Cloning and Response Analysis to Abiotic Stress of GbR2R3-MYB1 Gene from Ginkgo biloba

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

Abstract

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

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.

Figures/Tables 11

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

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

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

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

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

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

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

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

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

Fig. 9 Subcellular localization of GbR2R3-MYB1 protein

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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