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中国农学通报

中国农学通报 ›› 2016, Vol. 32 ›› Issue (10): 162-168.doi: 10.11924/j.issn.1000-6850.casb15100099

所属专题： 生物技术

• 生物技术科学 • 上一篇    下一篇

中国水仙NtFT2 正义基因表达载体的构建与愈伤组织遗传转化体系的建立

冯 莹,朱里莹,丁安琪,潘东明   

  1. 泉州师范学院资源与环境科学学院,福建农林大学园艺产品贮运保鲜研究所,福建农林大学园艺产品贮运保鲜研究所,福建农林大学园艺产品贮运保鲜研究所
  • 收稿日期:2015-10-23 修回日期:2016-03-17 接受日期:2015-11-18 出版日期:2016-04-19 发布日期:2016-04-19
  • 通讯作者: 潘东明
  • 基金资助:
    国家十一五科技支撑计划项目“台湾农业新品种、新技术引进创新研究与示范”(2007BAD07B01)；福建农林大学创新团队项目“园艺植物种质与高优生产技术创新”(cxtd12013)。

Construction of Expression Vector with NtFT2 Sense Gene and Establishment of Transgenic Procedure from Callus in Narcissus tazetta var. chinensis

  • Received:2015-10-23 Revised:2016-03-17 Accepted:2015-11-18 Online:2016-04-19 Published:2016-04-19

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摘要： 构建中国水仙NtFT2 正义基因表达载体，为进一步研究NtFT2 基因对中国水仙芽休眠调控作用提供参考；建立中国水仙愈伤组织遗传转化体系，为中国水仙愈伤组织遗传转化提供技术平台。笔者以中国水仙主芽为材料，通过PCR扩增获得NtFT2 基因；通过PCR、双酶切、连接等方法将NtFT2 基因正向插入到pCAMBIA1301 双GUS植物表达载体构建NtFT2 正义基因表达载体P1301-FT2，再通过冻融法将重组质粒P1301-FT2 导入根癌农杆菌。采用根癌农杆菌介导法将NtFT2 正义基因转化中国水仙愈伤组织，采用GUS组织化学染色法检测中国水仙愈伤组织遗传转化效率。结果表明，试验成功构建了中国水仙NtFT2 正义基因表达载体P1301-NtFT2；中国水仙愈伤组织与0.5 mol/L 甘露醇共培养6 h 后，置于1.0 OD600的农杆菌重悬液中侵染20 min，在25℃、黑暗条件下，共培养6 天，其GUS瞬时表达率达到64.28%。构建了中国水仙NtFT2正义基因表达载体，建立了高效的中国水仙愈伤组织遗传转化体系

关键词: 作物发育模式, 作物发育模式, 持续性温强, 土壤水分

Abstract: The expression vector of NtFT2 sense gene was constructed to study the regulation effect of NtFT2 gene on the bud dormancy; and transgenic procedure was established to provide a foundation for gene transformation in Narcissus tazetta var. Chinensis. NtFT2 gene was cloned from buds by PCR, NtFT2 sense gene was inserted into plant expression vector pCAMBIA1301 with double GUS by PCR, double digestion and connection method to obtain expression vector P1301-FT2 with NtFT2 sense gene. Then recombinant plasmid P1301-FT2 was transformed into Agrobacterium through freeze-thawing method. NtFT2 sense gene was transformed into callus by Agrobacterium-mediated transformation method, genetic transformation efficiency was detected by GUS histochemical staining method. Results showed that: expression vector with NtFT2 sense gene was obtained; the best transient expression of GUS reached 64.28% under the conditions as follows: callus which were pretreated with 0.5 mol/L mannitol for 6 h; and the Agrobacterium suspension was with OD600 value of 1.0, callus were infected for 20 min, and then they were transferred onto medium for co-cultivation for 6 days at 25℃ in the dark. Expression vector with NtFT2 gene was constructed, and a high-efficiency transgenic procedure mediated by Agrobacterium tumefaciens with NtFT2 sense gene was developed from callus in Narcissus tazetta var. Chinensis.