【作者】 朱辉；

【导师】 张忠明；

【作者基本信息】 华中农业大学 ， 微生物学， 2008， 博士

【摘要】 根瘤菌与豆科植物能形成一种特殊的共生体根瘤。根瘤菌与宿主植物共生体系的建立过程涉及到两者之间复杂的信号识别、基因表达调控、能量物质的交换等。根瘤菌与宿主植物之间的分子对话首先是在低氮或缺氮的环境下,宿主植物分泌一种类黄酮类化合物或相关化合物,该类物质能诱导根瘤菌一系列结瘤基因(nod)的表达并合成根瘤菌信号分子—结瘤因子(Nod factor)。结瘤因子反过来引起宿主植物根毛发生卷曲,侵入线的形成,开启与根瘤形成和发育相关基因的表达,直至根瘤成熟。然而,根瘤菌结瘤因子信号在宿主植物中的传递过程及其调控机制还不清楚,本文以模式豆科植物—百脉根(Lotus japonicus)共生固氮体系为材料,利用生物化学及分子生物学研究技术,开展了根瘤菌共生信号在宿主植物中传递途径和作用机制的研究。其主要研究结果如下:1.利用酵母双杂交技术鉴定了百脉根结瘤因子受体Li-NFR1、Lj-NFR5(NFR:Nodfactor receptor kinase)、共生受体激酶Lj-SymRK(SymRK:Symbiosis Receptor-likeKinase)、离子通道蛋白CASTOR等共生功能蛋白间的相互关系。结果表明,这几个蛋白质在酵母双杂交技术条件下并无相互作用。2.以接种百脉根根瘤菌2天、4天、6天、8天、12天的百脉根植株的根组织混合物为材料,抽提总RNA,利用RT-PCR合成cDNA,构建了百脉根AD-cDNA酵母双杂文库。其库容量达到2.5×10~6克隆/3μg DNA,其外源DNA片段大小为0.5-2.0 kb。3.SymRK基因编码一个类受体激酶蛋白。它由富含亮氨酸重复序列结构域(LRR,Leucine rich repeat),中间的跨膜结构域(Transmembrane),C-末端的蛋白激酶结构域(PK,Protein kinase)所组成。本研究分别以百脉根SymRK蛋白的LRR结构域和PK结构域为诱饵,筛选百脉根酵母双杂交AD-cDNA文库,得到了15种不同类型的与百脉根结瘤因子信号转导途径相关的基因。其中包括SIP1((?)ymRK-(?)nteracting(?)rotein)基因。根据Blast及生物信息学分析,SIP1蛋白质第126到第217氨基酸残基为ARID结构域,该结构域由8个α-螺旋和2个β-折叠组成。4.通过酵母双杂交以及免疫共沉淀等方法对SIP1蛋白与SymRK-PK蛋白相互作用的研究表明,SIP1与SymRK-PK蛋白相互作用的区域定位于SIP1蛋白C-末端,其蛋白N端和ARID结构域均不能与SymRK-PK相互作用。对SIP1蛋白自身相互作用的研究结果表明,SIP1蛋白能形成同源二聚体,而且,SIP1蛋白通过自身C-末端的184个氨基酸残基相互作用形成同源二聚体,其N-末端的223个氨基酸残基不参与对二聚体的形成。5.通过酵母单杂交以及凝胶阻滞的方法,我们证实SIP1蛋白具有结合DNA的能力,但是它没有转录激活的功能,由此确定SIP1蛋白为一个具有DNA结合功能的转录因子。SIP1蛋白能结合起始结瘤基因(NIN,nodule inception)基因的启动子序列,不能结合百脉根钙离子结合蛋白(CBP1,calcium-binding protein 1)基因的启动子序列,这一结果说明NIN蛋白是SIP1蛋白所调控的下游基因。我们截去NIN基因启动子的-59到-70区域的序列后,SIP1蛋白不能与NIN基因启动子剩余区域结合,这一结果表明NIN基因启动子-59到-70区域的序列是SIP1蛋白结合NIN基因启动子区域的关键序列。6.利用Real time RT-PCR检测了SIP1和NIN基因在百脉根中的表达,结果显示SIP1在百脉根不同组织(根、茎、叶)中均有表达,但是接种根瘤菌使其表达量显著增加。而NIN基因在百脉根中的表达则明显受到接种根瘤菌的影响,在接种根瘤菌5h后NIN基因的表达量就会倍增。7.利用基因枪轰击使SIP1-红色荧光(Ds-Red)融合蛋白导入洋葱表皮细胞,瞬间表达结果显示,SIP1蛋白的细胞中定位于细胞核。SymRK-PK和SIP1蛋白的相关激酶活性实验表明,SIP1蛋白并不是SymRK蛋白的磷酸化底物。更多还原

【Abstract】 The rhizobia develop root nodules with legumes,the symbiotic relationship is specified. Establishing this symbiosis requires a response of molecular signals,gene expression and nutrient exchange between the plant and the bacteria.The first step in the molecular dialogue between the plant and the bacteria is the detection by rhizobia of FLAVANOIDS and related molecules that are secreted from the legume roots.Production of Nod factor molecule is activated by the release of plant predominantly flavonoids,into the rhizosphere,where they activate Nod factor production through induction of a set of nod genes in the appropriate rhizobial strain.Nod factors induce root hair deformation,formation of infection threads and expression of early nodulin genes(ENOD) till nodulation was formation.However,we did not clear the mechanism of the signal transduction pathway which activated by Nod Factor in host plant.To determine this mechanism of the signal transduction pathway and explore the unknown molecules in this cascade we study the model legume Lotus japonicus by method of biological chemistry and molecular biology.In current work,we have obtained these results as follows:1.The Nod factor receptor kinase 1(NFR1),Nod factor receptor kinase 5(NFR5), Symbiosis Receptor-like Kinase(SymRK) and CASTOR genes were identification from Lotus japonicus which were key factor in the Nod Factor signal transduction pathway.We check the interaction of LjNFR1,LjNFR5,LjSymRK and LjCASTOR proteins by yeast two hybrid systems.The result indicates that proteins could not interact with each other.2.Cultivate Lotus japonicus for the plant tissue;harvest the root of Lotus japonicus after inoculation by 2d,4d,6d,8d and 12d.Combine all the tissues and extract the total RNA Synthesize the ds cDNA by RT-PCR.We constructed a yeast two hybrid(Y2H) AD-cDNA library with root tissue of Lotus japonicus.The library capacity is 2.5×10~6 transformants/3 ug DNA.The PCR result of random chosen library colonies shows that the average length of cDNA inserts is 0.5 kb to 2.0kb.3.Lotus SymRK gene comprises the classical hallmarks of an RLK;a signal peptide, three leucine-rich repeats(LRRs) of the extracellular type are found in the predicted extracellular domain,a transmembrane domain(TM) and an intracellular protein kinase domain(PK).We screened the library using SymRK extracellular domain(LRR) and protein kinase domain(PK) as the bait proteins.The result indicates that 15 types of genes were characterization.We describe here a novel DNA-binding protein from Lotus japonicus, referred to as SIP1,because it was identified as a SymRK-interacting protein 1.BLAST analysis of SIP1 protein contained conserved AT Rich Interaction Domain(ARID)(residues 126-217).The predicted 3-D model of the ARID domain of SIP1 consists of 8α-helices,twoβ-strands,and four structure-undefined loops.4.The N-terminal half of SIP1(SIP1N) containing the N-terminus and the ARID domain was not found to interact with SymRK-PK.The C-terminus of SIP1(SIP1C) was found to be responsible for its interaction with the kinase domain of SymRK。The interactions were also detected when the C-terminus of SIP1 was used SIP1C with SIP1;SIP1C with SIP1C,suggesting that the C-terminal 184 residues are responsible for SIP1 dimerization in the absence was not required for SIP1 dimerization.In contrast,the N-terminus(223 residues) of SIP1,which contains the ARID domain,was not required for SIP1 dimerization.5.SIP1 specifically binds to the promoter of LjNIN,but not to that of LjCBP1(a calcium-binding protein gene),both of which are known to be inducible by Nod factors.SIP 1 recognizes two of the three AT-rich domains present in the NIN gene promoter.Deletion of one of the AT-rich domain at the NIN promoter diminishes the binding of SIP1 to the NIN promoter.The sequence -59 to -70 is the most important site for SIP1 Bingding with NIN promoter.6.Using quantitative PCR,we examined the SIP1 and NIN mRNA levels in different tissues of L.japonicus.The SIP1 gene is expressed constitutively in leaves and the uninfected roots,and its expression levels are elevated after infection by Mesorhizobium loti.This expression pattern was distinct from that of NIN,which exhibited significant induction 5 h after rhizobial inoculation and maintained a high expression level in inoculated roots.It is proposed that SIP1 may be required for the expression of NIN and involved in the initial communications between the rhizobia and the host root cells.The protein is localized to the nucler when expressed as a red fluorescence fusion protein in the onion epidermal cells. Using the kinase assay,we confirmed that SIP1 protein was not the phosphorylation substrate of SymRK protein.更多还原