The Establishment of the Gene Transfer System of Higher Plants Using an Auxotrophic Mutant.

测定了大肠杆菌K-12硫胺素合成酶thiB基因的核苷酸序列。分析使我们能够分配的转录控制的网站，并推导出的酶的氨基酸序列。因此，细菌控制区被删除，并用花椰菜花叶病毒35 S RNA启动子取代，该启动子已显示出作为植物启动子的功能。将得到的携带融合体（35 S启动子和thiB编码区）的质粒导入根癌农杆菌（Agrobacterium tumefasciens），其用于感染thi 1拟南芥（Arabidopsis thalina）外植体。首先利用质粒中的潮霉素磷酸转移酶基因进行筛选，筛选出具有潮霉素抗性的转化子。在添加有硫胺素的平板中，生长潮霉素抗性愈伤组织并再生芽。然后，建立了抗潮霉素的转基因植株。将转基因植株自花授粉，获得转基因植株的下一代（T2代）。当T2种子播种在没有添加硫胺素的植物上时，约75%的植物生长良好，25%的植物表现出坏死和死亡。因此，携带CaMV 35 S启动子和thiB序列的融合基因恢复了拟南芥thi 1突变的缺陷。这些转基因植株的生长表现出比添加硫胺素的thi 1突变体的生长稍差，显示出与E.大肠杆菌酶。该thiB基因现在被操纵以成为高等植物转化的选择标记。

The nucleotide sequence was determined of the Escherichaia coli K-12 thiB gene coding for thiamine synthetic enzyme, thiamine-phosphate pyrophosphorylase. The analysis permitted us to assign the site of transcriptional control and to deduce the amino acid sequence of the enzyme. Thus, the bacterial controlling region was deleted and replaced with the Cauliflower Mosaic Virus 35S RNA promoter, which has been shown to function as a plant promoter. The resulting plasmid carrying the fusion (35S promoter and thiB coding region) was introduced into Agrobacterium tumefasciens, which was used for the infection of explants of thi1 Arabidopsis thalina. At first, the gene of the hygromycin phosphotransferase in the plasmid was used for selection and trasformants carrying hygromycin resistant character were selected. In plates with the addition of thiamine, hygromycin-resistant calli were grown and shoots were regenerated. Then, transgenic plants were established carrying hygromycin-resitant trait. These plants of transgenic generation (T1 generation) were self-pollinated and we got seeds of next generation (T2 generation). When T2 seeds were sown on plants without the addition of thiamine, about 75% plants grew happily and 25% showed necrosis and died. Accordingly, the fused gene carrying CaMV 35S promoter and thiB sequence recovered the defect of thi1 mutati on of Arabidopsis thaliana. The growth of these transgenic plants were shown to be a little poorer than that of thi1 mutants growt with the addition of thiamine, showing the difference of E. coli enzyme. This thiB gene is now manipulated to become a selectable marker of transformations of higher plants.