【作者】 刘金灵；

【导师】 王国梁； 戴良英； 刘雄伦；

【作者基本信息】 湖南农业大学 ， 作物遗传育种， 2009， 硕士

【摘要】 水稻假病斑突变体spl11(spotted leaf 11)来源于籼稻IR68 EMS处理后的突变体,受一对隐性基因控制。其突变体表现出组成型细胞坏死和对稻瘟病菌和白叶枯病菌的非小种特异性抗性,采用图位克隆,Spl11基因已被成功分离,其编码一个新的U-box/ARM重复蛋白,具有泛素蛋白连接酶E3活性,为植物细胞死亡和防御反应的负调控因子,表明SPL11很有可能是通过参与泛素蛋白降解途径来调节的植物细胞死亡和防御反应。最近的研究表明Spl11还作为一个水稻开花调节的正调节因子参与着水稻光周期介导的开花。为了进一步研究和分析SPL11介导的植物细胞程序化死亡,防御反应和开花调节信号机制。通过采用酵母双杂筛选,获得8个SPL11互作蛋白(SPL11 interacting proteins,SPINs),并分别命名为SPIN1-8,以期通过分析这些候选互作因子的功能来进一步认识SPL11的调控途径。其中,SPIN1编码一个RNA/DNA结合蛋白,功能分析表明SPIN1是一个水稻开花的负调节因子,SPL11通过负调节SPIN1来促进水稻开花。本研究将报道另一个SPL11互作基因Spin6的功能,主要研究结果如下:1.Spin6编码一个Rho GTPase激活蛋白,表明SPIN6很有可能是通过调节GDP和GTP交换循环来参与SPL11介导的信号调节。2.SPIN6亚细胞定位于整个水稻原生质体细胞,与SPL11具有同样的定位;3.GST-pull down离体(in-vitro)互作和水稻原生质体BiFC(in-vivo)互作分析结果表明,SPIN6和SPL11在离体和原位条件下都是互作的;4.对Spin6 RNAi转基因株系的表型分析,发现Spin6 RNAi导致了水稻细胞死亡的形成,同时,对Spin6 RNAi植株中Spl11表达水平分析表明,Spl11的表达受到了明显的抑制,这与Spl11突变导致了水稻细胞死亡的结果一致,表明SPIN6,与SPL11一样,是一个植物细胞死亡调节的负调控因子。5.Spin6 RNAi株系同时还表现出在短日照和长日照条件下的晚花表型,表明SPIN6是一个水稻光周期介导开花调节的正调节因子。6.Spin6 RNAi株系表现出矮化,结实率低等表型,这可能是Spin6 RNAi植株细胞死亡后导致的连锁反应的结果。7.Spin6 RNAi导致了SPL11表达的组成型的下调,而SPIN1却表现出与野生型相反的生物节律表达型,表明SPIN6很有可能正调节SPL11的表达进而抑制SPIN1的表达来调节下游信号的。8.对Spin6 RNAi株系中光周期开花调节相关基因表达分析表明,在短日照条件下,Spin6 RNAi抑制了Hd1/Hd3a介导的开花调节途径,而在长日照条件下,Hd1的表达则没有受到影响,但水稻开花调节的关键基因Hd3a的表达则受到明显地抑制,这表明长日照条件下SPIN6/SPL11介导的开花途径可能是通过调节另外一个未知因子进而调节Hd3a而实现的。综上所述,本研究表明Rho GTPase激活蛋白编码基因Spin6以物理互作的方式实现对Spl11的正调节,进而实现对水稻细胞死亡的负调控和对水稻光周期介导开花的正调节作用。更多还原

【Abstract】 The rice lesion mimic mutant spl11(spotted leaf 11) is controlled by a pair of recessive gene orginated from EMS treated IR68 mutants,spl11 mutant displayed a phenotye with spontaneous cell death and also enhanced non-race specific resistance to both rice blast fungus Magnaporthe grisea and bacterial blight Xoo.To better understand the molecular mechanism of SPL11-mediated cell death and defense response,the Spl11 gene was cloned by map-base cloning strategy.Spl11 encodes an U-box/ARM repeat containing protein and is a novel ubiquitin E3 ligase,suggesting that spl11 possibly participates in ubiquination-mediated protein modification to negatively regulate plant PCD and defense.Recently,Spl11 was also found to be a positive regulator for rice photoperiodic flowering.One of eight SPINs(SPL11 interacting proteins),that we identified in a yeast two-hybrid screening,SPIN1 plays a negative role in SPL11-mediated rice photperiodic flowering controling through physically interacting with SPL11 and negatively regualted by SPL11.Here,we reported that SPIN6,encoding a Rho GTPase-activating protein and also interacting with SPL11,plays multiple roles in negatively regulating plant programmed cell death,plant architecture and positively controlling photoperiodic flowering time in rice.The main results are showed as below:SPIN6 encodes a Rho GTPase-activating protein,suggesting a possible role in small GTPase protein mediated exchange between GDP and GTP.The subcelluar localization experiment showed that SPIN6 is a whole cellualr localized protein in rice protoplast with the same localization as SPL11.We aslo confirmed the in-vitro and in-vivo interaction through GST-pull down and protoplast BiFC interaction assay.Our phenotype evaluation for Spin6 RNAi lines showed that(ⅰ) Knock-down of SPIN6 leaded cell death formation; (ⅱ) Knock-down of Spin6 RNAi also displayed delayed flowering time phenotype under both long day and short day conditions.(ⅲ) As a linkage consquence companied with Spin6 RNAi cell death phenotype,the Spin6 RNAi lines also showed that an obvious dwarf and low seed seeting rate phenotype.(ⅳ) Spin6 RNAi lines also enhanced resistance to two Xoo races J12 and J22,but did not show significant phenotype to rice blast fungus.To futher understand the mechanisms of SPIN6/SPL11-mediated cell death and flowering regulation.We found that knock-down of Spin6 down-regulated Spl11 expression at transcriptional level,that suggests that SPIN6 is a positive regulator of SPL11,the down-regulated Spl11 should be the main cause of the cell death phenotype of Spin6 RNAi lines.We aslo found that SPIN1 displayed reverse circadian clock expression pattern in Spin6 RNAi lines comparing with wild type,suggesting that SPIN6 assocaited with SPL11 negatively regulates SPIN1 to controll rice flowering time.In addition,our data also showed that in short day condition the Hd1-mediated pathways was suppressed, however,although Hd1 expression was not affected in long day condition,Hd3a was significantly inhibited that is consisted with the delayed flowering time phenotype and the results of over-expression SPIN1 in long day conditon.Therefore,we speculated that there is still an unknown factor involved in this pathway,which is need futher investigation. Taking together,our data suggests that SPIN6 associated with SPL11 negatively regulates rice cell death but positively controls rice photoperiodic flowering time.更多还原