甘蔗AP2/ERF转录因子基因ShERF3的克隆、表达及其编码蛋白的定位

doi:10.13560/j.cnki.biotech.bull.1985.2022-1400

摘要/Abstract

摘要：

AP2/ERF转录因子在调控植物的生长发育、抵抗生物和非生物胁迫方面发挥重要作用。克隆ShERF3，并分析其功能，为甘蔗抗逆遗传改良提供基因资源。基于转录组数据从‘新台糖22号’甘蔗中克隆ShERF3，并通过Real time PCR、生物信息学分析、水稻原生质体亚细胞定位等技术对ShERF3编码的蛋白特性、亚细胞定位及基因表达模式进行分析。结果显示，克隆获得1 142 bp ShERF3的cDNA序列，包含1个1 053 bp的完整开放阅读框，编码350个氨基酸；ShERF3蛋白含有一个AP2结构域，属于AP2/ERF家族ERF亚家族成员，与高粱和柳枝稷ERF3、谷子和哈氏黍ERF118蛋白同源性较高；ShERF3蛋白是一种不稳定的疏水性蛋白，亚细胞定位结果显示其定位于细胞核；ShERF3主要在甘蔗成熟茎节中表达，在叶片和根系中相对表达较低；在PEG处理下，ShERF3表达先下调后上调，在NaCl处理下，随着胁迫时间延长ShERF3表达量降低。甘蔗ShERF3积极响应干旱和盐逆境胁迫，可能在甘蔗茎秆发育、干旱以及盐胁迫应答方面发挥关键作用。

关键词: 甘蔗, ShERF3, 非生物胁迫, 表达分析

Abstract:

AP2/ERF transcription factors play an important role on regulating plant growth and development and involving in biotic and abiotic stresses resistances in plants. Cloning and gene function analysis of ShERF3 may provide gene resource for sugarcane resistance genetic improvement. The ShERF3 gene was cloned from‘ROC22’sugarcane plants based on the transcriptome data. Then the structure, subcellular location and gene expression pattern of ShERF3 were analyzed with real time PCR, bioinformation and rice protoplast subcellular location methods. The results showed that 1 142 bp cDNA sequence of ShERF3 was obtained. It contained a 1 053 bp integral opening reading frame encoding 350 amino acids. The predicted encoding protein of ShERF3 containing a conserved AP2 domain belonged to the ERF subgroup of AP2/ERF family. The homology analysis indicated that ShERF3 was highly homologous with ERF3 from Sorghum bicolor and Panicum virgatum and ERF118 from Panicum hallii and Setaria italica. It predicted that ShERF3 is a unstable hydrophobic protein. The subcellular location analysis showed that ShERF3 located in the nulear of rice protoplast cells. ShERF3 mainly expressed in sugarcane mature internodes and relatively lower expressed in the leaves and roots. In addition, the expression of ShERF3 was firstly down-regulated and then up-regulated under PEG treatment. And the expression of ShERF3 decreased with the extension of stress time under NaCl treatment. These results indicates that ShERF3 actively responds to draught and salt stresses. It may play a key role on regulating sugarcane stem development, draught and salt stresses responses.

Key words: sugarcane, ShERF3, abiotic stress, expression analysis

赵雪婷, 高利燕, 王俊刚, 沈庆庆, 张树珍, 李富生. 甘蔗AP2/ERF转录因子基因ShERF3的克隆、表达及其编码蛋白的定位[J]. 生物技术通报, 2023, 39(6): 208-216.

ZHAO Xue-ting, GAO Li-yan, WANG Jun-gang, SHEN Qing-qing, ZHANG Shu-zhen, LI Fu-sheng. Cloning and Expression of AP2/ERF Transcription Factor Gene ShERF3 in Sugarcane and Subcellular Localization of Its Encoded Protein[J]. Biotechnology Bulletin, 2023, 39(6): 208-216.

图/表 8

图1 甘蔗ShERF3 cDNA序列PCR扩增电泳图

Fig. 1 PCR amplification electrophoresis of sugarcane ShERF3 cDNA M：DSMT marker 2000；1：ShERF3

图2 甘蔗ShERF3蛋白保守结构域

Fig. 2 Conserved domains of protein ShERF3 in sugarcane

图3 ShERF3氨基酸序列比对

Fig. 3 Amino acid sequence alignment of ShERF3

图4 ShERF3与其他AP2/ERF家族成员的系统进化树

Fig. 4 Phylogenetic tree of ShERF3 and other AP2/ERF family members

图5 甘蔗ShERF3蛋白质二级结构的预测蓝色为α-螺旋；红色为延伸链；绿色为β-转角；紫色为无规卷曲

Fig. 5 Prediction of secondary structure of ShERF3 in sugarcane The blue is α-helix; red is extended- strand; green is beta turn; purple is random coil

图6 甘蔗ShERF3亚细胞定位结果

Fig. 6 Subcellular localization results of ShERF3

图7 ShERF3在‘新台糖22号’不同部位的表达分析 IL：未成熟叶；ML：成熟叶；S1：茎节1-2；S2：茎节4-5；S3：茎节7-8；S4：茎节10-11；S5：茎节12-13；S6：茎节14-15；RS：根；不同小写字母表示在0.05水平差异显著。下同

Fig. 7 Expression analysis of ShERF3 gene in different parts of ‘ROC22’ IL: Immature leaves; ML: mature leaves; S1: stem node 1-2; S2: stem node 4-5; S3: stem node 7-8; S4: stem node 10-11; S5: stem node 12-13; S6: stem node 14-15; Rs: root system. Different lowercases indicate significant differences at the 0.05 level. The same below

图8 ShERF3在干旱、盐胁迫下的表达

Fig. 8 Expressions of SheRF3 gene under drought and salt stress

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