秋石斛遗传转化筛选标记基因及筛选剂的筛选

Selection of marker genes and screening agents for genetic transformation of Phalaenopsis-type Dendrobium hybrids

关键词:  秋石斛, 筛选标记基因, 筛选剂, 遗传转化, 筛选浓度

Key words:  Phalaenopsis-type Dendrobium hybrids, selection marker gene, screening agent, genetic transformation, selection concentration

摘要:

秋石斛(Phalaenopsis-type Dendrobium hybrids)是热带地区重要观赏植物,其因花形奇特、花色丰富,在花卉市场中具有极高的产业价值。分子育种是突破传统育种局限性的重要手段,筛选标记基因辅助转化可以加速转基因进程。为获得适合于秋石斛遗传转化的筛选标记基因和筛选剂,该研究以‘三亚阳光'和‘诺贝尔'的胚性愈伤组织(ECs)和原球茎为外植体,将其置于含不同浓度卡那霉素、遗传霉素(G418)、潮霉素和双丙氨膦的培养基中,测试其对不同筛选剂的敏感性,观察其生长状态,统计存活率,并以最优筛选标记基因和筛选剂进行遗传转化测试。结果表明:(1)2个品种的外植体对不同筛选剂的敏感性表现一致,即对潮霉素、G418、双丙氨膦较为敏感,而对卡那霉素不敏感; 在卡那霉素浓度为700 mg·L^-1的培养基中,外植体仍能正常生长。(2)潮霉素、G418和双丙氨膦的最低致死浓度分别为30、50、7 mg·L^-1。(3)当以潮霉素作为筛选剂、hpt作为筛选标记基因时,初次筛选采用30 mg·L^-1的浓度效果最佳,随着筛选周期的延长,适当提高筛选剂浓度有利于获得转基因株系。该研究明确了秋石斛2个栽培种对不同筛选剂的敏感性及其最低致死浓度,验证潮霉素抗性基因(hpt)、新霉素磷酸转移酶基因(npt)和除草剂抗性基因(bar)都可以作为秋石斛遗传转化体系构建的筛选标记基因,对应筛选剂分别为潮霉素、G418和双丙氨膦。此外,在遗传转化筛选过程中采用筛选剂浓度梯度递增策略,有利于转基因外植体的获得。

Abstract:

Phalaenopsis-type Dendrobium hybrids(Den-Phals)are important ornamental plants in tropical areas, with unique flower shapes and rich colors, and have extremely high industrial value in flower market. Molecular breeding is an important means to break through the limitations of traditional breeding, and screening marker genes assisted transformation can accelerate transgenic process. In order to obtain suitable selection marker genes and agents for genetic transformation of Den-Phals, the study took the embryogenic calluses(ECs)and protocorms of Dendrobium ‘Sonia Hiasakul' and Dendrobium ‘Nobile Lindl.' as explants, and placed them on media containing different concentrations of kanamycin, geneticin(G418), hygromycin and bialaphos to test the sensitivity of two varieties to different screening agents observe the growth status of them, and calculate the survival rate. Furthermore, conducted genetic transformation test using the optimal screening marker gene and screening agent. The results were as follows:(1)The explants of the two varieties had consistent sensitivity to different screening agents, that is, they were relatively sensitive to hygromycin, G418, bialaphos, but insensitive to kanamycin and could still growth normally in the culture medium with a concentration of 700 mg·L^-1.(2)The lethal concentrations of hygromycin, G418 and bialaphos to explants were 30, 50, 7 mg·L^-1, respectively.(3)Using hygromycin as the screening agent and hpt as the screening marker gene, the initial screening concentration of 30 mg·L^-1 was optimal, with the screening cycle extended, increasing the concentration of the screening agent was beneficial for obtaining transgenic lines. The study has determined the sensitivity and minimum lethal concentrations of two varieties of Den-Phals to different screening agents; hpt, npt, and bar can all be used as screening marker genes for establishment of genetic transformation systems of Den-Phals, with corresponding screening agents are hygromycin, G418, and bialaphos. In addition, during genetic transformation screening process, increasing the concentration gradient of the screening agent is beneficial for obtaining transgenic explants.