玉米转录因子NAC78的克隆及表达特性分析

doi:10.13560/j.cnki.biotech.bull.1985.2020-1155

摘要/Abstract

摘要：

NAC（NAM,ATAF1/2,CUC2）家族转录因子在调控植物生长发育、响应逆境胁迫及激素信号转导等方面起着重要作用,探究其功能可为解析玉米对逆境的响应机制提供基础。通过RT-PCR克隆得到了玉米转录因子NAC78的CDS序列,通过生物信息学方法分析NAC78的特点,用荧光定量检测了NAC78的组织表达特性及对逆境胁迫的响应情况。结果显示,NAC78基因的开放阅读框长1 830 bp,编码609个氨基酸,蛋白分子量为67.45 kD,等电点5.19。NAC78蛋白质N端含有一个保守的NAM结构域,由8个α螺旋和7个β片层构成。进化树分析结果表明NAC78与谷子NAC86亲缘关系最近。NAC78在玉米各组织中均表达,在胚乳中的表达量显著高于其他组织;在胚乳发育的过程中,NAC78的表达变化呈现先增加后降低的趋势,授粉后15 d的相对表达量较第5天增加52.52倍;NAC78的表达受到ABA、NaCl和PEG的诱导,在3种处理下均呈现先增加后降低的趋势,在ABA处理籽粒6 h后相对表达量较0 h增加了11.4倍,NaCl和PEG处理植株6 h后在叶片中的相对表达量分别增加了1.87和2.69倍。NAC78在玉米胚乳中表达量显著高于其他组织,并对ABA、NaCl和PEG处理有不同程度的响应。

关键词: NAC, 转录因子, 非生物胁迫, ABA

Abstract:

NAC（NAM,ATAF1/2,CUC2）family transcription factors play an important role in regulating plant growth and development,responding to stress and hormone signal transduction. Exploring their functions will provide bases for analyzing the stress response mechanism in maize. The CDS sequence of the NAC78 was cloned by RT-PCR,the characteristics of the NAC78 were analyzed by bioinformatics method,and the expression patterns of NAC78 in different tissues and its responses to stresses were detected by real-time quantitative PCR. The length of NAC78’s open reading frame was 1 830 bp,encoding 609 amino acids. Its molecular weight was 67.45 kD,and isoelectric point was 5.19. The N-terminal of the NAC78 protein contained a conserved NAM domain,consisting of 8 α-helices and 7 β-sheets. The results from phylogenetic tree analysis showed that NAC78 was most closely related to the NAC86 in Setaria italica. The results of real-time quantitative PCR showed that NAC78 expressed in all tissues of maize,and the expression in the endosperm was significantly higher than that in other tissues;the expression of NAC78 increased first and then decreased,and the relative expression increased by 52.52 times compared with that on the day 5. The expression of NAC78 was induced by ABA,NaCl and PEG,which increased first and then decreased under three stress treatments,and the relative expression in the seeds increased by 11.4 times after 6 h compared with that of 0 h under ABA treatment,and the those in the leaves treated with NaCl and PEG increased by 1.87 and 2.69 times after 6 h,respectively. In sum,the expression of NAC78 in the endosperm of maize is significantly higher than that in other tissues,and presents varied responses to ABA,NaCl and PEG treatment.

Key words: NAC, transcription factor, abiotic stress, ABA

何姗, 王智康, 杨阳, 牟玥薇, 黄玉碧, 余国武. 玉米转录因子NAC78的克隆及表达特性分析[J]. 生物技术通报, 2021, 37(5): 1-10.

HE Shan, WANG Zhi-kang, YANG Yang, MU Yue-wei, HUANG Yu-bi, YU Guo-wu. Gene Cloning and Expression Analysis of Transcription Factor NAC78 in Maize[J]. Biotechnology Bulletin, 2021, 37(5): 1-10.

图/表 8

表1 试验所用引物

Table 1 Primers used in the experiment

引物名称Primer name	引物序列Primer sequence（5'-3'）
NAC78-F	ATGGCGCCGGTCAGTTTGCCTC
NAC78-R	TCACTCACTGCCGCTATTTC
NAC78-RT-F	GGCCGTTAGGTTGGTTGAGA
NAC78-RT-R	TGAAGCCGCACACCTCTAAG
Actin-F	TCACTACGACTGCCGAGCGAG
Actin-R	GAGCCACCACTGAGGACAACATTAC

图1 NAC78蛋白的生物信息学分析 A：NAC78蛋白的亲疏水性预测;B：NAC78蛋白结构域;C：NAC78蛋白的三维结构预测

Fig.1 Bioinformatics analysis of NAC78 protein A: Prediction of hydrophilicity and hydrophobicity of NAC78 protein. B: NAC78 protein domain. C: Three dimensional structure prediction of NAC78 protein

图2 不同物种NAC78同源基因多序列比对高粱（SORBI_3001G529200）,谷子（SETIT_034541mg）,玉米（Zm00001d027395）,水稻（Os03t0119966）,普通小麦（Traes_CS5A02G519300）,香蕉（GSMUA_Achr1T02820）,毛果杨（POPTR_017G139500v3）,木薯（MANES_02G081200）,棉花（B456_007G188800）,马铃薯（XP_015165583.1）,番茄（Solyc02g036430）,烟草（NW_017670850.1）,猕猴桃（CEY00_Acc24239）,向日葵（NC_035448.1）,黄瓜（Csa_5G324000）,欧洲油菜（BnaA05g22540D）,拟南芥（AT5G17260）,小立碗藓（Pp3c8_6080V3）,卷柏（SELMODRAFT_100973）|,地钱（OAE31278.1);A-E：NAC结构域的5个亚结构域;字母TT表示转角,螺旋图形代表蛋白的α螺旋,箭头表示β片层

Fig.2 Multiple sequence alignment of NAC78 homologous genes in different species Sorghun bicolor (SORBI_3001G529200), Setaria italica (SETIT_034541mg), Zea mays (Zm00001d027395), Oryza sativa japonica group (Os03t0119966), Triticum aestivum (Traes_CS5A02G519300), Musa acuminata (GSMUA_Achr1T02820), Populus trichocarpa (POPTR_017G139500v3), Manihot esculenta (MANES_02G081200), Gossypium raimondii (B456_007G188800), Solanum tuberosum (XP_015165583.1), Solanum lycopersicum (Solyc02g036430), Nicotiana attenuata (NW_017670850.1), Actinidia chinensis (CEY00_Acc24239), Helianthus annuus (NC_035448.1), Cucumis sativus (Csa_5G324000), Brassica napus (BnaA05g22540D), Arabidopsis thaliana (AT5G17260), Physcomitrella patens (Pp3c8_6080V3), Selaginella moellendorffii (SELMODRAFT_100973), Marchantia polymorpha (OAE31278.1). A-E: Five subdomains of NAC domain; squiggles represents α-helices, arrows represents β-sheet and TT letters represents turns

图3 NAC78同源基因的进化分析

Fig.3 Evolutionary analysis of NAC78 homologous genes

图4 NAC78在不同组织（A）及授粉后不同时期（B）胚乳中的相对表达量误差线表示标准偏差,n=3

Fig.4 Relative expression of NAC78 in different tissues (A) and endosperm at different stages after pollination (B) The error line represents the standard deviation, n=3

表2 NAC78启动子中的顺式作用元件

Table 2 Cis-elements in the promoter of NAC78

元件名称Element name	序列Sequence	功能Function	数量Number	分类Classification
ABRE	ACGTG;TACGTG;CACGTA	响应脱落酸	4	激素响应元件
ABRE3a	TACGTG	响应脱落酸	1
ABRE4	CACGTA	响应脱落酸	1
CGTCA-motif	CGTCA	响应茉莉酸	3
GARE-motif	TCTGTTG	响应赤霉素	1
TGA-box	TGACGTAA	响应生长素	1
TGACG-motif	TGACG	响应茉莉酸	3
G-Box	TACGTG	响应光照	3	响应光照
GT1-motif	TACGTG	响应光照	1
Sp1	GGGCGG	响应光照	1
MBS	CAACTG	MYB转录因子结合位点	2
MYB	CAACCA;TAACCA;CCGTTG;TAACCA	MYB转录因子结合位点	7	转录因子结合位点
O2-site	GATGACATGG	O2转录因子结合位点	1
CCAAT-box	CAACGG	MYBHv1转录因子结合位点	2
RY-element	CATGCATG	调控种子发育	3	调控植物发育
ARE	TGCTATCCG	响应缺氧条件	1	响应非生物胁迫
GC-motif	CCCCCG	响应缺氧条件	2	响应非生物胁迫
TATA-box	TATATA	转录起始必须	4	转录必须元件
CAAT-box	CAAT;CAAAT	转录增强元件	4	转录必须元件

图5 ABA处理对玉米籽粒中NAC78表达的影响误差线表示标准偏差;星号代表显著差异（*P<0.05,**P<0.01）

Fig.5 Effect of ABA treatment on NAC78 expression in maize kernel The error line refers to the standard deviation. sterisk refers to significant difference (*P<0.05, **P<0.01)

图6 NaCl和PEG处理对叶片中NAC78表达的影响误差线表示标准偏差;星号代表与0 h表达量相比显著差异（*P<0.05,**P<0.01）

Fig.6 Effects of NaCl and PEG treatment on NAC78 expression in leaves The error line represents the standard deviation; sterisk represents significant difference between 0 h. (*P<0.05, **P<0.01）

参考文献 43

[1]	Souer E, Houwelinggen VA, Kloos D, et al. The noapical meris-tem gene of petunia is required for pattern formation in embryosand flowers and is expressed at meristem and primordial bound aries[J]. Cell, 1996,85(2):159-170. pmid: 8612269
[2]	Duval M, Hsieh T, Km SY, et al. Molecular characterization of AtNAM:a member of the Arabidopsis NAC domain superfamily[J]. Plant Molecular Biology, 2002,50(2):237-248. doi: 10.1023/A:1016028530943 URL
[3]	Aida M, Ishida T, Fukaki H, et al. Genes involved in organ separation in Arabidopsis, analysis of the cup-shaped cotyledon mutant[J]. The Plant Cell, 1997,9(6):841-857. doi: 10.1105/tpc.9.6.841 URL
[4]	Olsen AN, Ernst HA, Leggio LL, et al. NAC transcription factors:structurally distinct, functionally diverse[J]. Trends in Plant Science, 2005,10(2):80-87.
[5]	Ooka H, Satoh K, Doi K, et al. Comprehensive analysis of NAC family genes in Oryza sativa and Arabidopsis thaliana[J]. DNA Research, 2003,10(6):239-247. doi: 10.1093/dnares/10.6.239 URL
[6]	Nuruzzaman M, Manimekalai R, Sharoni AM, et al. Genome-wide analysis of NAC transcription factor family in rice[J]. Gene, 2010,465(1-2):30-44. doi: 10.1016/j.gene.2010.06.008 pmid: 20600702
[7]	Shiriga K, Sharma R, Kumar K, et al. Genome-wide identification and expression pattern of drought-responsive members of the NAC family in maize[J]. Meta Gene, 2014,2:407-417. doi: 10.1016/j.mgene.2014.05.001 URL
[8]	Aida M, Ishida T, Tasaka M. Shoot apical meristem and cotyledon formation during Arabidopsis embryogenesis:interaction among the CUP-SHAPED COTYLEDON and SHOOT MERISTEMLESS genes[J]. Development, 1999,126(8):1563-1570. pmid: 10079219
[9]	Guan Q, Yue X, Zeng H, et al. The protein phosphatase RCF2 and its interacting partner NAC019 are critical for heat stress-responsive gene regulation and thermotolerance in Arabidopsis[J]. The Plant Cell, 2014,26(1):438-453. doi: 10.1105/tpc.113.118927 URL
[10]	Bu Q, Jiang H, Li CB, et al. Role of the Arabidopsis thaliana NAC transcription factors ANAC019 and ANAC055 in regulating jasmonic acid-signaled defense responses[J]. Cell Reseach, 2008,18(7):756-767.
[11]	Maugarny-Calès A, Gonçalves B, Jouannic S, et al. Apparition of the NAC transcription factors predates the emergence of land plants[J]. Molecular Plant, 2016,9(9):1345-1348. doi: 10.1016/j.molp.2016.05.016 URL
[12]	Wu Y, Hou J, Yu F, et al. Transcript profiling identifies NAC-domain genes involved in regulating wall ingrowth deposition in phloem parenchyma transfer cells of Arabidopsis thaliana[J]. Front Plant Science, 2018,9:341. doi: 10.3389/fpls.2018.00341 URL
[13]	Lin YJ, Chen H, Li Q, et al. Reciprocal cross-regulation of VND and SND multigene TF families for wood formation in Populus trichocarpa[J]. Proc Natl Acad Sci U S A, 2017,114(45):E9722-E9729. doi: 10.1073/pnas.1714422114 URL
[14]	Xu B, Ohtani M, Yamaguchi M, et al. Contribution of NAC transcription factors to plant adaptation to land[J]. Science, 2014,343(6178):1505-1508. doi: 10.1126/science.1248417 URL
[15]	Puranik S, Sahu PP, Srivastava PS, et al. NAC proteins:regulation and role in stress tolerance[J]. Trends in Plant Science, 2012,17(6):369-381. doi: 10.1016/j.tplants.2012.02.004 pmid: 22445067
[16]	Hu P, Zhang K, Yang C. BpNAC012 Positively regulates abiotic stress responses and secondary wall biosynjournal[J]. Plant Physiology, 2019,179(2):700-717. doi: 10.1104/pp.18.01167 URL
[17]	Hu H, Dai M, Yao J, et al. Overexpressing a NAM, ATAF, and CUC(NAC)transcription factor enhances drought resistance and salt tolerance in rice[J]. Proc Natl Acad Sci U S A, 2006,103(35):12987-12992. doi: 10.1073/pnas.0604882103 URL
[18]	He X, Zhu L, Xu L, et al. GhATAF1, a NAC transcription factor, confers abiotic and biotic stress responses by regulating phytohormonal signaling networks[J]. Plant Cell Report, 2016,35(10):2167-2179. doi: 10.1007/s00299-016-2027-6 URL
[19]	Fujita Y, Fujita M, Shinozaki K, et al. ABA-mediated transcriptional regulation in response to osmotic stress in plants[J]. Journal of Plant Research, 2011,124(4):509-525. doi: 10.1007/s10265-011-0412-3 URL
[20]	Bari R, Jones J D. Role of plant hormones in plant defence responses[J]. Plant Molocular Biology, 2009,69(4):473-488.
[21]	He L, Bian J, Xu J, et al. Novel maize NAC transcriptional repressor ZmNAC071 confers enhanced sensitivity to ABA and osmotic stress by downregulating stress-responsive genes in transgenic Arabidopsis[J]. Journal of Agricultural and Food Chemistry, 2019,67(32):8905-8918. doi: 10.1021/acs.jafc.9b02331 URL
[22]	Zhu Y, Yan J, Liu W, et al. Phosphorylation of a NAC transcription factor by a calcium/calmodulin-dependent protein kinase regulates abscisic acid-induced antioxidant defense in maize[J]. Plant Physiology, 2016,171(3):1651-1664. doi: 10.1104/pp.16.00168 URL
[23]	Shen J, Lv B, Luo L, et al. The NAC-type transcription factor OsNAC2 regulates ABA-dependent genes and abiotic stress tolerance in rice[J]. Science Report, 2017,7:40641. doi: 10.1038/srep40641 URL
[24]	杨欢. 灌浆期高温干旱胁迫影响糯玉米籽粒产量形成的生理机制[D]. 扬州:扬州大学, 2017.
	Yang H. Physiological of high temperature and drought stress during grain filling on yield formation in waxy maize[D]. Yangzhou:Yangzhou University, 2017.
[25]	李叶蓓, 陶洪斌, 王若男, 等. 干旱对玉米穗发育及产量的影响[J]. 中国生态农业学报, 2015,23(4):383-391.
	Li YB, Tao HB, Wang RN, et al. Effect of drought on ear development and yield of maize[J]. Chinese Journal of Eco-Agriculture, 2015,23(4):383-391.
[26]	匡朴. 盐胁迫对不同耐盐性玉米品种萌发、苗期生长及产量的影响[D]. 泰安:山东农业大学, 2018.
	Kuang P. Effects of salt stress on seed germination, seeding growth and yield of maize variaties with different salt tolerance[D]. Tai’an:Shandong Agricultural University, 2018.
[27]	Zhang L, Li XH, Gao Z, et al. Regulation of maize kernel weight and carbohydrate metabolism by abscisic acid applied at the early and middle post-pollination stages in vitro[J]. Journal of Plant Physiology, 2017,216:1-10. doi: S0176-1617(17)30125-6 pmid: 28544894
[28]	Chen J, Zeng B, Zhang M, et al. Dynamic transcriptome landscape of maize embryo and endosperm development[J]. Plant Physiology, 2014,166(1):252-264. doi: 10.1104/pp.114.240689 URL
[29]	Ernst HA, Olsen AN, Larsen S, et al. Structure of the conserved domain of ANAC, a member of the NAC family of transcription factors[J]. EMBO Report, 2004,5(3):297-303. doi: 10.1038/sj.embor.7400093 URL
[30]	Mathew IE, Agarwal P. May the fittest protein evolve:favoring the plant-specific origin and expansion of NAC transcription factors[J]. Bioessays, 2018,40(8):e1800018.
[31]	Kang M, Kim S, Kim HJ, et al. The C-domain of the NAC transcription factor ANAC019 is necessary for pH-tuned DNA binding through a histidine switch in the N-domain[J]. Cell Report, 2018,22(5):1141-1150. doi: 10.1016/j.celrep.2018.01.002 URL
[32]	Pereira-Santana A, Alcaraz LD, Castaño E, et al. Comparative Genomics of NAC transcriptional factors in angiosperms:implications for the adaptation and diversification of flowering plants[J]. PLoS One, 2015,10(11):e0141866. doi: 10.1371/journal.pone.0141866 URL
[33]	Furuta KM, Yadav SR, Lehesranta S, et al. Plant development. Arabidopsis NAC45/86 direct sieve element morphogenesis culminating in enucleation[J]. Science, 2014,345(6199):933-937. doi: 10.1126/science.1253736 URL
[34]	Eleftheriou EP. Ultrastructural studies on protophloem sieve elements in Triticum aestivum L. nuclear degeneration[J]. Journal of Ultrastructure Research & Molecular Structure Research, 1986,95(1):47-60.
[35]	Groover A. Tracheary element differentiation uses a novel mechanism coordinating programmed cell death and secondary cell wall synjournal[J]. Plant Physiology, 1999,119(2):375-384. pmid: 9952432
[36]	Young TE, Gallie DR. Analysis of programmed cell death in wheat endosperm reveals differences in endosperm development between cereals[J]. Plant Molecular Biology, 1999,39(5):915-926. doi: 10.1023/A:1006134027834 URL
[37]	李睿. 稻麦淀粉胚乳细胞程序性死亡和淀粉质体的发生发育[D]. 武汉:华中农业大学, 2004.
	Li R. The programmed cell death of starchy endosperm and the genesis and development of amyloplast in rice and wheat[D]. Wuhan:Huazhong. Agricultural University, 2004.
[38]	Yoshida T, Mogami J, Yamaguchi-Shinozaki K. ABA-dependent and ABA-independent signaling in response to osmotic stress in plants[J]. Current Opinion in Plant Biology, 2014,21:133-139. doi: S1369-5266(14)00106-X pmid: 25104049
[39]	Shao HB, Liang ZS, Shao MG. Roles of ABA signal transduction during higher plant seed development and germination[J]. Forest Ecosystems, 2003,5(4):45-53.
[40]	Rook F, Corke F, Card R, et al. Impaired sucrose-induction mutants reveal the modulation of sugar-induced starch biosynthetic gene expression by abscisic acid signaling[J]. Plant Journal, 2001,26(4):421-433. pmid: 11439129
[41]	Agarwal PK, Jha B. Transcription factors in plants and ABA dependent and indepedent abiotic stress signaling[J]. Biologia Plantarum(Prague), 2010,54(2):201-212.
[42]	Bossi F, Cordoba E, Patricia D, et al. The Arabidopsis ABA-INSENSITIVE(ABI)4 factor acts as a central transcription activator of the expression of its own gene, and for the induction of ABI5 and SBE2. 2 genes during sugar signaling[J]. The Plant Journal, 2009,59(3):359-374. doi: 10.1111/tpj.2009.59.issue-3 URL
[43]	Ramon M, Rolland F, Thevelein JM, et al. ABI4 mediates the effects of exogenous trehalose on Arabidopsis growth and starch breakdown[J]. Plant Molecular Biology, 2007,63(2):195-206. doi: 10.1007/s11103-006-9082-2 URL