Control of gene expression by gene engineering technique and induction mechanism of the enzyme activity

1.烟草中L-半乳糖酸-1，4-内酯脱氢酶基因表达的调控在高等植物中，L-抗坏血酸（阿萨）生物合成的末端步骤由酶L-半乳糖酸-1，4-内酯脱氢酶（EC 1.3.2.3，GLDH）催化。我们通过PCR从烟草BY-2细胞中扩增GLDH cDNA，通过反义表达获得AsA缺陷型转基因烟草BY-2细胞系。对反义RNA表达显著的两个转基因细胞系AS 1 -1和AS 2 -2进行了分析。GLDH mRNA的反义抑制导致GLDH活性显著下降。发现转基因细胞系中的阿萨水平比野生型BY-2细胞低30%。在同步培养中，AS 1 -1和AS 2 -2细胞的分裂受到抑制，伴随着有丝分裂指数的下降，这可能是由于阿萨水平的下降。还发现细胞生长速率低于野生型细胞。AS 1 -1和AS 2 -2的愈伤组织粘软。显微镜下观察发现，AS 1 -1和AS 2 -2细胞异常细长。因此，我们观察到阿萨水平的下降对植物细胞的分裂、生长和结构有不利影响.甘薯伤后根和块茎中GLDH的诱导机制甘薯GLDH cDNA在大肠杆菌中得到高效表达。免疫家兔制备了抗GLDH蛋白的多克隆抗体。该抗体可识别马铃薯块茎和绿豆中的GLDH蛋白以及甘薯蛋白。伤后1 d内甘薯根和块茎中GLDH活性升高，随后组织中维生素C含量升高。GLDH活性和GLDH蛋白的增加模式相似，表明GLDH蛋白在创伤后重新合成，并导致GLDH活性增加。

1. Control of gene expression of L-galactono-1, 4-lactone dehydrogenase in tobacco.In higher plants, the terminal step of L-ascorbic acid (AsA) biosynthesis is catalyzed by the enzyme L-galactono-1, 4-lactone dehydrogenase (EC 1.3.2.3, GLDH). We generated AsA-deficient transgenic tobacco BY-2 cell lines by antisense expression of the GLDH cDNA that was amplified from BY-2 cells using PCR. Tow transgenic cell-lines, AS1-1 and AS2-2, having a marked expression of antisense RNA were analyzed. Antisense suppression of GLDH mRNA led to a significant decline in the GLDH activity. The AsA levels in the transgenic cell lines were found to be 30% lower than the wild-type BY-2 cells. In synchronous cultures, division of AS1-1 and AS2-2 cells was restrained with a concomitant decrease in mitotic index that was probably due to a decline in AsA levels. The rate of cell growth was also found to be less than that of the wild-type cells. The calli of AS1-1 and AS2-2 appeared to be sticky and soft. Microscopic analysis revealed that AS1-1 and AS2-2 cells were abnormally slender. Thus, we observed that decline in the AsA levels has an adverse effect on the division, growth and structure of a plant cell.2. Induction mechanism of GLDH in sweet potato root and potato tuber after wounding.cDNA of GLDH from sweet potato was over expressed in Escherichia coli. The specific polyclonal antibody against the GLDH protein was prepared by immunization of a rabbit. The antibody recognized GLDH protein from potato tuber and vigna mungo as well as the sweet potato protein. GLDH activity in sweet potato root and potato tuber increased in one-day after wounding and followed by the increase in the level of vitamin C in the tissue. Increased patterns of both GLDH activity and GLDH protein measured by western blotting were similar, suggesting that GLDH protein was synthesized de novo in response to wounding, and resulted in the increase in GLDH activity.