAMURO J K. Control of Arabidopsis flower and seed development by the homeotic gene APETALA2. The Plant Cell, 1994,6(9):1211-1225. doi: 10.1105/tpc.6.9.1211 pmid: 7919989
[5]	ALLEN M D, YAMASAKI K, OHME-TAKAGI M, TATENO M, SUZUKI M. A novel mode of DNA recognition by a beta- sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA. The EMBO Journal, 1998,17(18):5484-5496. doi: 10.1093/emboj/17.18.5484 pmid: 9736626
[6]	NAKANO T, SUZUKI K, FUJIMURA T, SHINSHI H. Genome-wide analysis of the ERF gene family in Arabidopsis and rice. Plant Physiology, 2006,140(2):411-432. doi: 10.1104/pp.105.073783 pmid: 16407444
[7]	SAKUMA Y, LIU Q, DUBOUZET J G, ABE H, SHINOZAKI K, YAMAGUCHI-FHINOZAKI K. DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression. Biochemical and Biophysical Research Communications, 2002,290(3):998-1009. doi: 10.1006/bbrc.2001.6299 pmid: 11798174
[8]	KRIZEK B A. AINTEGUMENTA utilizes a mode of DNA recognition distinct from that used by proteins containing a single AP2 domain. Nucleic Acids Research, 2003,31(7):1859-1868. doi: 10.1093/nar/gkg292 pmid: 12655002
[9]	YAMAGUCHI-FHINOZAKI K, SHINOZAKI K. Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annual Review of Plant Biology, 2006,57(1):781-803. doi: 10.1146/annurev.arplant.57.032905.105444
[10]	HATTORI Y, NAGAI K, FURUKAWA S, SONG X J, KAWANO R, SAKAKIBARA H, WU J H, MATSUMOTO T, YOSHIMURA A, KITANO H, MATSUOKA M, MORI H, ASHIKARI M. The ethylene response factors SNORKEL1 and SNORKEL2 allow rice to adapt to deep water. Nature, 2009,460(7258):1026-1030. doi: 10.1038/nature08258 pmid: 19693083
[11]	NIE J, WEN C, XI L, LV S H, ZHAO Q C, KOU Y P, MA N, ZHAO L J, ZHOU X F. The AP2/ERF transcription factor CmERF053 of chrysanthemum positively regulates shoot branching, lateral root, and drought tolerance. Plant Cell Reports, 2018,37(7):1049-1060. doi: 10.1007/s00299-018-2290-9 pmid: 29687169
[12]	翟莹, 赵艳, 杨晓杰, 孙天国, 张军, 姚雅男. 大豆乙烯响应因子GmERF6的功能分析. 生物技术通报, 2013(12):73-77.
	ZHAI Y, ZHAO Y, YANG X J, SUN T G, ZHANG J, YAO Y N. Functional analysis of soybean ethylene-response factor GmERF6. Biotechnology Bulletin, 2013(12):73-77. (in Chinese)
[13]	LI Y, ZHANG H, ZHANG Q, LIU Q C, ZHAI H, ZHAO N, HE S Z. An AP2/ERF gene, IbRAP2-12, from sweet potato is involved in salt and drought tolerance in transgenic Arabidopsis. Plant Science, 2019,281:19-30. doi: 10.1016/j.plantsci.2019.01.009 pmid: 30824052
[14]	SEO Y J, PARK J B, CHO Y J, JUNG C, SEO H S, PARK S K, NAHM B H, SONG J T. Overexpression of the ethylene-responsive factor gene BrERF4 from Brassica rapa increases tolerance to salt and drought in Arabidopsis plants. Molecule Cells, 2010,30(3):271-277. doi: 10.1007/s10059-010-0114-z
[15]	PARK H Y, SEOK H Y, WOO D H, LEE S Y, TARTE V N, LEE E H, LEE C H, MOON Y H. AtERF71/HRE2 transcription factor mediates osmotic stress response as well as hypoxia response in Arabidopsis. Biochemical and Biophysical Research Communications, 2011,414(1):135-141. doi: 10.1016/j.bbrc.2011.09.039
[16]	LEE J H, HONG J P, OH S K, LEE S, CHOI D, KIM W T. The ethylene-responsive factor like protein 1 (CaERFLP1) of hot pepper (Capsicum annuum L.) interacts in vitro with both GCC and DRE/CRT sequences with different binding affinities: possible biological roles of CaERFLP1 in response to pathogen infection and high salinity conditions in transgenic tobacco plants. Plant Molecular Biology, 2004,55(1):61-81. doi: 10.1007/s11103-004-0417-6
[17]	YI S Y, KIM J H, JOUNG Y H, LEE S, KIM W T, YU S H, CHOI D. The pepper transcription factor CaPF1 confers pathogen and freezing tolerance in Arabidopsis. Plant Physiology, 2004,136(1):2862-2874. doi: 10.1104/pp.104.042903 pmid: 15347795
[18]	HUANG Z J, ZHANG Z J, ZHANG X L, ZHANG H B, HUANG D F, HUANG R F. Tomato TERF1 modulates ethylene response and enhances osmotic stress tolerance by activating expression of downstream genes. FEBS Letter, 2004,573(1):110-116. doi: 10.1016/j.febslet.2004.07.064
[19]	ZHANG H W, HUANG Z J, XIE B Y, CHEN Q, TIAN X, ZHANG X L, ZHANG H B, LU X Y, HUANG D F, HUANG R F. The ethylene-, jasmonate-, abscisic acid- and NaCl-responsive tomato transcription factor JERF1 modulates expression of GCC box-containing genes and salt tolerance in tobacco. Planta, 2004,220(2):262-270. doi: 10.1007/s00425-004-1347-x
[20]	XU Z S, XIA L Q, CHEN M, CHENG X G, ZHANG R Y, LI L C, ZHAO Y X, LU Y, NI Z Y, LIU L, QIU Z G, MA Y Z. Isolation and molecular characterization of the Triticum aestivum L. ethylene- responsive factor 1 (TaERF1) that increases multiple stress tolerance. Plant Molecule Biology, 2007,65(6):719-732.
[21]	LI H, WANG Y, WU M, LI L H, LI C, HAN Z P, YUAN J Y, CHEN C B, SONG W Q, WANG C G. Genome-wide identification of AP2/ERF transcription factors in cauliflower and expression profiling of the ERF family under salt and drought stresses. Frontiers in Plant Science, 2017,8:946. doi: 10.3389/fpls.2017.00946 pmid: 28642765
[22]	MIZOI J, SHINOZAKI K, YAMAGUCHI-SHINOZAKI K. AP2/ERF family transcription factors in plant abiotic stress responses. Biochimica Biophysica Acta, 2012,1819(2):86-96.
[23]	WU L J, CHEN X L, REN H Y, ZHANG Z J, ZHANG H W, WANG J Y, WANG X C, HUANG R F. ERF protein JERF1 that transcriptionally modulates the expression of abscisic acid biosynthesis-related gene enhances the tolerance under salinity and cold in tobacco. Planta, 2007,226(4):815-825. doi: 10.1007/s00425-007-0528-9
[24]	CHENG M C, LIAO P M, KUO W W, LIN T P. The Arabidopsis ETHYLENE RESPONSE FACTOR1 regulates abiotic stress- responsive gene expression by binding to different cis-acting elements in response to different stress signals. Plant Physiology, 2013,162(2):1566-1582. doi: 10.1104/pp.113.221911
[25]	王佳慧, 顾凯迪, 王楚堃, 由春香, 胡大刚, 郝玉金. 苹果乙烯响应因子MdERF72对非生物胁迫的响应. 中国农业科学, 2019,52(23):4374-4385. doi: 10.3864/j.issn.0578-1752.2019.23.017
	WANG J H, GU K D, WANG C K, YOU C X, HU D G, HAO Y J. Analysis of apple ethylene response factor MdERF72 to abiotic stresses. Scientia Agricultura Sinica, 2019,52(23):4374-4385. (in Chinese) doi: 10.3864/j.issn.0578-1752.2019.23.017
[26]	ZHUO C L, LIANG L, ZHAO Y Q, GUO Z F, LU S Y, ZHOU C L. A cold responsive ethylene responsive factor from Medicago falcata confers cold tolerance by up-regulation of polyamine turnover, antioxidant protection, and proline accumulation. Plant Cell and Environment, 2018,41(9):2021-2032.
[27]	BOLT S, ZUTHER E, ZINTL S, HINCHA D K, SCHMÜLLING T. ERF105 is a transcription factor gene of Arabidopsis thaliana required for freezing tolerance and cold acclimation. Plant Cell and Environment, 2017,40(1):108-120. doi: 10.1111/pce.12838
[28]	ILLGEN S, ZINTL S, ZUTHER E, HINCHA D K, SCHMÜLLING T. Characterisation of the ERF102 to ERF105 genes of Arabidopsis thaliana and their role in the response to cold stress. Plant Molecule Biology, 2020,103(3):303-320.
[29]	BIELSA B, HEWITT S, REYES-CHIN-WO S, DHINGRA A, RUBIO-CABETAS M J. Identification of water use efficiency related genes in ‘garnem’ almond-peach rootstock using time-course transcriptome analysis. PLoS ONE, 2018,13(10):e0205493. doi: 10.1371/journal.pone.0205493 pmid: 30308016
[30]	KARABA A, DIXIT S, GRECO R, AHARONI A, TRIJATMIKO K R, MARSCH-MARTINEZ N, KRISHNAN A, NATARAJA K N, UDAYAKUMAR M, PEREIRA A. Improvement of water use efficiency in rice by expression of HARDY, an Arabidopsis drought and salt tolerance gene. Proceedings of the National Academy of Sciences of the USA, 2007,104(39):15270-15275.
[31]	张军辉. 葡萄灰霉病抗性QTL定位及候选基因研究[D]. 沈阳: 沈阳农业大学, 2018.
	ZHANG J H. QTL mapping and candidate gene research of grapevine Botrytis cinerea resistance[D]. Shenyang: Shenyang Agricultural University, 2018. (in Chinese)
[32]	XIE Z L, NOLAN T, JIANG H, TANG B Y, ZHANG M C, LI Z H, YIN Y H. The AP2/ERF transcription factor TINY modulates brassinosteroid-regulated plant growth and drought responses in Arabidopsis. The Plant Cell, 2019,31(8):1788-1806. doi: 10.1105/tpc.18.00918 pmid: 31126980
[33]	QI W W, SUN F, WANG Q J, CHEN M L, HUANG Y Q, FENG Y Q, LUO X J, YANG J S. Rice ethylene-response AP2/ERF factor OsEATB restricts internode elongation by down-regulating a gibberellin biosynthetic gene. Plant Physiology, 2011,157(1):216-228. doi: 10.1104/pp.111.179945
[34]	NAKANO T, FUJISAWA M, SHIMA Y, ITO Y. The AP2/ERF transcription factor SlERF52 functions in flower pedicel abscission in tomato. Journal of Experimental Botany, 2014,65:3111-3119. doi: 10.1093/jxb/eru154
[35]	SCARPECI T E, FREA V S, ZANOR M I, VALLE E M. Overexpression of AtERF019 delays plant growth and senescence, and improves drought tolerance in Arabidopsis. Journal of Experimental Botany, 2017,68(3):673-685. doi: 10.1093/jxb/erw429 pmid: 28204526
[36]	潘健, 温海帆, 何欢乐, 连红莉, 王刚, 潘俊松, 蔡润. 黄瓜ERF基因家族鉴定及其在雌花芽分化中的表达分析. 中国农业科学, 2020,53(1):133-147. doi: 10.3864/j.issn.0578-1752.2020.01.013
	PAN J, WEN H F, HE H L, LIAN H L, WANG G, PAN J S, CAI R. Genome-wide identification of cucumber ERF gene family and expression analysis in female bud differentiation. Scientia Agricultura Sinica, 2020,53(1):133-147. (in Chinese) doi: 10.3864/j.issn.0578-1752.2020.01.013

引物名称 Primer name	引物序列 Primer sequence (5′-3′)
BraERF023a-F	ATGCTGAGTACAGTGAGTGAAACC
BraERF023a-R	TCACAGACACGCCATGAACTG
Nco I-BraERF023a-F	CCATGGATGCTGAGTACAGTGAGTG
BstE II-BraERF023a-R	GGTCACCTCACAGACACGCCAT
qBraERF023a-F	CCACCACTTACCAACGAAC
qBraERF023a-R	AAGATCCGAGCCAAATCCG
35S-F	AACAGAACTCGCCGTAAAG
BraActin-F	GCTCCTCTTAACCCAAAGGC
BraActin-R	CACACCATCACCAGAATCCAGC
AtActin-F	GGTAACATTGTGCTCAGTGGTGG
AtActin-R	AACGACCTTAATCTTCATGCTGC

表型Phenotype	对照CK	L4	L8	L9
株高 Stem length (cm)	43.125±2.780c	56.750±3. 862a	51.125±1.0307ab	49.625±3.0923b
抽薹时间 Bolting time (d)	26.00±2.4495b	32.00±2.4495a	29±1.9148a	31.00±1.2583a
茎粗 Stem diameter (mm)	1.2375±0.0727b	1.7800±0.2191a	1.6300±0.1551a	1.7125±0.1059a
主茎 Stem (number)	5.75±0.9574c	6.75±1.0708ab	6.25±0.5000b	7.775±0.5774a
一级分枝 Primary branch (number)	13.75±2.630b	15.00±2.449ab	16.00±3.742a	13.75±5.132b
二级分枝 Secondary branch (number)	5.00±0.816a	5.00±1.155a	5.75±2.602a	6.00±1.15a
果荚长 Pod length (mm)	18.000±1.0475a	17.153±1.3553a	17.600±1.2035a	18.028±0.8587a