玉米NF-Y转录因子基因ZmNF-YB13响应干旱和盐胁迫的功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-0066

摘要/Abstract

摘要：

玉米NF-Y家族是一类重要的转录因子，在调控植物发育和逆境胁迫响应中起重要作用，探究该家族基因功能将为玉米抗逆育种提供重要的基因资源。本研究克隆获得ZmNF-YB13基因，使用生物信息学、实时荧光定量PCR等技术对该基因的基本特性、组织表达特性及响应逆境胁迫表达模式等进行分析。结果显示，该基因全长537 bp，编码蛋白含有178个氨基酸，分子量为18.9 kD，理论等电点为6.83，具有NF-Y家族特有的保守结构域。qPCR分析表明，ZmNF-YB13基因在玉米花丝中的表达量最高；同时ZmNF-YB13基因在不同逆境胁迫以及激素处理条件下均有不同程度的上调表达。在分别含有甘露醇、NaCl、ABA和JA的1/2 MS培养基上，转ZmNF-YB13基因拟南芥的根长于野生型，差异显著。在干旱和高盐处理下，土壤中生长的转基因拟南芥比野生型绿叶数多，MDA含量低，差异显著；同时干旱处理下，转基因植株的POD活性显著高于野生型。由此推测，ZmNF-YB13基因可能参与玉米耐旱、抗盐非生物胁迫的应答。

关键词: 玉米, 转录因子, 异源表达, 干旱胁迫, 盐胁迫

Abstract:

Maize NF-Y family is a class of important transcription factors，they play an important role in regulating plant development and stress response. Exploring the gene function of this family will provide important gene resources for maize stress-resistant breeding. In this study，ZmNF-YB13 gene was cloned，and its basic characteristics，the relative tissue-specific expressions and expression patterns under different stresses were analyzed by bioinformatics and real-time quantitative PCR. The gene was 537 bp in length and encoded 178 amino acids. The molecular weight of ZmNF-YB13 protein was 18.9 kD，and the theoretical isoelectric point was 6.83. ZmNF-YB13 protein had a unique conserved domain of NF-Y family. qPCR analysis showed that ZmNF-YB13 gene expression was the highest in maize silk；meanwhile ZmNF-YB13 gene expression was up-regulated under different abiotic stress and hormone stress. On 1/2 MS medium containing mannitol，NaCl，ABA and JA，the root length of ZmNF-YB13 transgenic Arabidopsis thaliana was significantly longer than that of the wild type. Under drought and high salt treatments，transgenic A. thaliana grown in soil had more green leaves and lower MDA content than wild-type. Concurrently，the POD activity of transgenic plants was significantly higher than that of wild type under drought treatment. It is speculated that ZmNF-YB13 gene may be involved in response to drought and salt stress in maize.

Key words: maize, transcription factor, heterologous expression, drought stress, salt stress

张彤彤, 郑登俞, 吴忠义, 张中保, 于荣. 玉米NF-Y转录因子基因ZmNF-YB13响应干旱和盐胁迫的功能分析[J]. 生物技术通报, 2022, 38(10): 115-123.

ZHANG Tong-tong, ZHENG Deng-yu, WU Zhong-yi, ZHANG Zhong-bao, YU Rong. Functional Analysis of ZmNF-YB13 Responding to Drought and Salt Stress[J]. Biotechnology Bulletin, 2022, 38(10): 115-123.

图/表 7

表1 本实验所用的引物

Table 1 Primers used in this study

引物名称 Primer name	引物序列 Primer sequence（5'-3'）
pZmNF-YB13-FP	ATGGCGGAAGCTCCGGCGAGCC
pZmNF-YB13-RP	TTAGTTTGAGATATCCCCGTTA
pZmNF-YB13RT-FP	AATGGCGACGATCTGCTGTGG
pZmNF-YB13RT-RP	GCTATCACCCTGCACCTCTCTG
pGAPDH-FP	CCCTTCATCACCACGGACTAC
pGAPDH-RP	AACCTTCTTGGCACCACCCT
pZmNF-YB13T-FP	GAGAAGCGGAAGACCATCA
pZmNF-YB13T-RP	GTATAATTGCGGGACTCTAATC

图1 ZmNF-YB13基因的系统进化分析

Fig.1 Phylogenetic analysis of gene ZmNF-YB13

图2 ZmNF-YB13基因在玉米不同组织中的差异表达分析图中误差线表示标准偏差。不同字母表示在0.05水平上有显著差异。下同

Fig.2 Tissue-specific expression pattern of gene ZmNF-YB13 in maize The error line in the figure refers to the standard deviation. Different letters indicate significant differences at the 0.05 level. The same below

图3 逆境和激素胁迫下ZmNF-YB13基因的表达模式分析 A：脱水处理；B：冷（4℃）处理；C：高盐（200 mmol/L NaCl）处理；D：PEG处理。数据为3个生物学重复±标准差

Fig.3 Expressions of ZmNF-YB13 in response to abiotic and hormone stress in maize A：Dehydration treatment；B：cold（4℃）treatment；C：high salt（200 mmol/L NaCl）treatment；D：PEG treatment. The error bar represents± SD of triplicate experiments

图4 T3代转基因拟南芥的鉴定 A：T3代转基因拟南芥的PCR鉴定；B：T3代转基因拟南芥的qPCR检测

Fig.4 Identification of T3 transgenic A. thaliana A：Identification of T3 generation A. thaliana by PCR. B：Identification of T3 generation A. thaliana by qPCR

图5 不同胁迫处理下转ZmNF-YB13基因拟南芥的表型分析 A：逆境处理下拟南芥的表型观察；B：逆境处理下拟南芥根长统计。*表示 0.05 水平上显著差异，**表示 0.01 水平上极显著差异，下同

Fig.5 Phenotypic analysis of ZmNF-YB13 transgenicA. thaliana under different stress treatments A：Phenotypic observation of A. thaliana under stress. B：Root length statistics of A. thaliana under stress treatment. *indicates significant difference at the 0.05 level. ** indicates very significant difference at the 0.01 level, the same below

图6 土壤中高盐和干旱处理下拟南芥表型分析 A：转ZmNF-YB13基因拟南芥在高盐、干旱以及覆水后的表型拍照分析；B，C：高盐和干旱处理下转基因和野生型拟南芥绿叶数量的统计分析；D：MDA含量的测定；E：POD活性的测定

Fig.6 Phenotypic analysis of A. thaliana under high salt and drought treatment in soil A：Analysis of phenotype of ZmNF-YB13 transgenic A. thaliana after high salt，drought and rewatering. B and C：Statistical analysis of green leaf number of transgenic and wild-type A. thaliana under high salt and drought treatment. D：Measurement of MDA content. E：Measurement of POD activity

参考文献 34

[1]	陈彦锋. 玉米高产栽培技术论述[J]. 世界热带农业信息, 2021(11):20-21.
	Chen YF. Discussion on high yield cultivation technology of maize[J]. World Trop Agric Inf, 2021(11):20-21.
[2]	田敬园, 刘荣, 杨谦, 等. 玉米种植保护性耕作技术及其应用研究[J]. 世界热带农业信息, 2021(11):7-8.
	Tian JY, Liu R, Yang Q, et al. Study on conservation tillage technology of maize planting and its application[J]. World Trop Agric Inf, 2021(11):7-8.
[3]	徐昆, 朱秀芳, 刘莹, 等. 气候变化下干旱对中国玉米产量的影响[J]. 农业工程学报, 2020, 36(11):149-158.
	Xu K, Zhu XF, Liu Y, et al. Effects of drought on maize yield under climate change in China[J]. Trans Chin Soc Agric Eng, 2020, 36(11):149-158.
[4]	王全忠, 薛超, 周宏. 气候变化对中国玉米生产的影响及应对研究[J]. 科技与经济, 2016, 29(5):45-49.
	Wang QZ, Xue C, Zhou H. An empirical study of effect & coping strategies about climate change on Chinese maize production[J]. Sci Technol Econ, 2016, 29(5):45-49.
[5]	闫振华, 刘东尧, 贾绪存, 等. 花期高温干旱对玉米雄穗发育、生理特性和产量影响[J]. 中国农业科学, 2021, 54(17):3592-3608.
	Yan ZH, Liu DY, Jia XC, et al. Maize tassel development, physiological traits and yield under heat and drought stress during flowering stage[J]. Sci Agric Sin, 2021, 54(17):3592-3608.
[6]	姚超, 杨敏婕, 许梅花. 盐胁迫下玉米种子萌发及影响[J]. 农家参谋, 2021(8):61-62.
	Yao C, Yang MJ, Xu MH. Effects of salt stress on seed germination of maize[J]. The Farmers Consultant, 2021(8):61-62.
[7]	刘畅, 孙璐. 盐碱胁迫对玉米苗期生理指标影响的研究进展[J]. 种子科技, 2020, 38(11):18-19.
	Liu C, Sun L. Research progress on effects of salt-alkali stress on physiological indexes of maize seedling stage[J]. Seed Sci Technol, 2020, 38(11):18-19.
[8]	袁海, 何鹏飞, 武君洁, 等. 盐胁迫对耐盐和盐敏感玉米幼苗生长和生理特性的影响[J]. 江苏农业科学, 2019, 47(19):86-89.
	Yuan H, He PF, Wu JJ, et al. Effects of salt stress on growth and biological traits of salt-tolerant and salt-sensitive maize seedlings[J]. Jiangsu Agric Sci, 2019, 47(19):86-89.
[9]	李世贵, 马瑞, 王芳芳, 等. 植物NF-Y转录因子研究进展[J]. 植物生理学报, 2021, 57(2):248-256.
	Li SG, Ma R, Wang FF, et al. Research progresses on plant NF-Y transcription factors[J]. Plant Physiol J, 2021, 57(2):248-256.
[10]	丁慧霞, 刘凤, 等. 植物中NF-Y转录因子的结构和功能研究进展[J]. 分子植物育种, 2017, 15(5):1691-1701.
	Ding HX, Liu F, et al. The structure and function of NF-Y in plants[J]. Mol Plant Breed, 2017, 15(5):1691-1701.
[11]	Li WX, Oono Y, Zhu JH, et al. The Arabidopsis NFYA5 transcription factor is regulated transcriptionally and posttranscriptionally to promote drought resistance[J]. Plant Cell, 2008, 20(8):2238-2251. doi: 10.1105/tpc.108.059444 URL
[12]	Leyva-González MA, Ibarra-Laclette E, Cruz-Ramírez A, et al. Functional and transcriptome analysis reveals an acclimatization strategy for abiotic stress tolerance mediated by Arabidopsis NF-YA family members[J]. PLoS One, 2012, 7(10):e48138. doi: 10.1371/journal.pone.0048138 URL
[13]	Das S, Parida SK, Agarwal P, et al. Transcription factor OsNF-YB9 regulates reproductive growth and development in rice[J]. Planta, 2019, 250(6):1849-1865. doi: 10.1007/s00425-019-03268-2 pmid: 31482329
[14]	Xiong YF, Ren Y, Li W, et al. NF-YC12 is a key multi-functional regulator of accumulation of seed storage substances in rice[J]. J Exp Bot, 2019, 70(15):3765-3780. doi: 10.1093/jxb/erz168 pmid: 31211389
[15]	Alam MM, Tanaka T, Nakamura H, et al. Overexpression of a rice heme activator protein gene(OsHAP2E)confers resistance to pathogens, salinity and drought, and increases photosynthesis and tiller number[J]. Plant Biotechnol J, 2015, 13(1):85-96. doi: 10.1111/pbi.12239 URL
[16]	Ma XY, Zhu XL, Li CL, et al. Overexpression of wheat NF-YA10 gene regulates the salinity stress response in Arabidopsis thaliana[J]. Plant Physiol Biochem, 2015, 86:34-43. doi: 10.1016/j.plaphy.2014.11.011 URL
[17]	Xu L, Lin ZY, Tao Q, et al. Multiple NUCLEAR FACTOR Y transcription factors respond to abiotic stress in Brassica napus L[J]. PLoS One, 2014, 9(10):e111354. doi: 10.1371/journal.pone.0111354 URL
[18]	Maheshwari P, Kummari D, Palakolanu SR, et al. Genome-wide identification and expression profile analysis of nuclear factor Y family genes in Sorghum bicolor L. (Moench)[J]. PLoS One, 2019, 14(9):e0222203. doi: 10.1371/journal.pone.0222203 URL
[19]	Wang PJ, Zheng YC, Guo YC, et al. Identification, expression, and putative target gene analysis of nuclear factor-Y(NF-Y)transcription factors in tea plant(Camellia sinensis)[J]. Planta, 2019, 250(5):1671-1686. doi: 10.1007/s00425-019-03256-6 URL
[20]	Feng C, Wang Y, Sun Y et al. Expression of the Malus sieversii NF-YB21 encoded gene confers tolerance to osmotic stresses in Arabidopsis thaliana[J]. Int J Mol Sci, 2021, 22(18):9777. doi: 10.3390/ijms22189777 URL
[21]	Sun XD, Lian HF, Liu XC, et al. The garlic NF-YC gene, AsNF-YC8, positively regulates non-ionic hyperosmotic stress tolerance in tobacco[J]. Protoplasma, 2017, 254(3):1353-1366. doi: 10.1007/s00709-016-1026-3 URL
[22]	Su HH, Cao YY, Ku LX, et al. Dual functions of ZmNF-YA3 in photoperiod-dependent flowering and abiotic stress responses in maize[J]. J Exp Bot, 2018, 69(21):5177-5189. doi: 10.1093/jxb/ery299 pmid: 30137393
[23]	Mei XP, Liu CX, Yu TT, et al. Identification and characterization of paternal-preferentially expressed gene NF-YC8 in maize endosperm[J]. Mol Genet Genomics, 2015, 290(5):1819-1831. doi: 10.1007/s00438-015-1043-5 URL
[24]	Zhang ZB, Li XL, et al. Isolation, structural analysis, and expression characteristics of the maize nuclear factor Y gene families[J]. Biochem Biophys Res Commun, 2016, 478(2):752-758. doi: 10.1016/j.bbrc.2016.08.020 URL
[25]	殷龙飞, 王朝阳, 吴忠义, 等. 玉米ZmGRAS31基因的克隆及功能研究[J]. 作物学报, 2019, 45(7):1029-1037. doi: 10.3724/SP.J.1006.2019.83070
	Yin LF, Wang ZY, Wu ZY, et al. Cloning and functional analysis of ZmGRAS31 gene in maize[J]. Acta Agron Sin, 2019, 45(7):1029-1037.
[26]	Seo PJ, Park MJ, Park CM. Alternative splicing of transcription factors in plant responses to low temperature stress:mechanisms and functions[J]. Planta, 2013, 237(6):1415-1424. doi: 10.1007/s00425-013-1882-4 URL
[27]	Zhao H, Wu D, et al. The Arabidopsis thaliana nuclear factor Y transcription factors[J]. Front Plant Sci, 2017, 7:2045.
[28]	Sato H, Suzuki T, et al. NF-YB2 and NF-YB3 have functionally diverged and differentially induce drought and heat stress-specific genes[J]. Plant Physiol, 2019, 180(3):1677-1690. doi: 10.1104/pp.19.00391 pmid: 31123093
[29]	Wang BM, et al. ZmNF-YB16 overexpression improves drought resistance and yield by enhancing photosynthesis and the antioxidant capacity of maize plants[J]. Front Plant Sci, 2018, 9:709. doi: 10.3389/fpls.2018.00709 pmid: 29896208
[30]	Zhang T, Zhang D, Liu YJ, et al. Overexpression of a NF-YB3 transcription factor from Picea wilsonii confers tolerance to salinity and drought stress in transformed Arabidopsis thaliana[J]. Plant Physiol Biochem, 2015, 94:153-164. doi: 10.1016/j.plaphy.2015.05.001 URL
[31]	Han X, et al. Overexpression of the poplar NF-YB7 transcription factor confers drought tolerance and improves water-use efficiency in Arabidopsis[J]. J Exp Bot, 2013, 64(14):4589-4601. doi: 10.1093/jxb/ert262 URL
[32]	Wang T, Wei Q, Wang Z, et al. CmNF-YB8 affects drought resistance in Chrysanthemum by altering stomatal status and leaf cuticle thickness[J]. J Integr Plant Biol, 2021:2021 Dec 10.
[33]	Tsikas D. Assessment of lipid peroxidation by measuring malondialdehyde(MDA)and relatives in biological samples:analytical and biological challenges[J]. Anal Biochem, 2017, 524:13-30. doi: 10.1016/j.ab.2016.10.021 URL
[34]	Nelson DE, Repetti PP, Adams TR, et al. Plant nuclear factor Y(NF-Y)B subunits confer drought tolerance and lead to improved corn yields on water-limited acres[J]. Proc Natl Acad Sci USA, 2007, 104(42):16450-16455. doi: 10.1073/pnas.0707193104 URL