【作者】 王文秀；

【导师】 杨洪全；

【作者基本信息】 上海交通大学 ， 生物学， 2016， 博士

【摘要】 光信号作为植物生长的重要的环境因子,不仅为植物生长提供能量来源,同时在植物生长过程中提供必要的信号来源。隐花色素(Cryptochrome,CRY)作为蓝光受体在植物和动物中介导多种光控反应。以拟南芥为模式植物的研究表明,CRY(CRY1和CRY2)的C端结构域(CCT1和CCT2)已经被证明是主要功能结构域,因为过量表达与β-葡糖苷酸酶(β-glucuronidase,GUS)融合的CCT1或CCT2的转基因植株(CCT1或CCT2)呈现出组成型光形态建成(Constitutive Photomorphogenesis,COP)的表型。另外,CCT1和CCT2参与CRY介导的蓝光信号通路是通过与COP1直接蛋白互作来完成的。CRY1的N端结构域(CNT1)的功能主要是介导CRY1的二聚化,而且这种二聚化是激活CRY1光受体活性所必需的。最新的研究发现,CNT1可以独立于CCT1参与CRY1介导的蓝光信号通路,并且发现CNT1参与CRY1介导的对赤霉素(gibberellin acids,GA)/油菜素内酯(brassinosteroids,BR)/生长素(auxin)响应基因表达的影响。虽然目前的研究已经表明CCT1和CNT1都参与了CRY1介导的蓝光信号通路,但是CCT1和CNT1在蓝光信号通路中功能的区别还未见报道。因此在全基因组范围分析CCT1的功能,并探索CCT1和CNT1在介导CRY1调控的蓝光信号通路的相同点和不同点至关重要。本研究对CCT1过量表达转基因植株进行了RNA-seq测序分析,并得到了6780个CCT1调控的基因。将得到的CCT1测序结果与之前得到的cry1cry2突变体和具有增强的蓝光反应的转基因植株35S::CNT1-NLS-YFP(CNT1)的RNA-seq结果进行比对。结果发现2903个(67.85%)CRY调控基因被CCT1调控,且其中的1095个基因也被CNT1调控。与GA/BR/auxin调控基因比对后发现CCT1与CNT1一起参与了CRY1介导的对激素响应基因表达的调控,而且大约有一半的GA/BR/auxin上调的基因被CCT1和CNT下调表达,这与CRY1和这些激素拮抗调控下胚轴伸长一致。另外,本研究发现在影响CRY1调控的激素响应基因时,CCT1和CNT1既有各自特异的也有它们共同调控的基因。qRT-PCR实验验证了转录组分析得到的一些已经报道的、参与调控下胚轴伸长的、而且被激素上调表达同时被CRY1、CCT1和CNT1下调的基因的表达情况。结果发现这些基因被蓝光、CRY1、CCT1和CNT1下调;而且在蓝光下,CCT1特异调控的基因(比如ACS5),在CCT1中的表达量比在CNT1中低;同样的,CNT1特异调控的基因(比如SAUR15),在蓝光照射下,CNT1中的表达量比CCT1中更低,与转录组分析结果一致。对CCT1和CNT1进行的GA敏感性实验发现CCT1和CNT1都参与了CRY1介导的抑制GA引起的下胚轴伸长。为了探明CCT1和CNT1抑制GA引起的下胚轴伸长的机制,我们进行了免疫印迹实验。结果发现是CCT1而不是CNT1参与了CRY1介导的抑制GA促进HY5蛋白降解过程的。为了在全基因组转录水平研究CCT1、CNT1和COP1调控激素响应基因表达的关系,我们还利用COP1的突变体cop1-4进行了RNA-seq测序分析,并将COP1调控的基因与CCT1、CNT1及GA/BR/auxin结果进行比对。结果发现CCT1、CNT1与COP1以相反的方向共同调控很多GA/BR/auxin响应基因表达。另外,我们还分析了蓝光和CRY1对BR信号通路及BR响应基因表达的影响。对BR的敏感性实验发现蓝光抑制BR对拟南芥下胚轴的促进是通过CRY1来实现的,而qRT-PCR实验也表明CRY1介导了蓝光对BR响应基因的抑制。本文通过转录组学,植物生理学及生物化学方法在全基因转录水平揭示了关于CRY1介导的蓝光信号通路机制,提出了CRY1的C端和N端可能参与了CRY1调控GA、BR或其它激素应答基因表达的生物学问题,同时为进一步研究CRY1信号与植物激素信号互作调控植物发育的机制研究提供了线索。更多还原

【Abstract】 Light not only provides plants with energy to maintain life,but also supplies plants with signal to regulate their developmental processes in the whole life cycle.Cryptochromes(CRY)are blue-light photoreceptors that mediate various light responses in plants and animals.The C-termini of CRY1 and CRY2(CCT1 and CCT2)are known to mediate CRY1 signaling,since transgenic plants expressing CCT1 or CCT2 fused to β-glucuronidase(GUS)(GUS-CCT1 or GUS-CCT2,abbreviated as CCT1 or CCT2)show a constitutively photomorphogenic phenotype.It has long been demonstrated that Arabidopsis CCT1 and CCT2 mediate light signaling through direct interaction with COP1.Most recently,CRY1 N terminus(CNT1)has been found to be involved in CRY1 signaling independent of CCT1,and implicated in the inhibition of gibberellin acids(GA)/brassinosteroids(BR)/auxin-responsive gene expression.Although it has been found that CNT1 and CCT1 are both involved in mediating CRY1 signaling,it is worth investigating how they are involved in regulating phytohormone-responsive gene expression at the transcriptomic level.Here,we performed RNA-Seq assay using CCT1,and compared the results with those obtained previously from cry1cry2 mutant and the transgenic plants expressing CNT1 fused to nuclear localization signal sequence(NLS)-tagged YFP(CNT1-NLS-YFP,abbreviated as CNT1),which display enhanced responsiveness to blue light.We found that 2,903(67.85%)of the CRY-regulated genes are regulated by CCT1 and that 1,095 of these CCT1-regulated genes are also regulated by CNT1.After annotating the gene functions,we found that CCT1 is involved in mediating CRY1 regulation of phytohormone-responsive genes,like CNT1,and that about half of the up-regulated genes by GA/BR/auxin are down-regulated by CCT1 and CNT1,consistent with the antagonistic role for CRY1 and these phytohormones in regulating hypocotyl elongation.qRT-PCR analyzes the genes that are relative to hypocotyl elongation and up-regulated by GA/BR/auxin,and found these genes were indeed down-regulated by blue light,CRY,CCT1 and CNT1.We further found that the identified genes specifically regulated by CCT1 indeed expressed at a lower level in CCT1 than in CNT1 seedlings exposed to blue light,such as ACS5.Similarly,the identified CNT1-specific genes expressed at a lower level in CNT1 than in CCT1 seedlings exposed to blue light,such as SAUR15.Physiological studies showed that both CCT1 and CNT1 are likely involved in mediating CRY1 reduction of seedlings sensitivity to GA under blue light.Furthermore,protein expression studies demonstrate that the inhibition of GA promotion of HY5 degradation by CRY1 is likely mediated by CCT1,but not by CNT1.To further examine the functional relevance of CCT1 and CNT1 with COP1,we also performed RNA-seq analysis of cop1 mutant and compared the genes,particularly those responsive to GA/BR/auxin,which are related to CCT1,CNT1 and COP1.We found that CCT1 and CNT1 co-regulate the expression of a large number of genes responsive to GA/BR/auxin with COP1 in an opposite direction.We also found that blue light inhibit seedlings sensitivity to BR in a CRY1 dependent manner,and qRT-PCR analyses reveal that the expression of BR response genes are also inhibited by CRY1.These results provide genome-wide transcriptome information concerning the signaling mechanism of CRY1,unraveling possible involvement of its C and N termini in its regulation of response of GA,BR and likely other phytohormones,and provide clue to further explore the molecular mechanisms of CRY1 signaling,which may be involve in regulation of phytohormone-responsive gene expression.更多还原