【作者】 李雪梅；

【导师】 张忠明；

【作者基本信息】 华中农业大学 ， 微生物学， 2013， 硕士

【摘要】 植物SUPERMAN类锌指蛋白属于C2H2型锌指蛋白,这类蛋白只含一个锌指结构域,其N端具有保守序列QALGGH, C端有一段富含亮氨酸的保守序列L(I)DLXLR(K)L,研究表明SUPERMAN基因在植物花发育、种子和胚的发育以及侧根和分枝发育过程中起重要作用。本研究以百脉根为实验材料,利用生物信息学方法在百脉根数据库中找到了一个参与根和根瘤发育的SUPERMAN类基因ZFP3,通过序列比对、同源性分析、启动子分析、和遗传转化等方法研究了该基因的蛋白结构、表达模式和生理功能。主要的研究结果如下：1.利用已经报道的拟南芥、矮牵牛、大豆等物种中的SUPERMAN类蛋白序列比对百脉根数据库,找到了一个可能参与豆科植物根瘤发育过程的SUPERMAN类基因LjZFP3。SUPERMAN类锌指蛋白属于TFIIIA型转录因子,有一个典型的C2H2型锌指结构域和一段高度保守的氨基酸序列QALGGH,在植物生长发育过程中起着重要作用。2.在PLACE数据库中分析ZFP3基因上游的1.82kb的序列,发现启动子区域内含有结瘤基因启动子中特有的典型的调控结瘤基因表达的保守作用元件5’-AAAGAT-3’,四个晚期结瘤素基因特有的启动子元件5’-CTCTT-3’和四个根特异性表达元件5’-ATATT-3’,分析结果暗示该基因可能在根与根瘤中特异表达。3.为了检测ZFP3基因的时空表达,我们构建了ZFP3Pro:GUS重组质粒,利用毛根转化体系,将该重组质粒转入豆科植物百脉根的根中,进行组织化学定位分析。结果显示ZFP3主要在根的维管束中表达,在根瘤原基中不表达,在发育的根瘤中ZFP3基因的表达量较高,而成熟和衰老的根瘤中ZFP3的表达量很低。Real-time PCR结果显示ZFP3基因在接种和未接种的根中均有表达,在接种根瘤菌2天后表达明显上升,而后则趋于稳定；ZFP3基因在不同时期的根瘤中均有表达,表达水平没有明显差异。启动子实验结果与Real-time PCR结果证实了ZFP3基因的在根与根瘤中都有表达,说明这个基因可能参与根及根瘤发育的调控。4.利用RNA干扰(RNAi)技术下调表达ZFP3基因,结果显示ZFP3的RNAi不仅导致了RNAi植株结瘤数目明显减少而且影响了植株的根的正常生长。这个结果证实ZFP3在根与根瘤的生长发育中发挥正调控作用。5.利用毛根转化技术将LjZFP3导入百脉根中超量表达,观察接种根瘤菌后根瘤的表型,结果显示超表达LjZFP3植株的结瘤数没有明显的变化,但是超表达植株中成熟根瘤的比例要比对照组大,说明其可能参与根瘤的发育。6.已有的研究表明C2H2型锌指蛋白可以作为有丝分裂原激活蛋白激酶的目标底物存在,本论文利用酵母双杂交技术发现ZFP3不仅能和多个MAPK蛋白相互作用,而且可以和MAPKK蛋白作用,表明ZFP3有可能以MAPK级联反应方式参与根和根瘤的生长发育。更多还原

【Abstract】 The SUPERMAN-like zinc finger protein is a kind of single zinc finger protein that belong to theCys2His2-type zinc finger protein family, the conserved domains are the QALGGH amino acid residues atN-terminus and a leucine-rich motif L(I)DLXLR(K)L in the carboxy-terminal region. The superman family play animportant role in flora organ morphogenesis, embryogenesis, seed development, lateral root formation and branchingof plant.In this study, using plant Lotus japonicus as subjects, we found a new SUP-family gene, named as ZFP3involving in the regulation of root and nodule development, in Lotus japonicas databaseAnd then we used sequence comparison, homology analysis, promoter analysis and genetic transformation method to study its protein structure,, expression patterns and physiological functions, The main research results are as follows:1. Using the SUPERMAN-like protein sequences in Arabidopsis thaliana soybean, petunia and other plant organisms to blast the L. japonicasdatabase, we found a new SUP-family gene, designated as ZFP3, possibly involved in the regulation of nodule development.The SUPERMAN-like zinc finger protein belong to the TFIIIA transcription factor with a classical C2H2-type zinc finger domain and conserved QALGGH amino acid residues,closely related to plant growth and development.2. Sequence analysis in PLACE database showed that LjZFP3gene contained al.82kbnucleotides promoter region, and several important upstream promoter elements. The conserved cis-acting sequence5’-AAAGAT-3’controlling nodulin gene expression is present at its promoter region. There are four cis-acting element motifs5’-CTCTT-3’related to late nodulin genes and four regulatory elements5’-ATATT-3’responsible for root-specific expressionin the5’-upstream of the ZFP3.The analysisseems to imply that ZFP3may be a root-specific and nodule-specific gene.3. To investigate the spatial pattern of ZFP3expression,we produce transgenic hairy roots containing fusions of the promoter region of ZFP3to the β-glucuronidase (GUS) reporter gene by hair root transformation system of L. japonicus. Histochemical staining shows that GUS reporter activity is observed mainly in root vascular bundles,and no expression of ZFP3was observed in nodule primordial The intensity of blue reaction is gradually increased in the developing nodules,and is very weak in mature and senescentnodules.The Real-Time PCRassay shows ZFP3mRNA is expressed in the roots of L. japonicus before and after inoculation with M. loti, the level of2d after inoculation is much higher, as compared with that in uninoculated control roots loti and then it tends to be stable; the expression level of ZFP3was elevated in nodules from different time remained similar level. These observations confirm that the ZFP3gene is indeed expressed in roots and nodules, and indicates that ZFP3maybe involve in the process of root and nodule development in L. japonicus.4.Significant decreases in Nodulation and root growth defects were observed in ZFP3RNAi roots compared with the control hairy roots.Thus, ZFP3may act as a positive regulator in the process of root growth and nodule development in L. japonicus.5. ZFP3was expressed under the control of the maize ubiquitin promoter (ZFP3OX) in transgenic hairy roots of L. japonicus. The differences of nodule numbers per root system between the control and ZFP3OX hairy roots were not shown to be statistically significant. But the proportion of mature nodulesin ZFP3OX was higher than the control.These results suggest that ZFP3may play an important role in the nodule development.6. Recent studies have shown that C2H2-type zinc finger protein can be the target substrates of MPKS.Using yeast two-hybrid assay in vivo, we find that ZFP3are not only able to interact with several MAPKs, but also with MAPKK, implying that ZFP3may play an important role in the nodule and root development through MAPK cascades.更多还原