乔松根特异启动子PmPgPR10驱动Na+/H+逆转运蛋白提高转基因水稻的耐盐性

摘要

为获得抗盐水稻,将小麦液泡膜Na+/H+逆向转运蛋白基因TaNHX2与乔松(Pinus griffithii)根诱导型特异表达启动子PmPgPR10融合(PmPgPR10∶∶TaNHX2)并转化水稻,以研究PmPgPR10启动子对TaNHX2基因表达的影响以及转基因植物的耐盐性.PCR、Southern和实时PCR试验结果表明,PmPgPR10∶∶TaNHX2基因已通过农杆菌介导法整合进水稻基因组,而且外源基因已在受体细胞中正确表达.在盐胁迫处理时,转PmPgPR10基因植株的耐盐性以及外源基因的表达量显著高于对照植株,说明PmPgPR10启动子可以调控TaNHX2基因在根中特异表达.为了进一步分析转基因植株耐盐机理,比较了日本晴和转基因T3代植株中液泡腺苷三磷酸酶(V-ATPase)和液泡焦磷酸酶(V-PPase)活性,发现转PmPgPR10∶∶TaNHX2基因水稻的V-ATPase和V-PPase酶活性显著高于非转基因对照,说明V-ATPase和V-PPase活性提高在转TaNHX2基因水稻耐盐性过程中发挥重要作用.在盐胁迫处理时,V-ATPase和V-PPase活性只能在转基因植株的根中但不能在叶片中被检测到,进一步说明PmPgPR10启动子在根中特异性表达.因此,PmPgPR10具有在根中增强下游TaNHX2基因表达,并显著提高转基因植株耐盐性的能力.%To obtain the salt-resistant rice varieties) Na' /H' antiporter gene was cloned from Triticum aestivum (TaNHX2) and transformed into rice driven by root-specific promoter of PR10 gene from Pinus griffithii (PmPgPR10::TaNHX2) , which hold the classical promoter features like TATA-box motif. Results of PCR, Southern and real-time PCR showed that the target gene had been integrated into rice genome successfully. The salt-tolerance assay showed that the salt-tolerance of the PmPgPRlO '.'. TaNHX2 transgenic rice lines was significantly increased compared with non-transgenic rice. This result was consistent with the enhancement of the expression level of TaNHXi gene under salt stress and indicated that the PmPgPRlO promoter was able to regulate the root-specific expression of TaNHX2. To further explore the salt-tolerance mechanism of transgenic lines, the activity of V-ATPase and V-PPase were measured and the higher enzyme activity in PmPgPRlO '.'. TaNHX2 transgenic line was attributed to the healthy growth status under salt stress. Furthermore, the activity improvement of the V-ATPase and V-PPase were detected under salinity stress only in root, not in leaf, further indicating the root-specific function of PmPgPRlO promoter. Based on those results, we conclude that the PmPgPRlO promoter from Pinus griffithii has the strong ability to enhance the expression of down-stream TaNHX2 gene in rice to resist the salt damage.