Genetic transformation of Prunus domestica L. using the hpt gene coding for hygromycin resistance as the selectable marker
Genetic transformation of Prunus domestica L. using the hpt gene coding for hygromycin resistance as the selectable marker
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DOI：
10.1016/j.scienta.2008.08.024
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发表时间：
2009-02
影响因子：
4.3
通讯作者：
L. Tian;F. A. Canli;Xinhua Wang;S. Sibbald
中科院分区：
文献类型：
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作者：
L. Tian;F. A. Canli;Xinhua Wang;S. Sibbald
研究和开发具有新的选择方案的转化技术对于各种基础研究和通过基因工程进行作物性状改良具有重要意义。本研究表明潮霉素抗性体系是李遗传转化的有效体系。将含有潮霉素磷酸转移酶（hpt）和β-葡萄糖醛酸酶（GUS）基因的质粒pC 1381的农杆菌菌株LBA 4404和含有相同标记和报告基因的质粒pC 1301的菌株EHA 105与成熟种子的胚轴共培养。将含有携带新霉素磷酸转移酶基因（npt II）基因的pC 2301质粒的后一种菌株用作对照。将感染的外植体置于含有5 mgL-1潮霉素或75 mgL-1卡那霉素的芽诱导培养基上进行选择。在潮霉素压力下，外植体发育出绿色芽。这些芽在转移到新鲜的潮霉素培养基上时显示出持续和旺盛的生长和发育。使用hpt序列引物的PCR和使用来自hpt基因的探针的Southern印迹分析证实了转基因的存在及其在再生植物中的稳定整合。在温室中获得完整的转基因植物。潮霉素选择是非常有效的，没有观察到逃逸。研究表明，潮霉素抗性可以作为一种有效的选择标记用于李的遗传转化。该体系对李的多基因转化具有重要意义。
The study and development of transformation technology with new selection schemes is important for various fundamental studies and for crop trait improvement via genetic engineering. Here we have shown that hygromycin resistance is an effective system for plum genetic transformation. Embryonic axes of mature seeds were co-cultivated with Agrobacterium strain LBA4404 containing the pC1381 plasmid carrying the hygromycin phosphotranferase gene (hpt) and β-glucuronidase (GUS) gene or with strain EHA105 containing the plasmid pC1301 carrying the same marker and reporter genes. The latter strain containing a pC2301 plasmid carrying the neomycin phosphotransferase gene (nptII) gene was used as a control. Infected explants were placed on shoot induction medium containing either 5mgL−1hygromycin or 75mgL−1kanamycin for selection. Green shoots developed from the explants under hygromycin pressure. These shoots showed continued and vigorous growth and development upon transfer onto fresh hygromycin medium. PCR using hpt sequence primers, and Southern blot analysis using a probe from the hpt gene, confirmed the presence of the transgenes and their stable integration in regenerated plants. Full transgenic plants were obtained in a greenhouse. Hygromycin selection was very effective and no escapes were observed. The study demonstrated that hygromycin resistance can be used as an effective selectable marker for plum transformation. The new system developed here is important and useful for multiple gene transformation in plum.