花生AhPLDα基因植物过表达载体构建及对拟南芥的遗传转化

发布时间：2018-06-29 08:51

本文选题：花生 + AhPLDα基因　；参考：《华北农学报》2016年06期

【摘要】：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。
[Abstract]:In order to clarify the function and mechanism of peanut AhPLD alpha gene in response to drought stress signal transduction, the whole long CDS fragment of AhPLD alpha 1 and AhPLD alpha 2 was amplified by RT-PCR method with C DNA of peanut leaf No. 4, which was treated by ABA, and the 2 genes were cloned to the plant expression vector p Bar-F3 respectively by the method of enzyme cut connection. The recombinant plasmid was transferred into Agrobacterium tumefaciens GV3101 by freezing and thawing method. The overexpressed plasmid was transferred into Arabidopsis by modified Floral-dip method. The result of colony PCR and enzyme digestion showed that the recombinant plasmid P Bar-AhPLD a, which was driven by Ca MV35S promoter, was constructed correctly. It was verified by RT-PCR and qRT-PCR that AhPLD alpha 1 and AhPLD alpha 2 gene were integrated into Arabidopsis thaliana. The test of drought stress showed that the drought resistance of the transgenic plants was obviously stronger than that of the wild type. The AhPLD a gene was involved in the response process of drought stress, and it was a potential candidate gene for improving the drought tolerance of the crops.
【作者单位】：河北省农林科学院粮油作物研究所河北省作物遗传育种实验室;
【基金】：国家自然科学基金项目(31201239;31301354) 河北省重点基础研究项目(10960122D) 河北省博士基金项目(F14E055610) 现代农业产业技术体系建设专项资金(CARS-14)
【分类号】：S565.2;Q943.2

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