水稻条纹斑马叶基因STZL的克隆与功能分析

Leaf color marker technology is one way to improve the hybrid rice seed purity, to prevent significant loss that may be caused to the production. We found in a stripe-zebra (stzl) mutant in Shennong 265 groups, and completed the morphological observation, mutant traits genetic analysis. The results show that the mutational characters were controlled by a single recessive nuclear gene (stzl), and a uroporphyrinogen Ⅲ Synthase was identified candidate genes for traits by fine mapping and sequencing analysis. On the basis of this, we will finish the cloning of the STZL gene. To confirm whether the candidate gene was in fact STZL, we will carry out a genetic complementation test and RNA interference test. The STZL expression pattern will be analyzed by quantitative RT-PCR, GUS fusion gene and GFP gene. The interacting protein with STZL is obtained by yeast two-hybrid system screening. The content of chlorophyll synthesis precursors, ultrastructure of chloroplast in leaf blade, agronomic characters and physiological and biochemical characteristics analysis will be done in order to further reveal the STZL gene function and mechanism of action. This study can not only deepen the people to molecular mechanism of the chlorophyll metabolism, and there is a provided marker gene with important application value for the hybrid rice breeding.

叶色标记可作为提高杂交水稻的种子纯度的途径之一，防范可能给生产造成的重大损失。我们在沈农265的群体中发现了一个条纹斑马突变体stzl (stripe zebra leaf)，遗传分析表明，突变性状由隐性单基因控制，并且通过精细定位和测序分析将一个尿卟啉原Ⅲ合酶确定为该性状的候选基因。本研究拟在现有基础上，通过功能互补实验验证STZL的功能；通过转基因及原位杂交等技术研究STZL基因的表达模式；利用RNA-seq技术鉴定STZL的下游候选基因；利用酵母双杂交系统筛选与STZL相互作用的蛋白；并通过叶绿素合成相关中间产物测定，酶学实验，叶绿体显微结构观察和生理特性分析等，了解该基因的功能。本研究将全面阐述STZL基因在水稻叶色遗传发育与形态建成中的调控机理，同时为叶色标记基因在杂交水稻育种中的应用提供理论参考。

叶片是水稻进行光合作用的主要器官，叶色突变不仅直接影响光合作用，还将影响水稻的产量和品质。该项目以条纹斑马突变体stzl为材料，遗传分析发现该突变表型受隐性单基因控制。我们利用图位克隆的方法克隆了STZL，该基因编码镁离子螯合酶H亚基，是四吡咯生物合成途径中叶绿素合成过程中的关键酶，突变体基因主要在胆色素原合酶至原卟啉Ⅸ的合成过程中起作用。电镜观察发现，突变体叶片的黄色部分的叶绿体数目相对较少，且叶绿体内类囊体膜呈现纵向排列，堆叠较少，淀粉粒饱满度较差。突变体花粉粒充实较差，导致其结实率显著低于野生型。通过对突变体和野生型气体交换参数和叶绿素荧光特征的分析表明，突变体stzl的黄叶部分由于非气孔因素导致CO2同化速率显著降低。突变体stzl的黄叶部分PSII天线色素对光能的吸收传递能力高于野生型，但由于突变体叶绿素含量的降低，导致单位面积对光能的吸收低于野生型，同时也导致了突变体需要通过增加热耗散的方式消耗掉多余的光能。基因的调控网络还在进一步研究。