Welcome to Acta Agronomica Sinica,

Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (03): 343-356.doi: 10.3724/SP.J.1006.2018.00343


Cloning of the Key Gene ZmCYP90B1 in Brassinosteroids Biosynthesis from Zea mays and Its Response to Adversity Stresses

Fang-Meng DUAN1(), Qiu-Lan LUO2, Xue-Li LU3, Na-Wei QI1, Xian-Shun LIU1, Wen-Wen SONG1,*()   

  1. 1 College of Plant Health and Medicine, Qingdao Agricultural University, Qingdao 266109, Shandong, China
    2 Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science / Shenzhen Engineering Laboratory of Marine Algal Biotechnology / College of Life Science, Shenzhen University, Shenzhen 518060, Guangdong, China
    3 Tobacco Research Institute of Chinese Academy, Agricultural Sciences, Qingdao 266101, Shandong, China
  • Received:2017-07-05 Accepted:2017-11-21 Online:2018-03-12 Published:2017-12-18
  • Contact: Wen-Wen SONG E-mail:fmduan@hotmail.com;songwenwen2002@163.com
  • Supported by:
    This study was supported by Qingdao Applied Basic Research Project (16-5-1-54-jch), Shandong Province Natural Science Foundation (ZR2013CQ028), Shenzhen Grant Plan for Science & Technology (JCYJ20150324141711583), and the Doctor Program of Higher Education (663/1111316).


Brassinosteroids (BRs) as an important phytohormone, play essential roles in plant growth, development and responses to adversity stresses. The key enzyme encoded by CYP90B1 is involved in the BRs biosynthesis. However, the characteristic of CYP90B1 gene response to stress has not been reported in maize. In this study, we cloned ZmCYP90B1 gene from Zea mays, GenBank accession number KY242373, via RT-PCR in combination of RACE technique. The full-length sequence of this gene is 2058 bp and the the complete open reading frame is 1518 bp, encoding 506 amino acid peptides. The predicted protein has the molecular weight of 57.66 kD and isoelectric point of 9.54, containing one transmembrane domain and one conserved domain of p450. Multiple sequences alignment analysis indicated the predicted protein shared high similarities with CYP90B1 from other plant species. The phylogenetic tree revealed a notable difference in the evolution of CYP90B1 between dicotyledons and monocotyledons. The qRT-PCR result showed that the expression of ZmCYP90B1 was induced by drought, high salt, low temperature, ABA, Spodoptera exigua attack, and methyl jasmonate (MeJA), suggesting that ZmCYP90B1 was involved in various abiotic stresses, insect resistance and response to MeJA. Overexpressing ZmCYP90B1 in tobacco seedlings could enhance drought resistance with less water loss rate and higher SPAD value in the transgenic lines. In addition, the activities of SOD, CAT, and POD and the content of proline increased significantly in all transgenic tobacco lines than in the wild type; both MDA and ABA contents were obviously lower in the transgenic lines than in the wild type. Through expression analysis of the down-stream stress-responsive genes, we demonstrated that drought tolerance enhancement by overexpressing ZmCYP90B1 might be involved in ABA-independent pathway and related to transcriptional regulation of antioxidant- related genes.

Key words: brassionsteroids, ZmCYP90B1 gene, real-time quantitative PCR, adversity stresses

Table 1

Primers used in cloning gene"

Sequence (5°-3°)

Table 2

Specific primers used for real-time PCR analysis"

Gene name
GenBank number
Primer sequence (5°-3°)
Product size
temperature (°C)
262 60
151 60
238 62
131 62
366 70
350 62
106 60
104 62
133 62

Fig. 1

PCR amplified products of ZmCYP90B1 A: The part fragment amplification of ZmCYP90B1; B: The 5´-RACE amplification of ZmCYP90B1; C: The 3´-RACE amplification of ZmCYP90B1; D: The ORF amplification of ZmCYP90B1; E: The identification of recombinant vector digested by double enzymes; M1: DL2000 marker; M2: 15000 marker."

Fig. 2

Sequence of amino acid encoded by ZmCYP90B1 The underline is the amino acid site of the transmembrane domain: 12-31; The grey shaded part is p450 conserved domain site: 2-491."

Fig. 3

Hydrophilicity and hydrophobicity analysis of predicted amino acid sequence encoded by ZmCYP90B1"

Fig. 4

Multiple sequence-alignments of CYP90B1 proteins from eight plants"

Fig. 5

Phylogenetic analysis between ZmCYP90B1 and CYP90B1 proteins from other plant species"

Table 3

Comparison of physical and chemical characteristics of amino acids of CYP90B1 from different plant species"

Molecular weight (kD)
等电点pI 负电荷氨基酸比例
Negatively charged residues (%)
Positively charged residues (%)
Instability index
Zea mays 506 57.66 9.54 10.47 13.44 46.74 -0.171
Dichanthelium oligosanthes 507 57.87 9.25 10.65 12.62 48.82 -0.161
Oryza brachyantha 504 57.12 9.27 10.52 12.70 52.69 -0.138
Aegilops tauschii 501 57.01 8.11 11.58 11.98 52.53 -0.133
Triticum urartu 525 59.75 8.09 11.62 12.00 51.61 -0.162
Arabidopsis thaliana 513 58.87 6.62 12.87 12.28 49.50 -0.301
Nicotiana attenuata 491 55.78 8.89 9.98 11.41 44.36 -0.166
Brassica napus 495 56.71 8.17 11.72 12.12 48.90 -0.213
Glycine soja 492 55.77 8.85 10.16 11.38 43.87 -0.128
Theobroma cacao 496 56.75 8.64 10.89 11.90 46.51 -0.219
Gossypium hirsutum 485 55.42 8.94 10.52 12.16 44.88 -0.163

Fig. 6

Relative expression levels of ZmCYP90B1 in different tissues The different lowercase letters in each column indicate significant difference at the 0.05 probability level."

Fig. 7

Expression profiles of ZmCYP90B1 under abiotic stresses The different lowercase letters in each column indicate significant difference at the 0.05 probability level."

Fig. 8

Expression profiles of ZmCYP90B1 under Spodoptera exigua and MeJA The different lowercase letters in each column indicate significant difference at the 0.05 probability level."

Fig. 9

Molecular identification of transgenic tobacco lines M: marker; P: positive plasmid; N: empty vector; W: double distilled water; WT: wild types; 1-10: transgenic lines."

Fig. 10

Functional analysis of wild types and transgenic tobacco seedlings under drought stress A: The phenotype of WT and transgenic lines (L4 and L9) before drought stress; B: The phenotype of WT and transgenic lines (L4 and L9) after 20% PEG treatment for 10 h; C: The water loss rate of WT and transgenic lines (L4 and L9) after 20% PEG treatment for 10 h; D: The SPAD value of WT and transgenic lines (L4 and L9) before and after 20% PEG treatment. The different lowercase letters in each column indicate significant difference at the 0.05 probability level."

Fig. 11

Physiological indices in both wild types and transgenic tobacco before and after drought stress The different lowercase letters in each column indicate significant difference at the 0.05 probability level."

Fig. 12

Expression profiles of stress-responsive genes before and after drought stress The different lowercase letters in each column indicate significant difference at the 0.05 probability level."

[1] Arteca J M, Arteca R N.Brassinosteroid-induced exaggerated growth in hydroponically grown Arabidopsis plants. Physiol Plant, 2001, 112: 104-112
[2] Hu Y X, Bao F, Li J Y.Promotive effect of brassinosteroids on cell division involves a distinct CycD3-induction pathway in Arabidopsis. Plant J, 2000, 24: 693-701
[3] Yu J Q, Huang L F, Hu W H, Zhou Y H, Mao W H, Nogués S.A role for brassinosteroids in the regulation of photosynthesis in Cucumis sativus. J Exp Bot, 2004, 55: 1135-1143
[4] Divi U K, Krishna P.Brassinosteroid: a biotechnological target for enhancing crop yield and stress tolerance.New Biotechnol, 2009, 26: 131-136
[5] Kurepin L V, Qaderi M M, Back T G, Reid D M, Pharis R P.A rapid effect of applied brassinolide on abscisic acid concentrations in Brassica napus leaf tissue subjected to short-term heat stress. Plant Growth Regul, 2008, 55: 165-167
[6] El-Mashad A A A, Mohamed H I. Brassinolide alleviates salt stress and increases antioxidant activity of cowpea plants (Vigna sinensis). Protoplasma, 2012, 249: 625-635
[7] Nakashitaetal H, Yasuda M, Nitta T, Asami T, Fujioka S, Arai Y, Sekimata K, Takatsuto S, Yamaguchi I, Yoshida S.Brassinosteroid function in a broad range of disease resistance in tobacco and rice.Plant J, 2003, 33: 887-898
[8] 李艳楠. 油菜素内酯处理对番茄根结线虫的防治效果研究. 河南科技大学硕士学位论文, 河南洛阳, 2012
Li Y N.The Research of Control Effects of Brassinosteroids Treatment on Tomato Root-knot Nematode Caused by Meloidogyne Incognita. MS Thesis of Henan University of Science and Technology, Luoyang,China, 2012 (in Chinese with English abstract)
[9] 张丹丹. 油菜素内酯提高黄瓜对根结线虫抗性的机制研究及田间效应. 浙江大学硕士学位论文, 浙江杭州, 2012
Zhang D D.Field Effect and Molecular Mechanisms of Brassinosteroids-increased the Resistance to Root-knot Nematode in Cucumis sativus. MS Thesis of Zhejiang University, Hangzhou,China, 2012 (in Chinese with English abstract)
[10] 孙超, 黎家. 油菜素甾醇类激素的生物合成、代谢及信号转导. 植物生理学报, 2017, 53: 291-307
Sun C, Li J.Biosynthesis, catabolism, and signal transduction of brassinosteroids.J Plant Physiol, 2017, 53: 291-307 (in Chinese with English abstract)
[11] Sahni S, Prasad B D, Liu Q, Grbic V, Sharpe A, Singh S P, Krishnaa P.Overexpression of the brassinosteroid biosynthetic gene DWF4 in Brassica napus simultaneously increases seed yield and stress tolerance. Sci Rep, 2016, 6: 28298
[12] Shimada Y, Fujioka S, Miyauchi N, Kushiro M, Takatsuto S, Nomura T, Yokota T, Kamiya Y, Bishop G J, Yoshida S.Brassinosteroid-6-oxidases from Arabidopsis and tomato catalyze multiple C-6 oxidations in brassinosteroid biosynthesis. Plant Physiol, 2001, 126: 770-779
[13] 申强. 过量表达ZmDWF4基因改良玉米产量性状的研究. 山东农业大学硕士学位论文,山东泰安, 2011
Shen Q.Overexpression of ZmDWF4 Improves Yield Traits in Maize. MS Thesis of Shandong Agricultural University, Tai’an,China, 2011 (in Chinese with English abstract)
[14] Choe S, Fujioka S, Noguchi T, Takatsuto S, Yoshida S, Feldmann K A.Overexpression of DWARF4 in the brassinosteroid biosynthetic pathway results in increased vegetative growth and seed yield in Arabidopsis. Plant J, 2001, 26: 573-582
[15] 武海军, 司建萍, 王新宇. 胡杨DWF4基因的克隆及其在拟南芥中的异源表达和功能分析. 见: 中国细胞生物学学会主编. 中国细胞生物学学会2013年全国学术大会论文摘要集. 湖北武汉: 2013. p 247
Wu H J, Si J P, Wang X Y.Cloning of the DWF4 gene from Populus euphratica, its heterologous expression and functional analysis in Arabidopsis. In: Chinese Academy of Cell Biology, eds. Proceedings of Academic Conference of Chinese Academy of Cell Biology in 2013. Wuhan, China, 2013. p 247 (in Chinese)
[16] Wu C Y, Trieu A, Radhakrishnan P, Kwok S F, Harris S, Zhang K, Wang J L, Wan J M, Zhai H Q, Takatsuto S, Matsumoto S, Fujioka S, Feldmann K A, Pennella R I.Brassinosteroids regulate grain filling in rice.Plant Cell, 2008, 20: 2130-2145
[17] Maharjan P M, Schulz B, Choe S.BIN2/DWF12 antagonistically transduces brassinosteroid and auxin signals in the roots of Arabidopsis. J Plant Physiol, 2011, 54: 126-134
[18] 徐涛, 王建斌, 洛世明. 玉米oxylipins信号途径关键基因的克隆及其在虫害诱导防御中的作用. 科学通报, 2005, 50: 2217-2225
Xu T, Wang J B, Luo S M.Cloning of key genes involved in oxylipins signaling pathway and their roles in insect induced defense in maize.Chin Sci Bull, 2005, 50: 2217-2225 (in Chinese)
[19] Xu F, Zhao Y X, Wang F, Guo D L, Wei Y L, Huang X Z.Cloning of a vacuolar H+-pryophosphatase gene from emphemeral plant olimarabidopsispumila whose overexpression improve salt tolerance in tobacco.Afr J Biotechnol, 2013, 12: 6817-6825
[20] Horsch R, Fry J, Hoffmann N, Eichholtz D, Rogers S A, Fraley R.A simple and general method for transferring genes into plants.Science, 1985, 227: 1229-1231
[21] Du H, Wang N, Cui F, Li X, Xiao J, Xiong L.Characterization of the beta-carotene hydroxylase gene DSM2 conferring drought and oxidative stress resistance by increasing xanthophylls and abscisic acid synthesis in rice. Plant Physiol, 2010, 154: 1304-1318
[22] 苍晶, 赵会杰. 植物生理学实验教程. 北京: 高等教育出版社, 2013. pp 151-153
Cang J, Zhao H J.Experimental Guide of Plant Physiology. Beijing: Higher Education Press, 2013. pp 151-153 (in Chinese)
[23] Liu T, Zhang J, Wang M, Wang Z, Li G, Qu L, Wang G.Expression and functional analysis of ZmDWF4, an ortholog of Arabidopsis DWF4 from maize(Zea mays L.). Plant Cell Rep, 2007, 26: 2091-2099
[24] Divi U K, Krishna P.Overexpression of the brassinosteroid biosynthetic gene AtDWF4 in Arabidopsis seeds overcomes abscisic acid-induced inhibition of germination and increases cold tolerance in transgenic seedlings. Plant Growth Regul, 2010, 29: 385-393
[25] Wang Y, Chen Y F, Chang X H, Wang X, Li N, Gao Y F.Heterologous expression of Populus euphratica DWF4 (PeDWF4) enhanced tolerance to abiotic stress in tobacco plants. Genomics Appl Biol, 2017, Heterologous expression of Populus euphratica DWF4 (PeDWF4) enhanced tolerance to abiotic stress in tobacco plants. Genomics Appl Biol, 2017, (in Chinese with English abstract)
[26] Miller G, Suzuki N, Ciftci-Yilmaz S, Mittler R.Reactive oxygen species homeostasis and signaling during drought and salinity stresses.Plant Cell Environ, 2010, 33: 453-457
[27] Kessler A, Baldwin I T.Plant response to insect herbivory: The emerging molecular analysis.Annu Rev Plant Biol, 2002, 53: 299-328
[28] Ryan C A.The systemin signaling pathway: differential activation of plant defensive genes.Biochim Biophy Acta, 2000, 1477: 112-121
[29] Li L, Li C, Lee G I, Howe G A.Distinct roles for jasmonate synthesis and action in the systemic wound response of tomato.Proc Natl Acad Sci USA, 2002, 99: 6416-6421
[1] WANG Dan-Dan, LIU Hong-Juan, WANG Hong-Xia, ZHANG Peng, SHI Chun-Yu. Cloning and functional analysis of the sweet potato sucrose transporter IbSUT3 [J]. Acta Agronomica Sinica, 2020, 46(7): 1120-1127.
[2] ZHAI Yu-Shan, DENG Yu-Qing, DONG Meng, XU Qian, CHENG Guang-Yuan, PENG Lei, LIN Yan-Quan*,XU Jing-Sheng*. Cloning and Characterization of Light Harvesting Chlorophyll a/b-Binding Protein Coding Gene (ScLhca3) in Sugarcane [J]. Acta Agron Sin, 2016, 42(09): 1332-1341.
[3] SU Wei-Hua**,LIU Feng**,HUANG Long,SU Ya-Chun,HUANG Ning,LING Hui,WU Qi-Bin,ZHANG Hua,QUE You-Xiong*. Cloning and Expression Analysis of a Ca2+/H+ Antiporter Gene from Sugarcane [J]. Acta Agron Sin, 2016, 42(07): 1074-1082.
[4] LIU Feng**,SU Wei-Hua**,HUANG Long,XIAO Xin-Huan,HUANG Ning,LING Hui,SU Ya-Chun,ZHANG Hua,QUE You-Xiong. Isolation and Characterization of a Na+/H+Antiporter Gene from Sugarcane [J]. Acta Agron Sin, 2016, 42(04): 501-512.
[5] HAO Jun-Jie,HU Yu-Wei,GUO Xiao-Qin,ZhAO Fu-An,JIA Xin-He,GUO Li-Juan,ZHANG Zhi-Xing,WANG Qing-Dong. Analysis of Resistance to Verticillium Wilt in Cotton by Reciprocal Grafting and Real-time Quantitative PCR [J]. Acta Agron Sin, 2013, 39(07): 1179-1186.
[6] DI Ying, LEI Ting-Ting, YAN Fan, HUANG Kai-Meng, LI Xiao-Wei, ZHANG Qiang-Lin, ZHANG Hai-Jun, SU Lian-Tai, SUN Cuan, WANG Yang, LI Jing-Wen, WANG Qiang-Yu. Cloning and Expression of a Stress-induced GmPRP Gene in Soybean (Glycine max) [J]. Acta Agron Sin, 2011, 37(12): 2152-2157.
[7] YANG Jing-Jing, LI Ya-Ning, LI Xing, LIU Da-Qun. Involvement of Heterotrimeric G Protein α and β Subunits in Defense Responses of Wheat to Puccinia triticina [J]. Acta Agron Sin, 2010, 36(12): 2028-2034.
[8] MA Xiong-Feng,YU Chun-Ming,TANG Shou-Wei,ZHU Ai-Guo,WANG Yan-Zhou,ZHU Si-Yuan,. Cloning and Tissue Expression of Acting1 Gene in Different Fiber Development Phases of Ramie [Boehmeria nivea (Linn.) Gaud] [J]. Acta Agron Sin, 2010, 36(1): 101-108.
[9] LIU Jian-Xin;YU Chun-Ming;TANG Shou-Wei;ZHU Ai-Guo;WANG Yan-Zhou;ZHU Si-Yuan;MA Xiong-Feng;XIONG He-Ping. Cloning and Tissue Expression of Important Enzyme Gene UGlcAE in Ramie Pectin Biosynthesis [J]. Acta Agron Sin, 2008, 34(11): 1938-1945.
[10] YU Shun-Wu;LIU Hong-Yan;LUO Li-Jun. Analysis of Relative Gene Expression Using Different Real-time Quantitative PCR [J]. Acta Agron Sin, 2007, 33(07): 1214-1218.
[11] CHEN Ying;SU Ning;XU Bo-Liang;GE Yi-Qiang;WANG Shu-Guang. Real-time Quantitative PCR Detection of Genetically Modified Maximizerâ Maize and YieldGardâ Maize [J]. Acta Agron Sin, 2004, 30(06): 602-607.
Full text



No Suggested Reading articles found!