本文選題：玉米 + LysM基因�。� 參考：《安徽農(nóng)業(yè)大學》2016年碩士論文

【摘要】：植物與微生物在長期協(xié)同進化過程中,逐漸形成一種高度復雜的互作識別機制,細胞表面識別受體可識別保守的病原菌相關分子模式,進而激活下游信號通路中的關鍵功能蛋白,誘導共生或防御反應。近期研究發(fā)現(xiàn),LysM結構域蛋白是一類重要的植物模式識別受體,在植物和真菌互作中起著極其重要的的角色。為進一步明確LysM受體基因如何特異性識別不同的配體,誘導下游共生或防御信號中的作用。本研究以報道的百脈根和苜蓿中已知的LysM基因為模板,結合生物信息學的方法,對玉米(B73)全基因進行比對,并結合相關生物軟件對基因的結構域進行預測,篩選玉米中調(diào)節(jié)共生或防御的LysM候選基因。借助RT-PCR技術從植株的根系中克隆出候選基因,分別命名為ZmLysM3和ZmLysM6。利用實時定量PCR技術對其時空表達進行分析,結果表明,這兩個LysM基因在不同組織中均有表達,尤其是根部具有較高的轉錄水平。用不同的誘導因子如細菌肽聚糖、脂多糖、鞭毛蛋白flg22、真菌幾丁質、AM真菌處理玉米根,發(fā)現(xiàn)ZmLysM3和ZmLysM6的轉錄水平可被顯著上調(diào)。由此可知該基因可能參與對細菌或真菌病原菌分泌的配體進行識別。結構預測可知ZmLysM6由LysM結構、跨膜結構域和胞內(nèi)激酶結構域組成,相比之下,ZmLysM3缺少胞內(nèi)的激酶結構域。為明確兩個LysM蛋白在細胞中的位置,ZmLysM3和ZmLysM6基因被連入表達載體pCAMBIA1305.1-GFP中,農(nóng)桿菌介導煙草葉片瞬時表達,對該基因的亞細胞定位進行檢測,結果可知,這兩個蛋白均位于細胞膜上。酵母雙雜實驗表明,ZmLysM3和ZmLysM6蛋白胞外結構域可能通過形成異源二聚體來識別胞外的配體,繼而誘導下游的信號轉導。為進一步明確二者的介導信號機制,本研究將其分別轉入和共轉入擬南芥cerk1-2(缺乏幾丁質感知系統(tǒng)的擬南芥突變體)中用于互補突變體的功能特征。對轉基因植株葉片進行真菌誘導處理72h后,由植株葉片的枯萎程度和對葉片中菌絲的抑制作用結果可知,同時轉ZmLysM3和ZmLysM6的轉基因擬南芥顯著增強植株的抗病性,而僅轉入一個LysM對真菌的抑制效果并不明顯。上述的研究結果,在理論上,將為闡明玉米LysM基因如何調(diào)節(jié)植株抗性機制的研究提供新的內(nèi)容與模式。在實踐上,可通過融合抗性品種受體激酶的胞外結構域,構建嵌合受體,提高感病植株的抗性。因此,開展LysM受體激酶的研究具有生產(chǎn)上的必要性,為雜交母本的選育、提高后代抗性有著重要意義。
[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.
【學位授予單位】：安徽農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S513;Q943.2

【參考文獻】

相關期刊論文 前4條

1 季東超;宋凱;邢晶晶;陳彤;田世平;;LysM蛋白介導植物免疫防衛(wèi)反應及其信號激發(fā)的研究進展[J];植物學報;2015年05期

2 Elya Masya Mohd Fishal;Sariah Meon;Wong Mui Yun;;Induction of Tolerance to Fusarium Wilt and Defense-Related Mechanisms in the Plantlets of Susceptible Berangan Banana Pre-Inoculated with Pseudomonas sp.(UPMP3) and Burkholderia sp.(UPMB3)[J];Agricultural Sciences in China;2010年08期

3 Brett J. Ferguson;Arief Indrasumunar;Satomi Hayashi;Yu-Hsiang Lin;Dugald E. Reid;Peter M. Gresshoff;;Molecular Analysis of Legume Nodule Development and Autoregulation[J];Journal of Integrative Plant Biology;2010年01期

4 李海燕,劉潤進,束懷瑞;叢枝菌根真菌提高植物抗病性的作用機制[J];菌物系統(tǒng);2001年03期

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