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國科大丨BjHMA4R通過結(jié)合胞質(zhì)Cd2+促植物耐鎘
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期刊：BMC Plant Biology
主題：BjHMA4R通過結(jié)合胞質(zhì)Cd2+促植物耐鎘
標(biāo)題：A repeat region from the Brassica juncea HMA4 gene BjHMA4R is specifically involved in Cd2+ binding in the cytosol under low heavy metal concentrations
影響因子：3.930
檢測指標(biāo)： Cd2+流速
檢測部位：大腸桿菌細(xì)胞，酵母細(xì)胞
Cd2+流實(shí)驗(yàn)處理方法：
大腸桿菌，酵母細(xì)胞，30μMCdCl2脅迫
Cd2+流實(shí)驗(yàn)測試液成份：
文獻(xiàn)無
推薦測試液成份：
0.03 mM CdCl2, 0.3 mM MES, 10 mM glucose, pH 6.0
通訊作者：中國科學(xué)院大學(xué)生命科學(xué)學(xué)院柴團(tuán)耀、梁爽
英文摘要
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.
中文摘要（谷歌機(jī)翻）
HMA4轉(zhuǎn)運(yùn)蛋白參與二價(jià)重金屬（Cd，Zn，Pb [鉛]和Co [鈷]）的轉(zhuǎn)運(yùn)和結(jié)合。通常，作為外排泵，HMA4轉(zhuǎn)運(yùn)蛋白可以增加酵母和大腸桿菌的重金屬耐受性。另外的研究表明，HMA4的C末端含有重金屬結(jié)合結(jié)構(gòu)域，并且酵母中來自該C末端結(jié)構(gòu)域的一部分肽的異源表達(dá)提供了高水平的Cd耐受性和Cd超積累。
我們從芥菜中克隆了BjHMA4，定量實(shí)時(shí)PCR分析表明BjHMA4在根，莖和葉中被Zn和Cd上調(diào)。BjHMA4的過表達(dá)顯著影響水稻和小麥幼苗的Zn / Cd分布。有趣的是，BjHMA4在C末端區(qū)域內(nèi)含有一個(gè)名為BjHMA4R的重復(fù)區(qū)域;這個(gè)重復(fù)區(qū)域距離后一個(gè)跨膜結(jié)構(gòu)域不遠(yuǎn)。我們通過酵母和大腸桿菌實(shí)驗(yàn)進(jìn)一步表征了BjHMA4R的詳細(xì)功能。值得注意的是，BjHMA4R極大地且特異性地改善了Cd耐受性，并且BjHMA4R轉(zhuǎn)化體均在含有500μMCdCl2的固體培養(yǎng)基上生長并且呈現(xiàn)出改善的Cd積累（大約是野生型[WT]菌株的兩倍）。另外，通過熒光顯微鏡觀察表明BjHMA4R清楚地定位于酵母的胞質(zhì)溶膠中。總體而言，這些發(fā)現(xiàn)表明BjHMA4R通過在低重金屬濃度下特異性結(jié)合胞質(zhì)溶膠中的Cd2+，特異性地改善了酵母中的Cd耐受性和Cd積累。此外，大腸桿菌實(shí)驗(yàn)中的類似結(jié)果證實(shí)了這種假設(shè)。
BjHMA4R可特異性結(jié)合胞質(zhì)溶膠中的Cd2+，從而在低重金屬濃度下基本上和特異性地改善Cd耐受性和積累。
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