Cloning ThPCS1 gene of Tamarix hispida to improve cadmium tolerance

doi:10.12302/j.issn.1000-2006.202011014

JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2021, Vol. 45 ›› Issue (3): 71-78.doi: 10.12302/j.issn.1000-2006.202011014

Previous Articles Next Articles

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 E-mail:peilongwang66@126.com;gaocaiqiu@nefu.edu.cn

Abstract

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

CLC Number:

Q943.2
S718

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, 2021, 45(3): 71-78.

Figures/Tables 4

Table 1

Fig.1

Fig.2

Fig.3

References 40

[1]	BUCHET J P, LAUWERYS R, ROELS H, et al. Renal effects of cadmium body burden of the general population[J]. Lancet, 1990,336(8717):699-702.DOI: 10.1016/0140-6736(90)92201-r. doi: 10.1016/0140-6736(90)92201-R
[2]	SCHÜTZENDÜBEL A, POLLE A. Plant responses to abiotic stresses:heavy metal-induced oxidative stress and protection by mycorrhization[J]. J Exp Bot, 2002,53(372):1351-1365.DOI: 10.1093/jxb/53.372.1351.
[3]	SANITÀ DI TOPPI L, GABBRIELLI R. Response to cadmium in higher plants[J]. Environ Exp Bot, 1999,41(2):105-130.DOI: 10.1016/S0098-8472(98)00058-6. doi: 10.1016/S0098-8472(98)00058-6
[4]	GHELIS T, DELLIS O, JEANNETTE E, et al. Abscisic acid plasmalemma perception triggers a calcium influx essential for RAB18 gene expression in Arabidopsis thaliana suspension cells[J]. FEBS Lett, 2000,483(1):67-70.DOI: 10.1016/s0014-5793(00)02088-3. doi: 10.1016/S0014-5793(00)02088-3
[5]	CLEMENS S. Evolution and function of phytochelatin synthases[J]. J Plant Physiol, 2006,163(3):319-332.DOI: 10.1016/j.jplph.2005.11.010. doi: 10.1016/j.jplph.2005.11.010
[6]	GRILL E, WINNACKER E L, ZENK M H. Phytochelatins:the principal heavy-metal complexing peptides of higher plants[J]. Science, 1985,230(4726):674-676.DOI: 10.1126/science.230.4726.674. doi: 10.1126/science.230.4726.674
[7]	WANG F, WANG Z, ZHU C. Heteroexpression of the wheat phytochelatin synthase gene (TaPCS1) in rice enhances cadmium sensitivity[J]. Acta Biochim Biophys Sin (Shanghai), 2012,44(10):886-893.DOI: 10.1093/abbs/gms073. doi: 10.1093/abbs/gms073
[8]	LI J C, GUO J B, XU W Z, et al. RNA interference-mediated silencing of phytochelatin synthase gene reduce cadmium accumulation in rice seeds[J]. J Integr Plant Biol, 2007,49(7):1032-1037.DOI: 10.1111/j.1672-9072.2007.00473.x. doi: 10.1111/jipb.2007.49.issue-7
[9]	VATAMANIUK O K, MARI S, LU Y P, et al. AtPCS1,a phytochelatin synthase from Arabidopsis:isolation and in vitro reconstitution[J]. PNAS, 1999,96(12):7110-7115.DOI: 10.1073/pnas.96.12.7110. doi: 10.1073/pnas.96.12.7110
[10]	HEISS S, WACHTER A, BOGS J, et al. Phytochelatin synthase (PCS) protein is induced in Brassica juncea leaves after prolonged Cd exposure[J]. J Exp Bot, 2003,54(389):1833-1839.DOI: 10.1093/jxb/erg205. doi: 10.1093/jxb/erg205
[11]	MEYER C L, PEISKER D, COURBOT M, et al. Isolation and characterization of Arabidopsis halleri and Thlaspi caerulescens phytochelatin synthases[J]. Planta, 2011,234(1):83-95.DOI: 10.1007/s00425-011-1378-z. doi: 10.1007/s00425-011-1378-z
[12]	VATAMANIUK O K, MARI S, LANG A, et al. Phytochelatin synthase,a dipeptidyltransferase that undergoes multisite acylation with γ-glutamylcysteine during catalysis[J]. J Biol Chem, 2004,279(21):22449-22460.DOI: 10.1074/jbc.m313142200. doi: 10.1074/jbc.M313142200
[13]	CLEMENS S, KIM E J, NEUMANN D, et al. Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast[J]. EMBO J, 1999,18(12):3325-3333.DOI: 10.1093/emboj/18.12.3325. doi: 10.1093/emboj/18.12.3325
[14]	CLEMENS S, SCHROEDER J I, DEGENKOLB T. Caenorhabditis elegans expresses a functional phytochelatin synthase[J]. Eur J Biochem, 2001,268(13):3640-3643.DOI: 10.1046/j.1432-1327.2001.02293.x. doi: 10.1046/j.1432-1327.2001.02293.x
[15]	LEE S, KORBAN S S. Transcriptional regulation of Arabidopsis thaliana phytochelatin synthase (AtPCS1) by cadmium during early stages of plant development[J]. Planta, 2002,215(4):689-693.DOI: 10.1007/s00425-002-0821-6. doi: 10.1007/s00425-002-0821-6
[16]	SEMANE B, CUYPERS A, SMEETS K, et al. Cadmium responses in Arabidopsis thaliana:glutathione metabolism and antioxidative defence system[J]. Physiol Plant, 2007,129(3):519-528.DOI: 10.1111/j.1399-3054.2006.00822.x. doi: 10.1111/ppl.2007.129.issue-3
[17]	GASIC K, KORBAN S S. Expression of Arabidopsis phytochelatin synthase in Indian mustard (Brassica juncea) plants enhances tolerance for Cd and Zn[J]. Planta, 2007,225(5):1277-1285.DOI: 10.1007/s00425-006-0421-y. doi: 10.1007/s00425-006-0421-y
[18]	RAMOS J, CLEMENTE M R, NAYA L, et al. Phytochelatin synthases of the model legume Lotus japonicus: a small multigene family with differential response to cadmium and alternatively spliced variants[J]. Plant Physiol, 2007,143(3):1110-1118.DOI: 10.1104/pp.106.090894. doi: 10.1104/pp.106.090894
[19]	LEE B D, HWANG S. Tobacco phytochelatin synthase (NtPCS1) plays important roles in cadmium and arsenic tolerance and in early plant development in tobacco[J]. Plant Biotechnol Rep, 2015,9(3):107-114.DOI: 10.1007/s11816-015-0348-5. doi: 10.1007/s11816-015-0348-5
[20]	张道远, 尹林克, 潘伯荣. 柽柳属植物抗旱性能研究及其应用潜力评价[J]. 中国沙漠, 2003,23(3):252-256.
	ZHANG D Y, YIN L K, PAN B R. Study on drought-resisting mechanism of Tamrix L.and assessing of its potential application[J]. J Desert Res, 2003,23(3):252-256.DOI: 10.3321/j.issn:1000-694X.2003.03.008.
[21]	姜廷波, 陈虹, 唐鑫华, 等. 转金属硫蛋白基因(MT1)烟草抗Cd²⁺胁迫的生理特性分析 [J]. 作物学报, 2007,33(11):1902-1905.
	JIANG T B, CHEN H, TANG X H, et al. Analysis of physiologic characteristics for Cd²⁺ tolerance on transgenic tobacco expressing metallothionein gene (MT1) [J]. Acta Agron Sin, 2007,33(11):1902-1905.DOI: 10.3321/j.issn:0496-3490.2007.11.027.
[22]	姚启超, 高彩球, 姜静, 等. 柽柳eIF1A基因耐重金属CdCl₂胁迫能力分析[J]. 东北林业大学学报, 2010,38(2):4-5,8.
	YAO Q C, GAO C Q, JIANG J, et al. Tolerance of eIF1A gene from Tamarix androssowii under CdCl₂ stress[J]. J Northeast For Univ, 2010,38(2):4-5,8.DOI: 10.3969/j.issn.1000-5382.2010.02.002.
[23]	贺琳, 蒋丽丽, 王玉成. 柽柳eIF1A基因耐重金属CdCl₂胁迫能力分析[J]. 东北林业大学学报, 2011, 39(4):101-104.
	HE L, JIANG L L, WANG Y C. Stress tolerance of thioredoxin peroxidase gene from Tamarix hispida(ThPrx1) inserted into yeast[J]. J Northeast For Univ, 2011,39(4):101-104.DOI: 10.13759/j.cnki.dlxb.2011.04.023.
[24]	GAO C, JIANG B, WANG Y, et al. Overexpression of a heat shock protein (ThHSP18.3) from Tamarix hispida confers stress tolerance to yeast[J]. Mol Biol Rep, 2012,39(4):4889-4897.DOI: 10.1007/s11033-011-1284-2. doi: 10.1007/s11033-011-1284-2
[25]	GAO C, WANG Y, JIANG B, et al. A novel vacuolar membrane H⁺-ATPase c subunit gene (ThVHAc1) from Tamarix hispida confers tolerance to several abiotic stresses in Saccharomyces cerevisiae [J]. Mol Biol Rep, 2011,38(2):957-963.DOI: 10.1007/s11033-010-0189-9. doi: 10.1007/s11033-010-0189-9
[26]	YANG G, WANG C, WANG Y, et al. Overexpression of ThVHAc1 and its potential upstream regulator,ThWRKY7,improved plant tolerance of Cadmium stress[J]. Sci Rep, 2016,6:18752.DOI: 10.1038/srep18752. doi: 10.1038/srep18752
[27]	LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[J]. Methods, 2001,25(4):402-408.DOI: 10.1006/meth.2001.1262. doi: 10.1006/meth.2001.1262
[28]	ZHANG T, ZHAO Y, WANG Y, et al. Comprehensive analysis of MYB gene family and their expressions under abiotic stresses and hormone treatments in Tamarix hispida[J]. Front Plant Sci, 2018,9:1303.DOI: 10.3389/fpls.2018.01303. doi: 10.3389/fpls.2018.01303
[29]	VIVARES D, ARNOUX P, PIGNOL D. A papain-like enzyme at work:native and acyl-enzyme intermediate structures in phytochelatin synjournal[J]. PNAS, 2005,102(52):18848-18853.DOI: 10.1073/pnas.0505833102. doi: 10.1073/pnas.0505833102
[30]	梅磊, 李玲, DAUD M K, 等. 棉花对重金属胁迫的应答反应与抗性机理研究进展[J]. 棉花学报, 2018,30(1):102-110.
	MEI L, LI L, DAUD M K, et al. Advances on response and resistance to heavy metal stress in cotton[J]. Cotton Sci, 2018,30(1):102-110.DOI: 10.11963/1002-7807.mlzsj.20171107.
[31]	DALCORSO G, FARINATI S, MAISTRI S, et al. How plants cope with cadmium:staking all on metabolism and gene expression[J]. J Integr Plant Biol, 2008,50(10):1268-1280.DOI: 10.1111/j.1744-7909.2008.00737.x. doi: 10.1111/jipb.2008.50.issue-10
[32]	TSUJI N, NISHIKORI S, IWABE O, et al. Comparative analysis of the two-step reaction catalyzed by prokaryotic and eukaryotic phytochelatin synthase by an ion-pair liquid chromatography assay[J]. Planta, 2005,222(1):181-191.DOI: 10.1007/s00425-005-1513-9. doi: 10.1007/s00425-005-1513-9
[33]	DAS U, RAHMAN M A, ELA E J, et al. Sulfur triggers glutathione and phytochelatin accumulation causing excess Cd bound to the cell wall of roots in alleviating Cd-toxicity in alfalfa[J]. Chemosphere, 2021,262:128361.DOI: 10.1016/j.chemosphere.2020.128361. doi: 10.1016/j.chemosphere.2020.128361
[34]	COBBETT C S. Phytochelatins and their roles in heavy metal detoxification[J]. Plant Physiol, 2000,123(3):825-832.DOI: 10.1104/pp.123.3.825. doi: 10.1104/pp.123.3.825
[35]	COBBETT C, GOLDSBROUGH P. Phytochelatins and metallothioneins:roles in heavy metal detoxification and homeostasis[J]. Annu Rev Plant Biol, 2002,53:159-182.DOI: 10.1146/annurev.arplant.53.100301.135154. doi: 10.1146/annurev.arplant.53.100301.135154
[36]	SU H X, ZOU T, LIN R Y, et al. Characterization of a phytochelatin synthase gene from Ipomoea pes-caprae involved in cadmium tolerance and accumulation in yeast and plants[J]. Plant Physiol Biochem, 2020,155:743-755.DOI: 10.1016/j.plaphy.2020.08.012. doi: 10.1016/j.plaphy.2020.08.012
[37]	BAI J Y, WANG X, WANG R, et al. Overexpression of three duplicated BnPCS genes enhanced Cd accumulation and translocation in Arabidopsis thaliana mutant cad1-3[J]. Bull Environ Contam Toxicol, 2019,102(1):146-152.DOI: 10.1007/s00128-018-2487-1. doi: 10.1007/s00128-018-2487-1
[38]	SASAKI A, YAMAJI N, MA J F. Overexpression of OsHMA3 enhances Cd tolerance and expression of Zn transporter genes in rice[J]. J Exp Bot, 2014,65(20):6013-6021.DOI: 10.1093/jxb/eru340. doi: 10.1093/jxb/eru340
[39]	ZHANG L X, GAO C, CHEN C, et al. Overexpression of rice OsHMA3 in wheat greatly decreases cadmium accumulation in wheat grains[J]. Environ Sci Technol, 2020,54(16):10100-10108.DOI: 10.1021/acs.est.0c02877. doi: 10.1021/acs.est.0c02877
[40]	YAN J L, WANG P T, WANG P, et al. A loss-of-function allele of OsHMA3 associated with high cadmium accumulation in shoots and grain of Japonicarice cultivars[J]. Plant Cell Environ, 2016,39(9):1941-1954.DOI: 10.1111/pce.12747. doi: 10.1111/pce.v39.9

Related Articles 15

[1]	SUN Jinwei, WANG Shengyan, FAN Diwu, ZHU Yongli. Effects of C, N and P additions on soil respiration in woodland under Cd stress [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2024, 48(1): 140-146.
[2]	HUANG Biyun, ZHUO Renying, QIAO Guirong. Comparative analysis of different types of callus in moso bamboo [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2023, 47(6): 141-149.
[3]	WANG Jianchao, QIU Wenmin, JIN Kangming, LU Zhuchou, HAN Xiaojiao, ZHUO Renying, LIU Xiaoguang, HE Zhengquan. Comprehensive analysis of WRKY gene family in Sedum plumbizincicola responding to cadmium stress [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2023, 47(2): 49-60.
[4]	WANG Yuanyuan, LIU Baichao, JIANG Bo, WANG Danni, GAO Caiqiu. Th2CysPrx gene enhanced abiotic stress tolerances of Saccharomyces cerevisiae [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2022, 46(4): 87-94.
[5]	WANG Zhanjun, WU Ziqi, WANG Zhaoxia, OU Zulan, LI Jie, CAI Qianwen, XU Zhongdong, ZHANG Zhaoliang. A comparative study of the evolution and codon usage bias in WOX gene family of three Camellia sinensis cultivars [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2022, 46(2): 71-80.
[6]	NA Xiaoying, LIU Gang, LIU Guifeng, WANG Xiuwei. Effects of the four genes expressions and photosynthetic parameters on seedlings growth of different birch clones [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2022, 46(2): 88-94.
[7]	LIN Lili, HU Anqi, CHEN Gang, ZHANG Jiyue, CAO Guangqiu, CAO Shijiang. Cloning and expression characteristics of ClWRKY44 gene from Cunninghamia lanceolata [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2022, 46(1): 203-209.
[8]	LI Mengjuan, ZHU Liming, HUO Junnan, ZHANG Jingbo, SHI Jisen, CHENG Tielong. Cloning and expression analyses of NtCBL1,NtCBL2 gene of Nitraria tangutorum [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2021, 45(3): 93-99.
[9]	ZHANG Xin, MA Miaomiao, LYU Wanqiu, LEE Joobin, YANG Jingli. Sequence characteristics and expression pattern analyses of PuZFP103 gene under abiotic stress in Populus ussuriensis [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2021, 45(1): 36-44.
[10]	ZHOU Xiaofeng, MO Zhenghai, SHANG Yangjuan, LUO Min, SU Wenchuan, TAN Pengpeng, HAN Minghui, DENG Qiuju, TIAN Zhaoxia, PENG Fangren. Cloning and expression analysis of the CiDGAT1 from Carya illinoinensis [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2020, 44(4): 55-62.
[11]	WANG Min, XI Dong, MO Zhenghai, CHEN Yu, ZHAO Yuqiang, ZHU Cancan. Cloning, subcellular localization and expression analysis of CiAGL6 gene in pecan [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2020, 44(4): 63-69.
[12]	TIAN Xueyao, ZHOU Jie, WANG Baosong, HE Kaiyue, HE Xudong. Cloning and expression pattern analysis of NAC genes in Salix [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2020, 44(1): 119-124.
[13]	ZHANG Qing, WEI Shuhe, DAI Huiping, JIA Genliang. The alleviating effects of selenium on cadmium-induced toxicity in tea leaves [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2020, 44(1): 200-204.
[14]	WANG Ying, QIU Wenmin, LI He, HE Xuelian, LIU Mingying, HAN Xiaojiao, QU Tongbao, ZHUO Renying. Research on the response of SaWRKY7 gene to cadmium stress in Sedum alfredii Hance [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2019, 43(03): 59-66.
[15]	LIU Zhongyuan, JIANG Bo, LÜ Jiaxin, LI Xinping, GAO Caiqiu. Interacting proteins of Tamarix hispida Th2CysPrx and their expression pattern analysis [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2019, 43(02): 86-92.

Metrics

Comments

www.nldxb.njfu.edu.cn

Edited ＆ Published by Editorial Department of Nanjing Forestry University(Natural Sciences Edition)
Address： No.159 Longpan Road,Nanjing 210037 Jiangsu,P.R.China
E-mail ：xuebao@njfu.edu.cn , xuebao@njfu.com.cn
Telephone +86-25-85428247,85427076
Distributed by China International Book Trading Corporation P.O. Box 399, Beijing ,P.R. China

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

Cloning ThPCS1 gene of Tamarix hispida to improve cadmium tolerance

RichHTML

PDF

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 4

References 40

Related Articles 15

Recommended Articles

Metrics

Comments

Author

Reader

Reviewer