本文選題：煙草 + 類胡蘿卜素�。� 參考：《河南農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：煙葉中的類胡蘿卜素以及它的降解生成物是引起煙葉揮發(fā)香味物質(zhì)的主要前體物質(zhì),其中降解產(chǎn)物的類型以及濃度值都能夠作為煙草質(zhì)量的評價(jià)指標(biāo),因此,研究煙葉內(nèi)部的類胡蘿卜素降解有關(guān)的基因情況,確定這些基因的重要功能,對于提升煙葉質(zhì)量具有重大的科研價(jià)值。類胡蘿卜素裂解雙加氧酶是植物類胡蘿卜素降解代謝途徑中的關(guān)鍵基因,CCDs家族有9個(gè)成員,其中包括NCED2,NCED3,NCED5,NCED6,NCED9等,本研究通過同源克隆方法獲得煙草品種K326類胡蘿卜素降解關(guān)鍵基因NCED3兩個(gè)c DNA全長序列,并對其進(jìn)行生物詳細(xì)的生物信息學(xué)分析,采用干旱脅迫研究基因的表達(dá)情況,同時(shí)采用in-fusion和gateway方法分別構(gòu)建了過表達(dá)和抑制表達(dá)載體,農(nóng)桿菌介導(dǎo)法轉(zhuǎn)化煙草植株,研究結(jié)果為進(jìn)一步分析NCED3在煙草抗旱性的功能研究方面提供一定的基礎(chǔ)。主要研究結(jié)果如下:1克隆得到烤煙品種K326類胡蘿卜素降解關(guān)鍵基因NCED3 2個(gè)拷貝NCED3-1、NCED3-2,并提交NCBI Genbank,登錄號(hào)KM605434和KM605435。序列分析表明:K326NCED3-1和NCED3-2基因分別包含一個(gè)1842bp和1830bp的開放讀碼框(ORF),各編碼613個(gè)和609個(gè)氨基酸。跨膜區(qū)預(yù)測、親水性和信號(hào)肽分析表明很可能是定位于線粒體膜上的親水性跨膜蛋白。二級結(jié)構(gòu)預(yù)測模型顯示此蛋白結(jié)構(gòu)以β折疊和無規(guī)則卷曲為主。蛋白三級結(jié)構(gòu)預(yù)測分析表明NCED3-1與NCED3-2空間結(jié)構(gòu)極為相似。2 PEG6000干旱脅迫分析表明,干旱脅迫可以誘導(dǎo)NCED3基因表達(dá)以及內(nèi)源ABA的積累。且在處理12h之前,NCED3表達(dá)與ABA累積速度均呈現(xiàn)升高趨勢,之后逐漸降低。3構(gòu)建了K326 NCED3基因的過表達(dá)和RNAi表達(dá)載體,并采用農(nóng)桿菌介導(dǎo)法轉(zhuǎn)化煙草植株。獲得了NCED3-1過表達(dá)和抑制表達(dá)陽性植株。
[Abstract]:Carotenoids in tobacco leaves and their degradation products are the main precursors causing volatile aroma substances in tobacco leaves. The types and concentrations of degradation products can be used as indicators of tobacco quality evaluation. It is of great scientific value to study the genes related to carotenoid degradation in tobacco leaves and to determine the important functions of these genes in order to improve the quality of tobacco leaves. Carotenoid cleavage dioxygenase is a key gene in the pathway of plant carotenoid degradation and metabolism. There are nine members of the CCDs family, including NCED2, NCED3, NCED5, NCED6, NCED9, and so on. In this study, the full-length sequence of NCED3 two c DNA, a key gene of carotenoid degradation in tobacco variety K326, was obtained by homologous cloning method, and its bioinformatics was analyzed in detail, and the expression of the gene was studied by drought stress. At the same time, the overexpression and inhibition expression vectors were constructed by in-fusion and gateway methods respectively. Agrobacterium tumefaciens mediated transformation of tobacco plants. The results provided a basis for further analysis of the function of NCED3 in the study of tobacco drought resistance. The main results were as follows: 1. The two copies of the key gene NCED3 of carotenoid degradation in flue-cured tobacco variety K326 were cloned as follows: NCED3-1, NCED3-2, and submitted to NCBI Genbank, accession numbers KM605434 and KM605435. Sequence analysis showed that the gene K326NCED3-1 and NCED3-2 contained an open reading frame of 1842bp and 1830bp, encoding 613 and 609 amino acids respectively. The hydrophilicity and signal peptide analysis indicated that the transmembrane proteins were probably located on the mitochondrial membrane. The prediction model of secondary structure showed that the protein structure was mainly 尾-folding and irregular crimp. Protein tertiary structure prediction analysis showed that the spatial structure of NCED3-1 and NCED3-2 was very similar to that of NCED3-2. 2 PEG6000 drought stress analysis showed that drought stress could induce the expression of NCED3 gene and the accumulation of endogenous ABA. The expression of NCED3 and the accumulation rate of ABA increased before treatment for 12 h, then the overexpression of K326 NCED3 gene and the expression of RNAi were gradually decreased. The vector was transformed into tobacco plants by Agrobacterium tumefaciens (Agrobacterium tumefaciens). NCED3-1 overexpression and inhibition expression positive plants were obtained.
【學(xué)位授予單位】：河南農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q943.2;S572

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本文編號(hào)：1911405