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| 秋石斛遗传转化筛选标记基因及筛选剂的筛选 |
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蒋琼海1,2, 李亚梅2,4, 李崇晖2, 尹俊梅2,3*
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1．海南大学 热带农林学院,海口570228；2．中国热带农业科学院热带作物品种资源研究所, 海口571101; 3．中国热带农业科学院三亚研究院,海南 三亚 572024； 4．中国热带农业科学院海口实验站,海口571101
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| 摘要: |
| 秋石斛（Phalaenopsis-type Dendrobium hybrids）是热带地区重要观赏植物，其花形奇特、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，筛选标记基因辅助筛选则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（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）两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，双丙氨膦较敏感，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和双丙氨膦的致死浓度分别为30 mg·L-1、50 mg·L-1和7 mg·L-1。（3）hpt为筛选标记基因，潮霉素为筛选剂的初次筛选浓度以30 mg·L-1最佳，随着筛选周期的延长，提高筛选剂浓度有利于获得转基因株系。该研究确定了秋石斛两个栽培种对不同筛选剂的敏感性和最低致死浓度，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，同时在遗传转化筛选过程中，筛选剂浓度梯度递增有利于转基因外植体的获得。 |
| 关键词: 秋石斛,筛选标记基因,筛选剂,遗传转化 |
| DOI：10.11931/guihaia.gxzw202410015 |
| 分类号:S682.31????? ? |
| 基金项目:海南省重点研发项目(No. ZDYF2024XDNY239)；中央级公益性科研院所基本科研业务费专项( No.1630032022) |
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| Selection of marker genes and screening agents for genetic transformation of Phalaenopsis-type Dendrobium hybrids |
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JIANG Qionghai1,2, LI Yamei2,4, LI Chonghui2, YIN Junmei2,3*
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1.School of Tropical Agriculture and Forestry, Hainan University, 570228, Haikou, China; 2. Tropical Crop Germplasm Research Institute, Chinese Academy of Tropical Agricultural Sciences, 572024, Haikou, China; 3.Sanya Research Institute, Chinese Academy of Tropical Agricultural Sciences, 571101, Sanya Hainan, China; 4. Haikou Experiment Station. CATAS, 571101, Haikou, China
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| Abstract: |
| Phalaenopsis-type Dendrobium hybrids (Den-Phals) are important ornamental plant 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 selection marker genes assisted screening 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 selection agents, and observe the growth status of them and calculate the survival rate. Furthermore, conducted genetic transformation test using the optimal marker gene and screening agent. The results were as follows: (1) The explants of the two varieties had consistent sensitivity to different selection agents and being more sensitive to hygromycin, G418, bialaphos, 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 the hygromycin, G418 and bialaphos to explants were 30 mg·L-1, 50 mg·L-1 and 7 mg·L-1, respectively. (3) Using hpt as the selection marker gene and hygromycin as the selection agent, the initial screening concentration of 30 mg·L-1 was optimal, with the screening cycle extended, increasing the concentration of the selection agent was beneficial for obtaining transgenic lines. The study has determinded 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 selection marker genes for establishment of genetic transformation systems of Den-Phals, with corresponding selection agents are hygromycin, G418, and bialaphos. At the same time, during genetic transformation, increasing the concentration gradient of the selection agent is beneficial for obtaining transgenic explants. |
| Key words: Phalaenopsis-type Dendrobium hybrids, selection marker gene, screening agent, genetic transformation |
