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Genetic Diversity and Alkaloid Biosynthesis in Somatic Hybrids of Duboisia and Nicotiana Tabacum

杜波伊西亚和烟草体细胞杂交种的遗传多样性和生物碱生物合成

基本信息

批准号：
63470142
负责人：
YAMADA Yasuyuki
金额：
$ 1.98万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1990
项目状态：
已结题

项目摘要

The genetic instability and alkaloid biosynthesis of hybrid cell lines, Duboisia B+Nicotiana tabacum, obtained by mechanical isolation of a single hybrid cell was studied.1) Ten subclones of calli derived from a single hybrid cell, Duboisia hopwoodii+Nicotiana tabacum were cultured for 3 years, and their genetic makeup clarified as to nuclear DNA content, chromosome constitution, and peroxidase isozymes. Nuclear DNA content differed in each subclone. In most subclones, mean DNA content was lower than the mean DNA content in the original hybrid cell line determined 1 year after fusion. This decrease in DNA content is partly attributable to the elimination of tobacco chromosomes that occurred in all subclones. The extent to witch tobacco chromosomes were eliminated varied among the subclones evidence that chromosome elimination occurred slowly. Peroxidase isozyme analysis indicated the loss of a tobacco-specific isozyme, thus confirming results obtained by chromosome analysis. Shoots regenerated from two hybird subclones after 2 years were also heterogeneous in morphology and nuclear DNA content.2) Somatic hybrids of Duboisia leichhardtii and Nicotiana tabacum were used to study the alkaloid bio-synthesis. The hybrid nature of the cloned cells and regenerated shoots was confirmed by cytological investigation and ribosomal-DNA analysis, respectively. The hybrid plantlets predominantly produced nicotine, while Duboisia plantlets produced both tropane and nicotine alkaloids. Activities involved in tropane-alkaloid biosynthesis were examined in a series of precursor-feeding experiments. The presence in the hybrid plants of activities responsible for the reduction of tropinone, the hydroxylation and epoxidation of hyoscyamine, and the conversion of nicotine to nonnicotine demonstrated the presence of the Duboisia genes for these enzyme activities.

对机械分离获得的杂交细胞系Duboisia B+Nictiana tabacum的遗传不稳定性和生物碱的生物合成进行了研究。1)对由Duboisia hop dii+Nictiana tabacum产生的10个愈伤组织亚克隆进行了3年的培养，并对它们的核DNA含量、染色体组成和过氧化物酶同工酶的遗传组成进行了阐明。各亚克隆的核DNA含量不同。在大多数亚克隆中，平均DNA含量低于融合后1年所测定的原始杂交细胞系的平均DNA含量。DNA含量的下降部分归因于所有亚无性系中发生的烟草染色体的消除。不同亚无性系的染色体消失程度不同，说明染色体消失的速度较慢。过氧化物酶同工酶分析表明，烟草特有的同工酶缺失，从而证实了染色体分析的结果。两个杂交亚无性系2年后再生的不定芽在形态和核DNA含量上也是不同的。2)以杜鹃和烟草的体细胞杂种为材料，研究其生物碱的生物合成。细胞学和核糖体DNA分析分别证实了克隆细胞和再生芽的杂种特性。杂交植株主要产生尼古丁，而杜波依斯植株同时产生托烷和尼古丁生物碱。在一系列前体喂养实验中检测了托烷生物碱生物合成的活性。在杂交植物中，负责托品酮还原、山梨胺羟化和环氧化以及尼古丁转化为降烟碱的活性的存在证明了这些酶活性的Duboisia基因的存在。