Approaches with Arabidopsis thaliana Mutants to the Regulatory Genes for Photosynthesis

A.拟南芥最适合从分子遗传学的角度来深入研究其转录调控机制.筛选光合突变体的模式试验A.即使在存在光合作用电子传递抑制剂3-（3，4-二氯苯基）-1，1-二甲基脲（DCMU）的情况下，拟南芥也在补充有葡萄糖或蔗糖的Gamborg B5琼脂培养基上生长，尽管在该条件下没有糖源它不能生长。由此产生的A.在抑制光合活性的条件下生长的拟南芥，在长波紫外光照射下产生较高的红色荧光.用报告基因和自杀基因对拟南芥进行诱变转化，尝试将P-葡萄糖醛酸酶（GUS）基因和氯霉素乙酰转移酶（CAT）基因作为报告基因，以及乙醇脱氢酶（ADH）基因作为自杀基因导入拟南芥。在用甲基磺酸乙酯（EMS）诱变之前，这一战略可能会使 ...更多信息 使目标突变体的筛选与高红色荧光筛选相结合，变得容易。本论文主要研究了核酮糖1，5 -二磷酸羧化酶/加氧酶小亚基（RuBisCO，rbcS-3B）和叶绿素a/结合蛋白（cabl）的基因成员。聚合酶链反应（PCR）使用与3 '-5'校正核酸外切酶活性相关的热稳定DNA聚合酶，快速获得覆盖rbcS-38和cab 1的启动子及其上游区域的约1.5-kbp的DNA片段，因为这些基因的核苷酸序列已经公布。从A.拟南芥DNA将具有rbcS-3B和cab 1启动子和调节活性的片段置于GUS、CAT和ADH基因的上游，并试图导入A. thaliana（哥伦比亚和本斯海姆，adhl.转基因A.用EMS诱变拟南芥，筛选在绿色叶中较少表达rbcS-3B或cabl的突变体。少

A. thaliana is most suitable for handling in the aspect of molecular generics to thoroughly Investigate mechanisms goveming the transcriptional regulation.1. A Model Experiment for Screening Photosynthetic MutantsA. thaliana grew on the agar medium of Gamborg B5 in supplementation with glucose or sucrose even in the presence of the inhibitor of photosynthetic electron transport, 3-(3, 4-dich ; orophenyQ-1, 1-dimethylurea(DCMU), although it did not grow without sugar sources under that condition. The resulting A. thaliana grown with inhibited level of photosynthetic activity, generated high red fluorescence upon exposure to a long wave UV light.2. Mutagenesis of A thaliana transformed with Reporter and Suicide GonesGenes for P-glucuronidase(GUS)and chloramphenicol acetyltransferase(CAT)as reporter genes, as well as a gene for alcohol dehydrogenase(ADH)as a suicide gene, were tried to introduce into A. thaliana prior to mutagenesis with ethylmethanesuffonate(EMS). This strategy may promi … More se to make the selection of objective mutants easily in combination with screening by high red fluorescence. We have focused on members of genes for small subunit of ribulose 1, 5 -bisphosphate carboxylase/oxygenase(RuBisCO, rbcS-3B)and chlorophyll a/binding proteins(cabl). Polymerase chain reaction(PCR)with thermostable DNA polymerases associated with a 3'-5' proofreading exonuclease activity, was used to quickly obtain approximately 1.5-kilobase pair(kbp)DNA fragments covering promoters and their upstream regions of rbcS-38and cabl, as nucleotide sequences of these genes have been published. A gene for ADH(approximately 2.2 kbp)was similarly amplilied from A. thaliana DNA. The fragments possessing the promoter and regulatory activities of rbcS-3B and cabl were-placed upstream of genes for GUS, CAT, and ADH, and tried to introduce Into A. thaliana(Columbia and Bensheim, adhl. The transgenic A. thaliana is mutagenized with EMS, and mutants which less express rbcS-3B or cabl in green leaves is being screened. Less