本文選題：凝集素 + 油菜素內(nèi)酯�。� 參考：《福建農(nóng)林大學(xué)》2017年碩士論文

【摘要】：凝集素(lectin)是一類從動物和植物中提取的糖蛋白或結(jié)合糖的蛋白,能夠可逆的、特異的結(jié)合糖類、糖蛋白和糖脂表面糖基而不改變糖類的結(jié)構(gòu)。在植物體中,糖蛋白起到信號傳遞或轉(zhuǎn)換作用,而凝集素可特異識別其糖鏈殘基,兩者相互作用,引發(fā)下游生化信號級聯(lián)反應(yīng)。木菠蘿凝集素(Jacalin)是一種植物中特有的具有對多種糖結(jié)合特異性的凝集素蛋白,其中擬南芥中的JAL30被報道參與植物對草食動物的化學(xué)防御功能。本實驗室的前期研究表明,JAL30蛋白在外源油菜素內(nèi)酯(BrassinosteroidsBRs)處理時含量增加且蛋白翻譯后修飾發(fā)生變化,然而JAL30應(yīng)答B(yǎng)R信號通路的分子機制仍不清楚。本文主要對擬南芥中JAL30基因的表達以及功能進行了研究。首先從擬南芥中克隆了JAL3 基因并且構(gòu)建了 35S啟動子驅(qū)動的JAL30的cDNA融合GFP的過表達載體,獲得了多個轉(zhuǎn)基因株系并對其進行表型觀察。結(jié)果表明,轉(zhuǎn)基因植株并沒有明顯的表型變化。我們還購買了JAL30的T-DNA突變體植株,也沒有發(fā)現(xiàn)植株有明顯表型�？赡苁怯捎谀氐鞍字饕ㄟ^一種短暫應(yīng)答響應(yīng)的方式調(diào)節(jié)植物生長,JAL30過量表達或者缺失對植物的長期生長表型無明顯影響。同時我們還將此載體轉(zhuǎn)入煙草進行亞細胞定位研究,瞬時轉(zhuǎn)化實驗顯示JAL30主要在細胞核以及細胞質(zhì)中大量表達。研究表明JAL30蛋白對BR以及其他激素誘導(dǎo)劑有應(yīng)答響應(yīng),因此我們對轉(zhuǎn)基因株系進行了 BR以及茉莉酮酸甲酯(JAMe)處理,并從轉(zhuǎn)錄和翻譯水平進行了表達分析。結(jié)果表明,在轉(zhuǎn)錄水平上,BR處理并沒有明顯增加JAL30的表達。在蛋白水平上,BR處理對JAL30有一定的上調(diào)作用,但外源施加BR抑制劑PPZ,再施加外源BR處理以后,上調(diào)作用就不再明顯。表明BR促進了 JAL30的蛋白表達,而BR抑制劑PPZ可能抑制了 JAL30蛋白表達。而JAMe處理則明顯上調(diào)JAL30蛋白表達。JAMe與植物的防御機制相關(guān),而BR則對植物生長起著重要的作用,后續(xù)實驗結(jié)果表明JAL30對兩者都有響應(yīng),在植物體內(nèi)有著重要的功能。本文中對過表達植株進行了免疫共沉淀以及質(zhì)譜分析(IP-MS)。提取過表達植株蛋白并進行蛋白免疫沉淀實驗,再經(jīng)質(zhì)譜分析其相互作用蛋白。結(jié)果表明,JAL30本身存在三個磷酸化修飾位點,并鑒定到53個相互作用蛋白。實驗結(jié)果證明JAL30參與到植物體內(nèi)多種信號通路,自身受到磷酸化翻譯后修飾調(diào)節(jié)。我們也鑒定到多個潛在的相互作用蛋白,為進一步研究JAL30蛋白的功能及調(diào)節(jié)機制奠定了基礎(chǔ)。
[Abstract]:Lectin is a class of glycoproteins or sugar binding proteins extracted from animals and plants, which can be reversible, specific binding carbohydrate, glycoprotein and glycolipid surface glycosyl without changing the structure of carbohydrates.In plant, glycoprotein plays the role of signal transduction or conversion, while lectin can specifically recognize its sugar chain residues, and the interaction between them leads to downstream biochemical signal cascade reaction.Pineapple lectin (Jacalin) is a unique lectin protein that is specific to many kinds of sugar binding in plants. JAL30 in Arabidopsis thaliana has been reported to be involved in plant chemical defense against herbivores.Previous studies in our laboratory showed that the content of JAL30 protein increased during BrassinosteroidsBRs treatment with exogenous BrassinosteroidsBRs, but the molecular mechanism of JAL30 response to Br signaling pathway was still unclear.In this paper, the expression and function of JAL30 gene in Arabidopsis thaliana were studied.Firstly, the JAL3 gene was cloned from Arabidopsis thaliana and the cDNA fusion vector of 35s promoter driven JAL30 was constructed. Several transgenic lines were obtained and their phenotypes were observed.The results showed that there was no obvious phenotypic change in transgenic plants.We also purchased JAL30 T-DNA mutant plants and found no obvious phenotype.It may be that lectin protein regulates the overexpression or absence of JAL30 mainly through a transient response to the growth of plants, and has no significant effect on the long-term growth phenotype of plants.At the same time, we also transferred the vector into tobacco for subcellular localization. Transient transformation experiments showed that JAL30 was mainly expressed in the nucleus and cytoplasm.The results showed that the JAL30 protein was responsive to Br and other hormone inducers, so we treated the transgenic lines with Br and jasmonate methyl ester, and analyzed their expression and expression at the transcriptional and translation levels.The results showed that Br treatment did not significantly increase the expression of JAL30 at the transcriptional level.At the protein level, Br treatment had a certain up-regulation effect on JAL30, but the upregulation was no longer obvious after exogenous Br treatment and exogenous Br treatment.The results showed that Br promoted the expression of JAL30 protein, while Br inhibitor PPZ inhibited the expression of JAL30 protein.JAMe treatment significantly upregulated the expression of JAL30 protein. JAMe was related to the defense mechanism of plants, while Br played an important role in plant growth. The results of follow-up experiments showed that JAL30 was responsive to both of them and had important functions in plants.In this paper, the over-expressed plants were analyzed by immunoprecipitation and mass spectrometry.The expressed plant protein was extracted and the protein immunoprecipitation assay was carried out, and then the interacting protein was analyzed by mass spectrometry.The results showed that there were three phosphorylation sites in JAL30 and 53 interacting proteins were identified.The results showed that JAL30 was involved in many signaling pathways in plants and was regulated by phosphorylation posttranslational modification.We also identified a number of potential interaction proteins, which laid a foundation for further study of the function and regulatory mechanism of JAL30 protein.
【學(xué)位授予單位】：福建農(nóng)林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q943.2

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