Scientia Agricultura Sinica ›› 2018, Vol. 51 ›› Issue (11): 2038-2048.doi: 10.3864/j.issn.0578-1752.2018.11.002

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning and Expression Analyses of the mate Gene in Buckwheat

CHANG XueLing1,2, ZHANG ZongWen2,3, LI YanQin1, GAO Jia2   

  1. 1Institute of Biotechnology of Shanxi University, Taiyuan 030006; 2Institute of Crop Science, Chinese Academy of Agricultural Science, Beijing 100081; 3Biodiversity International-China, Beijing 100081
  • Received:2017-12-05 Online:2018-06-01 Published:2018-06-01

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

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