摘要
为建立稳定的黄花蒿悬浮细胞系,以黄花蒿428–A植株的子叶、茎、腋芽和叶片为外植体,以MS为基础培养基,采用3因素(6–BA、NAA、2,4–D)4水平的L16(43)正交试验,优化黄花蒿愈伤组织诱导培养基,分析不同质量浓度(0、5、10、15、20、25、30 mg/L)潮霉素对黄花蒿悬浮细胞生长的影响,比较羧苄青霉素(0、100、150、200、250、300 mg/L)对农杆菌的抑制效果,并运用农杆菌介导法对黄花蒿悬浮细胞进行GUS报告基因及色氨酸单加氧酶iaaM基因的遗传转化。结果表明:黄花蒿叶片愈伤诱导率较高,叶片愈伤组织较优培养基为MS+6–BA 1.0 mg/L+NAA 0.5 mg/L,出愈率可达99.33%;潮霉素最适筛选质量浓度为30 mg/L,羧苄青霉素最适抑菌质量浓度为200 mg/L;转基因抗性愈伤GUS组织化学染色及PCR鉴定结果表明,外源基因iaaM已导入并整合至黄花蒿基因组中。
To establish cultured cell lines of Artemisia annua L.,we used its leaves and stems as explants,MS-based medium and performed three factors of6-BA,NAA,2,4-D mass concentration with each factor of4levels using L16(43)orthogonal table for orthogonal test.We also optimized different concentrations of hygromycin and carbenicillin on the growth of the suspension cells and the inhibition effect on Agrobacterium tumefaciens and applied Agrobacterium-mediated method to do genetic transformation of GUS reporter gene and tryptophan monooxygenase iaaM gene in the suspension cells.The results showed that the medium containing MS+6-BA1.0mg/L+NAA0.5mg/L,30mg/L hygromycin and200mg/L carbenicillin was optimal and the rate of callus formation is up to99.33%.And,GUS histochemical staining and PCR analysis of transgenic callus showed that the exogenous gene had been introduced and integrated into the genome of Artemisia annua L.,and the genetic transformation system of Artemisia annua L.suspension cells lines was successfully established.
作者
龙炎杏
张学文
罗莎
肖楠
赵燕
LONG Yanxing;ZHANG Xuewen;LUO Sha;XIAO Nan;ZHAO Yan(College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, Hunan 410128, China)
出处
《湖南农业大学学报（自然科学版）》
CAS
CSCD
北大核心
2018年第6期607-612,共6页
Journal of Hunan Agricultural University(Natural Sciences)
基金
湖南省大学生科技创新项目(DFCXS201206)
