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作物学报

作物学报 ›› 2024, Vol. 50 ›› Issue (11): 2731-2741.doi: 10.3724/SP.J.1006.2024.44024

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

大豆GmRSM1通过调节PIN基因表达促进顶端弯钩消失

付佳祺1,2(), 李世宽1,2, 谭萌慧1,2, 罗方3, 张传玲3, 刘祾悦3, 卢倩1,*(), 谷勇哲1,2,*()   

  1. 1哈尔滨师范大学生命科学与技术学院, 黑龙江哈尔滨 150025
    2中国农业科学院作物科学研究所, 北京 100081
    3阿荣旗农业事业发展中心, 内蒙古阿荣旗 162750
  • 收稿日期:2024-02-05 接受日期:2024-06-20 出版日期:2024-11-12 网络出版日期:2024-07-10
  • 通讯作者: *卢倩, E-mail: luqian@hrbnu.edu.cn; 谷勇哲, E-mail: guyongzhe@caas.cn
  • 作者简介:E-mail: 1440170828@qq.com
  • 基金资助:
    国家自然科学基金项目(32372190)

Soybean GmRSM1 promotes apical hook disappearance by regulating PIN gene expression

FU Jia-Qi1,2(), LI Shi-Kuan1,2, TAN Meng-Hui1,2, LUO Fang3, ZHANG Chuan-Ling3, LIU Ling-Yue3, LU Qian1,*(), GU Yong-Zhe1,2,*()   

  1. 1College of Life Sciences and Technology, Harbin Normal University, Harbin 150025, Heilongjiang, China
    2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    3Arong Banner Agricultural Development Center, Arong Banner 162750, Inner Mongolia, China
  • Received:2024-02-05 Accepted:2024-06-20 Published:2024-11-12 Published online:2024-07-10
  • Contact: *E-mail: luqian@hrbnu.edu.cn;E-mail: guyongzhe@caas.cn
  • Supported by:
    National Natural Science Foundation of China(32372190)

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摘要：

顶端弯钩是双子叶植物黄化幼苗短暂出现的独有结构, 尽管当前拟南芥顶端弯钩的模型网络已经相对透彻, 但在大豆中的研究却非常少。本试验构建pBAR-GmRSM1过表达载体, 通过遗传转化和阳性植株筛选, 得到3个拟南芥过表达纯合株系(OE-GmRSM1#64、#69和#70)和3个大豆纯合株系(OE-GmRSM1#103、#78和#95), 转化体的表达量均显著高于野生型。暗培养拟南芥和大豆, 过表达转化体的顶端弯钩消失或消失速度快于野生型。在扫描电镜下观察拟南芥野生型和转化体植株顶端弯钩部位细胞长度发现, 野生型在弯钩维持阶段弯钩内侧细胞长度小于外侧, 而转化体植株两侧细胞长度相同。分离大豆顶端弯钩内外两侧, 并通过检测PIN基因在大豆野生型与3个过表达转化体顶端弯钩的表达情况, 发现PIN1e、PIN3d和PIN6a三个基因在子叶出土时的顶端弯钩部位表达量显著高于顶端弯钩展开后的表达量, 而转化体中弯钩处这3个基因在弯钩维持阶段的表达量显著高于野生型, 但在展开后的表达量并无显著性差异。经过烟草瞬时表达, 确定GmRSM1蛋白定位在细胞核和细胞膜。因此, 顶端弯钩出现的原因是弯钩内外两侧细胞差异伸长, 因而导致下胚轴弯曲。GmRSM1可以通过正向调控生长素运输载体PIN1e、PIN3d和PIN6a缩短弯钩维持和展开时间, 验证了该基因对生长素转运的调控作用。本试验通过验证GmRSM1基因在顶端弯钩消失表型中的作用, 进一步完善了顶端弯钩的基因通路, 为后续研究奠定基础。

关键词: 顶端弯钩, 大豆, 拟南芥, PIN家族, 生长素

Abstract:

The apical hook is a transient structure found in etiolated dicot seedlings, and while the current model for the apical hook in Arabidopsis thaliana is relatively well-established, there is limited research on soybean. In this study, the pBAR- GmRSM1 overexpression vector was constructed, and three homozygous lines each of Arabidopsis thaliana (OE-GmRSM1#64, #69, and #70) and soybean (OE-GmRSM1#103, #78, and #95) were generated through genetic transformation and positive plant selection. The expression levels of the transgenic lines were significantly higher than the wild type. Both Arabidopsis thaliana and soybean cultures were grown in darkness, and the apical hooks in the overexpressed transformants disappeared or exhibited faster disappearance than the wild type. Scanning electron microscopy observations of cell length in the apical hook of Arabidopsis thaliana wild-type and transformants revealed that the inner hook cells were shorter than the outer hook cells during the hook maintenance stage, while the cell lengths were same on both sides in the transformant plants. The inner and outer sides of the soybean apical hook were separated, and the expression of the PIN gene in the wild type and three overexpressed transformants of soybean was analyzed. The results demonstrated that the expression levels of the PIN1e, PIN3d, and PIN6a genes were significantly higher in the apical hook after cotyledon unfolding, and during the hook maintenance stage, these three genes exhibited significantly higher expression levels in the transformants than the wild type, but there was no significant difference in expression levels after unfolding. Transient expression in tobacco confirmed than the GmRSM1 protein were localized in the nucleus and cell membrane. Thus, the apical hook formation is attributed to differential cell elongation between the inner and outer sides of the hook, leading to hypocotyl curvature. GmRSM1 regulates the expression of auxin transporters PIN1e, PIN3d, and PIN6a, thereby shortening the duration of hook maintenance and unfolding. These genes are positively regulated by GmRSM1 during the hook stage, and the study confirmed the regulatory effect of this gene on auxin transport. Overall, this study verified the role of GmRSM1 in the disappearance of the apical hook phenotype and further elucidated the gene pathway involved in apical curvature, laying a foundation for future research.

Key words: apical hook, soybean, Arabidopsis, PIN family, auxin

表1

生物信息学预测及分析所用网址"

网站或软件名
Website or software		 网址
Uniform resource locator		 用途
Purpose
ExPASy ProtParam http://us.expasy.org/tools 氨基酸组成及理化性质预测
Prediction of amino acid composition and physicochemical properties
Cell-PLoc 2.0 http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/ 蛋白亚细胞定位预测
Predict protein subcellular localization
SPOMA https://npsa.lyon.inserm.fr/cgi-bin/
secpred_sopma.pl		 蛋白二级结构预测
Protein secondary structure prediction
TMHMM-2.0 https://services.healthtech.dtu.dk/
services/TMHMM-2.0/		 蛋白跨膜分析
Protein transmembrane analysis
Phytozome 13 https://phytozome-next.jgi.doe.gov/ 获得GmRSM1的启动子序列、CDS序列、编码序列和蛋白序列, 并进行基因结构分析
The promoter sequence, CDS sequence, coding sequence and protein sequence of GmRSM1 were obtained, and the gene structure was analyzed
PlantCARE https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ 启动子区顺式作用元件分析
Analysis of cis-acting elements in the promoter region

图3

大豆GmRSM1过表达株系与野生型顶端弯钩表型差异 A: qRT-PCR对过表达大豆性株系进行相对表达量测定。图中**代表与野生型WT相比差异极显著(P < 0.01)。B: 大豆表型: 从上到下分别是野生型大豆、转化体OE-GmRSM1#103、#78和#95按培养时间进行表型观测的黄化幼苗的顶端弯钩。C: 大豆的顶端弯钩角度测量示意图。D: 按时间顺序检测并测量的顶端弯钩角度变化折线图。"

附表1

本试验所用引物及用途"

引物名称
Primer name		 上游引物
Forward primer (5°-3°)		 上游引物
Reverse primer (5°-3°)		 引物用途
Primer function
pBAR-GmRSM1 CTCGGTACCCGGGGATCCATGGCCT-CAAGCTCAGCTT GATGAAGGTTCTAAGCCTCCACTGA-TCTAGAGTCCGCAAATCACCAGTCT 基因克隆和构建过表达载体
Gene cloning and overexpression vector construction
qPCR-GmRSM1 GTAAGACTGTGGAGGAAGTGAA AGCATTTCTGTAATTGGGCAAG 荧光定量Real-time PCR
qPCR-GmActin GGTGGTTCTATCTTGGCATC CTTTCGCTTCAATAACCCTA 内参引物Reference primer
qPCR-AtUBQ1 TTCCTTGATGATGCTTGCTC TTGACAGCTCTTGGGTGAAG 内参引物Reference primer
qPCR-PIN1a GCAACCGAGGATCATAGCAT AGAGAACGCCTTTGAGTCCA 荧光定量Real-time PCR
qPCR-PIN1b TGTTGATTGCTTTGCCCATA TGTCTGATGCTCAACAAGCC 荧光定量Real-time PCR
qPCR-PIN1c ATGCAAAGCTTGGTTGAGGT GATCCTGGGGTTCTTCTTCC 荧光定量Real-time PCR
qPCR-PIN1d GCATAAAAAGTGGGACCGAA ATGACAACCTGTGCCATTCA 荧光定量Real-time PCR
qPCR-PIN1e GGGATGCTAATTGCTCTTCCTA GGTAGTTTGATCCACACTGCAA 荧光定量Real-time PCR
qPCR-PIN3a CCCCAACACTTACTCCAGTCTC ATAGCTATACGCAAGAGGGTGC 荧光定量Real-time PCR
qPCR-PIN3b AGAATTCGCAGACACAGCCT GTTGGCTTTGTTCCCACTGT 荧光定量Real-time PCR
qPCR-PIN3c GTGACGGTAGCTTCTCCTCG GAATTCTGGCTCTGGCTCTG 荧光定量Real-time PCR
qPCR-PIN3d ACACTTGCAAAATGGGGAAG GCCCAACTTGTTGAGTCCAT 荧光定量Real-time PCR
qPCR-PIN6a TGCAACTCGTGGTTCTTCAG CGTCGAATTTCGCTATGGAT 荧光定量Real-time PCR
qPCR-PIN6b AGTTCTTGACATGCCCTGCT TTCCCACAAGCTTTTCCAAC 荧光定量Real-time PCR

图1

GmRSM1的生物信息学分析 A: 进化树分析。图中红色圆形标注为GmRSM1的蛋白序列, 蓝色圆形标注为拟南芥ATRL1~ATRL6的蛋白序列和金鱼草中AmRAD蛋白序列。B: 蛋白序列保守性比对。图中数字表示每种肽中的氨基酸数, 标有星号的位置表示整个序列的100%一致, 三角形表示RAD-Like特有的残基。C: 基因结构分析; D: 蛋白的三级结构预测; E: 跨膜结构预测。"

表2

GmRSM1基因上游2000 bp的启动子元件分析"

元件
Motif		 数量
Number		 功能
Function
CAAT-box 47 启动子和增强子区域中常见的顺式作用元件
Common cis-acting element in promoter and enhancer regions
TATA-box 38 转录起始点−30左右的核心启动子元件 Core promoter element around −30 of transcription start
G-box 7 参与光响应的顺式作用调节元件 Cis-acting regulatory element involved in light responsiveness
ABRE 6 参与脱落酸反应的顺式作用元件 Cis-acting element involved in the abscisic acid responsiveness
MYC 5 MYC结合位点元件 MYC binding site element
ABRE3a 3 ABA (脱落酸)应答元件ABA (abscisic acid) transponder
ABRE4 3 ABA (脱落酸)应答元件ABA (abscisic acid) transponder
AT~TATA-box 3 核心启动子元件 Core promoter element
Box 4 3 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
ACE 2 参与光响应的顺式作用元件 Cis-acting element involved in light responsiveness
ERE 2 乙烯诱导顺式作用元件 Ethylene-induced cis-acting element
TCCC-motif 2 光响应元件的一部分 Part of a light responsive element
TCT-motif 2 光响应元件的一部分 Part of a light responsive element
ATCT-motif 1 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
Box II 1 光响应元件的一部分 Part of a light responsive element
Box III 1 蛋白结合位点 Protein binding site
CCAAT-box 1 MYB Hv1结合位点 MYB Hv1 binding site
GATA-motif 1 光响应元件的一部分 Part of a light responsive element
GT1-motif 1 光响应元件 Light responsive element
I-box 1 光响应元件的一部分 Part of a light responsive element
MBS 1 MYB结合位点参与干旱诱导 MYB binding site involved in drought-inducibility
MBSI 1 MYB结合位点参与类黄酮生物合成基因调控
MYB binding site involved in flavonoid biosynthetic genes regulation
MYB 2 MYB结合位点元件 MYB binding site element
MYB recognition site 1 MYB识别位点元件 MYB recognizes site-based elements
MYB-like sequence 1 MYB-like序列 MYB-like sequence
STRE 1 应激响应的顺式作用元件 Cis-acting element of stress response
TATC-box 1 参与赤霉素反应的顺式作用元件 Cis-acting element involved in gibberellin-responsiveness
TCA-element 1 参与水杨酸反应性的顺式作用元件 Cis-acting element involved in salicylic acid responsiveness

图2

拟南芥过表达GmRSM1株系与野生型顶端弯钩表型差异 A: qRT-PCR对过表达拟南芥阳性株系进行相对表达量测定: 图中**代表与野生型WT相比差异极显著(P < 0.01)。B: 拟南芥表型: 从左到右分别是野生型拟南芥、转化体OE-GmRSM1#64、#69和#70的黄化幼苗顶端弯钩。C: 拟南芥的过表达株系#69及野生型的顶端弯钩电子显微镜扫描图。"

图4

维持阶段和展开阶段PIN基因在顶端弯钩内外的相对表达量"

图5

GmRSM1蛋白的亚细胞定位 在绿色荧光、红色荧光、明场和叠加场4个视野下拍摄的照片。标尺为10 μm。"

图6

深播条件下大豆GmRSM1过表达株系与野生型顶端弯钩表型差异"

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