谷胱甘肽转移酶基因过量表达能加速盐胁迫下转基因拟南芥的生长

Overexpression of GST Gene Accelerates the Growth of Transgenic Arabidopsis Under Salt Stress

盐地碱蓬谷胱甘肽转移酶基因(glutathione stransferase gene,GST)克隆到植物表达载体pROKⅡ35S启动子的下游,通过农杆菌介导,利用花絮浸泡法转化拟南芥。转化子在含有卡那霉素的培养基上经过筛选以后,将初步验证为阳性的转基因植株通过PCR-Southern进一步证实。经过选育,筛选并分离到卡那霉素的抗性并且遗传稳定的T_3代纯合子转基因拟南芥品系。通过Northern杂交证实外源基因在转基因拟南芥中表达。在盐胁迫条件下,通过测量转基因植株(GT)和野生型植株(WT)的生物量和谷胱甘肽(氧化型:GSSG;还原型:GSH)发现:转基因植株的生物量较野生型有一定程度的提高;GSSG含量在转基因品系中比野生型的含量明显高。因此,过量表达GST能够提高转基因植株在盐胁迫条件下的生长,而且这很可能是由于还原型谷胱甘肽被氧化的结果。

The Suaeda salsa glutathione s-trans-ferase gene (GST) was inserted downstream of the 35S promoter in the plant expression vector pROK Ⅱ(Fig. 1) and then was introduced into Arabidopsis thaliana by Agrobacterium tumefaciens through floral dip method. Transformants were selected for their ability to grow on medium containing kanamycin (Figs.2, 3). The fact that the GST gene had been trans-ferred into the arabidopsis thaliana genome was confirmed by the PCR-Southern blotting analysis (Fig.4). After cultivation, independent homozygous transgenic lines were obtained after selection of T_3 progenies on MS medium containing kanamycin. The expression of the gene transferred into the Arabidopsis thaliana was confirmed by Northern blotting (Fig.5). During salt stress, analysis of total glutathione (both oxidized and reduced type) and biomass of transgenic and wild Arabidopsis.The biomass of transgenic lines (GT) was slightly but significantly greater than that of wild type line (WT) (Fig.6), and levels of oxidized glutathione (GSSG) were significantly higher in transgenic lines than in wild type (Figs.8, 9). Therefore, overexpression of GST can increase Arabidopsis growth under salt stress, and this effect can be caused by oxidation of the reduced glutathione(GSH).