白桦BpGRAS1基因的克隆及耐盐功能分析

doi:10.12302/j.issn.1000-2006.202010011

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南京林业大学学报（自然科学版） ›› 2021, Vol. 45 ›› Issue (5): 38-46.doi: 10.12302/j.issn.1000-2006.202010011

白桦BpGRAS1基因的克隆及耐盐功能分析

张群(), 及晓宇, 贺子航, 王智博, 田增智, 王超^*()

林木遗传育种国家重点实验室(东北林业大学),黑龙江哈尔滨 150040

收稿日期:2020-10-06 接受日期:2021-02-10 出版日期:2021-09-30 发布日期:2021-09-30
通讯作者: 王超
基金资助:
中央高校基本科研业务费专项资金项目(2572016DA01)

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

摘要/Abstract

摘要：

【目的】 GRAS转录因子是植物特有的转录因子家族之一,在植物响应盐、干旱等非生物胁迫中发挥重要的调控作用。从白桦(Betula platyphylla )中克隆GRAS转录因子基因,研究其耐盐功能,为研究木本植物GRAS转录因子的抗逆机制奠定理论基础。【方法】 在白桦转录组数据库中获得一个GRAS转录因子基因,命名为BpGRAS1 (GenBank 登录号: MN117546.1)。利用生物信息学进行多序列比对、构建进化树。分别构建植物过表达(pROKⅡ-BpGRAS1) 及抑制表达(pFGC5941-BpGRAS1) 载体。利用农杆菌介导高效瞬时遗传转化体系获得BpGRAS1基因瞬时过表达(OE)、抑制表达(IE) 及对照 (WT) 白桦植株。通过实时荧光定量RT-PCR(qRT-PCR) 技术分析盐胁迫下OE、IE及WT植株中BpGRAS1基因的表达情况,鉴定转基因植株中BpGRAS1的表达效率是否响应盐胁迫。在盐胁迫下比较了BpGRAS1基因瞬时过表达、抑制表达及对照白桦植株的电解质渗透率、失水率、丙二醛(MDA) 含量、过氧化物酶 (POD) 和超氧化物歧化酶 (SOD) 活性。【结果】 BpGRAS1基因的开放阅读框为1 425 bp,编码 474个氨基酸。BpGRAS1具有GRAS家族的序列特征,在C端的氨基酸序列相似度较高,与AtSHR亲缘关系较近。盐胁迫处理下,BpGRAS1的表达量升高,过表达植株中表达量高于对照,抑制表达植株中表达量低于对照,说明BpGRAS1受盐胁迫诱导,成功获得过表达及抑制表达植株。过表达BpGRAS1基因能降低白桦在盐胁迫下的电解质渗透率、失水率及 MDA 的积累,并显著增强了 POD 和 SOD 酶的活性,从而提高转基因植株的耐盐性。【结论】 BpGRAS1基因响应盐胁迫,过表达BpGRAS1基因降低了盐胁迫下植株细胞受损程度,通过增强POD 和 SOD 活性提高白桦的耐盐能力。

关键词: 白桦, GRAS转录因子, 耐盐性, 基因功能, 生理途径

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

中图分类号:

Q786
S722

张群,及晓宇,贺子航,等. 白桦BpGRAS1基因的克隆及耐盐功能分析[J]. 南京林业大学学报（自然科学版）, 2021, 45(5): 38-46.

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 (Natural Science Edition), 2021, 45(5): 38-46.DOI: 10.12302/j.issn.1000-2006.202010011.

图/表 7

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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

白桦BpGRAS1基因的克隆及耐盐功能分析

Cloning and salt tolerance analysis of BpGRAS1 gene in Betula platyphylla

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