【作者】 王慧梅；

【导师】 祖元刚；

【作者基本信息】 东北林业大学 ， 植物学， 2004， 博士

【摘要】 喜树是中国的特有树种，是长江流域以南各省（区）较为普遍的用材树种，从其体内提取的喜树碱是一种广谱抗癌和抗肿瘤药物。利用生物技术手段对这一药用植物和濒危物种进行组织培养，特别是通过遗传转化对其木材品质进行改良具有重要的意义。然而有关这方面对这一树种研究报道的较少，特别是以叶片外植体进行组织培养研究还未见报道。这一定程度上限制了对其进行遗传转化的研究。基于此，本文分别以腋芽增殖和愈伤组织再生途径建立了高频率的喜树再生体系。并对组培苗移栽适应过程中的光合生理特性与实生苗进行了对比研究。在此基础上，通过根癌农杆菌介导法将有关改良材质的纤维素合成前体—尿苷二磷酸葡萄糖代谢有关的尿苷二磷酸葡萄糖焦磷酸化酶基因（UGPase）导入喜树体内，通过目的基因的表达提高喜树的纤维素含量，解决我国造纸材资源紧缺问题，拓宽其利用范围和价值，是本论文研究的目的。主要结果如下： （1）确定最佳的喜树腋芽增殖的培养基：B5+BA1.0mg／l+suc30g／l+agar6g／l。再生频率达95％，平均每个腋芽增殖3.8个芽，经过两次继代培养，平均每个芽最多可增殖20个芽。 （2）确定叶片愈伤组织诱导和分化的最佳培养基：WPM+BA1.0mg／l+NAA0.2mg／l+suc30g／l+agar6g／l。愈伤组织诱导率为96％，分化率为70.3％。 （3）确立喜树的继代扩繁培养基：WPM+BA0.2-0.5mg／l+suc20g／l+agar6g／l和B5+BA0.2-0.5mg／l+suc20g／l+agar6g／l。 （4）确定喜树再生苗生根的最佳培养基：WPM+IBA0.5mg／l+20g／lsuc+agar6g／l。生根率为98％，根的平均数量为4-5条。 （5）组培苗的移栽成活率为90％以上，但是组培苗较实生苗的根系发育差，单位叶面积所对应根尖数目较少，致使叶片水分供应相对短缺，表现为组培苗气孔导度显著低于实生苗，并显著降低了组培苗的光合能力。 （6）建立了高效的喜树遗传转化体系：20mg／l卡那霉素为最佳的抗生素筛选浓度；500mg／l头孢霉素为抑菌剂最佳的抑菌浓度；叶片预培养时间为3天；菌液浓度为OD₆₀₀=0.1-0.5；侵染时间为5-10min；共培养时间为3天。经抗生素筛选得到的抗性植株通过PCR和Southern Blotting等系列实验检测，结果证明目的基因已完全整合到喜树的基因组中。遗传转化率为6％。更多还原

【Abstract】 Camptotheca acuminata Decaisne (Nyssaceae) is a tree species indigenous and unique to China. The secondary compound, camptothecin (CPT) from this species may sever to treat cancers, which made its worldwide fame. It is an important species for timber utilization in the regions of China southern to Changjiang River. But the poor timber quality limits its utilization in paper production. Therefore, gene transfer for improving timber quality of this species is significant since its increasing economic value. However, this kind of studies is limited, and to our knowledge, no reports on the propagation of this species by leaf explants and subsequent gene transfer are available until now. Moreover, the development of root system during the acclimation of rooted plantlet in green house as well as its influences on photosynthetic traits is also poor understood. This may influence the afforestration practices of the rooted plantlet in future. Under this background, present study was carried out. The aims of this study were to establish the high-frequency regeneration system and then to build a successful protocol for UGPase gene transformation system by Agrobacterium-mediated transformation. We found that,1. B5 medium supplemented with 3%(w/v)sucrose, 6%agar, l.Omg/lBA produced thehighest shoot regenerating rate, 95%. The average number of regenerated shoots was 3.8 per axillary bud. After two times of subculture, one axillary bud could successfully proliferate at most 20 shoots.2. The optimum medium for the callus induction and shoot regeneration from leaf derived from in vitro cultured individual was WPM medium supplemented with 1.0mg/l BA, 0.2mg/l NAA, 3%(w/v)sucrose and agar 6g/l. Callus induction rate was up to 96% and shoot regeneration rate was 70.3%.3. The optimal subculture medium for regenerated shoots were WPM or B5 mediumcontaining 2%(w/v)sucrose, 0.2-0.5 mg/1 BA and 6g/l agar .4. The optimum medium for rooting of the regenerated plantlets was WPM supplemented with 0.5mg/l IBA, 2%(w/v) sucrose and agar 6.0g/l. Root frequencies were 98% with high root number, 4 to 5.5. Survival rate of rooted plantlet was above 90%. Comparing to seedlings, root systemof rooted plantlet in soil was poorly developed. The roottips per unit leaf area were marked lower than that of seedlings, which may influence the supply of water. Thisroot status is demonstrated by the lower stomatal conductance and subsequent lower capacity in photosynthesis.6. The efficient system of genetic transformation was established. 20mg/l kanamycin was used in the shoot regeneration medium to select the resistant shoots. 500mg/l cefotaxine was used to kill the Agrobacteria after infection. Explants were pre-cultured for 3 days before infection. The pre-cultured explants were incubated for 5-10min in the bacterial suspension which concentration (OD600) was between 0.1 and 0.5. Length of co-cultivation period was 3 days. The target genes integrated into the genomic DNA of C. acuminata were detected and confirmed by PCR and Southern blotting analysis. Genetic transformation rate was 6%.更多还原