银杏GbR2R3-MYB1基因的克隆及非生物胁迫应答分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-1546

摘要/Abstract

摘要：

R2R3-MYB转录因子是MYB家族中成员数量较多的亚家族成员之一，在植物生长发育、激素信号传导、次生代谢产物形成及逆境胁迫调控等方面具有重要的作用。以银杏为材料，克隆获得GbR2R3-MYB1基因，并利用生物信息学方法分析GbR2R3-MYB1蛋白理化性质、结构与功能；通过构建pCAMBIA1300- R2R3MYB1-GFP融合表达载体及农杆菌介导烟草浸染实验，观察GbR2R3-MYB1基因亚细胞定位情况；利用RT-qPCR方法检测GbR2R3-MYB1基因在非生物逆境胁迫下的表达水平。结果表明，GbR2R3-MYB1基因编码区全长为819 bp，共编码272个氨基酸；蛋白质理论等电点为6.59，相对分子量大小为30 001.60 Da，此蛋白为不稳定亲水蛋白，其二级结构中含有28.31%的α-螺旋、4.78%的β-转角、61.03%的无规卷曲和5.88%的延伸链；GbR2R3-MYB1蛋白与火炬松、白云杉R2R3-MYB蛋白的氨基酸序列相似性较高，亲缘关系较近，与系统发育树进化分析结果基本相符；亚细胞定位检测发现GbR2R3-MYB1蛋白定位于细胞核。RT-qPCR分析表明，GbR2R3-MYB1基因对盐、干旱、低温及高温胁迫均有响应，其相对表达量在盐和干旱胁迫下出现先上升后下降的波动，而低温和高温胁迫下表现出先下降后上升再下降的趋势。GbR2R3-MYB1基因的克隆及功能分析可为进一步阐述银杏抗逆分子机理及其他植物的品种改良提供资源和依据。

关键词: 银杏, GbR2R3-MYB1基因, 克隆, 亚细胞定位, 非生物胁迫, 表达分析

Abstract:

R2R3-MYB transcription factor is one of the subfamily members having big number in MYB family. It plays an important role in growth and development，hormone signal transduction，secondary metabolite and stress regulation of plant. In this study，the GbR2R3-MYB1 gene from Ginkgo biloba was cloned，and bioinformatics software was applied to analyze the physicochemical properties，structure and function of the GbR2R3-MYB1 proteins. After the plant fusion expression vector pCAMBIA1300-R2R3MYB1-GFP was constructed and injected to tobacco epidermal cells through agrobacterium-mediated method，the subcellular localization of GbR2R3-MYB1 gene was observed. Finally RT-qPCR was sued to analyze the GbR2R3-MYB1 gene expressions in G. biloba leaves under abiotic stress. The results showed that the total length of GbR2R3-MYB1 coding region was 819 bp，encoding a 272 amino acid protein. The relative molecular weight of GbR2R3-MYB1 protein was about 30 001.60 Da and its theoretical isoelectric point（PI）was 6.59，which was an unstably hydrophilic protein. In the secondary structure of GbR2R3-MYB1 protein，there were 28.31% of α-helix，4.78% of beta turn，61.03% of random coil and 5.88% of extended strand. GbR2R3-MYB1 protein had the highest amino acid sequence similarity with R2R3-MYB of Pinus taeda and Picea glauca，and genetic relationships were close among them，which were basically consistent with phylogenetic evolution tree analysis. Subcellular localization results showed that GbR2R3-MYB1 was located on the cell nucleus. The RT-qPCR results demonstrated that GbR2R3-MYB1 gene was induced by salt，drought，cold and heat stress，and its relative expression were up-regulated first，then down-regulated under salt and drought stress，respectively，while down-regulated first then up-regulated，finally down-regulated under cold and heat stress. The cloning and functional analysis of GbR2R3-MYB1 gene will help to clarify the molecular mechanism of stress resistance in G. biloba，and provide resources and basis for the variety improvement of other plants.

Key words: Ginkgo biloba, GbR2R3-MYB1 gene, cloning, subcellular localization, abiotic stress, expression analysis

骆鹰, 谭智, 王帆, 刘晓霞, 罗小芳, 何福林. 银杏GbR2R3-MYB1基因的克隆及非生物胁迫应答分析[J]. 生物技术通报, 2022, 38(10): 184-194.

LUO Ying, TANG Zhi, WANG Fan, LIU Xiao-xia, LUO Xiao-fang, HE Fu-lin. Cloning and Response Analysis to Abiotic Stress of GbR2R3-MYB1 Gene from Ginkgo biloba[J]. Biotechnology Bulletin, 2022, 38(10): 184-194.

图/表 11

表1 实验所用引物序列信息

Table 1 Primer sequences used in this study

引物名称Primer name	引物序列Primer sequence（5'-3'）	用途Purpose
GbR2R3MYB1-F	AGAAGCTGCTCCTCGTTACAG	目的基因扩增引物
GbR2R3MYB1-R	GTCCTTGATCAAAATGGGGTA	目的基因扩增引物
pC007-F	TGTAAAACGACGGCCAGT	pClone007载体引物
pC007-R	CAGGAA ACAGCTATGACC	pClone007载体引物
GFP-F	AGAACACGGGGGACGAGCTCGGTACCATGGGTCGGTCTCCTTGCTG	亚细胞定位引物
GFP-R	CCCTTGCTCACCATGTCGACTCTAGATACCCGCAGTTGCCTGTAAT	亚细胞定位引物
Gb18S-F	CGAAGACGATCAGATACCG	qPCR内参基因
Gb18S-R	TCAGCCTTGCGACCATAC	qPCR内参基因
q-R2R3MYB1-F	ACGGCAGCTCCAGCTAATTC	qPCR检测引物
q-R2R3MYB1-R	GTGGTGGCTCTGCTTATGCT	qPCR检测引物

图1 GbR2R3-MYB1基因PCR扩增琼脂糖凝胶电泳 A：GbR2R3-MYB1目的片段PCR扩增；B：菌落PCR；M：DL2000 DNA marker；1-3：PCR鉴定的阳性克隆

Fig.1 Agarose gel electrophoresis of PCR amplified prod-ucts of GbR2R3-MYB1 A：PCR amplification of target fragment of GbR2R3-MYB1. B：Bacteria liquid PCR；M：DL2000 DNA marker；1-3：positive clones of colony PCR identification

图2 GbR2R3-MYB1基因CDS区核苷酸序列及编码氨基酸序列

Fig. 2 Nucleotide and amino acid sequence in the CDS region of GbR2R3-MYB1 gene

图3 银杏GbR2R3-MYB1蛋白保守域

Fig. 3 Conservative domain of GbR2R3-MYB1 protein from G. biloba

图4 银杏GbR2R3-MYB1蛋白与其他植物氨基酸序列的多重比对

Fig. 4 Multiple alignment of the amino acid sequences of GbR2R3-MYB1 in G. biloba and other plants

图5 植物MYB同源基因氨基酸序列系统进化树

Fig. 5 Phylogenetic tree of deduced amino acid sequences of plant MYB homologous genes

图6 GbR2R3-MYB1蛋白亲水/疏水性预测分析

Fig. 6 Prediction of hydrophilic/hydrophobicity for GbR2R3-MYB1 protein

图7 GbR2R3-MYB1蛋白二级结构预测蓝色：α-螺旋，绿色：β-转角，紫色：无规卷曲，红色：延伸链

Fig. 7 Prediction of secondary structure for GbR2R3-MYB1 protein Blue：Alpha helix. Green：Beta turn. Purple：Random coil. Red：Extended strand. D：Tertiary structure analysis of GbR2R3-MYB1 protein

图8 GbR2R3-MYB1蛋白三级结构预测

Fig. 8 Prediction of tertiary structure of GbR2R3-MYB1 protein

图9 GbR2R3-MYB1蛋白亚细胞定位结果

Fig. 9 Subcellular localization of GbR2R3-MYB1 protein

图10 GbR2R3-MYB1基因在盐、干旱、低温及高温胁迫下的表达分析图中柱状图上不同字母表示显著性差异，P < 0.05显著差异，P < 0.01极显著差异；数值为平均值$\bar{x}$±SE

Fig.10 Expression analysis of GbR2R3-MYB1 gene under salt，drought，cold，and heat stresses Each value was calculated as the mean of three samples standard error（SE）. The different letters above the bar indicate significant difference（P < 0.05）and very significant difference（P < 0.01）

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