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作物学报 ›› 2018, Vol. 44 ›› Issue (03): 343-356.doi: 10.3724/SP.J.1006.2018.00343

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

玉米油菜素甾醇生物合成关键酶基因ZmCYP90B1的克隆及其对逆境胁迫的响应

段方猛1(), 罗秋兰2, 鲁雪莉3, 齐娜伟1, 刘宪舜1, 宋雯雯1,*()   

  1. 1青岛农业大学植物医学学院, 山东青岛266109
    2深圳市海洋生物资源与生态环境重点实验室 / 深圳市海洋藻类开发与应用工程实验室 / 深圳大学生命科学学院, 广东深圳 518060
    3中国农业科学院烟草研究所, 山东青岛266101
  • 收稿日期:2017-07-05 接受日期:2017-11-21 出版日期:2018-03-12 网络出版日期:2017-12-18
  • 通讯作者: 宋雯雯
  • 作者简介:

    fmduan@hotmail.com

  • 基金资助:
    本研究由青岛市应用基础研究计划项目(16-5-1-54-jch), 山东省自然科学基金项目(ZR2013CQ028), 深圳市科技计划项目(JCYJ2015 0324141711583)和青岛农业大学博士基金项目(663/1111316)资助

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 Published:2018-03-12 Published online:2017-12-18
  • Contact: Wen-Wen SONG
  • 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).

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摘要：

油菜素甾醇作为一类重要植物激素, 在植物生长发育和抵御逆境胁迫等过程中发挥重要作用。CYP90B1基因编码酶是其合成途径中关键限速酶。本研究针对迄今有关玉米CYP90B1基因应答逆境胁迫特征尚未见报道现状, 采用RT-PCR结合RACE技术, 从玉米中克隆了油菜素甾醇生物合成关键基因ZmCYP90B1 (GenBank登录号KY242373)。ZmCYP90B1全长2058 bp, 开放阅读框为1518 bp, 编码506个氨基酸。ZmCYP90B1蛋白预测分子量为57.66 kD, 等电点为9.54, 含有1个跨膜结构域及1个p450保守结构域。序列比对表明, ZmCYP90B1与其他物种CYP90B1蛋白高度相似, 但在单双子叶植物中进化上具有明显差异。实时荧光定量PCR结果表明, 非生物胁迫(干旱、高盐、低温和脱落酸)、虫害(甜菜夜蛾取食)和茉莉酸甲酯均诱导ZmCYP90B1表达, 表明该基因响应多种非生物胁迫, 参与植物对虫害和茉莉酸甲酯的响应。进一步构建过量表达ZmCYP90B1基因的烟草株系并检测表明该烟草株系抗旱性增强, 叶片失水率下降, SPAD值增大。此外, 干旱胁迫下转基因烟草超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)的活性以及游离脯氨酸(Pro)的积累量均显著高于野生型, 丙二醛(MDA)和脱落酸(ABA)含量明显低于野生型。通过检测下游胁迫响应基因表达, 表明ZmCYP90B1提高植物抗旱性可能不依赖ABA途径, 而与其对抗氧化相关途径相关基因的转录调控有关。

关键词: 油菜素甾醇, ZmCYP90B1基因, 实时荧光定量PCR, 逆境胁迫

Abstract:

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

表1

克隆基因所用引物"

引物
Primer		 序列
Sequence (5°-3°)
DP-F GGCGGCATCCTGGGCAAGT
DP-R CCCGCGAAGAGCAGGCTCA
5°-GSP TGGACCACTTGCCCAGGATGCC
3°-GSP GAAGATGGAGGACAGGCTTGAGA
FP-F CTCTCTCCAACCCATCCGTC
FP-R GGGAGGTAATGTTGGGGTTTG

表2

荧光定量PCR所需引物信息"

基因名称
Gene name		 GenBank登录号
GenBank number		 引物序列
Primer sequence (5°-3°)		 产物长度
Product size
(bp)		 退火温度
Annealing
temperature (°C)
NtAPX NM_001324874.1 F: CAAATGTAAGAGGAAACTCAGAGGA
R: AGCAACAACTCCAGCTAATTGATAG		 262 60
NtCAT NM_001326196.1 F: AGGTACCGCTCATTCACACC
R: AAGCAAGCTTTTGACCCAGA		 151 60
NtSOD AF443178.1 F: CCGTCGCCAAATTGCATAG
R: CGATAGCCCAACCAAGAGAAC		 238 62
NtNCED1 NM_001326185.1 F: CTATTTCCACTTCAAAACCAACCAC
R: GGCACTTTCCACGGCATCT		 131 62
NtERD10C AB049337.1 F: ACGTGGAGGCTACAGATCGTGGTTT
R: TCTCCACTGGTACAGCCGTGTCCTCAC		 366 70
NtLEA5 AF053076.1 F: GAACCCAACAAGAGCGAGAGA
R: CGACAGGAAGCATTGACGAG		 350 62
Tob103 U60495.1 F: CAAGGAAATCACCGCTTTGG
R: AAGGGATGCGAGGATGGA		 106 60
ZmCYP90B1 KY242373 F: GGCTGTGGTCTCGTGTTTTATG
R: ACCATCCTCCCCTTTGCTCT		 104 62
Actin NM_00115473.1 F: CTGAGGTTCTATTCCAGCCATCC
R: CCACCACTGAGGACAACATTACC		 133 62

图1

ZmCYP90B1基因扩增结果 A: ZmCYP90B1部分片段扩增结果; B: ZmCYP90B1 5´-RACE扩增结果; C: ZmCYP90B1 3´-RACE扩增结果; D: ZmCYP90B1的ORF区扩增结果; E: 重组载体双酶切鉴定结果; M1: DL2000 marker; M2: 15000 marker。"

图2

ZmCYP90B1基因编码产物的氨基酸序列下画线部分为跨膜结构域氨基酸位点: 12~31; 灰色阴影部分为p450保守结构域位点: 2~491。"

图3

ZmCYP90B1预测的氨基酸亲水性和疏水性分析"

图4

8种植物CYP90B1蛋白的多序列比对"

图5

ZmCYP90B1与其他植物CYP90B1蛋白的系统发生分析"

表3

不同植物来源的CYP90B1氨基酸理化性质的比较"

类别
Category		 物种
Species		 氨基酸残基数
Residues		 分子量
Molecular weight (kD)		 等电点pI 负电荷氨基酸比例
Negatively charged residues (%)		 正电荷氨基酸比例
Positively charged residues (%)		 不稳定
系数
Instability index		 总平均亲水性
Hydropathicity
单子叶植物
Monocotyledons		 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
双子叶植物
Dicotyledons		 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

图6

ZmCYP90B1基因在不同组织中的相对表达水平图柱上不同小写字母表示在0.05水平上差异显著。"

图7

ZmCYP90B1在非生物胁迫下的表达模式图柱上不同小写字母表示在0.05水平上差异显著。"

图8

ZmCYP90B1在甜菜夜蛾取食和茉莉酸甲酯下的表达模式图柱上不同小写字母表示在0.05水平上差异显著。"

图9

转基因烟草植株的分子鉴定 M: marker; P: 阳性质粒; N: 空载体; W: 双蒸水; WT: 野生型; 1~10: 转基因株系。"

图10

野生型和转基因烟草植株在干旱胁迫下的功能鉴定 A: 干旱胁迫前野生型和转基因株系(L4和L9)的表型; B: 20% PEG处理10 h后野生型和转基因株系(L4和L9)的表型; C: 20% PEG处理10 h后野生型和转基因株系(L4和L9)的叶片失水率; D: 20% PEG处理前后野生型和转基因株系(L4和L9)的SPAD值。图柱上不同小写字母表示在0.05水平上差异显著。"

图11

干旱胁迫前后野生型和转基因烟草的生理指标图柱上不同小写字母表示在0.05水平上差异显著。"

图12

干旱胁迫前后胁迫相关基因的表达图柱上不同小写字母表示在0.05水平上差异显著。"

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