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

Abstract

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

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.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.chinaagrisci.com/EN/10.3864/j.issn.0578-1752.2024.05.012

https://www.chinaagrisci.com/EN/Y2024/V57/I5/980

Figures/Tables 5

Fig. 1

Table 1

Table 2

Genes identified from lotus cDNA library screening which mediate excessive metal stress tolerance"

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

Table 2

Fig. 2

Fig. 3

References 29

[1]	郭宏波, 柯卫东. 莲属分类与遗传资源多样性及其应用. 黑龙江农业科学, 2009(4): 106-109.
	GUO H B, KE W D. Advances on classification and genetic diversity of Lotus germplasm and its utilization. Heilongjiang Agricultural Sciences, 2009(4): 106-109. (in Chinese)
[2]	MING R, VANBUREN R, LIU Y L, YANG M, HAN Y P, LI L T, ZHANG Q, KIM M J, SCHATZ M C, CAMPBELL M, et al. Genome of the long-living sacred lotus (Nelumbo nucifera Gaertn.). Genome Biology, 2013, 14(5): R41. doi: 10.1186/gb-2013-14-5-r41
[3]	WANG Y, FAN G Y, LIU Y M, SUN F M, SHI C C, LIU X, PENG J, CHEN W B, HUANG X F, CHENG S F, et al. The sacred lotus genome provides insights into the evolution of flowering plants. The Plant Journal, 2013, 76(4): 557-567. doi: 10.1111/tpj.12313 pmid: 23952714
[4]	LIN Z Y, ZHANG C, CAO D D, DAMARIS R N, YANG P F. The latest studies on Lotus (Nelumbo nucifera)-an emerging horticultural model plant. International Journal of Molecular Sciences, 2019, 20(15): 3680. doi: 10.3390/ijms20153680
[5]	刘正位, 季群, 柯卫东, 朱红莲, 刘玉平, 彭静, 匡晶, 王芸, 郭丹丹. 莲基因组学和分子生物学研究进展. 长江蔬菜, 2017(18): 41-48.
	LIU Z W, JI Q, KE W D, ZHU H L, LIU Y P, PENG J, KUANG J, WANG Y, GUO D D. Research progress in molecular biology and genomics of Nelumbo. Journal of Changjiang Vegetables, 2017(18): 41-48. (in Chinese)
[6]	VATAMANIUK O K, MARI S, LU Y P, REA P A. AtPCS1, a phytochelatin synthase from Arabidopsis:Isolation and in vitro reconstitution. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96(12): 7110-7115.
[7]	SONG W Y, MARTINOIA E, LEE J, KIM D, KIM D Y, VOGT E, SHIM D, CHOI K S, HWANG I, LEE Y. A novel family of cys-rich membrane proteins mediates cadmium resistance in Arabidopsis. Plant Physiology, 2004, 135(2): 1027-1039. doi: 10.1104/pp.103.037739
[8]	CLEMENS S, KIM E J, NEUMANN D, SCHROEDER J I. Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast. The EMBO Journal, 1999, 18(12): 3325-3333. doi: 10.1093/emboj/18.12.3325
[9]	KIM Y Y, KIM D Y, SHIM D, SONG W Y, LEE J, SCHROEDER J I, KIM S, MORAN N, LEE Y. Expression of the novel wheat gene TM20 confers enhanced cadmium tolerance to bakers’ yeast. Journal of Biological Chemistry, 2008, 283(23): 15893-15902. doi: 10.1074/jbc.M708947200
[10]	SHIM D, HWANG J U, LEE J, LEE S, CHOI Y, AN G, MARTINOIA E, LEE Y. Orthologs of the class A4 heat shock transcription factor HsfA4a confer cadmium tolerance in wheat and rice. The Plant Cell, 2010, 21(12): 4031-4043. doi: 10.1105/tpc.109.066902
[11]	PAPOYAN A, KOCHIAN L V. Identification of Thlaspi caerulescens genes that may be involved in heavy metal hyperaccumulation and tolerance. characterization of a novel heavy metal transporting ATPase. Plant Physiology, 2004, 136(3): 3814-3823. doi: 10.1104/pp.104.044503
[12]	BLANVILLAIN R, KIM J H, WU S M, LIMA A, OW D W. OXIDATIVE STRESS 3 is a chromatin-associated factor involved in tolerance to heavy metals and oxidative stress. The Plant Journal, 2009, 57(4): 654-665. doi: 10.1111/tpj.2009.57.issue-4
[13]	彭佳师, 易红英, 龚继明. 超积累植物伴矿景天镉耐受基因SpMT2的分离及功能鉴定. 生物工程学报, 2020, 36(3): 541-548.
	PENG J S, YI H Y, GONG J M. Isolation and characterization of cadmium tolerant gene SpMT2 in the hyperaccumulator Sedum plumbizincicola. Chinese Journal of Biotechnology, 2020, 36(3): 541-548. (in Chinese)
[14]	PENG J S, DING G, MENG S, YI H Y, GONG J M. Enhanced metal tolerance correlates with heterotypic variation in SpMTL, a metallothionein-like protein from the hyperaccumulator Sedum plumbizincicola. Plant, Cell & Environment, 2017, 40(8): 1368-1378.
[15]	张雪洁, 张治远, 张仕泽, 白宁宁, 刘敏, 彭佳师. 甘蓝型油菜植物螯合肽合酶基因BnPCS1a的分离与功能验证. 植物生理学报, 2023, 59(5): 861-868.
	ZHANG X J, ZHANG Z Y, ZHANG S Z, BAI N N, LIU M, PENG J S. Isolation and functional characterization of phytochelatin synthase gene BnPCS1a in Brassica napus. Plant Physiology Journal, 2023, 59(5): 861-868. (in Chinese)
[16]	ZHANG P H, ZHANG X J, TANG T W, HU H L, BAI N N, ZHANG D W, MENG S, PENG J S. Isolation of three metallothionein genes and their roles in mediating cadmium resistance. Agronomy, 2022, 12(12): 2971. doi: 10.3390/agronomy12122971
[17]	GIETZ R D, SCHIESTL R H. Large-scale high-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nature Protocols, 2007, 2: 38-41. doi: 10.1038/nprot.2007.15 pmid: 17401336
[18]	LI H, YANG X Y, ZHANG Y, GAO Z Y, LIANG Y T, CHEN J M, SHI T. Nelumbo genome database, an integrative resource for gene expression and variants of Nelumbo nucifera. Scientific Data, 2021, 8: 38.
[19]	CHEN C J, CHEN H, ZHANG Y, THOMAS H R, FRANK M H, HE Y H, XIA R. TBtools: An integrative toolkit developed for interactive analyses of big biological data. Molecular Plant, 2020, 13(8): 1194-1202. doi: S1674-2052(20)30187-8 pmid: 32585190
[20]	ELBLE R. A simple and efficient procedure for transformation of yeasts. BioTechniques, 1992, 13(1): 18-20. pmid: 1503765
[21]	张培红, 张仕泽, 张治远, 李明悦, 刘奕君, 张雪洁, 白宁宁, 马敏, 彭佳师. 伴矿景天SpHIPP45基因特异介导镉耐受性. 植物生理学报, 2022, 58(7): 1346-1352.
	ZHANG P H, ZHANG S Z, ZHANG Z Y, LI M Y, LIU Y J, ZHANG X J, BAI N N, MA M, PENG J S. SpHIPP45 gene from Sedum plumbizincicola specifically mediates cadmium tolerance. Plant Physiology Journal, 2022, 58(7): 1346-1352. (in Chinese)
[22]	王垚, 胡博, 邓小秋, 蒋佳佳, 杜兰芳, 彭佳师. 伴矿景天SpPCR3基因提高酵母对镉的抗性. 植物生理学报, 2022, 58(7): 1353-1358.
	WANG Y, HU B, DENG X Q, JIANG J J, DU L F, PENG J S. SpPCR3 gene from Sedum plumbizincicola confers cadmium tolerance in yeast. Plant Physiology Journal, 2022, 58(7): 1353-1358. (in Chinese)
[23]	GONDAL A H, HUSSAIN I, IJAZ A B, ZAFAR A, CH B I, ZAFAR H, SOHAILMD, NIAZI H, TOUSEEF M, KHAN A A. Influence of soil pH and microbes on mineral solubility and plant nutrition: A review. Journal of Agriculture and Biological Sciences, 2021, 5: 71-81.
[24]	SUN C L, SUN N, OU Y Q, GONG B, JIN C W, SHI Q H, LIN X Y. Phytomelatonin and plant mineral nutrition. Journal of Experimental Botany, 2022, 73(17): 5903-5917. doi: 10.1093/jxb/erac289
[25]	DUAN Q N, LEE J C, LIU Y S, CHEN H, HU H Y. Distribution of heavy metal pollution in surface soil samples in China: A graphical review. Bulletin of Environmental Contamination and Toxicology, 2016, 97(3): 303-309. doi: 10.1007/s00128-016-1857-9 pmid: 27342589
[26]	HE J, RÖSSNER N, HOANG M T T, ALEJANDRO S, PEITER E. Transport, functions, and interaction of calcium and manganese in plant organellar compartments. Plant Physiology, 2021, 187(4): 1940-1972. doi: 10.1093/plphys/kiab122 pmid: 35235665
[27]	AUNG M S, MASUDA H. How does rice defend against excess iron?: Physiological and molecular mechanisms. Frontiers in Plant Science, 2020, 11: 1102. doi: 10.3389/fpls.2020.01102 pmid: 32849682
[28]	COBBETT C, GOLDSBROUGH P. Phytochelatins and metallothioneins: Roles in heavy metal detoxification and homeostasis. Annual Review of Plant Biology, 2002, 53: 159-182. pmid: 12221971
[29]	LESZCZYSZYN O I, IMAM H T, BLINDAUER C A. Diversity and distribution of plant metallothioneins: A review of structure, properties and functions. Metallomics, 2013, 5(9): 1146-1169. doi: 10.1039/c3mt00072a pmid: 23694960

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed

Comments

Recommended 0

No Suggested Reading articles found!

	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

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

RichHTML

PDF

Abstract

Cite this article

share this article

Figures/Tables 5

References 29

Related Articles 8

Metrics

Comments

Recommended 0

[1]	WANG XiaoBin, YAN Xiang, LI XiuYing. Environmental Safety Risk for Application of Anaerobic Fermentation Biogas Slurry from Livestock Manure in Agricultural Land in China [J]. Scientia Agricultura Sinica, 2021, 54(1): 110-139.
[2]	WANG XianDa, FAN GuoCheng, LI Jian. Optimum Content of Mineral Elements in the Leaves of Duweiwendan Pomelo (Citrus grandis (L.) Osbeck. cv. Duweiwendan) [J]. Scientia Agricultura Sinica, 2020, 53(17): 3576-3586.
[3]	XueFei MAO,JiXin LIU,YongZhong QIAN. Technical Review of Fast Detection of Heavy Metals in Soil [J]. Scientia Agricultura Sinica, 2019, 52(24): 4555-4566.
[4]	WANG XiaoBin,YAN Xiang,LI XiuYing,JI HongJie. Environmental Risks for Application of Phosphogysum in Agricultural Soils in China [J]. Scientia Agricultura Sinica, 2019, 52(2): 293-311.
[5]	WANG XiaoBin, YAN Xiang, LI XiuYing, CAI DianXiong, LEI Mei. Environment Risk for Application of Flue Gas Desulfurization Gypsum in Soils in China [J]. Scientia Agricultura Sinica, 2018, 51(5): 926-939.
[6]	YANG Zhen-xing, ZHOU Huai-ping, XIE Wen-yan, GUAN Chun-lin, CHE Li. Effect of Long-Term Fertilization on Pb, As Contents of Soil and Maize Grain [J]. Scientia Agricultura Sinica, 2015, 48(23): 4827-4836.
[7]	LIU Kai-Lou, LI Da-Ming, HUANG Qing-Hai, YU Xi-Chu, YE Hui-Cai, XU Xiao-Lin, HU Hui-Wen, WANG Sai-Lian. Ecological Benefits and Environmental Carrying Capacities of Red Paddy Field Subjected to Long-Term Pig Manure Amendments [J]. Scientia Agricultura Sinica, 2014, 47(2): 303-313.
[8]	MAO Xue-Fei-1, LIU Ji-Xin-2, WANG Min-1, QIAN Yong-Zhong-1. Applications of Solid Sampling Analytical Technologies of Elements for Quality and Safety of Agri-Products [J]. Scientia Agricultura Sinica, 2013, 46(16): 3432-3443.