Reduction of As Accumulation by Heterologous Expression of Halomonas Peripheral Phosphate-binding Protein Gene PBP in Transgenic Tobaccos

doi:10.11924/j.issn.1000-6850.casb2023-0114

Abstract

Abstract:

In order to explore the biological function of PBP gene in transgenic tobaccos and select the low arsenic (As) accumulation parent tobacco germplasm, taking T₂ generation seeds of transgenic tobacco with PBP gene and wild-type tobacco (Nicotiana tabacum K326) seeds as materials, Real-time PCR was used to study the spatial and temporal expression profile of exogenous PBP in transgenic tobaccos, and the activities of antioxidant enzymes (AOEs) in transgenic tobacco lines under As stress were also determined. AOEs activity showed that SOD and POD activities reached the maximum value of 80.25 U/g and 152.02 U/g in CK plants at 5 d after As stress, which were significantly higher than those in transgenic plants. Also, H₂O₂ and MDA contents in CK plants were significantly higher than those in transgenic plants at the later stage of As stress (3-5 d). Additionally, at 7 d and 14 d after stress, the content of As in transgenic tobacco plants was significantly reduced. Based on the expression profiling of ion transporter genes, the results showed that As stress not only induced significant up-regulation of PBP in transgenic plants, but also caused significant up-regulation of channel protein genes such as HAK1 and PHT4 in transgenic and CK plants, and the average expression levels of HAK1 and PHT4 in CK were 1.5 and 3.75 times of that in transgenic plants. The introduction of exogenous PBP gene can effectively reduce the accumulation of As in transgenic tobaccos.

Key words: Halomonas, periplasmic phosphate-binding protein, PBP gene, transgenic tobacco, low-As accumulation

HUANG Renquan, YUAN Ting, SONG Lala, QIN Lijun. Reduction of As Accumulation by Heterologous Expression of Halomonas Peripheral Phosphate-binding Protein Gene PBP in Transgenic Tobaccos[J]. Chinese Agricultural Science Bulletin, 2024, 40(12): 70-79.

Figures/Tables 8

References 32

[1]	FAROOQ M A, ISLAM F, ALI B, et al. Arsenic toxicity in plants: cellular and molecular mechanisms of its transport and metabolism[J]. Environmental and experimental botany, 2016, 8(4):42-52.
[2]	FINNEGAN P M, CHEN W. Arsenic toxicity: the effects on plant metabolism[J]. Frontiers in physiology, 2012, 3:1-18.
[3]	ZANELLA L, FATTORINI L, BRUNETTI P, et al. Overexpression of AtPCS1 in tobacco increases arsenic and arsenic plus cadmium accumulation and detoxification[J]. Planta, 2016, 243:605-622. doi: 10.1007/s00425-015-2428-8 URL
[4]	ASTA M P, CAMA J, MARTÍNEZ M, et al. Arsenic removal by goethite and jarosite in acidic conditions and its environmental implications[J]. Journal of hazardous materials, 2009, 171(1-3):965-972. doi: 10.1016/j.jhazmat.2009.06.097 pmid: 19628332
[5]	ZHU Y G, YOSHIAGA M, ZHAO F J, et al. Earth abides arsenic biotranformations[J]. Anual review of earth and planetary sciences, 2014, 42:443-467.
[6]	ASHER C J, REAY P F. Arsenic uptake by barley seedlings[J]. Functional plant biology, 1979, 6:459-466. doi: 10.1071/PP9790459 URL
[7]	BIENERT G P, THORSEN M, SCHÜSSLER M D, et al. Asubgroup of plant aquaporins facilitate the bidirectional diffusion of As(OH)₃ and Sb(OH)₃ across membranes[J]. BMC molecular and cell biology, 2008, 6:26.
[8]	常思敏. 砷对烤烟碳氮代谢的影响及铁和黄腐酸的减害效应[D]. 郑州: 河南农业大学, 2006.
[9]	ABDUL K S M, JAYASINGHE S S, CHANDANA E P S, et al. Arsenic and human health effects: a review[J]. Environmental toxicology and pharmacology, 2015, 40(3):828-846. doi: 10.1016/j.etap.2015.09.016 pmid: 26476885
[10]	朱学书, 尹努寻, 杨秀丽. 贵州矿产资源开发利用现状分析[J]. 贵州地质, 2012, 3:220-224.
[11]	陈卫平, 杨阳, 谢天, 等. 中国农田土壤重金属污染防治挑战与对策[J]. 土壤学报, 2018, 55(2):261-272.
[12]	张清海, 林绍霞, 林昌虎. 贵州农业非点源污染土壤重金属含量特征及区域差异性研究[J]. 中国环境监测, 2011, 27(1):88-91.
[13]	魏成熙, 谭丽娟, 常德荣. 贵阳市主要土壤重金属状况研究[J]. 中国土壤与肥料, 2006(4):20-24.
[14]	莫昌琍, 肖超. 贵州独山锑矿区农用土壤中砷污染的研究[J]. 贵阳学院学报(自然科学版), 2015, 10(2):43-46.
[15]	HU H, XIONG L. Genetic engineering and breeding of drought-resistant crops[J]. Annual review of plant biology, 2014, 65:715-741. doi: 10.1146/annurev-arplant-050213-040000 pmid: 24313844
[16]	曹婷婷. 假单胞菌砷甲基转移酶功能研究及转基因根瘤菌的构建和应用[D]. 南京: 南京农业大学, 2015.
[17]	ELIAS M, WELLNER A, GOLDIN-AZULAY K, et al. The molecular basis of phosphate discrimination in arsenate-rich environments[J]. Nature, 2012, 491(7422):134-137. doi: 10.1038/nature11517
[18]	CHAUDHARY K, AGARWAL S, KHAN S. Role of phytochelatins (PCs), metallothioneins (MTs), and heavy metal ATPase (HMA) genes in heavy metal tolerance[J]. (Part of the Fungal Biology) Mycoremediation and environmental sustainability, 2018:39-60.
[19]	WANG P, YIN N, CAI X, et al. Assessment of arsenic distribution, bioaccessibility and speciation in rice utilizing continuous extraction and in vitro digestion[J]. Food chemistry, 2021, 346:128969. doi: 10.1016/j.foodchem.2020.128969 URL
[20]	帅鸿, 欧阳迪庆, 陈玉成. 基于文献计量的我国农地重金属研究热点分析[J]. 农业环境科学学报, 2018, 37(4):688-695.
[21]	徐松, 唐芬. 采矿区土壤环境污染及其修复研究[J]. 科技创新与应用, 2018(21):81-82.
[22]	ARIF N, YADAV V, SINGH S, et al. Influence of high and low levels of plant-beneficial heavy metal ions on plant growth and development[J]. Frontiers in environmental science, 2016, 4:1-11.
[23]	王科. 不同污染条件下烟株对几种重金属的吸收分配规律及前茬作物对其影响的研究[D]. 长沙: 湖南农业大学, 2012.
[24]	尹文珂, 程金凤, 肖婉露, 等. 四尾栅藻对重金属镉胁迫的响应[J]. 农业环境科学学报, 2015, 34(4):633-638.
[25]	LI Y, ZHANG S, JIANG W, et al. Cadmium accumulation, activities of antioxidant enzymes, and malondialdehyde (MDA) content in Pistia stratiotes L.[J]. Environmental science and pollution research, 2013, 20:1117-1123. doi: 10.1007/s11356-012-1054-2 URL
[26]	GAJEWSKA E, SKŁODOWSKA M, SŁABA M, et al. Effect of nickel on antioxidative enzyme activities, proline and chlorophyll contents in wheat shoots[J]. Biologia plantarum, 2006, 50:653-659. doi: 10.1007/s10535-006-0102-5 URL
[27]	杨居荣, 贺建群, 张国祥, 等. 不同耐性作物中几种酶活性对Cd胁迫的反应[J]. 中国环境科学, 1996(2):113-117.
[28]	陈洋尔. 苔藓植物中重金属含量的测定以及重金属胁迫对苔藓生理指标的影响[D]. 成都: 四川大学, 2007.
[29]	吕冬梅, 朱广龙, 王玥, 等. 苗期重金属胁迫下蓖麻生长、生理和重金属积累效应[J]. 作物学报, 2021, 47(4):728-737. doi: 10.3724/SP.J.1006.2021.04146
[30]	封鹏雯. 甜菜碱提高烟草镉胁迫抗性机理的研究[D]. 济南: 山东农业大学, 2018.
[31]	LEBLANC M S, MCKINNEY E C, MEAGHER R B, et al. Hijacking membrane transporters for arsenic phytoextraction[J]. Journal of biotechnology, 2013, 163(1):1-9. doi: 10.1016/j.jbiotec.2012.10.013 pmid: 23108027
[32]	MENDOZA-CÓZATL D G, JOBE T O, HAUSER F, et al. Long-distance transport, vacuolar sequestration, tolerance, and transcriptional responses induced by cadmium and arsenic[J]. Current opinion in plant biology, 2011, 14(5):554-562. doi: 10.1016/j.pbi.2011.07.004 URL

基因	GenBank登录号	引物序列(5’→3’)
Actin	AB158612	GATCTTGCTGGTCGTGATCT ACTTCCGGACATCTGAACCT
PBP	CP016490.1	AATTCAAGGAGCTGGATCTGG CTCATAGCTCTAGACATTGGTCC

基因	GenBank登录号	引物序列(5’→3’)
Actin	AB158612	GATCTTGCTGGTCGTGATCT ACTTCCGGACATCTGAACCT
PBP	CP016490.1	AATTCAAGGAGCTGGATCTGG CTCATAGCTCTAGACATTGGTCC

基因	GenBank登录号	引物序列(5’→3’)
Actin	AB158612	GATCTTGCTGGTCGTGATCT ACTTCCGGACATCTGAACCT
PBP	CP016490.1	AATTCAAGGAGCTGGATCTGG CTCATAGCTCTAGACATTGGTCC
HAK1	DQ841950	ATCCACACCGAGCTTGTTTCAGGA TGGGTCCAATTCTTCCCACCAAGA
HMA	HF675181.1	TTCAAAAGCAGCAACGTCCG GCATCGCGGTTTTGATACCC
PHT4	AB042956.1	CATGGGAATTCGTTCGTCGC TCTTTATGCCGATACCCGCC
ATQ1	KF938567.1	CAAGTGGCATCGGTTTGCAA CAATCTTGAGGTCTCCCGGG

基因	GenBank登录号	引物序列(5’→3’)
Actin	AB158612	GATCTTGCTGGTCGTGATCT ACTTCCGGACATCTGAACCT
PBP	CP016490.1	AATTCAAGGAGCTGGATCTGG CTCATAGCTCTAGACATTGGTCC
HAK1	DQ841950	ATCCACACCGAGCTTGTTTCAGGA TGGGTCCAATTCTTCCCACCAAGA
HMA	HF675181.1	TTCAAAAGCAGCAACGTCCG GCATCGCGGTTTTGATACCC
PHT4	AB042956.1	CATGGGAATTCGTTCGTCGC TCTTTATGCCGATACCCGCC
ATQ1	KF938567.1	CAAGTGGCATCGGTTTGCAA CAATCTTGAGGTCTCCCGGG

株系	胁迫处理前		胁迫处理7 d		胁迫处理14 d
株系	As含量/(mg/kg)	P含量/(mg/kg)	As含量/(mg/kg)	P含量/(mg/kg)	As含量/(mg/kg)	P含量/(mg/kg)
CK	1.102±0.15a	0.158±0.01c	4.562±0.86a	0.152±0.01b	9.508±1.02a	0.165±0.01c
L3	1.011±0.12a	0.116±0.02d	3.215±0.77b	0.127±0.03c	5.071±0.87b	0.139±0.01d
L4	0.975±0.05a	0.187±0.01a	2.226±0.82c	0.204±0.01a	4.534±0.94c	0.239±0.03a
L5	1.004±0.14a	0.154±0.03c	3.031±0.88b	0.162±0.01b	4.823±0.87c	0.180±0.01b
L10	0.885±0.02a	0.175±0.02b	1.534±0.04d	0.192±0.02a	1.647±0.06f	0.228±0.02a
L13	0.972±0.01a	0.173±0.01b	1.305±0.13e	0.206±0.02a	2.095±0.23e	0.195±0.01a
L15	1.031±0.16a	0.152±0.02c	2.217±0.86c	0.158±0.02b	4.223±0.59c	0.174±0.02b
L21	0.875±0.04a	0.122±0.01d	1.223±0.08e	0.132±0.01c	3.524±0.44d	0.154±0.01d
L25	0.952±0.01a	0.141±0.01c	1.676±0.17d	0.167±0.03b	1.821±0.78e	0.182±0.02b