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)前体基因3‘末端的选择性剪接事件产生四种类型的mRNA变体，一种形式(tAPX-I)编码类囊体结合的APX(TAPX)，三种形式(sAPX-I，-II，-III)编码基质APX(SAPX)。在叶片中，SAPX-I、-II和-III与tAPX-I的mRNAs水平之比接近于1，而在根中，由于sAPX-III的增加和内含子11和12的交替切除导致tAPX-I的减少，SAPX-I的水平显著升高。在高等植物chlAPX基因序列的上游，发现了一个高度保守的剪接调控顺式元件(SRE)。凝胶漂移分析表明，SRE与来自叶片的核蛋白有很强的相互作用，而与菠菜和烟草根的核蛋白没有相互作用。这些结果表明，chlAPX Pre-mRNA的组织特异性选择性剪接受剪接增强子SRE的调控。获得了以叶绿体为靶标的高表达类囊体膜结合抗坏血酸过氧化酶(TAPX)的转基因烟草植株(TpTAP-12)。TbTAP-12植株的tAPX活性约为野生型的37倍。野生型和TpTAP-12植株的CO2固定能力和PSII活性没有变化，Calvin循环中的硫醇调节酶活性、抗氧化酶活性和抗氧化剂水平也是相同的。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植株。

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.