刚毛柽柳ThPCS1基因克隆与镉胁迫应答分析

doi:10.12302/j.issn.1000-2006.202011014

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南京林业大学学报（自然科学版） ›› 2021, Vol. 45 ›› Issue (3): 71-78.doi: 10.12302/j.issn.1000-2006.202011014

刚毛柽柳ThPCS1基因克隆与镉胁迫应答分析

王培龙(), 杨妮, 张傲然, 唐努尔·塞力克, 李爽, 高彩球^*()

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:2020-11-07 修回日期:2021-01-07 出版日期:2021-05-30 发布日期:2021-05-31
通讯作者: 高彩球
基金资助:
国家自然科学基金面上项目(31670679);黑龙江省大学生创新创业训练计划项目(201910225243)

Cloning ThPCS1 gene of Tamarix hispida to improve cadmium tolerance

WANG Peilong(), YANG Ni, ZHANG Aoran, Tangnver•Sailike , LI Shuang, GAO Caiqiu^*()

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

Received:2020-11-07 Revised:2021-01-07 Online:2021-05-30 Published:2021-05-31
Contact: GAO Caiqiu

摘要/Abstract

摘要：

【目的】探究刚毛柽柳(Tamarix hispida)植物络合素合酶(phytochelatin synthase,PCS)基因ThPCS1的镉胁迫应答功能。【方法】通过逆转录聚合酶链式反应(RT-PCR)对刚毛柽柳ThPCS1基因进行克隆;通过BioEdit、MEGA 5.0等软件对ThPCS1蛋白进行生物信息学分析;通过实时荧光定量PCR(RT-qPCR)技术分析镉(Cd)胁迫条件下ThPCS1基因在柽柳根和叶中的表达水平;构建pROKII-ThPCS1过表达载体,通过瞬时侵染技术获得转ThPCS1基因柽柳,分析比较转基因和对照柽柳Cd胁迫应答相关生理指标和生理染色情况。【结果】从刚毛柽柳转录组数据中分离出ThPCS1基因的全长转录组序列,预测该基因的开放阅读框为1 581 bp,编码526个氨基酸,编码蛋白质的分子质量为130.75 ku,理论等电点pI为5.0。保守结构域的多重比对分析结果均显示ThPCS1蛋白在N端具有PCS结构域。RT-qPCR结果显示在镉胁迫条件下,ThPCS1基因在柽柳根中被诱导表达,且呈现出组织特异性的表达模式。对镉胁迫处理后的刚毛柽柳ThPCS1转基因植株和对照植株相关生理指标进行测定,结果显示,同对照植株相比,转基因植株的活性氧和镉离子含量降低,细胞膜损伤减小。【结论】刚毛柽柳ThPCS1基因在根中的表达明显受到镉胁迫诱导,可能参与了刚毛柽柳对镉胁迫的应答。本研究初步证明刚毛柽柳ThPCS1基因可能提高了转基因植物的耐镉能力,是一个耐镉分子育种的候选基因。

关键词: 刚毛柽柳, 植物络合素合酶, 镉胁迫, 基因表达, 实时荧光定量PCR

Abstract:

【Objective】 This study aimed to improve the cadmium tolerance of Tamarix hispida. 【Method】The cadmium stress response of the phytochelatin synthase gene (ThPCS1) in T. hispida was explored. We cloned the ThPCS1 gene of T. hispida by using reverse transcription-polymerase chain reaction (RT-PCR). The bioinformatics information of the ThPCS1 protein was analyzed using BioEdit, MEGA 5.0 and other software. The expression of ThPCS1 in the roots and leaves of T. hispida under cadmium stress was analyzed by real-time quantitative (RT-q) PCR. We constructed a pROKII-ThPCS1 overexpression vector and used it to obtain ThPCS1 transgenic T. hispida by transient infection. Physiological indicators and staining of transgenic and control T. hispida under the Cd stress response were compared. 【Result】We sequenced the full length of the ThPCS1 gene from T. hispida transcriptome data. The open reading frame (ORF) of ThPCS1 gene was 1 581 bp, encoding 526 amino acids with a relative molecular mass of 130.75 ku, and the theoretical isoelectric point pI was 5.0. Conserved domains and multiple sequence alignment analyses showed that the ThPCS1 protein has a phytochelatin synthase (PCS) domain at the N-terminus. The RT-qPCR results revealed that ThPCS1 expression was induced in the roots of T. hispida under cadmium stress, and the expression was obviously tissue-specific. Furthermore, ThPCS1 was overexpressed in T. hispida and under Cd stress. The results of relevant physiological determination showed less reactive oxygen species and cadmium ions in plants overexpressing ThPCS1 than in the control. Accordingly, the cell membrane was less damaged in transgenic as compared to the control plants. 【Conclusion】The response of the T. hispida ThPCS1 gene to cadmium stress was significant, confirming its role in the response of T. hispida to cadmium stress. The ThPCS1 gene might improve the cadmium tolerance of T. hispida and serve as a candidate gene for the molecular breeding of T. hispida with excellent cadmium tolerance.

Key words: Tamarix hispida, phytochelatin synthase, cadmium stress, gene expression, RT-qPCR

中图分类号:

Q943.2
S718

王培龙,杨妮,张傲然,等. 刚毛柽柳ThPCS1基因克隆与镉胁迫应答分析[J]. 南京林业大学学报（自然科学版）, 2021, 45(3): 71-78.

WANG Peilong, YANG Ni, ZHANG Aoran, Tangnver•Sailike , LI Shuang, GAO Caiqiu. Cloning ThPCS1 gene of Tamarix hispida to improve cadmium tolerance[J].Journal of Nanjing Forestry University (Natural Science Edition), 2021, 45(3): 71-78.DOI: 10.12302/j.issn.1000-2006.202011014.

图/表 4

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用途 application	引物名称 prime name	引物序列 prime sequences (5'-3')
RT- qPCR	ThPCS1-DL-F	CAGCTCTTCACTCTGAGCTGC
	ThPCS1-DL-R	CTCCTCTTGACTTAGACTTG
	Actin-F	AAACAATGGCTGATGCTG
	Actin-R	ACAATACCGTGCTCAATAGG
	α-tubulin-F	CACCCACCGTTGTTCCAG
	α-tubulin-R	ACCGTCGTCATCTTCACC
	β-tubulin-F	GGAAGCCATAGAAAGACC
	β-tubulin-R	CAACAAATGTGGGATGCT
基因克隆 gene clone	ThPCS1-F	CGCGGATCCATGGCGATGGCTGGACTGTAC
基因克隆 gene clone	ThPCS1-R	CGGGGTACCAGAGGATAGCGTAGCTGCTGAGTTCTC
载体引物 carrier primers	ThPCS1-T-F	ATGGCGATGGCTGGACTGTAC
	ThPCS1-T-R	AGAGGATAGCGTAGCTGCTGAGTTCTC
	pROKⅡ-F	AGACGTTCCAACCACGTCTT
	pROKⅡ-R	CCAGTGAATTCCCGATCTAG

刚毛柽柳ThPCS1基因克隆与镉胁迫应答分析

Cloning ThPCS1 gene of Tamarix hispida to improve cadmium tolerance

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