农杆菌介导灰绿藜NHX1基因转化新疆大叶苜蓿的研究

利用转基因技术能够快速有效地获得抗盐碱苜蓿的新品种,但苜蓿的品种直接影响着其再生能力和遗传转化,因此建立农杆菌介导的大叶苜蓿转基因体系对于大叶苜蓿抗逆新品种培育具有重要的促进作用.将新疆盐生植物灰绿藜(Chenopodium glaucum Linn.)的Na+/H+反向运输体基因Cg-NHX1构建在植物表达载体pBIN438上,并转化入根瘤农杆菌EHA105,以5～7 d苗龄的新疆大叶紫花苜蓿(Medicago.sativa)子叶为转化受体,通过重组EHA105介导的子叶浸染法将Cg-NHX1基因导入新疆大叶紫花苜蓿中,获得了抗卡那霉素的再生植株.提取转基因植株基因组DNA,进行PCR检测和Southern blot分析,结果表明Cg-NHX1基因已经被整合到新疆大叶紫花苜蓿基因组中.研究成功地获得灰绿藜NHX1基因转化的新疆大叶紫花苜蓿植株,并在新疆大叶紫花苜蓿遗传转化影响因素的研究中,发现菌液浓度OD600nm为0.2～0.3,浸染时间20 min适宜于子叶愈伤诱导,有利于基因转化植株的获得.

Transformation of Cg-NHX1 Gene in Xinjiang Big Leaf Alfalfa (Medicago.sativa)by Agrobacterium Mediated Gene Transfer

The new variety of salt tolerance alfalfa can be quickly and effectively obtained by transgenic technology.But the ability of regeneration and transformation of alfalfa is greatly affected by its different varieties.So,establishing transgenic system of Xinjiang big leaf alfalfa by Agrobacterium mediated gene transfer has important promoting effect on breeding new variety.CgNHX1 gene was cloned from the Chenopodium glaucum.Chenopodium glaucum is the kind of halophyte that has the salt tolerance.The Cg-NHX1 gene was ligated with pBIN438 plant expression vector and transformed into Agrobacterium tumefaciens EHA105.Transgenic Big Leaf alfalfa was gotten by co-cultivating 5-7 d old cotyledon with recombinant Agrobacterium tumefaciens.The genomic DNA of transgenic Big Leaf alfalfa was extracted and analyzed by PCR and Southern blot techniques.The results indicated that the Cg-NHX1 gene had been transformed into Xinjiang Big Leaf Alfalfa.The study of optimizing transformation showed that Agrobacterium tumefaciens's concentration of OD600 in 0.2～0.3,and time of cotyledons immersed in 20min are favorable to inducing calli and obtaining the transgenic big Leaf alfalfa successfully.