甘薯盐胁迫响应基因IbMYB3的表达特征及生物信息学分析

doi:10.11983/CBB19094

摘要/Abstract

摘要： MYB转录因子具有多种生物学功能, 在植物响应生物和非生物胁迫中发挥重要作用。该文从盐胁迫后的甘薯(Ipomoea batatas)水培苗转录组数据(RNA-seq)中筛选出2个受盐胁迫显著上调表达的MYB基因, 分别命名为IbMYB3和IbMYB4。多种非生物胁迫和植物生长物质处理下的基因表达分析显示, IbMYB3受逆境诱导显著上调表达, 暗示其可能参与甘薯非生物胁迫响应。生物信息学分析表明, IbMYB3开放阅读框长度为1 059 bp, 编码353个氨基酸残基, 蛋白分子量为39.41 kDa, 理论等电点(PI)为5.26, 为酸性带负电的亲水性蛋白。亚细胞定位结果表明, IbMYB3蛋白定位于细胞核, 具有较强的转录激活活性。上述结果表明, IbMYB3转录因子可能在甘薯非生物胁迫响应过程中发挥重要调控作用, 研究结果为进一步探明IbMYB3基因的功能奠定了基础。

关键词: 生物信息学分析, 表达特征, IbMYB3, 甘薯, 转录激活

Abstract: MYB transcription factors have multiple biological functions and play important roles in mediating plant responses to biotic and abiotic stresses. In this paper, two MYB genes, named IbMYB3 and IbMYB4, which were significantly induced by salt stress, were screened from RNA-seq data of salt-stressed sweetpotato (Ipomoea batatas) plantlets. Gene expression analysis showed that the expression of IbMYB3 was significantly up-regulated by various abiotic stresses and plant growth substances treatments, suggesting that IbMYB3 might be involved in the abiotic stress responses of sweetpotato. Further bioinformatic analysis showed that the open reading frame of IbMYB3 is 1 059 bp in length, encoding 353 amino acids, with a predicted molecular weight of 39.41 kDa and the theoretical isoelectric point (PI) of 5.26, which is an acidic negatively charged hydrophilic protein. Subcellular localization showed that the IbMYB3 protein localizes to the nucleus, and has strong transcriptional activation activity. Taken together, these results demonstrated that the IbMYB3 transcription factor might play an important role in regulating the abiotic stress responses of sweetpotato. This study thus lays the foundation for further illustration of IbMYB3 function.

Key words: bioinformatic analysis, expressional patterns, IbMYB3, Ipomoea batatas, transcriptional activation

李格,孟小庆,李宗芸,朱明库. 甘薯盐胁迫响应基因IbMYB3的表达特征及生物信息学分析. 植物学报, 2020, 55(1): 38-48.

Ge Li,Xiaoqing Meng,Zongyun Li,Mingku Zhu. Expression Patterns and Bioinformatic Analyses of Salt Stress Responsive Gene IbMYB3 in Ipomoea batatas. Chinese Bulletin of Botany, 2020, 55(1): 38-48.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB19094

https://www.chinbullbotany.com/CN/Y2020/V55/I1/38

图/表 9

表1 实验所用引物

Table 1 Primers used in the experiment

Primer name	Primer sequence (5'-3')
FIbMYB3-F	ATGGGAAGATCTCCATGCTG
FIbMYB3-R	TTTACAGCAAATCTTCGAAATCTA
FIbMYB4-F	ATGGGGAGATCACCATGCT
FIbMYB4-R	TTGTTCTGGACAAATATCTACAGAA
QIbMYB3-F	GCCAGCCAACTTGAGTACCG
QIbMYB3-R	AAGAACCGAGTCCAACCCG
QIbMYB4-F	ATCCACTCCCACCTTGTACGAC
QIbMYB4-R	TCCAAAACAGCCGCCATAGT
QIbARF-F	CTTTGCCAAGAAGGAGATGC
QIbARF-R	TCTTGTCCTGACCACCAACA
IbMYB3-EGFP- XbaI-F	CCGTCTAGAATGGGAAGATCTCCATGCTGT
IbMYB3-EGFP- BamHI-R	CGCGGATCCCAGCAAATCTTCGAAATCTAAG
IbMYB3-BD-F	CCGGAATTCATGGGAAGATCTCCATGCTGT
IbMYB3-BD-R	CGCGGATCCTTACAGCAAATCTTCGAAATCTAAG

图1 亚细胞定位载体pBI121:IbMYB3:EGFP的构建 (A) pBI121:IbMYB3:EGFP融合蛋白载体图谱; (B) 转化的农杆菌检测(1-8泳道分别代表载体转入农杆菌后的菌落PCR鉴定结果)。

Figure 1 Construction of pBI121:IbMYB3:EGFP subcellular localization vector (A) Vector map of pBI121:IbMYB3:EGFP fusion protein construction; (B) Detection in Agrobacterium (Lanes 1-8 represent the PCR identification results after the vector was transferred into Agrobacterium, respectively).

图2 用于转录激活活性分析的pGBKT7:IbMYB3载体的构建

Figure 2 Construction of pGBKT7:IbMYB3 vector for transcriptional activation

表2 IbMYB3和IbMYB4基因的RNA-seq数据信息

Table 2 The RNA-seq data information of IbMYB3 and IbMYB4 genes

Gene name	Gene ID	RNA-seq of Xu22 root control (FPKM)	RNA-seq of Xu22 root after salt-treatment (FPKM)	Log₂ FC (Fold change)	Swiss protein annotation (OS=Arabidopsis thaliana)
IbMYB3	c57279.graph_c0	0.07874256	44.64485107	7.975299063	Myb-like DNA-binding domain
IbMYB4	c53945.graph_c0	1.095722132	20.97216423	4.085231265	Myb-like DNA-binding domain

图3 不同处理下水培甘薯苗根中IbMYB3和IbMYB4基因的相对表达 (A) 100 mmol∙L-1 NaCl; (B) 20% PEG6000; (C) 100 μmol∙L-1乙烯合成前体氨基环丙烷羧酸(ACC); (D) 100 μmol∙L-1脱落酸(ABA); (E) 100 μmol∙L-1赤霉素(GA); (F) 100 μmol∙L-1茉莉酸(JA)。*和**分别表示在P<0.05和P<0.01水平差异显著。

Figure 3 Relative expression analysis of IbMYB3 and IbMYB4 in roots of hydroponic sweetpotato plantlets under different treatments (A) 100 mmol∙L-1 NaCl; (B) 20% PEG6000; (C) 100 μmol∙L-1 ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC); (D) 100 μmol∙L-1 abscisic acid (ABA); (E) 100 μmol∙L-1 gibberellic acid (GA); (F) 100 μmol∙L-1 jasmonate acid (JA). * and ** indicate significant differences at P<0.05 and P<0.01, respectively.

图4 IbMYB3蛋白氨基酸序列分析 (A) 疏水性分析; (B) 跨膜区(TM)预测; (C) 功能域预测

Figure 4 Amino acid sequence analysis of IbMYB3 protein (A) Hydrophobic analysis; (B) Prediction of transmembrane (TM) region; (C) Domain prediction

图5 IbMYB3蛋白结构预测 (A), (B) SOPMA预测的二级结构; (C) 预测的四级结构

Figure 5 Structural prediction of IbMYB3 protein (A), (B) Secondary structure predicted by SOPMA; (C) Predicted tertiary structure

图6 IbMYB3蛋白在洋葱表皮细胞中的亚细胞定位与转录激活活性分析 (A) IbMYB3与EGFP融合蛋白在洋葱下表皮细胞中的亚细胞定位; (B) IbMYB3的转录激活活性分析。Bars=4 μm

Figure 6 Subcellular localization in onion epidermal cells and transcriptional activation activity analysis of IbMYB3 protein (A) Subcellular localization of IbMYB3 and EGFP fusion protein in the onion lower epidermis cells; (B) Transcriptional activation activity analysis of IbMYB3 protein. Bars=4 μm

图7 MYB蛋白序列比对及系统发生树 (A) IbMYB3与其它非甘薯物种中盐胁迫耐受相关MYB蛋白序列比对; (B) IbMYB3与其它物种中盐胁迫耐受相关MYB蛋白的系统发生树

Figure 7 MYB proteins sequence alignment and phylogenetic tree (A) Proteins sequence alignment between IbMYB3 and the MYB proteins associated with salt stress tolerance in species other than sweetpotato; (B) Phylogenetic tree of IbMYB3 and the MYB proteins associated with salt stress tolerance in other species

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