摘要
构建中国水仙Nt FT2正义基因表达载体,为进一步研究Nt FT2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Nt FT2基因;通过PCR、双酶切、连接等方法将Nt FT2基因正向插入到p CAMBIA1301双GUS植物表达载体构建Nt FT2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Nt FT2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Nt FT2正义基因表达载体P1301-Nt FT2;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Nt FT2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。
The expression vector of Nt FT2 sense gene was constructed to study the regulation effect of Nt FT2 gene on the bud dormancy, and transgenic procedure was established to provide a foundation for genetransformation in Narcissus tazetta var. chinensis. Nt FT2 gene was cloned from buds by PCR, Nt FT2 sensegene was inserted into plant expression vector p CAMBIA1301 with double GUS by PCR, double digestion andconnection method to obtain expression vector P1301-FT2 with Nt FT2 sense gene. Then recombinant plasmidP1301- FT2 was transformed into Agrobacterium through freeze- thawing method. Nt FT2 sense gene wastransformed into callus by Agrobacterium- mediated transformation method, the genetic transformationefficiency was detected by GUS histochemical staining method. Results showed that： expression vector with Nt FT2 sense gene was obtained, the best transient expression of GUS reached 64.28% under the condition asfollows： callus was pretreated with 0.5 mol/L mannitol for 6 h, the Agrobacterium suspension was with OD600 value of 1.0, callus was infected for 20 min, and then they were transferred onto medium for co-cultivation for6 days at 25℃ in the dark. Expression vector with Nt FT2 gene was constructed, and a high- efficiencytransgenic procedure mediated by Agrobacterium tumefaciens with Nt FT2 sense gene was developed fromcallus in Narcissus tazetta var. chinensis.
出处
《中国农学通报》
2016年第10期162-168,共7页
Chinese Agricultural Science Bulletin
基金
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)
关键词
中国水仙
NtFT2基因
愈伤组织
遗传转化
Narcissus tazetta var.chinensis
Nt FT2 gene
callus
genetic transformation
