苦荞FtDELLA基因的克隆与表达分析

doi:10.3969/j.issn.1004-1524.20230913

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (8): 1709-1718.DOI: 10.3969/j.issn.1004-1524.20230913

苦荞FtDELLA基因的克隆与表达分析

孙培媛(), 冉彬, 王佳蕊, 李洪有()

贵州师范大学生命科学学院,荞麦产业技术研究中心,贵州贵阳 550001

收稿日期:2023-07-26 出版日期:2024-08-25 发布日期:2024-09-06
作者简介:*李洪有,E-mail: lihongyouluod@163.com
孙培媛(1998—),女,贵州铜仁人,硕士研究生,主要从事分子逆境胁迫研究。E-mail: sunpeiyuan779@163.com
通讯作者: 李洪有
基金资助:
贵州省科技计划项目(黔科合基础-ZK〔2021〕重点035);国家自然科学基金项目(31701494);国家自然科学基金项目(32260461);国家燕麦荞麦现代农业产业技术体系专项资金(CARS-07-A5)

Cloning and expression analysis of Fagopyrum tataricum FtDELLA gene

SUN Peiyuan(), RAN Bin, WANG Jiarui, LI Hongyou()

Research Center of Buckwheat Industry Technology, College of Life Sciences, Guizhou Normal University, Guiyang 550001, China

Received:2023-07-26 Online:2024-08-25 Published:2024-09-06
Contact: LI Hongyou

摘要/Abstract

摘要：

DELLA蛋白是赤霉素(GA)传导途径中的负调控蛋白,其在植物生长发育和胁迫响应中起着重要作用。该实验利用RT-PCR技术从苦荞中克隆到一个DELLA基因,命名为FtDELLA。克隆得到FtDELLA基因CDS全长1 452 bp,编码483个氨基酸。蛋白多序列比对显示,FtDELLA蛋白含有DELLA蛋白家族保守的DELLA保守结构域;系统进化分析表明,FtDELLA蛋白与拟南芥中AtRGA1亲缘关系最近。qRT-PCR结果显示,FtDELLA在苦荞不同组织部位均有表达,其中在茎和灌浆前的种子中表达量最高,在苦荞种子发育过程中,FtDELLA表达量呈逐渐下降趋势。在干旱胁迫下,FtDELLA的表达明显受到抑制,表明其可能是苦荞干旱胁迫应答的一个负调控基因。该研究结果为进一步研究FtDELLA在苦荞中的功能和作用机制提供了理论基础。

关键词: 苦荞, FtDELLA, 基因克隆, 赤霉素, 表达分析

Abstract:

DELLA protein is a negative regulatory protein in the gibberellin (GA) transduction pathway, which plays an important role in plant growth and development and stress response. In this experiment, a DELLA gene, named FtDELLA, was cloned from buckwheat using RT-PCR. The gene is 1 452 bp in CDS length and encodes 483 amino acids. Phylogenetic analysis showed that the FtDELLA protein contained the conserved DELLA conserved structural domains of the DELLA family. The qRT-PCR results showed that FtDELLA was highly expressed in stems and pre-grouting seeds, and its expression showed a decreasing trend during seed development. Under drought stress, the expression of FtDELLA showed a decreasing trend, indicating that it may be a negative regulatory gene for drought stress response in buckwheat. The results suggest that FtDELLA may play an important regulatory role in the growth and development of buckwheat and drought stress response. The results provide a theoretical basis for further research on the function and mechanism of action of FtDELLA in buckwheat.

Key words: Fagopyrum tataricum, FtDELLA, gene cloning, gibberellin, expression analysis

中图分类号:

S519

孙培媛, 冉彬, 王佳蕊, 李洪有. 苦荞FtDELLA基因的克隆与表达分析[J]. 浙江农业学报, 2024, 36(8): 1709-1718.

SUN Peiyuan, RAN Bin, WANG Jiarui, LI Hongyou. Cloning and expression analysis of Fagopyrum tataricum FtDELLA gene[J]. Acta Agriculturae Zhejiangensis, 2024, 36(8): 1709-1718.

图/表 10

表1 本研究所用引物序列

Table 1 Primer sequences used in this study

引物名称 Primer name	引物序列 Primer sequence(5'-3')
FtDELLA-kF	ATGCCTGCAGGTCCGACGAT
FtDELLA-kR	TCAAGTCTCAGCCGAGTTTCG
FtDELLA-qF	TTCCATCACAGAGCTTCACC
FtDELLA-qR	GTTGTGGTTCGCCTCTTGTT
FtHLK-qF	TTCACGGGCACCATTCATGG
FtHLK-qR	AGGTGGAAGCTGAAGGAAGC
FtUPL7-qF	ATGTTGTGGGACGTGTGGTT
FtUPL7-qR	AAGCGATCCTCTGGTGATAG

图1 PCR扩增结果 M,DL2 000 marker;1,PCR扩增产物。

Fig.1 PCR amplification result M, DL2 000 marker; 1, PCR amplification product.

图2 FtDELLA基因的CDS和氨基酸序列

Fig.2 Coding region and amino acid sequence of FtDELLA

图3 FtDELLA理化性质分析 A,氨基酸含量分布;B,FtDELLA蛋白磷酸化修饰;C,FtDELLA蛋白氨基酸亲、疏水预测;D,FtDELLA蛋白信号肽预测图。

Fig.3 Analysis of physical and chemical properties of FtDELLA A, Amino acid content distribution; B, FtDELLA phosphorylation modifications; C, FtDELLA amino acid pro- and hydrophobic predictions; D, FtDELLA signal peptide prediction map.

图4 FtDELLA结构预测 A,保守结构域;B,二级结构预测,其中蓝色为α螺旋,红色为β折叠,绿色为β转角,紫色为无规卷曲;C-a,FtDELLA蛋白N端蛋白三级结构示意图;C-b,FtDELLA蛋白C端蛋白三级结构示意图。

Fig.4 FtDELLA structure prediction A, Conserved structural domain; B, Prediction of secondary structure; the blue represents α-helix; the red represents β-sheet; the green represents β-turn; the purple represents random coil. C-a, Schematic representation of the N-terminal tertiary structure of the FtDELLA protein; C-b, Schematic representation of the C-terminal tertiary structure of the FtDELLA protein.

图5 FtDELLA保守基序分析

Fig.5 FtDELLA conserved motif analysis

图6 FtDELLA与其他物种DELLA蛋白多序列比对

Fig.6 Multiple sequence comparison between FtDELLA and DELLA proteins of other species

图7 FtDELLA系统进化树

Fig.7 Phylogenetic tree of FtDELLA

图8 FtDELLA在苦荞不同组织部位的表达种子1,开花后3 d的种子;种子2,开花后10 d的种子;种子3,开花后13 d的种子。

Fig.8 FtDELLA expression in different tissue parts of buckwheat Seed 1, Seeds at 3 days after flowering; Seed 2, Seeds at 10 days after flowering; Seed 3, Seeds at 13 days after flowering.

图9 FtDELLA在干旱胁迫下的响应程度

Fig.9 Level of FtDELLA response under drought stress

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苦荞FtDELLA基因的克隆与表达分析

Cloning and expression analysis of Fagopyrum tataricum FtDELLA gene

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