Abstract：Tamba Black Soybean (Glycine max 'Tamba') is an aluminum-resistant plant whose aluminum-resistant mechanism is that their root tips secrete the citric complex aluminum ions. In order to explore its excellent resistance to aluminum genetic resources, this study, with the foundation of transcriptome data about Tamba black soybean's aluminum stress, cloned one of Tamba black soybean's gene of citrate transporter by using reverse transcription PCR (RT-PCR) and this gene was named GmMATE2 (GenBank No. MF497433.1). CDS of GmMATE2 was 1 674 bp in length and GmMATE2 encoded 557 amino acids with a relative molecular mass of 60.38 kD, the isoelectric point of 9.54. GmMATE2, whose instability index was 28.38, was a stable protein. Amino acids sequence of GmMATE2 analysis showed that it contained nine transmembrane structures, one Polysacc_synt_C domain and two family-specific MatE structures of multidrug and toxic compound extrusion (MATE). Evolutionary tree analysis showed that GmMATE2 was closely related to MATE proteins of Medicago truncatula, Trifolium repens and Cicer arietinum. When Tamba black soybean subjected to aluminum stress (pH 4.5, 0.5 mmol/L CaCl2, 100 μmol/L AlCl3), the semi-quantitative RT-PCR analysis showed that GmMATE2 was up-regulated in the root tips, with the highest expression at 24 h and citric acid secretion in the root tips increased with time, which suggested that GmMATE2 was involved in response to the aluminum stress. The plant overexpression vector pBI121-GmMATE2 was constructed and 3 transgenic tobaccos (Nicotiana tabacun) were obtained by agrobacterium-mediated leaf disc method. When 3 transgenic tobaccos subjected to aluminum stress (pH 4.5, 0.5 mmol/L CaCl2, 50 μmol/L AlCl3), qRT-PCR analysis indicated that GmMATE2 was up-regulated in transgenic tobaccos. The citric acid secretion of transgenic tobacco root tips were 2.56~3.79 times that of wild types. The relative elongation of transgenic tobacco roots were 1.91~3.45 times that of wild types. The root tips of transgenic tobaccos were more stained lightly with chrome azurine S than that of the wild types. All the results above indicated that GmMATE2 of Tamba black soybean was a citrate transporter gene, which could improve citric acid secretion and aluminum resistance of transgenic tobaccos. This study provides genetic resources for improving aluminum resistance of aluminum sensitive plants.
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