本文選題：玉米 + LysM基因�。� 參考：《安徽農(nóng)業(yè)大學(xué)》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(fù)雜的互作識別機制,細(xì)胞表面識別受體可識別保守的病原菌相關(guān)分子模式,進而激活下游信號通路中的關(guān)鍵功能蛋白,誘導(dǎo)共生或防御反應(yīng)。近期研究發(fā)現(xiàn),LysM結(jié)構(gòu)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(dǎo)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(jié)合生物信息學(xué)的方法,對玉米(B73)全基因進行比對,并結(jié)合相關(guān)生物軟件對基因的結(jié)構(gòu)域進行預(yù)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(shù)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(shù)對其時空表達進行分析,結(jié)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(zhuǎn)錄水平。用不同的誘導(dǎo)因子如細(xì)菌肽聚糖、脂多糖、鞭毛蛋白flg22、真菌幾丁質(zhì)、AM真菌處理玉米根,發(fā)現(xiàn)ZmLysM3和ZmLysM6的轉(zhuǎn)錄水平可被顯著上調(diào)。由此可知該基因可能參與對細(xì)菌或真菌病原菌分泌的配體進行識別。結(jié)構(gòu)預(yù)測可知ZmLysM6由LysM結(jié)構(gòu)、跨膜結(jié)構(gòu)域和胞內(nèi)激酶結(jié)構(gòu)域組成,相比之下,ZmLysM3缺少胞內(nèi)的激酶結(jié)構(gòu)域。為明確兩個LysM蛋白在細(xì)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(dǎo)煙草葉片瞬時表達,對該基因的亞細(xì)胞定位進行檢測,結(jié)果可知,這兩個蛋白均位于細(xì)胞膜上。酵母雙雜實驗表明,ZmLysM3和ZmLysM6蛋白胞外結(jié)構(gòu)域可能通過形成異源二聚體來識別胞外的配體,繼而誘導(dǎo)下游的信號轉(zhuǎn)導(dǎo)。為進一步明確二者的介導(dǎo)信號機制,本研究將其分別轉(zhuǎn)入和共轉(zhuǎn)入擬南芥cerk1-2(缺乏幾丁質(zhì)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(zhuǎn)基因植株葉片進行真菌誘導(dǎo)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(jié)果可知,同時轉(zhuǎn)ZmLysM3和ZmLysM6的轉(zhuǎn)基因擬南芥顯著增強植株的抗病性,而僅轉(zhuǎn)入一個LysM對真菌的抑制效果并不明顯。上述的研究結(jié)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(jié)構(gòu)域,構(gòu)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(xué)位授予單位】：安徽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S513;Q943.2

【參考文獻】

相關(guān)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(dǎo)植物免疫防衛(wèi)反應(yīng)及其信號激發(fā)的研究進展[J];植物學(xué)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

，

本文編號：2029732