灰皮支黑豆GmPUB24基因的生物信息学与胞囊线虫诱导表达分析

doi:10.3969/j.issn.1004-1524.2022.06.03

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (6): 1124-1132.DOI: 10.3969/j.issn.1004-1524.2022.06.03

灰皮支黑豆GmPUB24基因的生物信息学与胞囊线虫诱导表达分析

李文辰(), 刘鑫, 齐泽铮, 于璐, 王芳()

齐齐哈尔大学生命科学与农林学院,黑龙江齐齐哈尔 161006

收稿日期:2021-08-21 出版日期:2022-06-25 发布日期:2022-06-30
作者简介:*王芳,E-mail： wangfangnd@hotmail.com
李文辰（1998—）,男,辽宁阜新人,硕士研究生,研究方向为植物抗病性。E-mail： liwenchen2021@163.com
通讯作者: 王芳
基金资助:
国家自然科学基金(31801725);黑龙江省教育厅基本科研业务费科研项目(135309361);齐齐哈尔大学研究生创新科研项目

Bioinformatics of Huipizhi Black soybean GmPUB24 and expression under Heterodera glycines infection

LI Wenchen(), LIU Xin, QI Zezheng, YU Lu, WANG Fang()

College of Life Sciences and Agroforestry, Qiqihar University, Qiqihar 161006, Heilongjiang, China

Received:2021-08-21 Online:2022-06-25 Published:2022-06-30
Contact: WANG Fang

摘要/Abstract

摘要：

为明确大豆U-box型E3泛素连接酶介导植物抗病性的机理,以抗胞囊线虫品种灰皮支黑豆为材料,利用RT-PCR技术克隆GmPUB24基因的蛋白质编码区序列（coding sequence,CDS）,对该基因进行生物信息学分析,并接种大豆胞囊线虫,进行诱导表达分析。结果表明,GmPUB24基因CDS总长1 254 bp,编码417个氨基酸,分子量为46.78 ku。蛋白质二级结构分析显示,GmPUB24编码的蛋白质含有α螺旋、无规则卷曲、延伸链、β折叠,α螺旋占比最高为58.9%,为亲水蛋白质且无跨膜结构域,无信号肽;蛋白质系统进化树表明,其与野生大豆亲缘性最高,亚细胞定位显示其定位于细胞质中。GmPUB24基因上游1 500 bp启动子区含有CGTCA-motif、TGACG-motif等响应抗病通路的作用元件,以及ABRE、WUN-motif、TATA-box等响应非生物胁迫的作用元件。实时荧光定量PCR（qRT-RCR）结果显示,GmPUB24在接种大豆胞囊线虫1~3 d持续上调表达,接种3 d时根部表达量为未接种线虫样本的6.14倍,表明GmPUB24基因可被大豆胞囊线虫诱导表达,可能参与大豆抵御胞囊线虫的过程。研究结果为阐明大豆U-box家族基因在抗大豆胞囊线虫病中的调控机制奠定了基础。

关键词: 大豆, 克隆, 启动子, 生物信息学, 表达

Abstract:

In order to clarify the mechanism of soybean U-box E3 ubiquitin ligase mediating plant disease resistance, coding sequence (CDS) of GmPUB24 gene from resistant cultivar Huipizhi black bean was cloned by RT-PCR. Bioinformatics and expression analysis of this gene after inoculation with soybean cyst nematode were carried out. The results showed that the length of CDS was 1 254 bp, encoded a total of 417 amino acids with a molecular weight of 46.78 ku. Protein secondary structure analysis showed that the protein encoded by GmPUB24 contained alpha helix, random coil, extended chain and beta turn, and the alpha helix accounted for the highest proportion of 58.9%. Protein encoded by GmPUB24 gene was a hydrophilic protein without transmembrane domain and signal peptide. Protein phylogenetic tree displayed that it had the highest affinity with wild soybean. Subcellular location predicted that it localized in the cytoplasm. Promoter analysis of 1 500 bp upstream of GmPUB24 gene showed that it contained CGTCA-motif, TGACG-motif and other elements that respond to disease resistance pathways, as well as ABRE, WUN-motif, TATA-box and other corresponding abiotic stress elements. Quantitative real-time PCR (qRT-RCR) results showed that the expression of GmPUB24 was induced by soybean cyst nematode, and the highest expression level in roots was 6.14 times compared with the uninoculated sample on the third day, indicating that GmPUB24 gene was induced by soybean cyst nematode and might be involved in the process of soybean defense against cyst nematode. These results provided a basis for clarifying the regulatory mechanism of soybean U-box family gene in resistance to soybean cyst nematode disease.

Key words: soybean, cloning, promoter, bioinformatics, expression

中图分类号:

李文辰, 刘鑫, 齐泽铮, 于璐, 王芳. 灰皮支黑豆GmPUB24基因的生物信息学与胞囊线虫诱导表达分析[J]. 浙江农业学报, 2022, 34(6): 1124-1132.

LI Wenchen, LIU Xin, QI Zezheng, YU Lu, WANG Fang. Bioinformatics of Huipizhi Black soybean GmPUB24 and expression under Heterodera glycines infection[J]. Acta Agriculturae Zhejiangensis, 2022, 34(6): 1124-1132.

图/表 9

图1 GmPUB24基因菌液PCR的琼脂糖凝胶电泳结果 M,DL2000 分子量标记物;1~3,菌液样品的3次重复。

Fig.1 Agarose gel electrophoresis results of bacterial liquid PCR of GmPUB24 geneM, DL2000 marker; 1-3, 3 replicates of bacterial fluid samples.

图2 GmPUB24基因编码的蛋白质U-box结构域

Fig. 2 U-box domain of protein encoded by GmPUB24 gene

图3 GmPUB24编码蛋白质的二级结构红色,延伸链;蓝色,α-螺旋;绿色,β折叠;紫色,无规则卷曲。

Fig. 3 Secondary structure of protein encoded by GmPUB24 Red, Extended strand; Blue, Alpha helix; Green, Beta turn; Purple, Random coil.

图4 GmPUB24蛋白质的亲水性与疏水性以0为界限,正值代表疏水氨基酸,负值代表亲水氨基酸。

Fig. 4 Hydrophilicity and hydrophobicity of GmPUB24 protein Taking 0 as the limit, positive values represented hydrophobic amino acids and negative values represented hydrophilic amino acids.

图5 灰皮支黑豆GmPUB24与其他植物相关蛋白的U-box结构域同源比对黑色阴影为氨基酸具有100%一致性,粉色表示一致性在75%以上,蓝色表示一致性在50%-75%之间,白色表示一致性在50%以下。KAG4974237,野生大豆;XP_003539150,栽培大豆;XP_027911296,豇豆;XP_014523587,小绿豆;XP_027348689,红豆;KYP39063,木豆;XP_004506267,鹰嘴豆;OAP04810,拟南芥;XP_004229574,番茄;XP_015635733,水稻;PWZ26684,玉米。下同。

Fig. 5 Homologous alignment of U-box domains of Huipizhi black bean GmPUB24 with other plant associated proteins Black shading meant amino acids with 100% identity, pink meant more than 75% identity, blue meant 50%-75% identity, and white means less than 50% identity. KAG4974237, Glycine soja; XP_003539150, Glycine max; XP_027911296, Vigna unguiculata; XP_014523587, Vigna radiata var. Radiata; XP_027348689, Abrus precatorius; KYP39063, Cajanus cajan; XP_004506267, Cicer arietinum; OAP04810, Arabidopsis thaliana; XP_004229574, Solanum lycopersicum; XP_015635733, Oryza sativa; PWZ26684, Zea mays. The same as below.

图6 灰皮支黑豆GmPUB24蛋白质与其他植物同源蛋白的系统进化树采用邻接法,自举检验1 000次;图中标尺为遗传距离。

Fig. 6 Phylogenetic tree of GmPUB24 protein from Huipizhi black bean and other plant homologous proteinsNeighbor Joining method with 1 000 bootstrap replicates. The scale bar represented the genetic distance.

表1 GmPUB24启动子顺式元件分析

Table 1 Analysis of cis element of GmPUB24 promoter

名称 Name	数量 Number	序列 Sequence	功能 Function
ABRE	3	ACGTG	参与脱落酸反应 Involved in the abscisic acid reaction
Box 4	7	ATTAAT	参与光反应有关的保守DNA Conserved DNA elements involved in photo reaction
Box II	2	CCACGTGGC	参与光响应Components involved in light response
CAAT-box	20	CCAAT	启动子和增强子区常见的顺式作用元件 Involved in promoter and enhancer regions
CAT-box	1	GCCACT	参与分生组织相关表达 Involved in the expression of meristems
CGTCA-motif	3	CGTCA	参与茉莉酸反应 Involved in the jasmonic acid reaction
TATA-box	35	ATATAT	参与核心启动子30位点转录 Transcription element involved in the 30 position of the core promoter
TGACG-motif	3	TGACG	参与茉莉酸反应 Involved in the jasmonic acid reaction
Unnamed-1	4	GAATTTAATTAA	60s蛋白质活性位点 Involved in 60s protein active site
WUN-motif	1	AAATTTCCT	参与植物伤口反应 Elements involved in plant wound response
ARE	1	AAACCA	无氧诱导 Necessary for anaerobic induction
AT-rich element	1	ATAGAAATCAA	结合蛋白（ATBP-1）的结合位点 Binding site element of binding protein (ATBP-1)
G-box	3	GCCACGTGGA	参与光响应Involved in light response
O2-site	1	GATGATGTGG	参与玉米醇溶蛋白代谢调节 Involved in the regulation of zein metabolism

图7 GmPUB24与TUA4 PCR电泳产物检测 M, DL2000 分子量标记物; 1, TUA4; 2, GmPUB24.

Fig. 7 Electrophoresis detection of GmPUB24 and TUA4 PCR products M, DL2000 marker; 1, TUA4; 2, GmPUB24.

图8 大豆GmPUB24基因在大豆胞囊线虫侵染不同时间的表达量 **代表P ≤ 0.01,***代表P ≤ 0.001,ns代表不显著。

Fig. 8 Expression of soybean GmPUB24 gene at different time of soybean cyst nematode infection ** represented P ≤ 0.01, *** represented P ≤ 0.001, ns represented the difference was not significant.

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灰皮支黑豆GmPUB24基因的生物信息学与胞囊线虫诱导表达分析

Bioinformatics of Huipizhi Black soybean GmPUB24 and expression under Heterodera glycines infection

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