硅调控特定基因提高水稻修复UV-B伤害的分子机制研究

Depletion of the stratospheric ozone (03) layer due to the emission of chlorofluorocarbons and other trace gases has resulted in increased solar ultraviolet-B (UV-B: 280-315 nm) radiation on the earth's surface, which would increase DNA damage,and reduce aboveground biomass and plant height. These effects of UV-B radiation take serious influence on plant growth and development. The previous studies showed that silicon (Si) contributed to enhance gene expression of photolyase (PL), which can repair UV-B damage on rice plant, however, there are 4 PL genes in rice genome, and which PL gene presents high effciency on this process is still unknown. In order to find the specific PL gene and explore the underlying mechanism,the present research will use qRT-PCR to determinate the differential expression of PL genes, which aims to obtain the specific PL gene with high efficiency on repairing UV-B damage on rice.The promoter of the specific PL gene will be amplified to screen their interacted transcription factors by using Solid-phase DNAse I footprinting method, and then EMSA and western-blot technologies will be used to confirm the positive interactions between gene and transcription factors. Further,both RNAi and overexpression technologies will also be performed to investigate the positive function of the transcription factor and specific PL gene, in order to illuminate their ability or relationship on repairing DNA damage. According to study, the underlying molecular mechanism of silicon in regulation of rice repairing UV-B damage will be presented, and these findings will provide theoretical basis and technological support for enhancing rice UV-B tolerance in the cropland.

氯氟烃等有害气体的大量排放严重破坏了大气臭氧层，由此导致地表紫外线UV-B(280-315nm)辐射增强，使得水稻等作物的DNA受损，生长受抑制，生物量减少，严重影响其生长发育。前期研究表明硅营养能够增强水稻光裂解酶(PL)基因表达进而提高修复UV-B伤害的能力，但水稻中具有4个PL基因，其中哪个基因在此过程中更为高效还未知。据此，本研究拟通过qRT-PCR检测明确高效参与硅提高水稻修复UV-B伤害的PL基因，克隆其启动子，采用固相DNAseI足迹法获得该PL基因启动子的转录因子，并利用EMSA及Western blot技术验证转录因子与启动子的有效互作。在此基础上，应用RNAi及Overexpression技术，验证转录因子及该PL基因对提高水稻修复UV-B伤害的作用，从而揭示硅营养通过特定PL基因提高水稻修复UV-B伤害的分子机制，为利用此技术提高水稻抗UV-B辐射提供理论依据。

近年来，氯氟烃等有害气体的大量排放严重破坏了大气臭氧层，由此导致了地表紫外线B(UV-B, 280-315nm)辐射不断增强。UV-B辐射可使水稻叶片DNA受损，细胞活动受抑制，生物量减少，最终影响其生长发育。前期研究表明施硅能够提高水稻光裂解酶(photolyase, PL)基因表达而有利于修复DNA损伤。水稻基因组中含有多个编码PL的基因，其中哪个基因在此修复过程中更为高效还未知。本项目以野生型抗UV-B辐射水稻品种Lemont及其硅吸收基因(Low silicon gene 1, Lsi1)分别被RNAi和过量表达的转基因植株为材料，对UV-B辐射前后各供试水稻叶片的硅含量、环丁烷嘧啶二聚体(CPDs)和6-4光产物(6-4 PPs)的含量进行测定。研究结果显示，在UV-B辐射后，各供试水稻叶片中的硅含量、CPDs含量和6-4-PPs含量都显著提高，但在Lsi1过量表达的转基因水稻中的CPDs含量增加程度较小，而在Lsi1被RNA干扰的Lemont转基因水稻中的增加程度最大。进一步采用荧光定量PCR(qRT-PCR)对水稻叶片中的4个PL基因表达进行检测，结果发现PL2(Os10g0167600)基因在UV-B辐射下的Lsi1过量表达的转基因水稻中的上表达倍数最大。通过启动子DNA-pulldown的方法筛选与Os10g0167600基因启动子结合的转录因子，结果发现编码methyl-CpG binding domain containing protein (MeCP)的转录因子可与Os10g0167600启动子结合。亚细胞定位结果也显示，OsMeCP编码的结构域蛋白定位在水稻的细胞核中，进一步证实其转录调控作用。在此基础上，应用RNAi和Overexpression分别增强和抑制水稻中OsMeCP基因的表达，结果也显示，过量表达OsMeCP后水稻的Os10g0167600表达降低，其在UV-B辐射下的DNA损伤较野生型增加，而OsMeCP-RNAi则结果相反，进一步证明了OsMeCP对Os10g0167600的调控作用。上述研究结果深入揭示硅调控水稻PL基因提高DNA损伤修复能力的过程，为利用栽培调控提高水稻耐受UV-B辐射提供了理论依据。