根癌农杆菌对云南红豆杉原生质体的转化

Transformation of Protoplast-derived Colonies by Agrobactenum tumefaciens in Taxus vunnanensis

本文介绍了根癌农杆菌T-DNA对云南红豆杉原生质体的转化,分析了转化系的紫杉醇合成能力。实验以生长20 d的云南红豆杉幼茎诱导的淡黄色愈伤组织为材料,经酶解可分离出大量有活力的原生质体。在由B5无机盐、KM有机成分、3.0 mg/L 2,4-D、0.1mg/L KT和0.45 mol/L果糖组成的培养基中培养1周后,原生质体再生细胞持续分裂形成细胞团。根癌农杆菌对原生质体的转化能力与原生质体生长状态及菌株相关。虽然刚分离的或已再生细胞壁的原生质体不能被根癌农杆菌转化,但由10个以上细胞组成的原生质体克隆和根癌农杆菌B6S3菌株共培养后可实现T-DNA转化,高压纸电泳检测转化系具有冠瘿碱的合成,转化率约为5％。HPLC证实转化系具有合成紫杉醇的能力,但不同转化系中紫杉醇含量变异很大,最高含量为0.076％,是对照愈伤组织的6倍,表明T-DNA的插入改变了培养细胞对紫杉醇的合成。获得的高产转化系细胞生长比对照低,但继代培养中其生长和紫杉醇积累基本保持稳定。尽管转化系合成紫杉醇能力远未达到商业化生产的要求,但研究通过T-DNA对原生质体的转化而实现插入诱变,可以为分离高产紫杉醇优良细胞系提供单细胞筛选系统。

In order to isolate cell lines with high paclitaxel productivity via molecular mutagenesis, the transformation of Taxus yunnanensis protoplasts by Agrobacterium tumefaciens was studied. Protoplasts were obtained from friable, light yellow callus of T. yunnanensis in enzyme solution. Callus in the exponential growth phase of 15-20 days after subculture produced the highest yield of viable protoplasts. When cultured in liquid medium composed of B5 salts, KM vitamin and organic components and 0.45 mol/L fructose and supplemented with 3.0 mg/L 2,4-D and 0.1 mg/L KT, protoplasts undenvent sustained divisions and formed cell colonies. Although A. tumefaciens failed to transform freshly isolated protoplasts and protoplasts with regenerated cell walls, transformants were obtained by co-cultivating protoplast-derived minicolonies consisting of 10 or more celis with the strain B6S3. The transformants were confirmed by opine analysis. The transformation frequency was about 5%. HPLC analysis showed that there was a significant difference in paclitaxel content among all transformants. The highest paclitasel content in transformants was found to be 0.076%, which was 6 times as high as in control callus. The transformant with high paclitaxel accumulation showed a lower cell growth as compared to its control. During subculture the transformant remained unchanged with respect to cell growth and paclitaxel accumulation. Although the paclitaxel accumulation in the transformants was not high enough to produce paclitaxel for commercial purposes, the method described in the present study would provide an opportunity to isolate cell lines with high paclitaxel productivity from mutagenized single cell culture via T-DNA insertion.