花生AhPLDα基因植物过表达载体构建及对拟南芥的遗传转化

Vector Construction to Overexpress AhPLDα Genes from Peanut and Genetic Transformation into Arabidopsis thaliana

为了明确花生AhPLDα基因在响应干旱胁迫信号传导中的功能和作用机制,以ABA处理的冀花4号花生叶片c DNA为模板,用RT-PCR方法扩增AhPLDα1和AhPLDα2的全长CDS片段,采用酶切-连接的方法分别将这2个基因定向克隆到植物表达载体p Bar-F3上,冻融法将重组子转入根癌农杆菌GV3101,利用改良Floral-dip法将过表达质粒转入拟南芥。菌落PCR和酶切结果表明,Ca MV35S启动子驱动的过表达载体重组质粒p Bar-AhPLDα构建正确。通过RT-PCR和qRT-PCR验证,表明AhPLDα1和AhPLDα2基因整合到拟南芥基因组中并能过量表达,获得了阳性转基因纯合株系。干旱胁迫试验表明,转基因植株的耐旱性较野生型明显增强。可见,AhPLDα基因参与了干旱胁迫应答过程,是转基因途径提高作物耐旱性的潜在候选基因。

This study provides a good foundation for further elucidating the function and mechanism of AhPLDα1 and AhPLDα2 in responding to drought stress in peanut.The full-length CDS sequences of AhPLDα1 and AhPLDα2 were cloned from Jihua 4 peanut leaf with ABA treatment using RT-PCR.The AhPLDα1 and AhPLDα2gene fragments were inserted into the expression vector p Bar-F3 by forward ways.These constructs were transformed into Agrobacterium tumefaciens GV3101 by freeze-thawing method and then introduced into wild-type Arabidopsis thaliana using modified floral-dip method.Under the condition of drought,the authors observed phenotypic changes of wild type and transgenic plants.Colony PCR and enzyme digestion results showed that,the plant overexpressing recombinant plasmid p Bar-AhPLDα driven by Ca MV35 S promoter was successfully constructed.Glyphosate resistance screening,PCR detection and gene expression analysis showed that the positive transgenic plants were obtained.Under water deprivation,overexpression of AhPLDα1 and AhPLDα2 in transgenic Arabidopsis plants resulted in significantly enhanced tolerance to drought stress.In conclusion,AhPLDα1 and AhPLDα2 have a certain relation with drought stress signal transduction in transgenic Arabidopsis plants,and are potential candidate genes on the way to modified crop drought resistance.