鱼腥草离体再生及农杆菌介导的遗传转化

In-Vitro Culture and the Agrobacterium-mediated Genetic Transformation of Houttuynia cordata Thunb.

【目的】建立鱼腥草离体再生系统和农杆菌介导的遗传转化方法。【方法】以鱼腥草叶片为外植体置于不同激素配比的MS培养基上,进行愈伤组织诱导和分化再生。以根癌农杆菌EHA105/pCAMB IA1305.2进行转化,将外源GUS(β-葡萄糖醛糖苷酶)基因、潮霉素抗性基因转入鱼腥草,以组织化学染色法检测GUS活性,以聚合酶链反应(PCR)法鉴定基因组中的GUS基因。【结果】MS培养基中添加0.1 mg/Lα-奈乙酸(NAA)、2.0 mg/L 6-苄基腺嘌呤(6-BA)、0.5 mg/L呋喃氨基嘌呤(KT)适于以鱼腥草叶片为外植体进行离体再生,出芽率达96.7%。潮霉素作为筛选试剂,使用浓度为2.5 mg/L;头孢霉素作为抑菌剂,使用浓度为250 mg/L。经感染的外植体中GUS反应呈阳性的占75.0%,8周统计抗性芽比率5.5%,PCR分析表明GUS基因已整合到鱼腥草基因组中。【结论】通过鱼腥草离体培养条件的研究及根癌农杆菌介导的遗传转化,获得了较高的离体再生率,实现了鱼腥草的遗传转化。

Objective To establish a plantlet regeneration system and an effective system for the Agrobacteriummediated genetic transformation of Houttuynia cordata Thunb. Methods The leaf blades of Houttuynia cordata Thunb. were used as the explants, and were cultured in MS medium with different somatotropins and cytokinins added to induce callus and plantlet regeneration. Agrobacterium tumefaciens strain EHA105, which contained vector pCAMBIA1305.2 carrying β-glucuronidase reporter gene （GUS） and hygromycin B phosphotransferase, was transformed into Houttuynia cordata Thunb. The expression of GUS gene in the transformed leaf blades was detected by histochemical assay. Presence of GUS was confirmed by genomic polymerase chain reaction （PCR） analysis. Results MS medium with α-naphthaleneacetic acid （NAA） 0. 1 mg/L, 6-benzyladenine （6-BA） 2.0 mg/L, and kinetin （KT） 0.5 mg/L added was effective for callus formation and plantlet regeneration, and the shoot formation rate was 96.7 %. The optimal concentration of hygromycin B for selection was 2.5 mg/L, and the optimal concentration of cefotaxime for inhibiting EHA105 was 250 mg/L. The histochemical localization of GUS and the genomic PCR analysis both confirmed the successful transformation of GUS into Houttuynia cordata Thunb. The expression of GUS gene being positive accounted for 75.0% , and the resistant-shoot regeneration rate was 5.5% of explants after culturing for 8 weeks. Conclusion Plant regeneration system and Agrobacterium- mediated genetic transformation for Houttuynia cordata Thunb. have been established successfully.