目的优化南方红豆杉愈伤组织诱导与继代培养条件,获得含紫杉醇量高的红豆杉愈伤组织,明确紫杉醇合成相关基因的表达模式与紫杉醇量的关系。方法探讨了适合南方红豆杉的愈伤组织诱导及继代培养的激素组成及浓度。比较了南方红豆杉(种胚、幼茎、幼芽)、曼地亚红豆杉(种胚)和太平洋红豆杉(种胚)不同外植体来源的愈伤组织的生长特点和紫杉醇的量,分析了不同愈伤组织紫杉醇合成相关基因的表达差异。结果添加3.0 mg/L 2,4-D的Murashige&Skoog(MS)培养基有利于南方红豆杉的诱导,诱导率高于92%。添加2.0 mg/L 2,4-D与1.0 mg/L 6-苄基腺嘌呤(6-BA)的继代培养基有利于南方红豆杉的紫杉醇积累。同一培养条件下,南方红豆杉种胚来源的愈伤组织紫杉醇量最高,达到干质量的0.027%。含紫杉醇量高的愈伤组织,其紫杉醇合成途径中的=牛儿基=牛儿基焦磷酸合成酶(GGPPS)、紫杉二烯合成酶(TASY)、紫杉烷-10β-羟化酶(T10βH)、10-去乙酰巴卡亭Ⅲ-β-O-乙酰转移酶(DBAT)、苯丙氨酸氨基变位酶基因(PAM)、去苯甲酰紫杉-N-苯甲酰转移酶(DBTNBT)等酶基因的表达水平高。结论 从南方红豆杉的种胚为外植体源获得了含紫杉醇量高的愈伤组织,提高GGPPS、TASY、T10βH、DBAT、PAM、DBTNBT的表达水平将促进紫杉醇的合成。

Objective The callus induction and subculture condition of Taxus chinensis var.mairei were optimized in the experiment.High Taxol-yielding callus was obtained,and the correlation between expression patterns of Taxol biosynthesis related genes and Taxol content was defined.Methods Optimal hormone composition and concentration for induction and subculture of callus derived from T.chinensis var.mairei were briefly studied.The growth characteristics and Taxol content of calli derived from T.chinensis var.mairei(including vitro embryos,juvenile stems,and juvenile buds),T.media(vitro embryos) and T.brevifolia(vitro embryos) were compared.Expression of Taxol biosynthesis related genes in the above different calli was analyzed.Results MS Medium supplemented with 3.0 mg/L 2,4-D was suitable for callus induction of T.chinensis var.mairei,the induced rate is up to 92%.Subculture medium supplemented with 2.0 mg/L 2,4-D and 1.0 mg/L 6-BA was suitable for Taxol biosynthesis of T.chinensis var.mairei.Under the same culture conditions,Taxol content of calli derived from vitro embryos of T.chinensis var.mairei was the highest and had reached 0.027% of callus dry weight.The genes of Taxol biosynthesis related enzymes— geranylgeranyl diphosphate synthase(GGPPS),taxadiene synthase(TASY),taxane-10β-hydroxylase(T10βH),10-deacetylbaccatinⅢ-β-10-O-acetyltransferase(DBAT),phenylalanine aminomutase(PAM),and 3′-N-debenzoyltaxol N-benzoyltransferase(DBTNBT),were expressed at the high level in the high Taxol-producing callus.Conclusion Vitro embryos can be used as the first choice explant source to obtain high Taxol-yielding callus.To improve gene expression level of GGPPS,TASY,T10βH,DBAT,PAM,and DBTNBT would promote Taxol biosynthesis.

国家自然科学基金资助项目(30771116,30970243);山东省杰出青年基金(QJ200810)