莲中介导矿质元素胁迫耐受性基因的筛选与功能验证

doi:10.3864/j.issn.0578-1752.2024.05.012

中国农业科学 ›› 2024, Vol. 57 ›› Issue (5): 980-988.doi: 10.3864/j.issn.0578-1752.2024.05.012

莲中介导矿质元素胁迫耐受性基因的筛选与功能验证

胡衡亮¹(), 顾天宇¹^,²^,³, 陈思颖¹^,²^,³, 王垚¹, 彭佳师¹^,²^,³()

¹ 湖南科技大学生命科学与健康学院，湖南湘潭411201
² 湖南科技大学经济作物遗传改良与综合利用湖南省重点实验室，湖南湘潭 411201
³ 湖南科技大学重金属污染土壤生态修复与安全利用湖南省高校重点实验室，湖南湘潭 411201

收稿日期:2023-08-11 接受日期:2023-11-09 出版日期:2024-03-01 发布日期:2024-03-06
通信作者:
彭佳师，E-mail：jspeng@hnust.edu.cn
联系方式: 胡衡亮，E-mail：2260113811@qq.com。
基金资助:
国家自然科学基金(U20A2024); 湖南省自然科学基金(2022JJ20022); 大学生创新创业训练计划(202110534065)

Isolation and Functional Verification of Genes Mediating Mineral Element Stress Tolerance in Lotus

HU HengLiang¹(), GU TianYu¹^,²^,³, CHEN SiYing¹^,²^,³, WANG Yao¹, PENG JiaShi¹^,²^,³()

¹ School of Life and Health Sciences, Hunan University of Science and Technology, Xiangtan 411201, Hunan
² Hunan Key Laboratory of Economic Crops Genetic Improvement and Integrated Utilization, Hunan University of Science and Technology, Xiangtan 411201, Hunan
³ Key Laboratory of Ecological Remediation and Safe Utilization of Heavy Metal-Polluted Soils, Hunan University of Science and Technology, Xiangtan 411201, Hunan

Received:2023-08-11 Accepted:2023-11-09 Published:2024-03-01 Online:2024-03-06

摘要/Abstract

摘要：

【目的】莲是一种传统的药食同源作物，但其蕴藏的丰富基因资源仍缺乏功能鉴定。分离鉴定莲中参与微量矿质营养元素及重金属元素积累或耐受过程的功能基因，丰富莲中介导营养高效以及逆境耐受的基因资源，并为莲的遗传改良提供理论依据。【方法】以湘莲代表品种‘寸三莲’为试验材料。首先构建莲的酵母表达cDNA文库，并将文库转化酿酒酵母后分别在含有过量镉（Cd）、锰（Mn）、锌（Zn）、铜（Cu）、铁（Fe）、铝（Al）胁迫的平板上筛选，分离阳性酵母克隆中介导相关胁迫耐受性的目的基因。结合生物信息学分析和酵母互补试验对筛选出的耐受基因进行功能验证。【结果】构建了库容量超过10⁶个单克隆、重组率100%、平均插入片段大于1 000 bp的酵母文库。通过筛选得到介导Cd耐受的基因13个、Mn耐受基因4个、Zn耐受基因4个、Cu耐受基因3个、Fe耐受基因7个、Al耐受基因1个，其中3个基因能够同时介导Fe和Mn的耐受性。这些基因在莲除6号染色外的其他染色体上都有分布。【结论】本研究构建了高质量的莲酵母表达cDNA文库，并筛选得到29个介导微量矿质营养元素或重金属元素胁迫抗性的基因，为莲的营养高效以及避免重金属污染的遗传改良积累了基因资源。

关键词: 寸三莲, 矿质营养元素, 重金属, 抗性基因

Abstract:

【Objective】 Lotus (Nelumbo nucifera) is a traditional edible and medicinal homologous crop in China, yet its abundant genetic resources lack functional characterization. This research aimed to functionally identify genes involving in the accumulation/tolerance of trace mineral nutrients and heavy metal elements in lotus, thereby accumulated gene resources that facilitated increased nutrient efficiency and stress tolerance, so as to provide a theoretical basis for the genetic improvement of lotus. 【Method】The representative Xianglian lotus variety, Cunsanlian was used as the experimental material. Initially, a yeast expression cDNA library of lotus was constructed and transformed into Saccharomyces cerevisiae. It was then screened on plates containing excessive cadmium (Cd), manganese (Mn), zinc (Zn), copper (Cu), iron (Fe), and aluminum (Al) stress to isolate target genes mediating stress tolerance in positive yeast clones. Finally, the functional verification of the selected tolerance genes was conducted through combining bioinformatics analysis and yeast complementation verification. 【Result】A yeast cDNA library of lotus was constructed with a capacity exceeding 10⁶ yeast monoclonal clones, a recombination rate of 100%, and an average insertion fragment size greater than 1 000 bp. Following the screening on stress plates, 13 genes for Cd tolerance, 4 for Mn tolerance, 4 for Zn tolerance, 3 for Cu tolerance, 7 for Fe tolerance, and 1 for Al tolerance were identified. Among these, 3 genes were able to mediate tolerance to both Fe and Mn. These genes were distributed on all lotus chromosomes except chromosome 6.【Conclusion】A high-quality yeast expression cDNA library of lotus was constructed, and screened 29 genes mediating resistance to excess trace mineral nutrients or heavy metal elements, accumulating gene resources for lotus genetic improvement for enhanced nutrient efficiency and the prevention of heavy metal accumulation.

Key words: Cunsanlian, mineral nutrients, heavy metals, stress tolerance gene

胡衡亮, 顾天宇, 陈思颖, 王垚, 彭佳师. 莲中介导矿质元素胁迫耐受性基因的筛选与功能验证[J]. 中国农业科学, 2024, 57(5): 980-988.

HU HengLiang, GU TianYu, CHEN SiYing, WANG Yao, PENG JiaShi. Isolation and Functional Verification of Genes Mediating Mineral Element Stress Tolerance in Lotus[J]. Scientia Agricultura Sinica, 2024, 57(5): 980-988.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2024.05.012

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图/表 5

图1

表1

表2

莲cDNA文库筛选得到的介导过量金属胁迫耐受的基因信息"

金属元素 Metal element	编号 Number	基因ID Gene ID	测序总克隆数 Total number of clones sequenced	占比 Proportion (%)	功能注释 Function annotation
Cd	Cd1	Nn8g41136	5	4.50	E3泛素蛋白连接酶 RNF144A-like E3 ubiquitin-protein ligase RNF144A-like
	Cd11	Nn1g06214	11	9.90	乙醇脱氢酶 Alcohol dehydrogenase 1
	Cd15	Nn5g27090	2	1.80	未鉴定基因LOC104593877 Uncharacterized LOC104593877
	Cd18	Nn4g22887	1	0.90	含LIM结构域的WLIM1-like LIM domain-containing protein WLIM1-like
	Cd19	Nn5g27723	1	0.90	类赤霉素调节蛋白2 Gibberellin-regulated protein 2-like
	Cd22	Nn1g04086	2	1.80	类金属硫蛋白3 Metallothionein-like protein 3
	Cd41	Nn7g37741	6	5.40	LSD1蛋白 Protein LSD1
	Cd42	Nn3g19712	41	36.90	类谷胱甘肽γ-谷氨酰半胱氨酸转移酶1 Glutathione gamma-glutamylcysteinyltransferase 1-like
	Cd47	Nn1g02879	29	26.10	类谷胱甘肽γ-谷氨酰半胱氨酸转移酶1 Glutathione gamma-glutamylcysteinyltransferase 1-like
	Cd51	Nn3g20095	1	0.90	热激转录因子A-5 Heat stress transcription factor A-5
	Cd54	Nn8g38524	5	4.50	E3泛素蛋白连接酶RNF217 Probable E3 ubiquitin-protein ligase RNF217
	Cd67	Nn5g28779	4	3.60	未鉴定基因 Uncharacterized
	Cd88	Nn1g04197	3	2.70	含锚蛋白重复序列的蛋白ITN1-like Ankyrin repeat-containing protein ITN1-like
Mn	Mn1		5	14.70	未知蛋白Unknown
	Mn10	Nn2g15732	19	55.90	金属耐受蛋白4 Metal tolerance protein 4
	Mn13	Nn3g18415	4	11.80	类液泡铁转运蛋白1.1 Vacuolar iron transporter 1.1-like
	Mn31	Nn7g38287	6	17.60	类金属耐受蛋白10 Metal tolerance protein 10-like
Zn	Zn1	Nn2g13145	4	21.00	含锌指CCCH结构域蛋白66类似蛋白 Zinc finger CCCH domain-containing protein 66-like
	Zn5	Nn1g06462	8	42.10	蛋白质磷酸酶2C 39 Probable protein phosphatase 2C 39
	Zn16	Nn5g26947	2	10.50	蛋白质磷酸酶2C 44 Probable protein phosphatase 2C 44
	Zn21	Nn8g40571	5	26.30	蛋白质磷酸酶2C 59 Probable protein phosphatase 2C 59
Cu	Cu8	Nn1g06836	2	50.00	广泛逆境胁迫蛋白PHOS34 Universal stress protein PHOS34
	Cu38	Nn1g03816	1	25.00	广泛逆境胁迫蛋白PHOS34 like Universal stress protein PHOS34-like
	Cu41	Nn8g40145	1	25.00	类γ-谷氨酰肽酶3 Gamma-glutamyl peptidase 3-like
Fe	Fe1	Nn2g13750	73	92.5	受体样蛋白激酶At5g47070 Probable receptor-like protein kinase At5g47070
	Fe19	Nn2g10992	1	1.25	未鉴定基因LOC104597278 Uncharacterized LOC104597278
	Fe32	Nn4g25766	1	1.25	60S核糖体蛋白L18-3 60S ribosomal protein L18-3
	Fe36		1	1.25	未知蛋白 Unknown
	Fe38	Nn2g15732	1	1.25	金属耐受蛋白4 Metal tolerance protein 4
	Fe56	Nn7g38287	1	1.25	类金属耐受蛋白10 Metal tolerance protein 10-like
	Fe68	Nn3g18415	1	1.25	类液泡铁转运蛋白1.1 Vacuolar iron transporter 1.1-like
Al	Al1	Nn8g38765	3	100.00	丝裂原激活蛋白激酶激酶2类似蛋白 Mitogen-activated protein kinase kinase 2-like

表2

图2

图3

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	初级文库 Primary library	次级文库 Secondary library	酵母文库 Yeast library
重组率 Recombination rate	100%	100%	100%
平均插入长度 Average insertion length (bp)	>1000 bp	>1000 bp	>1000 bp
库容量 Cfu	1.3×10⁷	1.3×10⁷	1.1×10⁶

莲中介导矿质元素胁迫耐受性基因的筛选与功能验证

Isolation and Functional Verification of Genes Mediating Mineral Element Stress Tolerance in Lotus

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