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作物学报 ›› 2022, Vol. 48 ›› Issue (4): 1017-1026.doi: 10.3724/SP.J.1006.2022.04274

• 研究简报 • 上一篇    下一篇

甘蔗赤霉素氧化酶基因ScGA20ox1的克隆及功能分析

周慧文1(), 丘立杭1, 黄杏, 李强2, 陈荣发1, 范业赓1, 罗含敏1, 闫海锋1, 翁梦苓1, 周忠凤1,*(), 吴建明1,*()   

  1. 1农业农村部甘蔗生物技术与遗传改良重点实验室 / 广西甘蔗遗传改良重点实验室/广西农业科学院甘蔗研究所, 广西南宁 530007
    2广东省湛江农垦科学研究所, 广东湛江 524086
  • 收稿日期:2021-04-07 接受日期:2021-09-09 出版日期:2022-04-12 网络出版日期:2021-10-18
  • 通讯作者: 周忠凤,吴建明
  • 作者简介:E-mail: windyrentmd@qq.com
  • 基金资助:
    国家重点研发计划项目(2019YFD1000503);广西自然科学基金项目(2021GXNSFAA220014);国家现代农业产业技术体系广西创新团队资助(nycytxgxcxtd-2021-03-02)

Cloning and functional analysis of ScGA20ox1 gibberellin oxidase gene in sugarcane

ZHOU Hui-Wen1(), QIU Li-Hang1, HUANG Xing, LI Qiang2, CHEN Rong-Fa1, FAN Ye-Geng1, LUO Han-Min1, YAN Hai-Feng1, WENG Meng-Ling1, ZHOU Zhong-Feng1,*(), WU Jian-Ming1,*()   

  1. 1Key Laboratory of Sugarcane Biotechnology and Genetic Improvement, Ministry of Agriculture and Rural Affairs / Guangxi Key Laboratory of Sugarcane Genetic Improvement, Sugarcane Research Institute / Guangxi Zhuang Autonomous Region Academy of Agri-cultural Sciences, Nanning 530007, Guangxi, China
    2Scientific Research Institute of Zhanjiang State Farms, Zhanjiang 524086, Guangdong, China
  • Received:2021-04-07 Accepted:2021-09-09 Published:2022-04-12 Published online:2021-10-18
  • Contact: ZHOU Zhong-Feng,WU Jian-Ming
  • Supported by:
    Sub-project of the National Key Research and Development Program of China(2019YFD1000503);Natural Science Foundation of Guangxi, China(2021GXNSFAA220014);Guangxi Innovation Team of China Agricultural Research System(nycytxgxcxtd-2021-03-02)

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摘要：

GA20-氧化酶(GA20-oxidase, GA20ox)是赤霉素(gibberellic acid, GA)合成过程中的关键限速酶, 但甘蔗GA20ox1基因(ScGA20ox1)的功能及其表达模式尚未明确。本研究利用RT-PCR和RACE技术克隆了甘蔗GA20-氧化酶基因(ScGA20ox1), ScGA20ox1基因全长1574 bp, 含有一个1125 bp的完整开放阅读框(open reading frame, ORF), 编码375个氨基酸。ScGA20ox1蛋白分子量为42.3 kD, 理论等电点为5.95, 不含信号肽, 不含跨膜结构域, 为可溶性蛋白。实时荧光定量PCR结果表明该基因在甘蔗幼苗中茎秆的表达量最高, 叶片次之, 根的表达量最低; 干旱、低温和赤霉素处理均会改变该基因在不同组织的表达模式。利用农杆菌介导法转化拟南芥, 获得过表达ScGA20ox1的转基因拟南芥植株, 转基因拟南芥植株存在株高增高, 节间长度增长的表型变异。本研究结果表明, ScGA20ox1参与甘蔗的生长发育并在响应非生物逆境中发挥重要调控作用, 过表达转基因拟南芥形态发生变化, 这为深入探究ScGA20ox1生物学功能, 解析甘蔗株型调控分子机制提供理论依据。

关键词: 甘蔗, 赤霉素氧化酶, 基因克隆, 表达水平

Abstract:

GA20-oxidase (GA20ox) is a key rate-limiting enzyme in the synthesis of gibberellic acid (GA), but the biological function and expression pattern of gene GA20ox1 in sugarcane remain unknown. In this study, we cloned GA20-oxidase gene (ScGA20ox1) from the sugarcane variety ROC22 sequence by RT-PCR and RACE, which had a total length of 1574 bp and a complete open reading frame (ORF) of 1125 bp that encoded 375 amino acids. The ScGA20ox1 protein is a hydrophilic protein with a molecular weight of 42.3 kD, an iscelectric point (pI) of 5.95, and no transmembrane structure or signal peptide. Real-time fluorescence quantitative analysis revealed that ScGA20ox1 had the highest expression levels in stems, medium in the leaves, and the lowest in roots of sugarcane seedlings. Drought stress, low temperature stress, and GA3 treatment change the expression patterns of ScGA20ox1 in different tissues. ScGA20ox1 was transformed into Arabidopsis via Agrobacterium-mediated transformation. Transgenic Arabidopsis thaliana plants with overexpressed ScGA20ox1 were obtained from the transformed Arabidopsis thaliana via Agrobacterium-mediated transformation, which showed a phenotypic variation of plant height and internode length increase. In conclusion, ScGA20ox1 was involved in sugarcane’s growth as a crucial regulator in response to abiotic stress and the phenotypic variation of overexpressed transgenic Arabidopsis thaliana changed, which would lay a theoretical basis for future in-depth studies on ScGA20ox1’s biological function and analysis of the molecular mechanism of sugarcane plant shape regulation.

Key words: sugarcane, gibberellin oxidase, gene cloning, expression level

表1

ScGA20ox1基因克隆与表达分析中使用的引物"

引物
Primer		 引物序列
Primer sequences (5'-3')		 用途
Function
F1 CATCCCGC(T)A(C)GCAGTTCATCT 中间片段 Intermediate fragment
R1 CAACATCGGCGACACCTTCAT
5R1 CCTCAACTACTACCCGCCGT 5' RACE
5R2 ACTTCCGCCGCTTCTTCCAG
3F1 TTCTTCACGCTCCCGCTGTC 3' RACE
3F2 ACTTCCGCCGCTTCTTCCAG
F2 ATGGTGCAGCAGGCTGCGC 编码区 Coding region
R2 GGCCTCATCACCTCCATGA
GA20-qF CGGCTTCTTCCAGGTGGTCAA RT-qPCR
GA20-qR TGGAAGGAGACGCTGTCGTTC
25S rRNA-qF GCAGCCAAGCGTTCATAGC RT-qPCR
25S rRNA-qR GGATTGTTCACCCACCAATAGG
knpGA20-F GGGGTACCATGGTGCAGCAGGCTGCGC 载体构建 Vector construction
bamGA20-R CGGGATCCTCATGGAGGTGATGAGGCC 载体构建 Vector construction

图1

PCR检测ScGA20ox1克隆产物电泳图 M: DL2000 marker; A: 中间片段; B: 3′端片段; C: 5′端片段; D: 开放阅读框。"

图2

ScGA20ox1基因的保守功能结构域分析"

图3

ScGA20ox1 蛋白的三级结构"

图4

不同植物的GA20ox蛋白序列比对分析 CBX45610: 小麦; NP 001241783: 玉米; AAB48239: 水稻; AAT49058: 大麦; KY416517: 甘蔗; XP-002463483: 高粱。黑色方框依次为GA20ox蛋白的LPWKET和NYYPPCQRP的保守结构域。"

图5

ScGA20ox1与其他植物GA20ox氨基酸序列的进化树分析"

图6

ScGA20ox基因在不同组织和不同处理下的差异表达 A: 不同组织; B: 干旱胁迫; C: 低温胁迫; D: 赤霉素处理。不同小写字母表示同一组织在处理0 h、24 h、48 h后表达水平的差异显著性(P < 0.05)。"

图7

抗性植株的PCR检测 M: DL2000 marker; 1: 纯水对照; 2: pEASY-ScGA20ox1质粒; 3: 野生型拟南芥; 4~13: 转基因拟南芥。"

图8

ScGA20ox1在野生型(WT)和转基因拟南芥(ga20-1~ga20-10)中的表达水平 不同小写字母表示处理间差异显著(P < 0.05)。"

图9

过表达转基因拟南芥株系的表型鉴定 A: 表型观察; B: 株高测量; C: 节间长度测量; D: 节间数测量; WT: 野生型拟南芥; ga20-3、ga20-5、ga20-6为过表达转基因拟南芥株系。不同小写字母表示处理间差异显著(P < 0.05)。"

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