耐碱GsCHX19基因的克隆及对苜蓿的遗传转化

doi:10.16035/j.issn.1001-7283.2016.03.008

摘要/Abstract

摘要：

基于实验室前期野生大豆G07256碱胁迫转录组数据,结合生物信息学方法筛选得到响应碱胁迫的cation/H ⁺逆向转运蛋白基因GsCHX19,克隆了GsCHX19基因全长CDS编码序列并将其构建到pCAMBIA330035Su植物超量表达载体中。以肇东紫花苜蓿为受体材料,通过农杆菌介导法将重组的植物表达载体转化其子叶节,用含0.5mg/L草铵膦的筛选培养基进行筛选,获得20株抗性植株。采用PCR方法检测抗性植株中bar基因的表达,获得16株PCR阳性植株。对获得的PCR阳性植株进行RT-PCR及real-time PCR检测,鉴定共获得11株RT- PCR阳性植株,且证明GsCHX19基因在各转基因植株中均有不同程度的表达。结果表明,GsCHX19基因成功转化苜蓿,并且得到表达,该转基因植株将为耐盐碱转基因苜蓿新品种的培育提供重要的试验材料。

关键词: Cation/H ⁺逆向转运蛋白, GsCHX19, 野生大豆, 苜蓿, 遗传转化

Abstract:

GsCHX19, a cation/H ⁺ antiporter gene, has been identified as a putative bicarbonate stress responsive gene in previous studies, through bioinformatic analysis of transcriptome sequencing data of the Glycine soja G07256 under alkali stress. In this study, the full length coding region of GsCHX19 gene was cloned and constructed into the plant expression vector pCAMBIA330035Su. The recombinant plasmid was then transferred into the cotyledon nodes of Medicago sative by the way of agrobacterium-mediated genetic transformation method. And 20 resistant seedlings were obtained after screening on the medium supplement with 0.5mg/L glufosinate-ammonium. By detecting selection marker of bar gene and PCR test, and then RT-PCR, 11 plants were further confirmed as transgenic plants. The real-time PCR results indicated that GsCHX19 showed different expression levels in different transgenic plants. All the results showed that the GsCHX19 was transferred into alfalfa and successfully expressed. The transgenic plants will provide important experimental materials for development of saline-alkali tolerant alfalfa cultivars.

Key words: Cation/H ⁺ antiporter, GsCHX19, Glycine soja, Alfalfa, Transformation

杨浩,朱延明. 耐碱GsCHX19基因的克隆及对苜蓿的遗传转化[J]. 作物杂志, 2016 (3): 37–44

Hao Yang,Yanming Zhu. Isolation of a Alkali Stress Responsive Gene GsCHX19 and Transformation into Medicago sativa L．[J]. Crops, 2016(3): 37–44

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