Cloning and salt tolerance analysis of BpGRAS1 gene in Betula platyphylla

doi:10.12302/j.issn.1000-2006.202010011

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

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Cloning and salt tolerance analysis of BpGRAS1 gene in Betula platyphylla

ZHANG Qun(), JI Xiaoyu, HE Zihang, WANG Zhibo, TIAN Zengzhi, WANG Chao^*()

State Key Laboratory of Tree Genetics and Breeding (Northeast Forestry University), Harbin 150040,China

Received:2020-10-06 Accepted:2021-02-10 Online:2021-09-30 Published:2021-09-30
Contact: WANG Chao E-mail:1207916213@qq.com;wangchao@nefu.edu.cn

Abstract

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

CLC Number:

Q786
S722

ZHANG Qun, JI Xiaoyu, HE Zihang, WANG Zhibo, TIAN Zengzhi, WANG Chao. Cloning and salt tolerance analysis of BpGRAS1 gene in Betula platyphylla[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2021, 45(5): 38-46.

Figures/Tables 7

Table 1

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用途 application	引物名称 primer name	序列 sequence(5'-3')
基因克隆 gene cloning	BpGRAS1-F BpGRAS1-R	ATGAAATTGACGCTTTGCAAAG TCATGGTTTCCATGCTGAAGC
载体构建 vector construction	pROKII-BpGRAS1-F	GCTCTAGAATGAAATTGACGCTTTGCAAAG
	pROKII-BpGRAS1-R	CGGGGTACCTCATGGTTTCCATGCTGAAGC
	pFGC5941-BpGRAS1-cisF	CCCATGGCACACACATGGACATAACTC
	pFGC5941-BpGRAS1-cisR	AGGCGCGCCTCTTGCACACTCGTTTAG
	pFGC5941-BpGRAS1-antiF	CTCTAGACACACACATGGACATAACTC
	pFGC5941-BpGRAS1-antiR	CGCGGATCCTCTTGCACACTCGTTTAG
实时荧光定量PCR quantitative real-time PCR	q-OE-BpGRAS1-F	GTAATAGCCAGCACATCC
	q-OE-BpGRAS1-R	TTCCTGCACTTCAACTCT
	q-IE-BpGRAS1-F	CACTACTCCACAAGAAGCACC
	q-IE-BpGRAS1-R	ACTCGTTTAGAAGACTCGAGG
	Actin-F	AAACAATGGCTGATGCTG
	Actin-R	ACAATACCGTGCTCAATAGC
	β-Tublin-F	GGAAGCCATAGAAAGACC
	β-Tublin-R	CAACAAATGTGGGATGCT

Cloning and salt tolerance analysis of BpGRAS1 gene in Betula platyphylla

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