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长春花毛状根再生植株的获得及抗癌生物碱的产生

来源：花匠小妙招时间：2025-11-03 10:06

[关键词]

[摘要]

目的建立长春花转基因毛状根再生植株快速繁育体系,获得生物碱量提高的长春花再生植株。方法研究外源植物生长调节剂对长春花毛状根愈伤组织诱导、不定芽分化、不定根分化和再生植株移栽的影响,采用HPLC法测定长春花再生植株生物碱量,并采用定量PCR技术分析目的基因的表达情况。结果转基因长春花毛状根愈伤组织诱导和不定芽诱导的最佳培养基均是MS+6-BA 2.0 mg/L+NAA 0.3 mg/L,不定根分化的最适培养基是1/2 MS+NAA 0.3 mg/L。采用炼苗的改进方法可使长春花再生植株的移栽成活率达90%。HPLC分析结果表明,长春花再生植株中长春碱、长春新碱和阿吗碱量分别比对照提高4.0、5.8、1.8倍,其中,优良单株OG30-3的长春新碱和长春碱量比对照提高7倍和10倍。定量PCR分析结果表明,转基因植株orca3基因和g10h基因的表达量比非转基因植株的高。结论转基因长春花毛状根再生植株可促进抗癌生物碱的产生。

[Key word]

[Abstract]

Objective To establish an efficient in vitro plant regeneration system in the transgenic Catharanthus roseus hairy roots and to obtain the high production of anticancer terpenoid indole alkaloids (TIAs) in C. roseus regeneration plantlets. Methods The effects of plant growth regulators on callus induction, adventitious shoot induction, and adventitious root induction were investigated. HPLC was used to determine the alkaloids in C. roseus and quantitative PCR (QPCR) to analyze the expression of target genes. ResultsThe results showed that the best medium for callus and shoot induction was MS + 6-BA 2.0 mg/L + NAA 0.3 mg/L and the best medium for root induction was 1/2 MS + NAA 0.3 mg/L. The improved transplant method could increase the survival rate of C. roseus plantlets to 90%. TIA profiling by HPLC revealed that the simultaneous introduction and overexpression of orca3 and g10h in transgenic plant significantly enhanced vinblastine, vincristine, and ajmalicine accumulation. The concentrations of vinblastine,vincristine, and ajmalicine were 4.0-, 5.8-, and 1.8-fold respectively more than those in non-transformed plants. QPCR results showed that the expressions of orca3 and g10h genes were greater than these of non-transformed root. Conclusion The regeneration plantlets from C. roseus transgenic hairy roots could significantly promote the antitumor alkaloid accumulation.

[基金项目]

中国博士后科学基金项目(20060390733);宁波市农业创新创业资金项目(2010C91050);宁波市农业科技攻关项目(2010C10051,2010C10057);宁波大学学科项目(XKL121,XKL11D2099)

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龚一富,王何瑜,卢鹏,袁泉,王桂林,刘建军.长春花毛状根再生植株的获得及抗癌生物碱的产生[J].中草药,2012,43(4):788-794

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收稿日期: 2011-09-06