玉米ZmLysM受体基因克隆及功能分析

发布时间：2018-06-17 04:38

  本文选题：玉米 + LysM基因　； 参考：《安徽农业大学》2016年硕士论文

【摘要】：植物与微生物在长期协同进化过程中,逐渐形成一种高度复杂的互作识别机制,细胞表面识别受体可识别保守的病原菌相关分子模式,进而激活下游信号通路中的关键功能蛋白,诱导共生或防御反应。近期研究发现,LysM结构域蛋白是一类重要的植物模式识别受体,在植物和真菌互作中起着极其重要的的角色。为进一步明确LysM受体基因如何特异性识别不同的配体,诱导下游共生或防御信号中的作用。本研究以报道的百脉根和苜蓿中已知的LysM基因为模板,结合生物信息学的方法,对玉米(B73)全基因进行比对,并结合相关生物软件对基因的结构域进行预测,筛选玉米中调节共生或防御的LysM候选基因。借助RT-PCR技术从植株的根系中克隆出候选基因,分别命名为ZmLysM3和ZmLysM6。利用实时定量PCR技术对其时空表达进行分析,结果表明,这两个LysM基因在不同组织中均有表达,尤其是根部具有较高的转录水平。用不同的诱导因子如细菌肽聚糖、脂多糖、鞭毛蛋白flg22、真菌几丁质、AM真菌处理玉米根,发现ZmLysM3和ZmLysM6的转录水平可被显著上调。由此可知该基因可能参与对细菌或真菌病原菌分泌的配体进行识别。结构预测可知ZmLysM6由LysM结构、跨膜结构域和胞内激酶结构域组成,相比之下,ZmLysM3缺少胞内的激酶结构域。为明确两个LysM蛋白在细胞中的位置,ZmLysM3和ZmLysM6基因被连入表达载体pCAMBIA1305.1-GFP中,农杆菌介导烟草叶片瞬时表达,对该基因的亚细胞定位进行检测,结果可知,这两个蛋白均位于细胞膜上。酵母双杂实验表明,ZmLysM3和ZmLysM6蛋白胞外结构域可能通过形成异源二聚体来识别胞外的配体,继而诱导下游的信号转导。为进一步明确二者的介导信号机制,本研究将其分别转入和共转入拟南芥cerk1-2(缺乏几丁质感知系统的拟南芥突变体)中用于互补突变体的功能特征。对转基因植株叶片进行真菌诱导处理72h后,由植株叶片的枯萎程度和对叶片中菌丝的抑制作用结果可知,同时转ZmLysM3和ZmLysM6的转基因拟南芥显著增强植株的抗病性,而仅转入一个LysM对真菌的抑制效果并不明显。上述的研究结果,在理论上,将为阐明玉米LysM基因如何调节植株抗性机制的研究提供新的内容与模式。在实践上,可通过融合抗性品种受体激酶的胞外结构域,构建嵌合受体,提高感病植株的抗性。因此,开展LysM受体激酶的研究具有生产上的必要性,为杂交母本的选育、提高后代抗性有着重要意义。
[Abstract]:During the long-term coevolution of plants and microorganisms, a highly complex mechanism of interaction recognition was gradually formed, and the cell surface recognition receptors could recognize conservative molecular patterns related to pathogens. In turn, it activates the key functional proteins in the downstream signaling pathway and induces symbiotic or defensive responses. Recently, it has been found that LysM domain protein is a kind of important plant pattern recognition receptor, which plays an extremely important role in the interaction between plants and fungi. To further clarify how LysM receptor genes specifically recognize different ligands and induce downstream symbiosis or defense signals. In this study, the reported Lys M gene was used as template and bioinformatics method was used to compare the whole gene of Corn B73, and the domain of the gene was predicted by using the related biological software. Screening of LysM candidate genes in maize to regulate symbiosis or defense. Candidate genes were cloned from plant roots by RT-PCR and named ZmLysM3 and ZmLysM6, respectively. The temporal and spatial expression of LysM gene was analyzed by real-time quantitative PCR. The results showed that the two LysM genes were expressed in different tissues, especially in the roots. Maize roots were treated with different inducible factors such as bacterial peptidoglycan, lipopolysaccharide, flagellin flg22 and fungal chitin AM fungi. It was found that the transcription levels of ZmLysM3 and ZmLysM6 could be upregulated significantly. It is suggested that the gene may be involved in the identification of ligands secreted by bacteria or fungal pathogens. Structural prediction showed that ZmLysM6 was composed of LysM structure, transmembrane domain and intracellular kinase domain, whereas ZmLysM3 lacked intracellular kinase domain. In order to identify the location of two LysM proteins, ZmLysM3 and ZmLysM6 genes were inserted into the expression vector pCAMBIA1305.1-GFP. Agrobacterium tumefaciens mediated the transient expression in tobacco leaves and detected the subcellular localization of the gene. The results showed that the two proteins were located on the cell membrane. Yeast double cross experiments showed that the extracellular domains of ZmLysM3 and ZmLysM6 proteins may recognize extracellular ligands by forming heterodimer, and then induce downstream signal transduction. In order to further elucidate the mechanism of signaling mediated by these two mutants, they were transferred and co-transferred into Arabidopsis cerk1-2 (Arabidopsis thaliana mutant lacking chitin sensitive system), respectively, and the functional characteristics of these mutants were used as complementary mutants in Arabidopsis thaliana (Arabidopsis thaliana). Transgenic Arabidopsis thaliana transformed with ZmLysM3 and ZmLysM6 could significantly enhance the resistance of transgenic Arabidopsis thaliana after 72 hours of fungal induction treatment, and the results showed that the wilting degree of leaves and the inhibition of hyphae in leaves were significantly enhanced by transgenic Arabidopsis thaliana transgenic with ZmLysM3 and ZmLysM6. The inhibitory effect of only one LysM on fungi was not obvious. The above results will provide a new content and model for elucidating how LysM gene regulates plant resistance mechanism in maize. In practice, chimeric receptors can be constructed by fusing the extracellular domain of receptor kinases of resistant varieties to enhance the resistance of susceptible plants. Therefore, it is necessary to study LysM receptor kinase in production, which is of great significance for the breeding of hybrid parent and the improvement of resistance of progeny.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S513;Q943.2

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