Analysis of molecular mechanisms of defense systems by ascorbate peroxidase against oxidative damages caused by excess light energy

抗坏血酸过氧化物酶防御过量光能引起的氧化损伤的分子机制分析

基本信息

批准号：
12660090
负责人：
SHIGEOKA Shigeru
金额：
$ 0.7万
依托单位：
Kinki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

Alternative splicing events in the 3'-terminal region of chloroplast ascorbate peroxidase (chlAPX) pre-mRNA in spinach and tobacco, which produced four types of mRNA variants, one form (tAPX-I) encoding thylakoid-bound APX (tAPX) and three forms (sAPX-I, -II, -III) encoding stromal APX (sAPX), were regulated in a tissue-specific manner. The ratio of the level of SAPX mRNAs (sAPX-I, -II, and -III) to tAPX-I mRNA was close to 1 in leaf, whereas the ratio in root was greatly elevated due to an increase in sAPX-III and a decrease in tAPX-I resulting from the alternative excision of intron 11 and intron 12, respectively. A putative splicing regulatory cis-element (SRE), which is highly conserved in the sequences of chlAPX genes of higher plants, was identified upstream of the acceptor site in intron 12. Gel-shift analysis showed that SRE interacts strongly with a nuclear protein from leaves, but not those from the roots of spinach and tobacco. These results indicate that the tissue-specific alternative splicing of chlAPX pre-mRNA is regulated by the splicing enhancer SRE.Transgenic tobacco plants (TpTAP-12) overexpressing thylakoid membrane-bound ascorbate peroixdase (tAPX) targeted to chloroplasts were generated. The tAPX activity in TbTAP-12 plants was approximately 37-fold higher than that of the wild-type. The abilities of CO_2 fixation and PSII activity in between wild-type and TpTAP-12 plants were not changed.The activities of thiol-modulated enzymes in the Calvin cycle, antioxidative enzymes, and levels of antioxidants were also same values in both plants. Transgenic tobacco plants -suppressing chloroplastic ascorbate peroixdase isoenzymes are generating using antisense technology.Five lines of those plants at T_1 generation were isolated.

Alternative splicing events in the 3'-terminal region of chloroplast ascorbate peroxidase (chlAPX) pre-mRNA in spinach and tobacco, which produced four types of mRNA variants, one form (tAPX-I) encoding thylakoid-bound APX (tAPX) and three forms (sAPX-I, -II, -III) encoding stromal APX (sAPX), were regulated以组织特异性的方式。 SAPX mRNA（SAPX-I，-II和-III）与TAPX-I mRNA的水平比叶片接近1，而root的比率大大升高，因为SAPX-III的增加，由于SAPX-III的增加，TAPX-I的降低是由于Intron 11 and Interon Intron Entron and Ontron Expiess and Intron 11和Interon 12和Interon 12和Interon 12的降低。假定的剪接调控顺式元素（SRE）在较高植物的Chlapx基因的序列中高度保守，在内含子12中的受体位点的上游鉴定出了12。凝胶移位分析表明，SRE与叶子中的核蛋白质相互作用，但没有来自叶子的核蛋白质，但没有来自菠菜和托巴科的根。这些结果表明，CHLAPX Pre-MRNA的组织特异性替代剪接受剪接增强剂SRE SRE。转运烟草植物（TPTAP-12）过表达的依克拉基膜结合的as虫抗焦油的perOixdase（TAPX）靶向靶向氯植物群。 TBTAP-12植物中的TAPX活性比野生型的TAPX活性高约37倍。野生型和TPTAP-12植物之间CO_2固定和PSII活性的能力没有改变。在加尔文周期中，硫醇调节的酶的活性，抗氧化酶，抗氧化剂的水平在两种植物中也相同。转基因烟草植物 - 抑制叶绿体抗坏血酸二糖酶同工酶正在使用反义技术产生。分离了T_1生成的这些植物的线。