Cloning and salt tolerance analysis of BpGRAS1 gene in Betula platyphylla

ZHANG Qun, JI Xiaoyu, HE Zihang, WANG Zhibo, TIAN Zengzhi, WANG Chao

JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2021, Vol. 45 ›› Issue (5) : 38-46.

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JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2021, Vol. 45 ›› Issue (5) : 38-46. DOI: 10.12302/j.issn.1000-2006.202010011

Cloning and salt tolerance analysis of BpGRAS1 gene in Betula platyphylla

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Abstract

【Objective】 The GRAS transcription factor is a plant-specific transcription factor family that plays a vital regulatory role in response to abiotic stresses, such as salt and drought. In this study, a GRAS transcription factor gene was cloned from Betula platyphylla, and its salt tolerance function was identified, which laid a theoretical foundation for studying the molecular mechanism of tolerance to woody plant GRAS transcription factors. 【Method】 A GRAS transcription factor gene was screened from the B. platyphylla transcriptome database, named BpGRAS 1(GenBank Number: MN117546.1). Bioinformatics analysis was performed for multiple sequence alignment and phylogenetic tree construction. The primers BpGRAS1-F and BpGRAS1-R were designed according to the gene sequence, and the BpGRAS1 gene was cloned using B. platyphylla cDNA as a template. Plant overexpression (pROKⅡ-BpGRAS1) and RNA-silencing expression (pFGC5941-BpGRAS1) vectors were constructed to further identify gene function. An Agrobacterium-mediated high-efficiency transient transformation system was used to obtain BpGRAS1 transient overexpression (OE), inhibitory expression (IE) and wild type (WT) B. platyphylla plants. The expression levels of the BpGRAS1 gene in the OE, IE and WT plants treated with 150 mmol/L NaCl were analyzed by real-time fluorescent quantitative RT-PCR (qRT-PCR) to test the expression efficiency of BpGRAS 1 in transgenic plants and to determine whether it responds to salt stress. The electrolyte leakage, water loss rate within 0-4 h, MDA content, peroxidase (POD) and superoxide dismutase (SOD) activities in OE, IE and WT plants under salt stress were measured and compared to identify the salt tolerance of the BpGRAS1 gene.【Result】 The open reading frame of BpGRAS1 gene is 1 425 bp, encoding 474 amino acids. Multiple sequence alignments results showed that the amino acid sequence in the C-terminal of BpGRAS1 is conserved and has the sequence characteristics of the GRAS family. Phylogenetic tree analysis showed that BpGRAS1 was closely related to AtSHR. Under salt stress treatment, the expression level of BpGRAS1 plants was higher than that in non-treatment plants. The expression level of BpGRAS1 was higher in OE plants and lower in IE plants, than in WT plants, indicating that BpGRAS1 was induced by salt treatment and that the OE and IE plants were obtained. Overexpression of BpGRAS1 reduced electrolyte leakage (P < 0.05), water loss rate and MDA content (P < 0.05) of B. platyphylla under salt stress; significantly enhanced the activities of POD and SOD enzymes (P < 0.05); and lowered the number of dead cells, improving the salt tolerance of transgenic plants.【Conclusion】 BpGRAS1 can respond to salt stress. Overexpression of the BpGRAS1 gene reduces the damage to plant cells in transgenic plants under salt stress and improves the salt tolerance of B. platyphylla by enhancing the activities of POD and SOD enzymes. The results of this study lay the foundation for further understanding the salt tolerance mechanism of B. platyphylla.

Key words

Betula platyphylla / GRAS transcription factor / salt tolerance / gene function / physiological pathway

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ZHANG Qun , JI Xiaoyu , HE Zihang , et al . Cloning and salt tolerance analysis of BpGRAS1 gene in Betula platyphylla[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY. 2021, 45(5): 38-46 https://doi.org/10.12302/j.issn.1000-2006.202010011

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