| 摘要: |
| 采用显微计数的方法获得潮霉素、氯霉素、卡那霉素、硫酸链霉素等抗生素对裙带菜雌、雄配子体的致死率,通过统计分析研究了雌雄配子体对这些抗生素的敏感性。结果表明,裙带菜雌雄配子体对潮霉素和氯霉素敏感,482.7μg/ml潮霉素分别在第9、10天使雄配子体、雌配子体全部致死;雌雄配子体对氯霉素的敏感性还存在明显的差异,第8天氯霉素对雌雄配子体的半致死剂量分别是218.78μg/ml和371.54μg/ml。配子体对卡那霉素,硫酸链霉素相对不敏感,6000kU/L卡那霉素和800μg/ml的硫酸链霉素作用10天后雌雄配子体均无明显死亡现象。潮霉素和氯霉素可以作为裙带菜配子体的筛选试剂。 |
| 关键词: 裙带菜 雌雄配子体 抗生素 选择标记 |
| DOI： |
| 分类号: |
| 基金项目:中国科学院知识创新工程项目(ZKCX2211)资助;国家863计划(2002AA628020)资助 |
附件 |
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| SENSITIVITIES OF GAMETOPHYTES OF BROWN SEAWEED UNDARIA PINNATIFIDA TO FOUR ANTIBIOTICS |
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LI Da-Peng1, XIONG Yan1,2
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1.Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071;2.Graduate School, Chinese Academy of Sciences, Beijing, 100039
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| Abstract: |
| Genetic transformation is an important method of breeding higher plants and crops. Some researches of genetic engineering have been done in seaweed. However, enormous genetic transformation of seaweed remains largely unknown. Screening of transgenic plants by antibiotics or herbicides is one of the tasks in the research.
To find an effective selectable marker for transgenic seaweed, especially gametophytes of Undaria pinnatifida, their sensitivity to four antibiotics which are hygromycin (Hm), chloramphenicol (Cm), kanamycin (Km), and streptomycin (Str), was tested. All of them are frequently used in genetic plant engineering.
Female and male U. pinnatifida gametophyte clones were from seaweed seedstock in our lab. Samples were blended first, then filtered through 100μm nylon net to remove broken cell pieces. The left-over on the net was transferred to PES medium dissolving antibiotics. Concentration of the antibiotics were set as follows:
Cm: 0μg/ml, 50μg/ml, 100μg/ml, 200μg/ml, 400μg/ml;
Hm: 0μg/ml, 50μg/ml, 100μg/ml, 200μg/ml, 300μg/ml, 400μg/ml;
Km: 0 kU/L, 100 kU/L, 400 kU/L, 1000 kU/L, 4000 kU/L, 6000 kU/L;
Str: 0μg/ml, 50μg/ml, 100μg/ml, 400μg/ml, 800μg/ml.
They were cultured at 25°C, 12h photoperiod at 2000–3000 lx. The samples were removed every day and observed under microscope. Death rate was determined in the ratio of number of dead cells to the number of total cells in the visible field.
Results showed that female and male gametophytes of U. pinnatifida were not sensitive to Km and Str. While, they were sensitive to both Hm and Cm. The lethal concentration of Hm in Day 10 was 482.7μg/ml for both female and male gametophytes. Female gametophytes died out after 6-day treatment with 329.5μg/ml Cm. 92% male gametophytes died after 9-day culture in 556.9μg/ml Cm. Meanwhile, LD50 of Hm at 5, 6, 7, 8, 9, 10d and LD50 of Cm at 6, 7, 8, 9d were calculated separately.
Further test indicated that sensitivity to Cm varied between female and male gametophytes. Significance test was conducted, and the difference was statistically significant at α= 0.1 and 90% confidence.
The gene CAT (Chloramphenicol acetyltransferase) could confer resistance of transformed cells to Cm in a medium and the untransformed cells would be killed. In the case of Hm, the resistance gene codes for a kinase (hygromycin phosphotransferase, HPT) inactivates hygromycin through phosphorylation.
The results above suggest that both HPT and CAT genes can be used as a selectable marker in transgenic gametophyte of U. pinnatifida. |
| Key words: Undaria pinnatifida, Female and male gametophytes, Antibiot ics, Selectable marker |
