受UV-B诱导的水稻叶片早衰基因ups的分离克隆及其功能研究

Rice is one of the world's most important food crops because it is a staple food for more than half of the world's population. Deletion of stratospheric ozone due to anthropogenic sources such as chlorofluorocarbons has lead to an increase in solar ultraviolet-B radiation (315-280 nm) reaching the Earth's surface, which has potentially deleterious consequences for agricultural production and natural ecosystems. Increased UV-B radiation causes significant direct and indirect biological effects including degradation and conformational changes in DNA, protein and lipids and alterations in photosynthesis, growth and morphology of plants. Studies indicated that increased UV-B radiation caused a significant reduction in dry matter and 20% yield, and affected seed quality as follows: protein, sugar and starch levels decreased in rice. So it is very important to develop UV-B-resistant rice and demonstrate the mechanisms of UV-B resistance in rice..Our previous studies showed that the ups mutant had recessive inheritance and was governed by a single locus. The ups gene determined to be on the long arm of chromosome 3 and is 0.1 cM and 0.2 cM away, respectively from the closest InDel markers ID15 and ID 19, and co-segregated with the SSLP marker RM3646. Further analysis revealed that this 90-kb sequence contains 12 putative genes including two cytochrome P450 genes. Considering that cytochrome P450s are ubiquitous heme proteins and known to play an important role in detoxification of natural and involve in biosynthesis of secondary metabolites such as flavonoid which is very important compounds for UV-B protectants in plants. We therefore considered those two cytochrome P450 genes as the primary candidates for the wild Ups gene.To address which of the two candidate genes corresponds to the ups locus present in the mutant, we compared the sequences of the two genes based on PCR products mplified from the mutant and the respective wild-type progenitors 93-11. Comparing ups to 93-11, it was observed that among the two genes, only one gene from ups mutant a single-base deletion of T in its coding sequence..In this study, we will clone the candidate gene and investigate its function and expression analysis, and reveal the molecular and physiological mechanism, and develop the UV-B resistance rice in the future. Furthermore, we also investigate its regulated network through yeast two-hybrid, microarray and widespread RNA Sequence

水稻是最重要的粮食作物之一，在粮食生产和消费中占有极其重要的地位。工业化的高速推进致使臭氧层不断变薄，到达地面的紫外辐射增强。研究显示，增强5%的UV-B将使水稻减产20%以上以及稻米品质将显著下降。因此，重视臭氧层变薄带来的各种生物学效应，揭示紫外线影响水稻叶片早衰的遗传分子机理，培育耐(抗)紫外线新材料,对于提高水稻产量，保障水稻生产的可持续发展及粮食安全具有重要意义。在前期的研究中，申请人以辐射诱变获得的ups早衰突变体为材料，已经将该隐性基因精细定位在第3染色体长臂上，测序分析表明在一个细胞色素P450基因的血红蛋白结合区发生了单碱基缺失，致使该基因功能丧失。本研究将在克隆该候选基因的基础上，分析其表达模式，弄清基本分子生理特征，阐明紫外线引起叶片衰老的遗传机制。此外，利用基因芯片技术、转录组高通量测序技术并结合酵母双杂，获得与该基因互作的关键基因并进行功能研究，确定其调控网络。

水稻是全球最重要的粮食作物之一，为世界50%的人口提供口粮。叶片是水稻进行光合作用的最重要器官，其功能叶片的早衰将直接影响水稻产量及其品质。一般认为，紫外线与极端温度是影响叶片衰老重要外因，而ROS累积、内源激素以及基因表达变化是影响叶片衰老重要内因。研究显示，每增加5%的UV-B将使水稻减产20%以上。因此，研究不同光质尤其是紫外线对叶片衰老的影响，将有助于揭示自然光影响水稻生长发育的分子生理机理，为培育延缓叶片衰老的水稻新材料奠定基础，对保障我国粮食安全具有重要意义。本研究将利用图位克隆技术分离克隆调控突变体ups/ospls4和ospls2叶片早衰基因，研究其调控网络及导致叶片早衰的主要原因，揭示其分子生理机理。结果显示：1）利用基因互补及RNAi抑制技术，证实UPS/OsPLS4基因(LOC_Os03g39650)就是控制突变体ups/ospls4叶片早衰的基因；2）扫描电镜显示，突变体ups/ospls4的叶表面刺毛明显更多，而透射电镜显示，抽穗初期突变体ups/ospls4叶绿体已开始降解；3）由于叶绿体的降解及ROS清除酶酶活降低，如SOD和CAT明显降低，导致突变体ups/ospls4叶片中ROS明显累积，且突变体ups/ospls4对外源H2O2更敏感；4）外源JA和SA显著抑制突变体ups/ospls4植株的生长；5）RNA-Seq显示，突变体ups/ospls4中与光合作用相关上调基因有48，下调基因有35个；6）UV-B明显促进突变体ups/ospls4离体叶片早衰；7）利用图位克隆技术，定位克隆了调控突变体ospls2叶片早衰基因LOC_Os03g38990，蛋白分析表明其编码一个DNA/RNA解旋酶，进一步分析表明该突变体对自然光敏感，且导致叶片衰老的主因是叶片中ROS累积。上述研究结果将有助于揭示不同光质调控水稻叶片衰老的分子生理机理。