期刊：BMC Plant Biology
标题：A repeat region from the Brassica juncea HMA4 gene BjHMA4R is specifically involved in Cd2+ binding in the cytosol under low heavy metal concentrations
0.03 mM CdCl2, 0.3 mM MES, 10 mM glucose, pH 6.0
HMA4 transporters are involved in the transport and binding of divalent heavy metals (Cd, Zn, Pb [lead] and Co [cobalt]). In general, as efflux pumps, HMA4 transporters can increase the heavy metal tolerance of yeast and Escherichia coli. Additional research has shown that the C-terminus of HMA4 contains a heavy metal-binding domain and that heterologous expression of a portion of peptides from this C-terminal domain in yeast provides a high level of Cd tolerance and Cd hyperaccumulation.
We cloned BjHMA4 from Brassica juncea, and quantitative real-time PCR analysis revealed that BjHMA4 was upregulated by Zn and Cd in the roots, stems and leaves. Overexpression of BjHMA4 dramatically affects Zn/Cd distribution in rice and wheat seedlings. Interestingly, BjHMA4 contains a repeat region named BjHMA4R within the C-terminal region; this repeat region is not far from the last transmembrane domain. We further characterized the detailed function of BjHMA4R via yeast and E. coli experiments. Notably, BjHMA4R greatly and specifically improved Cd tolerance, and BjHMA4R transformants both grew on solid media that contained 500 μM CdCl2 and presented improved Cd accumulation (approximately twice that of wild-type [WT] strains). Additionally, visualization via fluorescence microscopy indicated that BjHMA4R clearly localizes in the cytosol of yeast. Overall, these findings suggest that BjHMA4R specifically improves Cd tolerance and Cd accumulation in yeast by specifically binding Cd2+ in the cytosol under low heavy metal concentrations. Moreover, similar results in E. coli experiments corroborate this postulation.
BjHMA4R can specifically bind Cd2+ in the cytosol, thereby substantially and specifically improving Cd tolerance and accumulation under low heavy metal concentrations.
HMA4转运蛋白参与二价重金属（Cd，Zn，Pb [铅]和Co [钴]）的转运和结合。通常，作为外排泵，HMA4转运蛋白可以增加酵母和大肠杆菌的重金属耐受性。另外的研究表明，HMA4的C末端含有重金属结合结构域，并且酵母中来自该C末端结构域的一部分肽的异源表达提供了高水平的Cd耐受性和Cd超积累。
我们从芥菜中克隆了BjHMA4，定量实时PCR分析表明BjHMA4在根，茎和叶中被Zn和Cd上调。BjHMA4的过表达显着影响水稻和小麦幼苗的Zn / Cd分布。有趣的是，BjHMA4在C末端区域内含有一个名为BjHMA4R的重复区域;这个重复区域距离最后一个跨膜结构域不远。我们通过酵母和大肠杆菌实验进一步表征了BjHMA4R的详细功能。值得注意的是，BjHMA4R极大地且特异性地改善了Cd耐受性，并且BjHMA4R转化体均在含有500μMCdCl2的固体培养基上生长并且呈现出改善的Cd积累（大约是野生型[WT]菌株的两倍）。另外，通过荧光显微镜观察表明BjHMA4R清楚地定位于酵母的胞质溶胶中。总体而言，这些发现表明BjHMA4R通过在低重金属浓度下特异性结合胞质溶胶中的Cd2+，特异性地改善了酵母中的Cd耐受性和Cd积累。此外，大肠杆菌实验中的类似结果证实了这种假设。
Fig. 12. Detection of BjHMA4R activity by Cd2+ flux measurements. Transgenic BjHMA4R and control cells were exposed to 30 μM Cd2+ and measurements were taken for 600 s, using a vibrating probe, after the flux became ready. a yeast cells, b E. coli cells. The data are expressed as the mean ± SE of three replicates; * and ** indicate significant levels at 5 and 1% (evaluated by Student’s t test), respectively