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

菠菜和烟草叶绿体抗坏血酸过氧化物酶（chlAPX）前体mRNA 3 '端的选择性剪接事件以组织特异性方式受到调控，产生4种mRNA变体，即编码类囊体结合APX（tAPX）的tAPX-Ⅰ和编码基质APX（sAPX）的sAPX-Ⅰ、-Ⅱ、-Ⅲ。在叶片中，SAPXmRNA（sAPX-I，-II和-III）与tAPX-I mRNA的水平之比接近1，而在根中，该比率由于内含子11和内含子12的交替切除分别导致sAPX-III的增加和tAPX-I的减少而大大升高。在内含子12的受体位点上游发现了一个在高等植物chlAPX基因序列中高度保守的剪接调控顺式元件（SRE）。凝胶位移分析表明，SRE强烈相互作用的核蛋白从叶子，但不是那些从菠菜和烟草的根。这些结果表明，chlAPX前体mRNA的组织特异性选择性剪接受剪接增强子SRE的调控。TbTAP-12植物中的tAPX活性比野生型高约37倍。野生型和TpTAP-12植株的CO_2固定能力和PSII活性没有变化，卡尔文循环中巯基调节酶活性、抗氧化酶活性和抗氧化剂水平也没有变化。利用反义技术获得了抑制叶绿体抗坏血酸过氧化物酶同工酶的转基因烟草，并在T_1代获得了5个转基因烟草株系。

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.