發(fā)布時(shí)間：2018-04-03 16:11

  本文選題：馬鈴薯　切入點(diǎn)：StBAG3　出處：《內(nèi)蒙古農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：BAG蛋白(Bcl-2 associated athanogene)家族是一類多功能蛋白家族,其功能廣泛,在酵母、植物和動(dòng)物中都存在。在動(dòng)物細(xì)胞中,該蛋白既可調(diào)控細(xì)胞的凋亡,又參與腫瘤發(fā)生的過程。然而已有的研究表明,植物中的BAG蛋白介入植物的抗病防衛(wèi)反應(yīng)和逆境脅迫反應(yīng)。本研究通過同源比對(duì)設(shè)計(jì)引物從馬鈴薯中克隆得到StBAG3基因,并成功構(gòu)建了StBA4G的超表達(dá)載體和沉默載體。通過農(nóng)桿菌介導(dǎo)的瞬時(shí)表達(dá)方法初步研究了StBAG3在馬鈴薯晚疫病抗性建立過程中的作用,具體的研究結(jié)果如下:1.通過分析擬南芥中已經(jīng)克隆得到的AtBAG4的蛋白序列,找到BAG蛋白的功能域保守序列,以此序列在馬鈴薯基因庫(kù)中比對(duì)得到StBAG3基因序列。以此序列設(shè)計(jì)引物,通過PCR克隆得到StBAG3基因。該基因全長(zhǎng)2074bp,開放閱讀框1026bp,編碼341個(gè)氨基酸。蛋白質(zhì)二級(jí)結(jié)構(gòu)預(yù)測(cè)表明,該蛋白二級(jí)結(jié)構(gòu)由α螺旋、β-折疊和無規(guī)則卷曲組成,其中α螺旋89個(gè),β-折疊35個(gè),無規(guī)則卷曲217個(gè)。蛋白結(jié)構(gòu)的分析表明,StBAG3包含一個(gè)BAG功能域和一個(gè)UBQ功能域,沒有跨膜結(jié)構(gòu),不產(chǎn)生信號(hào)肽,是一種親水性的不穩(wěn)定蛋白。蛋白亞細(xì)胞定位預(yù)測(cè)的結(jié)果顯示StBAC3可能定位在細(xì)胞核。2.StBAG3在馬鈴薯中的表達(dá)譜分析結(jié)果表明不同馬鈴薯品種中的StBAG3表達(dá)量存在差異,其中冀張薯12號(hào)中的表達(dá)量最高,而夏坡蒂中StBAG3表達(dá)量最低,其他品種表達(dá)量居中。而StBAG3基因在馬鈴薯不同組織的相對(duì)表達(dá)量由高到低順序是:莖、嫩葉、根和老葉。3.晚疫菌接種實(shí)驗(yàn)的結(jié)果表明,不同品種的馬鈴薯葉片中的StBAG3基因的表達(dá)量隨著接種時(shí)間的推移而逐漸增加,并在48h達(dá)到峰值;接種72 h后,StBAG3基因的表達(dá)量開始下降。4.通過載體構(gòu)建,獲得了StBAG3在35S啟動(dòng)子驅(qū)動(dòng)下的超表達(dá)載體和沉默載體。5.農(nóng)桿菌介導(dǎo)的瞬時(shí)表達(dá)結(jié)果表明接種晚疫菌后瞬時(shí)表達(dá)StBAG3的馬鈴薯葉片的病斑擴(kuò)展速率明顯小于注射水和pCAMBIA1302-GFP空載體的葉片病斑擴(kuò)展度。預(yù)示著StBAG3可能參與了馬鈴薯對(duì)晚疫病的抗性建立過程。
[Abstract]:BAG protein Bcl 2 associated DNA family is a kind of multifunctional protein family, which has a wide range of functions and is found in yeast, plants and animals.In animal cells, the protein not only regulates cell apoptosis, but also participates in tumorigenesis.However, previous studies have shown that BAG proteins in plants are involved in disease resistance, defense response and stress response.In this study, StBAG3 gene was cloned from potato by homologous alignment design primer, and the superexpression vector and silencing vector of StBA4G were successfully constructed.The role of StBAG3 in the establishment of resistance to late blight in potato was studied by Agrobacterium tumefaciens mediated transient expression method. The results are as follows: 1.By analyzing the protein sequence of AtBAG4 cloned from Arabidopsis thaliana, the conserved sequence of functional domain of BAG protein was found, and the StBAG3 gene sequence was obtained by comparing the sequence in potato gene bank.The primers were designed and the StBAG3 gene was cloned by PCR.The total length of the gene is 2074 BP, open reading frame 1026 BP, encoding 341 amino acids.The prediction of protein secondary structure showed that the protein secondary structure was composed of 偽 helix, 尾 -fold and irregular crimp. Among them, 偽 helix 89, 尾 -fold 35, irregular crimp 217.The analysis of protein structure shows that StBAG3 contains a BAG functional domain and a UBQ functional domain. It has no transmembrane structure and does not produce signal peptide. It is a hydrophilic unstable protein.The results of protein subcellular localization prediction showed that StBAC3 might be located in the nucleus. 2. The results of expression profiling of StBAG3 in potato showed that there were differences in StBAG3 expression in different potato varieties, and the highest expression level was found in Jizhanshu 12.However, the expression of StBAG3 was the lowest in Charlotte, while the expression of StBAG3 in other varieties was the middle.The relative expression of StBAG3 gene in different tissues of potato was in the order of stem, young leaf, root and old leaf.The results of inoculation experiment showed that the expression of StBAG3 gene in potato leaves increased with the inoculation time and reached its peak at 48h, and the expression of STBAG3 gene began to decrease at 72h after inoculation.The overexpression vector and silencing vector of StBAG3 driven by 35s promoter were obtained by vector construction.The results of transient expression mediated by Agrobacterium tumefaciens showed that the disease spot spreading rate of potato leaves with transient expression of StBAG3 after inoculation was significantly lower than that of water injection and pCAMBIA1302-GFP empty vector.It suggests that StBAG3 may be involved in the establishment of resistance to late blight in potato.
【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S435.32

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 羅守進(jìn);;安徽省馬鈴薯主要病害的發(fā)生與防治[J];農(nóng)業(yè)災(zāi)害研究;2015年10期

2 盧肖平;;馬鈴薯主糧化戰(zhàn)略的意義、瓶頸與政策建議[J];華中農(nóng)業(yè)大學(xué)學(xué)報(bào)(社會(huì)科學(xué)版);2015年03期

3 夏昒p，

本文編號(hào)：1705932