Populus euphratica and Populuspruinosa, sister species in the Turanga Section （Salicaceae）, growing in semi-arid saline areas are known for their high salinity tolerance. In this study, by combining growth level wit...Populus euphratica and Populuspruinosa, sister species in the Turanga Section （Salicaceae）, growing in semi-arid saline areas are known for their high salinity tolerance. In this study, by combining growth level with Na＋ and K＋ contents, the expression level of vacuolar Na＋/H＋ antiporters was investigated for NaCl-induced changes in P. euphratica and t3. pru- inosa calli. Compared to R euphratica, P. pruinosa calli grew well in 200 mM NaC1 stress from 14. to 21 days. Increasing the stressed time caused an increase in Na＋ content concomitant with a decrease in K＋ content in P. euphratica calli, whereas, with the presence of 200 mM NaCI, K＋ content has a less increase in 14 and 21 days than in 7 days which was detected in R pruinosa calli. The transcript levels of six genes coding for NHX-type Na＋/H＋ antiporters suggest that vacuolar NHX1-NHX6 antiporters play important roles in responding to salt stress in R pruinosa. Our data suggest that there exists a higher salt tolerance for P. pruinosa than P. euphratica at the cellular level, Na＋ avoidance or accumulation is observed in cellular compartments, and that expression of NHX antiporters is linked to the accumulator phenotype.展开更多
NHXFS1基因是通过DNA家族改组(DNA family shuffling)技术,以拟南芥、水稻和菊花的液泡膜Na+/H+逆向转运蛋白基因(NHX1)为亲本获得的活性显著增强的新基因。为制备该蛋白的多克隆抗体,对该蛋白进行跨膜结构分析,选取跨膜蛋白的C末端为靶...NHXFS1基因是通过DNA家族改组(DNA family shuffling)技术,以拟南芥、水稻和菊花的液泡膜Na+/H+逆向转运蛋白基因(NHX1)为亲本获得的活性显著增强的新基因。为制备该蛋白的多克隆抗体,对该蛋白进行跨膜结构分析,选取跨膜蛋白的C末端为靶标,并将其克隆到原核表达载体pET32a中,成功构建了原核融合蛋白pET32a-NHXFS1-抗原表达载体,转化大肠杆菌BL21(DE3)并诱导表达。通过镍柱亲和层析纯化该融合表达蛋白,获得了纯度约为80%的纯化蛋白,用于免疫新西兰大白兔制备多克隆抗体。ELISA实验表明,该抗体的效价达到1:128 000,提取表达NHXFS1蛋白的酵母液泡经该多克隆抗体Western blot检测,证明该抗体具有较好的NHXFS1蛋白特异性。NHXFS1多克隆抗体的制备为进一步认识NHXFS1新蛋白结构与功能以及植物耐盐分子生物学的研究奠定了基础。展开更多
基金Financial support for this research was provided by the Program for New Century Excellent Talents in the Ministry of Education in China(NCET-09-0446),NSFC(31370396,30800865) and lzujbky-2012-k22 to YuXia Wu
文摘Populus euphratica and Populuspruinosa, sister species in the Turanga Section （Salicaceae）, growing in semi-arid saline areas are known for their high salinity tolerance. In this study, by combining growth level with Na＋ and K＋ contents, the expression level of vacuolar Na＋/H＋ antiporters was investigated for NaCl-induced changes in P. euphratica and t3. pru- inosa calli. Compared to R euphratica, P. pruinosa calli grew well in 200 mM NaC1 stress from 14. to 21 days. Increasing the stressed time caused an increase in Na＋ content concomitant with a decrease in K＋ content in P. euphratica calli, whereas, with the presence of 200 mM NaCI, K＋ content has a less increase in 14 and 21 days than in 7 days which was detected in R pruinosa calli. The transcript levels of six genes coding for NHX-type Na＋/H＋ antiporters suggest that vacuolar NHX1-NHX6 antiporters play important roles in responding to salt stress in R pruinosa. Our data suggest that there exists a higher salt tolerance for P. pruinosa than P. euphratica at the cellular level, Na＋ avoidance or accumulation is observed in cellular compartments, and that expression of NHX antiporters is linked to the accumulator phenotype.
文摘NHXFS1基因是通过DNA家族改组(DNA family shuffling)技术,以拟南芥、水稻和菊花的液泡膜Na+/H+逆向转运蛋白基因(NHX1)为亲本获得的活性显著增强的新基因。为制备该蛋白的多克隆抗体,对该蛋白进行跨膜结构分析,选取跨膜蛋白的C末端为靶标,并将其克隆到原核表达载体pET32a中,成功构建了原核融合蛋白pET32a-NHXFS1-抗原表达载体,转化大肠杆菌BL21(DE3)并诱导表达。通过镍柱亲和层析纯化该融合表达蛋白,获得了纯度约为80%的纯化蛋白,用于免疫新西兰大白兔制备多克隆抗体。ELISA实验表明,该抗体的效价达到1:128 000,提取表达NHXFS1蛋白的酵母液泡经该多克隆抗体Western blot检测,证明该抗体具有较好的NHXFS1蛋白特异性。NHXFS1多克隆抗体的制备为进一步认识NHXFS1新蛋白结构与功能以及植物耐盐分子生物学的研究奠定了基础。
