荞麦mate的克隆及表达分析

doi:10.3864/j.issn.0578-1752.2018.11.002

摘要/Abstract

摘要： 【目的】多药物和有毒化合物外排家族（multidrug and toxic compound extrusion family，MATE）是生物中5类解毒输出转运蛋白家族之一。作为植物中最大的转运蛋白家族之一，其主要功能为次生代谢物的积累、重金属转运和激素信号转导。通过分析荞麦mate的克隆与表达为MATE型转运蛋白成员在荞麦中的功能研究提供基础。【方法】通过3′和5′RACE克隆获得荞麦mate全长cDNA序列；利用在线网上工具预测MATE蛋白的分子量、等电点、二级结构及跨膜结构域；选取MEGA软件中的NJ法构建荞麦MATE蛋白的系统发育树以初步推测MATE蛋白在荞麦中的作用方式及功能；荧光定量试验测定mate在荞麦不同组织及不同发育阶段种子中的相对表达量，并与原花青素含量测定结合进一步验证MATE转运蛋白在荞麦中的功能。【结果】通过RACE克隆获得2条荞麦mate全长cDNA，分别命名为Fett12 （GenBank accession No. MG515589）和Femate3 （GenBank accession No. MG515590）。通过ExPASy中的protparam在线分析得到Fett12编码一个含有492个氨基酸残基的蛋白，其分子量为53.81 kD，等电点为6.75；Femate3编码一个含有516个氨基酸残基的蛋白，分子量为56.12 kD，等电点为6.52。通过构建系统进化树显示FeTT12属于原花青素MATE转运蛋白；FeMATE3属于柠檬酸盐MATE转运蛋白。将FeMATE3与其他物种中转运柠檬酸盐的MATE型蛋白氨基酸多序列比对及同源分析，发现其与Al离子胁迫相关的荞麦MATE蛋白FeMATE2的一致性达到96.33%；同时将FeTT12氨基酸序列与其他植物TT12编码的氨基酸序列进行比对和同源分析，得到荞麦FeTT12与其他物种的TT12蛋白的氨基酸序列具有极高的同源性，其中与MnTT12蛋白同源性最高，为77.3%；与拟南芥TT12蛋白同源性最低，为41.5%。进一步研究Fett12的表达特异性与原花青素累积之间的相关性，得到Fett12的表达具有明显的时空特异性，且在荞麦叶中的表达量最高。原花青素的含量测定中发现原花青素作为次级代谢产物，其分布具有明显的组织器官差异，且在荞麦花中的含量明显较高。在荞麦不同发育阶段的种子中，Fett12的表达量随着种子不断成熟而增高，PA的含量逐渐下降。【结论】在荞麦中成功克隆获得2条mate序列——Fett12和Femate3。其中，Fett12与荞麦中原花青素的转运和累积相关，Femate3可能与荞麦中Al离子胁迫相关。

关键词: MATE, 荞麦, 原花青素, RACE

Abstract: 【Objective】MATE (multidrug and toxic compound extrusion family) is one of the five detoxification output transporter families in organisms. MATEs as one of the largest transporter families in plants，its physiological functions reported to date are xenobiotic efflux, accumulation of secondary metabolites, the translocation and detoxification of metal ions, and plant hormone signaling transduction. The cloning and expression analyses of mate gene in Fagopyrum esculentum provide the basis for the functional study of MATE transporters in buckwheat species. 【Method】The full-length cDNA sequence of mate gene from buckwheat was obtained by 3’ and 5’ RACE cloning. The molecular weight, isoelectric point, secondary structure and transmembrane domain of MATE protein were analyzed using online analysis program. The nonrooted neighbor-joining tree of buckwheat MATE and other species MATE was generated by MEGA program to preliminarily determined the molecular structural model and the function of MATE protein in buckwheat. Quantitative RT-PCR assay were applied to determine the relative expression levels of mate in different tissues spanning developmental stages. In combination with the determination of proanthocyanidins content to investigation the physiological function of MATE transporters in buckwheat species. 【Result】Two full length cDNAs of mate genes from buckwheat were obtained by 3′ and 5′RACE cloning, named Fett12 (GenBank accession No. MG515589) and Femate3 (GenBank accession No. MG515590). Fett12 encodes a protein containing 492 amino acid residues with a molecular weight of 53.81 kD and isoelectric point of 6.75. Femate3 encodes a putative protein of 516 amino acid residues with a molecular weight of 56.12 kD and isoelectric point was 6.52. The phylogenetic tree showed that FeTT12 was belong to the proanthocyanidin MATE transporter and FeMATE3 was belong to the citrate MATE transporter. Multiple alignment and homology analysis revealed that the FeMATE3 had 96.33% identity to FeMATE2, and the FeTT12 was highly homologous to other TT12 proteins. The FeTT12 protein had the highest identity (77.3%) to MnTT12 and had the lowest identity (41.5%) to AtTT12. Furthermore, the gene expression patterns of Fett12 were correlated with the accumulation of proanthocyanidins. Results showed that the expression of Fett12 had obvious spatio-temporal specificity and the transcript of Fett12 was most abundant in buckwheat leaves. As for the accumulation pattern of PAs, the results showed significantly higher accumulation in buckwheat flower. In buckwheat seeds during the maturation, the expression levels of Fett12 increased with the development stages of seeds, and the content of PAs decreased gradually.【Conclusion】 We obtained two mate genes——Fett12 and Femate3. FeTT12 may be involved in the translocation and accumulation of procyanidins in common buckwheat. FeMATE3 may be involved in Al tolerance.

Key words: MATE, buckwheat, proanthocyanidins, RACE

常雪玲，张宗文，李艳琴，高佳. 荞麦mate的克隆及表达分析[J]. 中国农业科学, 2018, 51(11): 2038-2048.

CHANG XueLing, ZHANG ZongWen, LI YanQin, GAO Jia. Cloning and Expression Analyses of the mate Gene in Buckwheat[J]. Scientia Agricultura Sinica, 2018, 51(11): 2038-2048.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2018.11.002

https://www.chinaagrisci.com/CN/Y2018/V51/I11/2038

参考文献

[1] Omote H, Hiasa M, Matsumoto T, Otsuka M, Moriyama Y. The MATE proteins as fundamental transporters of metabolic and xenobiotic organic cations. Trends in Pharmacological Sciences, 2006, 27(11): 587-593.

[2] He F, Pan Q H, Shi Y, Duan C Q. Biosynthesis and genetic regulation of proanthocyanidins in plants. Molecules, 2008, 13(10): 2674-2703.

[3] Koes R, Verweij W, Quattrocchio F. Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. Trends in Plant Science, 2005, 10(5): 236-242.

[4] Feeny P. Seasonal changes in Oak leaf tannins and nutrients as a cause of spring feeding by winter moth caterpillars. Ecology, 1970, 51(4): 565-581.

[5] Bais H P, Vepachedu R, Gilroy S, Callaway R M, Vivanco J M. Allelopathy and exotic plant invasion: from molecules and genes to species interactions. Science, 2003, 301(5638): 1377-1380.

[6] rior R L, Gu L. Occurrence and biological significant of proanthocyanidins in the american diet. Phytochemistry, 2005, 66(18): 2264-2280.

[7] Zhao M, Yang B, Wang J, Liu Y, Yu L, Jiang Y. Immunomodulatory and anticancer activities of flavonoids extracted from litchi (Litchi chinensis Sonn) pericarp. International Immunopharmacology, 2007, 7(2): 162-166.

[8] Rao L J, Yada H, Ono H, Ohnishikameyama M, Yoshida M. Occurrence of antioxidant and radical scavenging proanthocyanidins from the Indian minor spice nagkesar (Mammea longifolia planch and triana syn). Bioorganic & Medicinal Chemistry, 2004, 12(1): 31-36.

[9] Subarnas A, Wagner H. Analgesic and anti-inflammatory activity of the proanthocyanidin shellegueain A from Polypodium feei METT. Phytomedicine International Journal of Phytotherapy & Phytopharmacology, 2000, 7(5): 401-405.

[10] Sato M, Maulik G, Ray P S, Bagchi D, Das D K. Cardioprotective effects of grape seed proanthocyanidin against ischemic reperfusion injury. Journal of Molecular & Cellular Cardiology, 1999, 31(6): 1289-1297.

[11] Sano T, Oda E, Yamashita T, Naemura A, Ijiri Y, Yamakoshi J, Yamamoto J. Anti-thrombotic effect of proanthocyanidin, a purified ingredient of grape seed. Thrombosis Research, 2005, 115(1/2): 115-121.

[12] Brown M H, Paulsen I T, Skurray R A. The multidrug efflux protein NorM is a prototype of a new family of transporters. Molecular Microbiology, 1999, 31(1): 394-395.

[13] Morita M, Shitan N, Sawada K, Van Montagu M C E, Inze D, Rischer H, Goossens A, Oksman-Caldentey K M, Moriyama Y, Yazaki K. NorM, a Putative Multidrug Efflux Protein, of Vibrio parahaemolyticus and Its Homolog in Escherichia coli. Antimicrobial Agents & Chemotherapy, 1998, 42(7): 1778-1782.

[14] Li L, He Z, Pandey G K, Tsuchiya T, Luanet S. Functional cloning and characterization of a plant efflux carrier for multidrug and heavy metal detoxification. Journal of Biological Chemistry, 2002, 277(7): 5360-5368.

[15] 沈知临, 许磊, 陈文, 吴楠, 蔡永萍, 林毅, 高俊山. 亚洲棉和雷蒙德氏棉MATE基因家族生物信息学及其同源基因在陆地棉中的表达分析. 棉花学报, 2016, 562(3): 215-226.

Shen Z L, Xu L, Chen W, Wu N, Cai Y P, Lin Y, Gao J S. Bioinformatic analysis of the multidrug and toxic compound extrusion gene family in Gossypium arboreum and Gossypium raimondii, and expression of orthologs in Gossypium hirsutum. Cotton Science,2016, 562(3): 215-226. (in Chinese)

[16] Takanashi K, Shitan N, Yazaki K. The multidrug and toxic compound extrusion (MATE) family in plants. Plant Tissue Culture Letters, 2014, 31(5): 417-430.

[17] Marinova K, Pourcel L, Weder B, Schwarz M, Barron D, Routaboul J M, Debeaujon I, Klein M. The Arabidopsis MATE transporter TT12 acts as a vacuolar flavonoid/H⁺-antiporter active in proanthocyanidin-accumulating cells of the seed coat. The Plant Cell, 2007, 19(6): 2023-2038.

[18] Sivaguru M, Liu J, Kochian L V. Targeted expression of SbMATE in the root distal transition zone is responsible for sorghum aluminum resistance. Plant Journal for Cell & Molecular Biology, 2013, 76(2): 297-307.

[19] Magalhaes J V, Liu J, Guimarães C T, Lana U G, Alves V M, Wang Y H, Schaffert R E, Hoekenga O A,Piñeros M A, Shaff J E, Klein P E, Carneiro N P, Coelho C M, Trick H N, Kochian L V. A gene in the multidrug and toxic compound extrusion (MATE) family confers aluminum tolerance in sorghum. Nature Genetics, 2007, 39(9): 1156-1161.

[20] Furukawa J, Yamaji N, Wang H, Mitani N, Murata Y, Sato K, Katsuhara M, Takeda K, Ma J F. An aluminum- activated citrate transporter in barley. Plant & Cell Physiology, 2007, 48(8): 1081-1091.

[21] Durrett T P, Gassmann W, Rogers E E. The FRD3-mediated efflux of citrate into the root vasculature is necessary for efficient iron translocation. Plant Physiology, 2007, 144(1): 197-205.

[22] Gao J, Wang T T, Liu M X, Liu J, Zhang Z W. Transcriptome analysis of filling stage seeds among three buckwheat species with emphasis on rutin accumulation. Plos One, 2017, 12(12):e0189672.

[23] Lei G J, Yokosho K, Yamaji N, Ma J F. Two MATE transporters with different subcellular localization are involved in Al tolerance in buckwheat. Plant & Cell Physiology, 2017, 58(12): 2179-2189.

[24] Gao J S, Wu N, Shen Z L, Lv K, Qian S H, Guo N, Sun X, Cai Y P, Lin Y. Molecular cloning, expression analysis and subcellular localization of a Transparent Testa 12 ortholog in brown cotton (Gossypium hirsutum L.). Gene, 2016, 576(2): 763-769.

[25] Wang L, Bei X, Gao J, Li Y, Hu Y. The similar and different evolutionary trends of MATE family occurred between rice and Arabidopsis thaliana. Bmc Plant Biology, 2016, 16(1): 207-225.

[26] Lepiniec L, Debeaujon I, Routaboul J M, Baudry A, Pourcel L, Nesi N, Caboche M. Genetics and biochemistry of seed flavonoids. Annual Review of Plant Biology, 2006, 57(1): 405-430.

[27] Zhao J, Dixon R A. MATE transporters facilitate vacuolar uptake of epicatechin 3'-O-glucoside for proanthocyanidin biosynthesis in Medicago truncatula and Arabidopsis. The Plant Cell, 2009, 21(8): 2323-2340.

[28] Frank S, Keck M, Sagasser M, Niehaus K, Weisshaar B, Stracke R. Two differentially expressed MATE factor genes from apple complement the Arabidopsis transparent testa12 mutant. Plant Biology, 2011, 13(1): 42-50.

[29] He N, Zhang C, Qi X, Zhao S, Tao Y, Yang G, Lee T H, Wang X, Cai Q, Li D, Lu M, Liao S, Luo G, He R, Tan X, Xu Y, Li T, Zhao A, Jia L, Fu Q, Zeng Q, Gao C, Ma B, Liang J, Wang X, Shang J, Song P, Wu H, Fan L, Wang Q, Shuai Q, J Zhu J, Wei C, Keyan ZS, Jin D, Wang J, Liu T, Yu M, Tang C, Wang Z, Dai F, Chen J, Y Liu Y, Zhao S, Lin T, Zhang S, J Wang J, Wang J, Yang H, Yang G, Wang J, Paterson A H, Xia Q, Ji D, Xiang Z. Draft genome sequence of the mulberry tree Morus notabilis. Nature Communications, 2013, 4(9): 2445-2454.

[30] Pérezdíaz R, Ryngajllo M, Pérezdíaz J, Peña-Cortés H, Casaretto J A, González-Villanueva E, Ruiz-Lara S. VvMATE1 and VvMATE2 encode putative proanthocyanidin transporters expressed during berry development in Vitis vinifera L. Plant Cell Reports, 2014, 33(7): 1147-1159.

[31] Zielińska D, Turemko M, Kwiatkowski J, Zieliński H. Evaluation of flavonoid contents and antioxidant capacity of the aerial parts of common and tartary buckwheat plants. Molecules, 2012, 17(8): 9668-9682.

[32] Zheng S J, Ma J F, Matsumoto H. Continuous secretion of organic acids is related to aluminium resistance during relatively long‐term exposure to aluminium stress. Physiologia Plantarum, 2010, 103(2): 209-214.

[33] Ma J F, Ryan P R, Delhaize E. Aluminium tolerance in plants and the complexing role of organic acids. Trends in Plant Science, 2001, 6(6): 273-278.

[34] Ma J. Detoxifying aluminum with buckwheat. Nature, 1997, 390(6660): 569-570.