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作物学报

作物学报 ›› 2023, Vol. 49 ›› Issue (9): 2462-2471.doi: 10.3724/SP.J.1006.2023.24236

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

马铃薯StCYP85A3促进萌芽及根系伸长的功能解析

刘洁1,2(), 蔡诚诚1,2, 刘石锋1,2, 邓孟胜3, 王雪枫2, 温和1,2, 李罗品1,2, 严奉君1,2, 王西瑶1,2,*()   

  1. 1四川农业大学西南作物基因资源发掘与利用国家重点实验室, 四川成都 611130
    2四川农业大学农学院马铃薯研究与开发中心, 四川成都 611130
    3四川轻化工大学生物工程学院, 四川宜宾 644000
  • 收稿日期:2022-10-19 接受日期:2023-02-21 出版日期:2023-09-12 网络出版日期:2023-03-10
  • 通讯作者: *王西瑶, E-mail: 1357664714@qq.com
  • 作者简介:刘洁, E-mail: 1196438854@qq.com
  • 基金资助:
    马铃薯种薯活力调控的分子机制与应用研究项目(SKL-ZY202203);国家现代农业技术体系四川薯类创新团队项目(sccxtd- 2023-09)

Function analysis of potato StCYP85A3 in promoting germination and root elongation

LIU Jie1,2(), CAI Cheng-Cheng1,2, LIU Shi-Feng1,2, DENG Meng-Sheng3, WANG Xue-Feng2, WEN He1,2, LI Luo-Pin1,2, YAN Feng-Jun1,2, WANG Xi-Yao1,2,*()   

  1. 1State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, Sichuan Agricultural University, Chengdu 611130, Sichuan, China
    2Potato Research and Development Center, College of Agriculture, Sichuan Agricultural University, Chengdu 611130, Sichuan, China
    3College of Biological Engineering, Sichuan University of Light Chemical Industry, Yibin 644000, Sichuan, China
  • Received:2022-10-19 Accepted:2023-02-21 Published:2023-09-12 Published online:2023-03-10
  • Supported by:
    Molecular Mechanism and Application of Seed Potato Vigor Regulation(SKL-ZY202203);Sichuan potato Innovation Team Project of National Modern Agricultural Industry Technology System(sccxtd- 2023-09)

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摘要：

油菜素内酯(BR)是一类植物甾醇类激素, 对植物生长发育及胁迫应答具有显著的调控作用, 马铃薯中StCYP85A3作为BR合成酶的编码基因, 功能仍待进一步探究。本研究从‘川芋10号’马铃薯中克隆出该基因, 生物信息学分析表明该蛋白具有催化BR合成的典型亲氧结合域, 属于CYP85As家族成员。通过组织表达分析发现该基因在块茎芽眼及根中表达量最高, 且芽眼中的表达量随着储藏时间的延长而增加, 在块茎芽眼休眠解除时表达迅速升高。同时发现外源BR可诱导马铃薯芽眼及根中StCYP85A3表达量升高, 从而显著促进了种薯萌芽及幼苗根的伸长。通过在拟南芥野生型及cyp85a2突变体中过表达StCYP85A3发现, 过表达株系种子萌发及根系伸长都早于野生型, 同时回补株系弥补了突变体种子萌发、植株根系生长迟缓的缺陷。此外, 外源BR对过表达株系种子萌发、根系伸长无显著促进作用, 但显著促进了野生型、突变体及回补株系种子萌发、根系伸长。上述马铃薯及拟南芥试验结果表明, StCYP85A3具有促进萌芽及根系伸长的功能。

关键词: 马铃薯, StCYP85A3, BR合成酶, 根系, 萌芽

Abstract:

Brassinolide (BR), a kind of phytosterol hormone, plays the significant role in regulating plant growth and development and stress response. The function of StCYP85A3 as the encoding gene of BR synthease in potato remains to be further explored. In this study, the gene was cloned from potato ‘Chuanyu 10’. Bioinformatics analysis showed that this protein had a typical aerobic binding domain for catalyzing BR synthesis and belonged to the CYP85As family. Tissue expression analysis indicated that the relative expression level of this gene was the highest in the bud eye of the tuber and the root, and the expression amount in the bud eye increased with the extension of storage time, and the expression increased rapidly when the dormancy of the tuber bud eye was released. At the same time, the exogenous BR could induce the relative expression level of StCYP85A3 in potato bud eye and root, which significantly promoted the germination of seed potato and the elongation of seedling root. The overexpression of StCYP85A3 in Arabidopsis wild-type and mutant cyp85a2 demonstrated that the seed germination and root elongation of the overexpression strains were earlier than wild type, and the defects of mutant seed germination and plant root growth retardation were remedied by supplementing strains. In addition, exogenous BR did not significantly promote seed germination and root elongation of over expressed strains, but significantly promoted seed germination and root elongation of wild type, mutant, and complementary strains. The above potato and Arabidopsis thaliana experiments showed that StCYP85A3 had the function of promoting germination and root elongation.

Key words: potato, StCYP85A3, BR synthetase, root system, germination

表1

本研究中所用的引物"

基因Gene name 编号Gene ID 引物Primer sequence (5°-3°)
EF1αL Soltu.DM.06G005580 F: CTTGTACACCACGCTAAGGAG
R: GTCAATGCAAACCATTCCTTG
qCYP85A3 Soltu.DM.02G009130.1 F: CCACATACTTGGTTGCCCTAC
R: TGCTCCTCTAATGTGCTTGTG
CYP85A3 Soltu.DM.02G009130.1 F: ATGGCCATTTTCTTGATTGTTTT
R: CTAGTAAGTTGAAACCTTAATC

图1

亚细胞定位预测、蛋白序列保守性Logo图、进化树分析 A: 亚细胞定位预测; B: 蛋白序列保守性Logo图; C: 进化树分析; D: 基因结构分析。蛋白序列保守型Logo图, 用来分析和展示序列保守性, 图中字母高度与该位置的相应碱基或氨基酸残基的出现频率决定, 以bit为单位, 每个位置字母由大到小排列, 可从顶端字母识别保守序列。"

表2

StCYP85A3启动子重要顺式作用元件"

调控元件
Cis-element		 核心序列
Core sequence		 元件功能描述
Functional description of cis-element		 位置
Position
ABRE CACGTG ABA响应元件
Cis-acting element involved in the abscisic acid responsiveness		 -6, -574
ARE AAACCA 顺式作用调节元件对无氧诱导至关重要
Cis-acting regulatory element essential for the anaerobic induction		 -1467
TGA-element AACGAC 生长素响应元件
Auxin-responsive element		 -205, -438
Box4 ATTAAT 参与光响应的保守DNA模块的一部分
Part of a conserved DNA module involved in light responsiveness		 -12, -112, -818, -1082, -1442
GT1-motif GGTTAA 光响应元件
Cis-acting element for light responsive		 -91
G-box TACGTG 光响应元件
Cis-acting element for light responsive		 -6, -574
CAT-box GCCACT 与分生组织表达相关的顺式作用调节元件
Cis-acting regulatory element related to meristem expression		 -245

图2

CYP85A3组织表达分析及C10贮藏期芽眼处CYP85A3表达量分析 A: CYP85A3组织表达量; B: C10贮藏期CYP85A3表达量分析。误差线代表标准差, 统计分析采用Student’s t检验, 不同小写字母表示组织间及贮藏后的显著差异(P < 0.05)。"

图3

BR外源施用对马铃薯块茎萌芽及根系生长的影响 A: 不同浓度BR处理对马铃薯块茎发芽率的影响; B: 不同浓度BR处理对马铃薯幼苗根长的影响; C: BR处理对马铃薯芽眼中StCYP85A3表达量的影响; D: BR处理对马铃薯幼苗根中StCYP85A3表达量的影响。误差线代表标准差, 统计分析采用Student’s t检验, 不同小写字母表示显著差异(P < 0.05)。"

图4

过表达、突变体及回补发芽率 A: MS培养基上不同株系拟南芥萌芽率统计; B: 含有50 nmol L-1 BR的MS培养基上不同株系拟南芥萌芽率统计; C: MS培养基上不同株系拟南芥种植3 d后萌芽情况; D: 含有50 nmol L-1 BR的MS培养基上不同株系拟南芥种植60 h的萌芽情况。误差线代表标准差, A, B两图60 h数据共同进行显著性分析, 统计分析采用Student’s t检验, 不同小写字母表示不同株系发芽的显著差异(P < 0.05)。"

图5

过表达、突变体及回补根长 A: MS及含50 nmol L-1 BR MS培养基上不同株系拟南芥根长统计; B: MS培养基上不同株系拟南芥根长表型; C: 50 nmol L-1 BR处理各株系拟南芥根中StCYP85A3表达量随时间变化; D: 50 nmol L-1 BR处理各株系拟南芥发芽10 d后根长表型。误差线代表标准差, 统计分析采用Student’s t检验, 不同小写字母表示根长及表达量的显著差异(P < 0.05)。"

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