拟南芥AMP1基因RNAi表达载体的构建及转基因株系的抗旱性分析

Construction of RNAi Vector for Arabidopsis AMP1 and Functional Analysis of the Transgenic Plants in Drought Resistance

利用Gateway克隆技术构建AMP1基因的RNAi表达载体p K7GWIWG2-AMP1转化拟南芥,获得AMP1下调表达的转基因株系,以拟南芥野生型为对照,研究AMP1基因在干旱胁迫中的作用。结果表明,甘露醇模拟干旱能提高RNAi株系的萌发率,增加相对根长,表现出了一定的干旱耐受性;丙二醛(MDA)、脯氨酸(Pro)、过氧化物酶(POD)、超氧化物歧化酶(SOD)的测定结果可以看出,与野生型相比,RNAi株系的MDA含量降低,Pro含量升高和POD、SOD酶活性增强;外源ABA可抑制RNAi株系种子萌发,减小叶片气孔开度。另外,Real-time PCR分析表明,正常条件下RNAi株系中AREB1基因表达上调。可见通过下调AMP1基因表达,可以提高转基因拟南芥株系抗干旱胁迫能力。

The RNAi vector for Arabidopsis AMP1 （pK7GWIWG2-AMP1） was constructed by Gateway gene cloning, and transgenic plants were obtained. Drought resistance and abscisic acid （ABA） sensitivity of the transgenic plants were examined in this study. Under mannitol treatment, the increase of seed germination and enhanced seedling root growth were observed in the transgenic plants of AMP1. In further, the decrease level of malondialdehyde （MDA） but increase of proline （Pro） was detected in the transgenic lines. As expected, higher activities of peroxidase （POD） and superoxide dismutase （SOD） were observed in the AMP1 transgenic plants compared to the wild type. These results suggested that the down-regulation of AMP1 gene expression signifi- cantly improveed drought tolerance in the transgenic plants. In addition, it was found that the ABA sensitivity and the seed germination rate of AMP1 RNAi transgenic lines were lower than that of wild-type plants, and the leaf stomatal aperture of the transgenic plants was smaller than control plants. By analyzing the expression of ABA responsive genes, we found that the AREB1 gene was up-regulated in the transgenic lines under normal growth condition. These observations provide new evidence supporting the conclusion that plant drought toler- ance could be enhanced when the expression of AMP1 is down-regulated in transgenic plants.