巨桉金属耐受蛋白EgMTP6的分子特征及功能

doi:10.11707/j.1001-7488.20220510

摘要/Abstract

摘要：

目的: 筛选巨桉金属耐受蛋白基因EgMTP6, 分析其生物信息学特征、亚细胞定位、锌转运功能和表达模式, 揭示丛枝菌根真菌、锌、磷与EgMTP6的相互关系, 探究其在缓解锌胁迫中的作用。方法: 通过生物信息学分析, 得到EgMTP6的核苷酸和氨基酸序列特征。利用农杆菌注射烟草叶表皮细胞分析EgMTP6的亚细胞定位, 酵母突变体互补分析EgMTP6转运锌的功能。利用荧光定量PCR技术, 分析锌(ZnSO₄)、磷(NaH₂PO₄)和异形根孢囊霉对EgMTP6表达模式的影响。结果: 基因预测显示: EgMTP6包含一个长度为1 524bp的开放阅读框, 编码507个氨基酸, 包含12个外显子和11个内含子。结构预测显示: EgMTP6蛋白氨基端位于膜内侧, 羧基端位于膜外侧, 包含5个跨膜螺旋和HD-HD基序。系统进化分析表明: EgMTP6属于Group6, 归为Fe/Zn-CDF类群。多序列比对发现, MTP6含有亚铁离子外排泵结构(Ferrous-iron efflux pump, FieF)。亚细胞定位显示EgMTP6定位在内质网, 酵母突变体互补表明EgMTP6是锌转运蛋白。荧光定量结果说明: EgMTP6的表达不受锌浓度影响, 但受高浓度磷和异形根孢囊霉诱导。结论: EgMTP6属于Fe/Zn-CDF类群, 参与锌的转运, 表达量不受锌浓度影响, 高浓度磷处理和接种异形根孢囊霉时表达量显著提高。EgMTP6在分子特征和功能方面的研究有助于进一步探讨该基因在巨桉菌根耐受锌胁迫时的功能。

关键词: 巨桉, EgMTP6, 锌, 磷, 丛枝菌根真菌

Abstract:

Objective: Eucalyptus can tolerate the heavy metal toxicity tosome extent, in which metal tolerance proteins play an important role. In this study, we screened a metal tolerance protein gene EgMTP6 from Eucalyptus grandis and analyzed its bioinformatics characteristics, subcellular localization, zinc transport function and expression pattern, in order to reveal the relationship between arbuscular mycorrhizal fungi, zinc, phosphorus and EgMTP6, and to explore the role of EgMTP6 in alleviating zinc stress. Method: Through bioinformatics analyses, the gene nucleotide and amino acid sequence characteristics of EgMTP6 were obtained. Subcellular localization of EgMTP6 was analyzed by expressing EgMTP6 inepidermal cells of tobacco leaves, and the zinc transport function was analyzed by yeast mutant complementation. The effects of zinc (ZnSO₄), phosphorus (NaH₂PO₄) and Rhizophagus irregularis on the expression of EgMTP6 were analyzed by qRT-PCR. Result: In this study, bioinformatics analyses displayed that EgMTP6 contained an open reading frame in a length of 1 524 bp, which encoded 507 amino acids, including 12 exons and 11 introns. The tructural prediction showed that the N-terminal of EgMTP6 protein was located in the cytoplasm and the C-terminal was located in the extracellular. The protein contained five transmembrane domains and the HD-HD motif. Phylogenetic analysis showed that EgMTP6 was in Group 6 and belonged to Fe/Zn-CDF. Multiple-sequence alignment showed that MTP6 contained FieF domain (Ferrous-iron efflux pump, FieF). Subcellular localization analysis of tobacco leaves illustrated that EgMTP6 was located in the endoplasmic reticulum. Yeast mutant complementation showed that EgMTP6 was a zinc transporter. Fluorescence quantitative results showed that the expression of EgMTP6 was not affected byzinc, but induced by high concentration of phosphorus, and R. irregularis. Conclusion: EgMTP6 belongs to Fe/Zn-CDFgroup andit is involved in the zinc transport. Its expression is unaffected by zinc concentration, but is induced by high concentration of phosphorus, and R. irregularis. This investigations on molecular characteristics and function of EgMTP6 will be helpful to future studies on the molecular mechanism of heavy metal tolerance of arbuscular mycorrhizal in Eucalyptus grandis.

Key words: Eucalyptus grandis, EgMTP6, Zn, phosphorus, arbuscular mycorrhizal fungi

中图分类号:

S718.46

韩丽娜,谢贤安,陈辉,唐明. 巨桉金属耐受蛋白EgMTP6的分子特征及功能[J]. 林业科学, 2022, 58(5): 93-101.

Lina Han,Xianan Xie,Hui Chen,Ming Tang. Molecular Characteristics and Function of the Metal Tolerant Protein, EgMTP6 in Eucalyptus grandis[J]. Scientia Silvae Sinicae, 2022, 58(5): 93-101.

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参考文献 0

	廖妤婕, 谈宇, 付旺, 等. 丛枝菌根真菌作用下桉树对铅的耐受机制研究. 基因组学与应用生物学, 2014, 33 (3): 633- 639.
	Liao Y J , Tan Y , Fu W , et al. Study on the Pb-tolerance mechanism of eucalyptus under the role of Arbuscular mycorrhizal fungi. Genomics and Applied Biology, 2014, 33 (3): 633- 639.
	宋福强, 孔祥仕, 李季泽, 等. 基于抑制消减杂交技术筛选AM真菌与紫穗槐共生相关基因. 林业科学, 2014, 50 (11): 64- 74.
	Song F Q , Kong X S , Li J Z , et al. Screening the related genes in the AM fungi and Amorpha fruticosa symbiosis with the suppression subtractive hybridization technique. Scientia Silvae Sinicae, 2014, 50 (11): 64- 74.
	唐明, 胡文涛, 陈辉. 2019. 一种分层培养富集丛枝菌根真菌孢子的方法. 发明专利, 中国, ZL201610532342. 7.
	Tang M, Hu W T, Chen H. 2019. A method of layered trap culture of arbuscular mycorrhizal fungi from the field. Invention Patent, China, ZL201610532342. 7. [in Chinese]
	吴平治, 陈键, 栾升, 等. 拟南芥CDF家族基因AtMTP6编码一个锌离子转运蛋白. 生命科学研究, 2006, 10 (3): 244- 247. doi: 10.3969/j.issn.1007-7847.2006.03.011
	Wu P Z , Chen J , Luan S , et al. A cation diffusion facilitator (CDF) gene AtMTP6 encodes a zinc transporter in Arabidopsis. Life Science Research, 2006, 10 (3): 244- 247. doi: 10.3969/j.issn.1007-7847.2006.03.011
	闫明, 钟章成. 铝胁迫对感染丛枝菌根真菌的樟树幼苗生长的影响. 林业科学, 2007, 43 (4): 59- 65.
	Yan M , Zhong Z C . Effects of aluminum stress on growth of Cinnamomum camphora seedlings inoculated with AMF. Scientia Silvae Sinicae, 2007, 43 (4): 59- 65.
	Ai P H , Sun S B , Zhao J N , et al. Two rice phosphate transporters, OsPht1;2 and OsPht1;6 have different functions and kinetic properties in uptake and translocation. Plant Journal, 2009, 57 (5): 798- 809. doi: 10.1111/j.1365-313X.2008.03726.x
	Arpat A B , Magliano P , Wege S , et al. Functional expression of PHO1 to the Golgi and trans-Golgi network and its role in export of inorganic phosphate. Plant Journal, 2012, 71 (3): 479- 491.
	Arrivault S , Senger T , Krämer U . The Arabidopsis metal tolerance protein AtMTP3 maintains metal homeostasis by mediating Zn exclusion from the shoot under Fe deficiency and Zn oversupply. Plant Journal, 2006, 46 (5): 861- 879. doi: 10.1111/j.1365-313X.2006.02746.x
	Barceló J , Poschenrieder C . Plant water relations as affected by heavy metal stress: A review. Journal of Plant Nutrition, 1990, 13 (1): 1- 37. doi: 10.1080/01904169009364057
	Bouain N , Kisko M , Rouached A , et al. Phosphate/zinc interaction analysis in two lettuce varieties reveals contrasting effects on biomass, photosynthesis, and dynamics of Pi transport. BioMed Research International, 2014, 3, 548254.
	Burleigh S H , Kristensen B K , Bechmann I E . A plasma membrane zinc transporter from Medicago truncatula is up-regulated in roots by Zn fertilization, yet down-regulated by arbuscular mycorrhizal colonization. Plant Molecular Biology, 2003, 52 (5): 1077- 1088. doi: 10.1023/A:1025479701246
	Cotrim C A , Jarrott R J , Martin J L , et al. A structural overview of the zinc transporters in the cation diffusion facilitator family. Acta Crystallographica Section D: Structural Biology, 2019, 75, 357- 367. doi: 10.1107/S2059798319003814
	Delhaize E , Gruber B D , Pittman J K , et al. A role for the AtMTP11 gene of Arabidopsis in manganese transport and tolerance. Plant Journal, 2007, 51 (2): 198- 210. doi: 10.1111/j.1365-313X.2007.03138.x
	Ding J , Lu L , Wang C , et al. High level of zinc triggers phosphorus starvation by inhibiting root-to-shoot translocation and preferential distribution of phosphorus in rice plants. Environmental Pollution, 2021, 277 (20): 116778.
	Eroglu S , Meier B , von Wirén N , et al. The vacuolar manganese transporter MTP8 determines tolerance to iron deficiency-induced chlorosis in Arabidopsis. Plant Physiology, 2016, 170 (2): 1030- 1045. doi: 10.1104/pp.15.01194
	Fan X N , Che X R , Lai W Z , et al. The auxin-inducible phosphate transporter AsPT5 mediates phosphate transport and is indispensable for arbuscule formation in Chinese milk vetch at moderately high phosphate supply. Environmental Microbiology, 2020, 22 (6): 2053- 2079. doi: 10.1111/1462-2920.14952
	Fu X Z , Tong Y H , Zhou X , et al. Genome-wide identification of sweet orange (Citrus sinensis) metal tolerance proteins and analysis of their expression patterns under zinc, manganese, copper, and cadmium toxicity. Gene, 2017, 629, 1- 8. doi: 10.1016/j.gene.2017.07.072
	Gustin J L , Zanis M J , Salt D E . Structure and evolution of the plant cation diffusion facilitator family of ion transporters. BMC Ecology and Evolution, 2011, 11, 76.
	Hewitt E J. 1966. Sand and water culture methods used in the study of plant nutrition. Farnham Royal, UK: Commonwealth Agricultural Bureaux.
	Huang L , Chen D Q , Zhang H Q , et al. Funneliformis mosseae enhances root development and Pb phytostabilization in Robinia pseudoacacia in Pb-contaminated soil. Frontiers in Microbiology, 2019, 10, 2591. doi: 10.3389/fmicb.2019.02591
	Kobae Y , Uemura T , Sato M H , et al. Zinc transporter of Arabidopsis thaliana AtMTP1 is localized to vacuolar membranes and implicated in zinc homeostasis. Plant and Cell Physiology, 2004, 45 (12): 1749- 1758. doi: 10.1093/pcp/pci015
	Li L , Kaplan J . Defects in the yeast high affinity iron transport system result in increased metal sensitivity because of the increased expression of transporters with a broad transition metal specificity. Journal of Biological Chemistry, 1998, 273 (35): 22181- 22187. doi: 10.1074/jbc.273.35.22181
	Lu M , Fu D . Structure of the zinc transporter YiiP. Science, 2007, 317 (5845): 1746- 1748. doi: 10.1126/science.1143748
	Ma G , Li J Y , Li J J , et al. OsMTP11, a trans-Golgi network localized transporter, is involved in manganese tolerance in rice. Plant science, 2018, 274, 59- 69. doi: 10.1016/j.plantsci.2018.05.011
	Maiti S K , Rana V . Assessment of heavy metals contamination in reclaimed mine soil and their accumulation and distribution in Eucalyptus hybrid. Bulletin of Environmental Contamination and Toxicology, 2017, 98 (1): 97- 104. doi: 10.1007/s00128-016-1966-5
	Maret W . Molecular aspects of human cellular zinc homeostasis: redox control of zinc potentials and zinc signals. Biometals, 2009, 22 (1): 149- 157. doi: 10.1007/s10534-008-9186-z
	Migocka M , Małas K , Maciaszczyk-Dziubinska E , et al. Cucumber Golgi protein CsMTP5 forms a Zn-transporting heterodimer with high molecular mass protein CsMTP12. Plant Science, 2018, 277, 196- 206. doi: 10.1016/j.plantsci.2018.09.011
	Montanini B , Blaudez D , Jeandroz S , et al. Phylogenetic and functional analysis of the cation diffusion facilitator (CDF) family: improved signature and prediction of substrate specificity. BMC Genomics, 2007, 8 (1): 107. doi: 10.1186/1471-2164-8-107
	Myburg A A , Grattapaglia D , Tuskan G A , et al. The genome of Eucalyptus grandis. Nature, 2014, 510 (7505): 356- 362. doi: 10.1038/nature13308
	Pan Q N , Cui B M , Deng F Y , et al. RTP1 encodes a novel endoplasmic reticulum (ER)-localized protein in Arabidopsis and negatively regulates resistance against biotrophic pathogens. New Phytologist, 2016, 209, 1641- 1654. doi: 10.1111/nph.13707
	Perotto S , Rodda M , Benetti A , et al. Gene expression in mycorrhizal orchid protocorms suggests a friendly plant-fungus relationship. Planta, 2014, 239 (6): 1337- 1349. doi: 10.1007/s00425-014-2062-x
	Shirazi Z , Abedi A , Kordrostami M , et al. Genome-wide identification and characterization of the metal tolerance protein (MTP) family in grape (Vitis Vinifera L.). 3 Biotech, 2019, 9 (5): 199. doi: 10.1007/s13205-019-1728-2
	Sinclair S A , Senger T , Talke I N , et al. Systemic upregulation of MTP2-and HMA2-mediated Zn partitioning to the shoot supplements local Zn deficiency responses. The Plant Cell, 2018, 30 (10): 2463- 2479. doi: 10.1105/tpc.18.00207
	Ton V K , Mandal D , Vahadji C , et al. Functional expression in yeast of the human secretory pathway Ca²⁺/Mn²⁺ ATPase defective in Hailey-Hailey disease. Journal of Biological Chemistry, 2002, 277 (8): 6422- 6427. doi: 10.1074/jbc.M110612200
	Trouvelot A, Kough J L, Gianinazzi-pearson V. 1986. Mesure du taux de mycorhization VA d'un système radiculaire. Recherche de méthode d'estimation ayant une signification fonctionnelle. Physiological and genetical aspects of mycorrhizae: proceedings of the 1st European symposium on mycorrhizae. Paris, France: INRA Press, 217-221.
	Van der Heijden M G A , Bardgett R D , Van Straalen N M . The unseen majority: soil microbes as drivers of plant diversity and productivity in terrestrial ecosystems. Ecology Letters, 2008, 11 (3): 296- 310. doi: 10.1111/j.1461-0248.2007.01139.x
	Xie X A , Lin H , Peng X , et al. Arbuscular mycorrhizal symbiosis requires a phosphate transceptor in the Gigaspora margarita fungal symbiont. Molecular Plant, 2016, 9 (12): 1583- 1608. doi: 10.1016/j.molp.2016.08.011
	Yu X Z , Fan W J , Lin Y J . Analysis of gene expression profiles for metal tolerance protein in rice seedlings exposed to both the toxic hexavalent chromium and trivalent chromium. International Biodeterioration and Biodegradation, 2018, 129, 102- 108.
	Yuan L , Yang S , Liu B , et al. Molecular characterization of a rice metal tolerance protein, OsMTP1. Plant Cell Reports, 2012, 31 (1): 67- 79.
	Zhang W , Chen X X , Liu Y M , et al. Zinc uptake by roots and accumulation in maize plants as affected by phosphorus application and arbuscular mycorrhizal colonization. Plant and Soil, 2017, 413, 59- 71.

引物名称 Primer names	引物序列(5′-3’) Primer sequences (5′-3′)	用途 Usage
EgMTP6-F	ATGGGCTTCAGATTTTGGAAC	片段扩增
EgMTP6-R	TCATTTCTGATTCATT TCTTCTAA	Fragment amplified
EgMTP6-TF	CGGGTTCGAAATCGATGGATCCATGGGCTTCAGATTTTGGAAC	烟草亚细胞定位
EgMTP6-TR	CGCGTCCTAGGCTACGTAGGATCCTCATTTCTGATTCATTTCTTCTAA	Subcellular localization in tobacco
EgMTP6-YF	TTGCGGCCGCATGGGCTTCAGATTTTGGAAC	酵母异源表达
EgMTP6-YR	TTGCGGCCGCTCATTTCTGATTCATTTCTTCTAA	Heterologous expression in yeast
EgMTP6-qF	TGGTTGCTCTTCTTGGTGTAG	实时荧光定量PCR
EgMTP6-qR	CCAGTTTCAAGTCCAGCCTTA	Quantitative Real-time PCR
EgUBI3-qF	TCACCTACGTCTACCAGAAGG	实时荧光定量PCR
EgUBI3-qR	TCCTCGAAAGCTGTAAACATGG	Quantitative Real-time PCR

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