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

测定了大肠杆菌编码硫胺素合成酶、硫胺素-磷酸焦磷酸化酶的k - 12thib基因的核苷酸序列。分析使我们能够确定转录控制位点并推断酶的氨基酸序列。因此，细菌控制区被删除，取而代之的是花椰菜花叶病毒35S RNA启动子，该启动子已被证明具有植物启动子的功能。将携带35S启动子和thb编码区的融合质粒导入根筋膜农杆菌，用于侵染th1拟南芥外植体。首先利用质粒中潮霉素磷酸转移酶基因进行筛选，筛选出携带潮霉素耐药性状的变形子。在添加硫胺素的培养皿中，生长出抗潮霉素愈伤组织，并再生出芽。然后，建立了携带抗潮霉素性状的转基因植株。这些转基因植株（T1代）自花授粉，我们得到了下一代（T2代）的种子。在不添加硫胺素的植株上播种T2种子时，约75%的植株生长良好，25%的植株出现坏死和死亡。因此，携带CaMV 35S启动子和thiB序列的融合基因恢复了拟南芥thi1突变的缺陷。这些转基因植株的生长比添加了硫胺素的thi1突变体稍差，说明了大肠杆菌酶的差异。这个thiB基因现在被操纵成为高等植物转化的一个可选择的标记。

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.