【作者】 郝亚琦；

【导师】 梁宗锁；

【作者基本信息】 西北农林科技大学 ， 药用植物学， 2012， 博士

【摘要】 油菜素甾醇类物质（Brassinosteroids）是继生长素，细胞分裂素，乙烯，赤霉素和脱落酸之后被广泛认定的第六大类植物内源激素，具有促进植物生长发育，细胞伸长和分裂，增强作物抗逆以及提高产量等作用。近年来，研究人员对油菜素内酯的重要功能进行了大量研究，尤其是在信号转导途径方面。但BZR1（BRASSINAZOLE-RESISTANT1）作为油菜素内酯信号通路中的关键转录因子，如何调控下游响应基因的表达目前还尚不清楚。所以，对BZR1靶基因的功能分析对于理解BR下游的信号通路以及阐明其与其他信号通路之间的关系有着很重要的意义。本研究通过对BZR1的靶基因进行初步筛选，将其克隆至含有35S启动子的载体中进行过表达，然后对转基因植株进行表型鉴定，由此，筛选到两个过表达植株具有明显表型的基因：庇荫响应的负调控因子PAR1（PHYTOCHROME RAPIDLYREGULATED1）以及一个功能未知的基因（暂命名为QQ1）。PAR1属于HLH转录因子家族，由于缺乏经典的DNA结合区域，所以被认为可能通过与能够结合DNA的转录因子形成异源二聚体，进而来调控基因的表达，但目前还未发现与PAR1相互作用的转录因子。本文利用酵母双杂交系统对可能与其相互作用的基因进行初步确认，并运用DNA pull down实验和染色质免疫共沉淀等实验对基因进行了体内以及体外验证，分析其机理，取得的主要研究结果如下：1.通过将候选靶基因构建到带有MYC标签的35s过表达载体中，筛选到两个具有明显表型的基因PAR1和QQ1。与野生型Col-0相比，PAR1，QQ1基因的过表达植株均出现植株矮小，叶柄缩短，颜色深绿等表型。2. PAR1与光敏色素相互作用因子PIF4在体内和体外均能相互作用。与野生型Col-0相比，PAR1OX转基因植株对光超敏感，增加光强，转基因植株的下胚轴缩短的更为明显。光照可明显增加PAR1的蛋白量，且这种增加量对红光，远红光和蓝光均有所响应，表明PAR1蛋白量的增加被多种光受体所调控。3.在qRT-PCR实验中，PAR1抑制PIF4的靶基因，与野生型Col-0相比，HFR1，PIL1和IAA29基因的表达在PAR1OX中较低。运用DNA pull－down实验以及染色质免疫共沉淀技术在体外和体内均证明了PAR1抑制PIF4结合DNA。将T1代的PAR1OX/PIF4OX双转基因植株与PAR1OX以及PIF4OX转基因植株进行比较，PIF4OX促进下胚轴的伸长，PAR1OX部分抑制PIF4OX下胚轴的伸长，表明PIF4在PAR1OX转基因植株中的活性受到抑制。同时，PAR1抑制PIF4所调节的部分生理响应，表现在PAR1OX转基因植株对赤霉素和高温响应不敏感，这与pifq的四突变体（缺失PIF1，PIF3，PIF4以及PIF5）相似，即下胚轴的伸长受到抑制，均不敏感。4.用酵母双杂交系统筛选与PAR1相互作用的因子时，除了PIF4，另外还发现PRE1也与PAR1相互作用，同时双分子荧光互补实验也证明了PAR1与PRE1的相互作用。PRE1属于bHLH家族，受油菜素内酯以及赤霉素的正调控。将PAR1OX与PREOX进行杂交，F1代转基因植株完全显示出PREOX的表型，即叶柄伸长，叶子伸展等，说明PRE1抑制了PAR1OX矮小的表型。以上的结果预示着PAR1-PRE1和PAR-PIF4可能形成HLH/bHLH的异源二聚体来共同响应光和植物激素，进而调控细胞伸长和植物的生长发育。5.此外，还筛选到另外一个过表达植株具有明显表型的BZR1靶基因，命名为QQ1，经过序列比对分析得出QQ1属于HLH家族，不含有DNA结合区域，与AIF1和PAR1位于较近的分支点上，亲缘关系较为密切。对其进行初步的研究后发现，QQ1可以和PRE1在酵母双杂交系统中相互作用，而且短时间的光照能很显著的增加其蛋白量，BL处理能够降低其蛋白量，这与QQ1的过表达植株的表型相一致，以上的结果可初步推测QQ1可能是另外一个连接光和油菜素内酯信号转导的一个桥梁分子。下一步的工作将集中在阐明QQ1在光与内源激素交叉信号中作用的分子机制。更多还原

【Abstract】 （Brassinosteroids）have been recognized as the sixth class of plant hormones aftercytokinin, auxin, gibberellins, ethylene and abscisic acid. Brassinosteroids regulate diverseplant growth and developmental processes such as promotion of stem elongation and celldivision, increase of tolerance to various stresses. Recently, more progresses were made infunctions analysis of Brassinosteroids, special in elucidating the brassinosteroid signalingpathway. BZR1（BRASSINAZOLE-RESISTANT1） is a key transcription factor in the BRsignaling pathway to regulate gene expression. How BZR1regulates the expression ofdownstream genes is still unclear. Analysis of the target genes of BZR1is a key forunderstanding the downstream of BR pathways and elucidating the transcriptional networkwith other signaling pathway, and also improving the bioactive substances of medicinal plant.In this study, we screened some BR regulated BZR1targets genes. We analyzed thegrowth phenotype of the over-expressing transgenic plants and their response to light andvarious hormones. Finally, we identified two genes, over-expression of which resulted inobvious phenotypes in the transgenic plants, PAR1（PHYTOCHROME RAPIDLYREGULATED1）and QQ1with unknown function. PAR1and its closest homolog PAR2arenegative regulators of shade-avoidance syndrome (SAS), they belong to the HLHtranscription factor family that lacks a typical basic domain required for DNA binding, andare believed to regulate gene expressions through DNA binding transcription factors that areyet to be identified. We used yeast-two-hybrid system to screen the possible genes which caninteract with the target genes. Further analysis of the mechanism by sorts of moleculartechniques such as DNA pull-down and chromatin immunoprecipitation assay identified thefunctions in vitro and vivo. The results show as follows:1. We generated overexpressing transgenic plants fused with MYC tag for candidategenes. Over expressing PAR1(PAR1OX) and QQ1(QQ1OX) transgenic plants displayeddwarfism with reduced petiole length and small leaves compared with wild-type Col-0.2. Here, we show that PAR1interacts with PIF4(phytochrome interaction factor4) inyeast-two-hybrid assay, bimolecular fluorescence complementation, in vitro pull down assayand Co-immunoprecipitation assay. Transgenic plants overexpressing PAR1arehypersensitive to light; the hypocotyl length of PAROX is shorter than wildtype Col-0understrong light density. PAR1protein level increased in etiolated seedlings under white light, and also under in red, far-red and blue light, indicating that PAR1protein stability is regulated bymultiple photoreceptors.3. PAR1inhibits PIF4direct target genes. The expression level of HFR1, PIL1andIAA29in PAR1OX are lower than wild type Col-0in qRT-PCR result. DNA pull-down andchromatin immunoprecipitation (ChIP) assays showed that PAR1inhibits PIF4DNA bindingin vitro and in vivo. We generated PAR1OX/PIF4OX double transgenic plants and thencompared hypocotyl length with each single transgenic plant. While PIF4OX dramaticallypromoted hypocotyl elongation, PAR1OX partially suppressed the long-hypocotyl phenotypeof PIF4OX, consistent with PIF4being less active in the PAR1OX background. In addition,PAR1is involved in the PIF4-mediated response, as transgenic plants overexpressing PAR1are insensitive to gibberellin (GA) or high temperature in hypocotyl elongation, similar to thepifq (PIF1, PIF3, PIF4and PIF5mutant).4. In addition to PIF4, PAR1also interacts with PRE1in yeast-two-hybrid andbimolecular fluorescence complementation assay. PRE1, belonging to HLH family, isactivated by brassinosteroids (BRs) and GA. We generated PAR1OX/PRE1OX doubletransgenic plants. The F1-generation plants showed long petioles and expended leaf blades,similar to PRE1OX plants. It indicated that overexpression of PRE1largely suppressed thedwarf phenotype of PAR1OX. These results indicate that PAR1–PRE1and PAR1–PIF4heterodimers form a complex HLH/bHLH network regulating cell elongation and plantdevelopment in response to light and hormones.5. In addition to PAR1, we also identified a BZR1directly target gene which hasobviously phenotype in transgenic plants, named QQ1. QQ1belongs to HLH family lacks atypical basic domain required for DNA binding. It is close to AIF1and PAR1after alignment.We found that QQ1interacts with PRE1in the yeast-two-hybrid assay. QQ1protein leveldramatically increased when exposed under short time of white light. BL treatment reducedits protein level which is consistent with the phenotype of the PAROX transgenic plant. Theresults indicated that QQ1might be another link for integrating the light and BR signalingpathway.更多还原