糜子PmDEP1和PmEP3基因的克隆与表达特征分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0689

生物技术通报 ›› 2025, Vol. 41 ›› Issue (2): 150-162.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0689

• 研究报告 • 上一篇

糜子PmDEP1和PmEP3基因的克隆与表达特征分析

许圆梦¹^,²^,³(), 毛娇¹^,²^,³, 王梦瑶¹^,²^,³, 王数¹^,²^,³, 任江陵¹^,²^,³, 刘宇涵¹^,²^,³, 刘思辰¹^,²^,³, 乔治军¹^,²^,³, 王瑞云¹(), 曹晓宁¹^,²^,³()

^1.山西农业大学农学院，太谷 030801
^2.山西农业大学农业基因资源研究中心，太原 030031
^3.农业农村部黄土高原作物基因资源与种质创制重点实验室，太原 030031

收稿日期:2024-07-18 出版日期:2025-02-26 发布日期:2025-02-28
通讯作者: 王瑞云，女，博士，教授，研究方向：糜子分子育种；E-mail: wry925@126.com
曹晓宁，男，博士，研究员，研究方向：谷子糜子种质资源创新利用及耐逆生理机制；E-mail: caoxiaoning2008@163.com
作者简介:许圆梦，女，硕士研究生，研究方向：糜子分子育种；E-mail: xuyuanmeng1229@163.com
基金资助:
山西省重点研发项目(2022ZDYF110);中央引导地方科技发展资金(YDZJSX2022A044);谷子高粱产业技术体系谷子糜子生理岗位(CARS-06-14.5-A16);山西省现代农业杂粮产业技术体系(2024CYJSTX03-23)

Cloning and Expression Characteristics Analysis of Millet Genes PmDEP1 and PmEP3

XU Yuan-meng¹^,²^,³(), MAO Jiao¹^,²^,³, WANG Meng-yao¹^,²^,³, WANG Shu¹^,²^,³, REN Jiang-ling¹^,²^,³, LIU Yu-han¹^,²^,³, LIU Si-chen¹^,²^,³, QIAO Zhi-jun¹^,²^,³, WANG Rui-yun¹(), CAO Xiao-ning¹^,²^,³()

^1.College of Agriculture, Shanxi Agricultural University, Taigu 030801
^2.Center for Agricultural Genetic Resources Research, Shanxi Agricultural University, Taiyuan 030031
^3.Key Laboratory of Crop Genetic Resources and Germplasm Development in Loess Plateau, Ministry of Agriculture and Rural Affairs, Taiyuan 030031

Received:2024-07-18 Published:2025-02-26 Online:2025-02-28

摘要/Abstract

摘要：

目的穗型是影响作物产量和机械化收割的重要因素，DEP1（Dense Erect Panicles 1）和EP3（Erect Panicle 3）基因是控制穗型形成的关键基因。旨在探究糜子（Panicum miliaceum L.）PmDEP1和PmEP3基因的结构与表达特征。方法以散穗型糜子笤帚糜子（ZM）、密穗型糜子M278和侧穗型糜子M350为材料，克隆获得PmDEP1和PmEP3，并对其开展生物信息学分析。采用RT-qPCR检测不同穗型糜子PmDEP1和PmEP3的表达模式。结果序列结果分析显示，PmDEP1 cDNA全长为1 044 bp，编码347个氨基酸，蛋白结构域预测为PAT1，二级结构以无规则卷曲为主，三级结构与水稻DEP1蛋白高度相似，预测定位于细胞核，与柳枝稷具有较高的同源性，拥有28个磷酸化位点，不具有跨膜结构和信号肽。PmEP3 cDNA全长1 215 bp，编码358个氨基酸，编码的蛋白属于F-box家族，二级结构以无规则卷曲和延伸链为主，三级结构与水稻Os02g0260200蛋白高度相似，预测定位于细胞质，与柳枝稷具有较高的同源性，不含信号肽，拥有32个磷酸化位点，具有少量的跨膜结构。RT-PCR结果显示，PmDEP1和PmEP3在不同时期的不同部位中表达，PmDEP1在拔节期的根中表达量最高，在抽穗期的笤帚糜子和M278中的叶部表达量最高，在M350中的茎部表达量最高。PmEP3在拔节期的叶中表达量最高，在笤帚糜子和M278抽穗期的叶部表达量最高，在M350的穗部表达量最高。结论 PmDEP1和PmEP3为穗型基因，可能参与调控糜子穗型形成。

关键词: 糜子, 穗型基因, PmDEP1, PmEP3, 基因克隆, 表达分析

Abstract:

Objective Panicle type is an important factor affecting crop yield and mechanized harvesting. DEP1 (Dense Erect Panicles 1) and EP3 (Erect Panicles 3) genes are key ones controlling spike type formation. This study aims to explore the structure and expression characteristics of PmDEP1 and PmEP3 ones in proso millet (Panicum miliaceum L.). Method PmDEP1 and PmEP3 genes were cloned and subjected to bioinformatics analysis using the scattered ear type millet varieties, broomstick millet (ZM), dense ear type millet M278, and lateral ear type millet M350 as materials. RT-qPCR was used to detect the expression patterns of PmDEP1 and PmEP3 genes in different ear types of proso millet. Result Sequence result analysis shows that, the full-length cDNA of PmDEP1 is 1 044 bp, encoding 347 amino acids. The protein domain is predicted to be PAT1. The secondary structure is dominated by random coils. The tertiary structure is highly similar with that of rice DEP1 protein. It is predicted to be located in the nucleus and has high homology with switchgrass. It has 28 phosphorylation sites and does not have transmembrane structure and signal peptide. The full-length cDNA of PmEP3 is 1 215 bp, encoding 358 amino acids. The encoded protein belongs to the F-box family. The secondary structure is dominated by random coils and extended chains. The tertiary structure is highly similar with that of rice Os02g0260200 protein. It is predicted to be located in the cytoplasm. It has high homology with switchgrass, does not contain signal peptides, has 32 phosphorylation sites, and has a small amount of transmembrane structure. The RT-PCR results shows that PmDEP1 and PmEP3 genes are expressed in different parts of the plant at different stages. PmDEP1 gene is mainly expressed in the roots at the jointing stage, with the highest expression in the leaves of broomstick millet and M278 at the heading stage, and in the stems of M350. The expressions of PmEP3 gene is the highest in the leaves at the jointing stage, in the leaves of broomstick millet and M278 at the heading stage, and in the ears of M350. Conclusion PmDEP1 and PmEP3 genes are spike type genes that may be involved in regulating the formation of proso millet spike types.

Key words: Panicum miliaceum L., spike type gene, PmDEP1, PmEP3, gene cloning, expression analysis

许圆梦, 毛娇, 王梦瑶, 王数, 任江陵, 刘宇涵, 刘思辰, 乔治军, 王瑞云, 曹晓宁. 糜子PmDEP1和PmEP3基因的克隆与表达特征分析[J]. 生物技术通报, 2025, 41(2): 150-162.

XU Yuan-meng, MAO Jiao, WANG Meng-yao, WANG Shu, REN Jiang-ling, LIU Yu-han, LIU Si-chen, QIAO Zhi-jun, WANG Rui-yun, CAO Xiao-ning. Cloning and Expression Characteristics Analysis of Millet Genes PmDEP1 and PmEP3[J]. Biotechnology Bulletin, 2025, 41(2): 150-162.

图/表 13

表1 PmDEP1和PmEP3的引物序列

Table 1 Primer sequences for PmDEP1 and PmEP3

引物 Primer	引物序列 Primer sequence （5′-3′）	功能 Primer function
actin	TCTGTTTTTCTCTATGTGCAGGA	基因表达分析 Analysis of gene expression
PmDEP1-Q	TGCTGCTTCCATTGTTCTTGCTG
PmEP3-Q	GAGCCTCCGCAAGTGGTATGG
PmDEP1	AGTGAATTCGAGCTCGGTACATGGGGGAGGAGGTGGCG	基因克隆 Gene cloning
PmEP3	AGTGAATTCGAGCTCGGTACATGGGGTCGGAAGAGTGGGAG	基因克隆 Gene cloning

图1 PmDEP1和PmEP3基因的克隆和结构分析A：PmDEP1的凝胶电泳图；B：PmEP3的凝胶电泳图；C：PmDEP1基因结构；D：PmEP3基因结构

Fig. 1 Cloning and structural analysis of PmDEP1 and PmEP3 genesA: Gel electrophoresis map of PmDEP1 gene. B: Gel electrophoresis map of PmEP3 gene. C: PmDEP1 gene structure. D: PmEP3 gene structure

图2 PmDEP1和PmEP3的核苷酸及氨基酸序列和基因结构域A：PmDEP1的核苷酸及氨基酸序列；B：PmEP3的核苷酸及氨基酸序列；C：PmDEP1基因结构域；D：PmEP3基因结构域

Fig. 2 Nucleotide and amino acid sequences and gene domains of PmDEP1 and PmEP3A: Nucleotide and amino acid sequence of PmDEP1. B: Nucleotide and amino acid sequence of PmEP3. C: PmDEP1 gene domain. D: PmEP3 gene domain

图3 PmDEP1和PmEP3编码蛋白质中各氨基酸组成

Fig. 3 Amino acid composition of the protein encoded by PmDEP1 and PmEP3

图4 PmDEP1和PmEP3蛋白的疏水性/亲水性和磷酸化预测A：PmDEP1蛋白的疏水性/亲水性预测；B：PmDEP1蛋白的磷酸化预测；C：PmEP3蛋白的疏水性/亲水性预测；D：PmEP3蛋白的磷酸化预测

Fig. 4 Prediction of hydrophobicity/hydrophilicity and phosphorylation of PmDEP1 and PmEP3 proteinsA: Hydrophobic/hydrophilic prediction of PmDEP1 protein. B: Phosphorylation prediction of PmDEP1 protein. C: Hydrophobic/hydrophilic prediction of PmEP3 protein. D: Phosphorylation prediction of PmEP3 protein

图5 糜子蛋白PmDEP1（A）和PmEP3（B）系统进化树

Fig. 5 Phylogenetic tree of PmDEP1 （A） and PmEP3 （B） proteins in broomcorn millet

图6 PmDEP1蛋白（A）和PmEP3蛋白（B）与其他物种同源序列比对

Fig. 6 PmDEP1 protein （A） and PmEP3 （B） protein aligned with homologous sequences of other species

图7 PmDEP1蛋白和PmEP3蛋白二级结构和三级结构预测A：PmDEP1蛋白的二级结构；B：PmEP3蛋白的二级结构；C：PmDEP1蛋白的三级结构；D：PmEP3蛋白的三级结构

Fig. 7 Prediction of secondary and tertiary structures of PmDEP1 and PmEP3 proteinsA: Secondary structure of PmDEP1 protein. B: Secondary structure of PmEP3 protein. C: Tertiary structure of PmDEP1 protein. D: Tertiary structure of PmEP3 protein

图8 PmDEP1蛋白和PmEP3蛋白信号肽和跨膜结构域预测A：PmDEP1蛋白信号肽预测；B：PmEP3蛋白信号肽预测；C：PmDEP1蛋白跨膜结构域预测；D：PmEP3跨膜结构域预测

Fig. 8 Signal peptide and transmembrane domain prediction of PmDEP1 protein and PmEP3 proteinA: Prediction of PmDEP1 protein signal peptide. B: Prediction of PmEP3 protein signal peptide. C: Prediction of PmDEP1 protein transmembrane domain. D: Prediction of PmEP3 transmembrane domain

表2 PmDEP1和PmEP3蛋白的亚细胞定位预测结果

Table 2 Prediction results of subcellular localizations for PmDEP1 and PmEP3 proteins

亚细胞位置 Subcellular location	可能性 Possibility/%
亚细胞位置 Subcellular location	PmDEP1	PmEP3
细胞核	78.3	26.1
细胞质	8.7	47.8
质膜	4.3	-
过氧化物酶体	4.3	-
线粒体	4.3	26.1

图9 PmDEP1（A）和PmEP3（B）启动子顺式作用元件

Fig. 9 Cis-acting elements of PmDEP1 （A） and PmEP3 （B） promoters

表3 PmDEP1和PmEP3启动子顺式作用元件分析

Table 3 Analysis of cis-acting elements of PmDEP1 and PmEP3 promoters

基因名称 Gene name	作用元件 Functional component	核心序列 Core sequence	数量 Number	功能 Function
PmDEP1	CGTCA-motif	CGTCA	1	MeJA作用调控元件
	TGACG-motif	TGACG	1	MeJA作用调控元件
	G-box	CACGAC	4	光响应应答元件
	chs-CMA2a	TCACTTGA	1	光响应应答元件
	TCA-element	CCATCTTTTT	1	水杨酸作用元件
	P-box	CCTTTTG	1	赤霉素反应元件
	ARE	AAACCA	1	厌氧诱导调控元件
	GC-motif	CCCCCG	2	参与缺氧特异性诱导元件
	MBS	CAACTG	1	干旱诱导元件
	CCGTCC-box	CCGTCC	1	与分生组织特异性激活调节元件
	STRE	AGGGG	4	应激反应元件
PmEP3	CGTCA-motif	CGTCA	1	MeJA作用调控元件
	TGACG-motif	TGACG	1	MeJA作用调控元件
	ATCT-motif	AATCTAATCC	1	光响应应答元件
	TCT-motif	TCTTAC	2	光响应应答元件
	MRE	AACCTAA	1	MYB参与光反应结合位点
	GARE-motif	TCTGTTG	1	赤霉素反应元件
	TC-rich repeats	ATTCTCTAAC	1	赤霉素反应元件
	AuxRR-core	GGTCCAT	1	生长素反应调控元件
	AAGAA-motif	GAAAGAA	1	脱落酸反应性相关性元件
	ARE	AAACCA	2	厌氧诱导调控元件
	MBS	CAACTG	1	干旱诱导元件
	CAT-box	GCCACT	1	分生组织表达调控元件
	GCN4_motif	TGAGTCA	1	参与胚乳表达的顺式调控元件

图10 糜子PmDEP1和PmEP3在ZM、M350和M278的拔节期和抽穗期的不同部位表达量A：PmDEP1在拔节期各个部位表达情况；B：PmDEP1在抽穗期各个部位表达情况；C：PmEP3在拔节期各个部位表达情况；D：PmEP3在抽穗期各个部位表达情况；不同小写字母表示表达量在P<0.05水平上差异显著

Fig. 10 Expressions of PmDEP1 and PmEP3 genes in different parts of ZM， M350 and M278 at jointing and heading stagesA: Expressions of PmDEP1 in different parts at jointing stage. B: Expressions of PmDEP1 in different parts at heading stage. C: Expressions of PmEP3 in different parts at jointing stage. D: Expressions of PmEP3 in various parts at heading stage. Different lowercase letters indicate that the expression was significantly different at the P<0.05 level

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糜子PmDEP1和PmEP3基因的克隆与表达特征分析

Cloning and Expression Characteristics Analysis of Millet Genes PmDEP1 and PmEP3

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