本文選題：PPR蛋白 + GhPPR��； 參考：《陜西師范大學(xué)》2016年碩士論文

【摘要】：PPR(Pentatricopeptide repeat)蛋白是近年來在植物中新發(fā)現(xiàn)的一類蛋白質(zhì)超家族,廣泛存在于陸生植物中,在植物生長(zhǎng)發(fā)育過程中具有重要作用。PPR蛋白大多定位在質(zhì)體或線粒體中,參與基因表達(dá)調(diào)控的各項(xiàng)進(jìn)程,包括轉(zhuǎn)錄、翻譯、RNA編輯及RNA剪接等。研究表明植物中PPR基因缺失后會(huì)導(dǎo)致植株生長(zhǎng)遲緩、葉片黃化、種子敗育、花期延遲等一系列不良表型。本研究以陸地棉徐州142為實(shí)驗(yàn)材料,利用棉花EST庫(kù)設(shè)計(jì)引物,通過PCR技術(shù)克隆得到GhPPR9、GhPPR11基因的cDNA及DNA全長(zhǎng)序列；應(yīng)用生物信息學(xué)方法分析其結(jié)構(gòu)特征；利用實(shí)時(shí)熒光定量PCR研究基因在不同組織部位及不同逆境環(huán)境下的表達(dá)模式；用葉肉細(xì)胞原生質(zhì)體研究?jī)蓚�(gè)PPR蛋白在細(xì)胞中的定位,在此基礎(chǔ)上,構(gòu)建VIGS載體,轉(zhuǎn)化棉花,分別干涉GhPPR9和GhPPR11基因的表達(dá),檢測(cè)轉(zhuǎn)基因棉花的各項(xiàng)生理生化指標(biāo),以期深入研究這兩個(gè)基因在棉花中的功能。取得的主要研究結(jié)果如下：1、GhPPR9的cDNA全長(zhǎng)3072bp,編碼1023個(gè)氨基酸,沒有內(nèi)含子,分子量114.22kD,等電點(diǎn)7.17。GhPPR9屬于PPR基因家族的P亞族,有25個(gè)PPR基序；與可可(XP_007038120.1),蓖麻(XP 002511099.1)等有較高的同源性。GhPPR11的cDNA全長(zhǎng)1914bp,編碼637個(gè)氨基酸,沒有內(nèi)含子,分子量71.53kD,等電點(diǎn)7.79。GhPPR11屬于PPR基因家族的PLS亞族,有13個(gè)PPR基序；GhPPR11與可可中的PPR蛋白(XP_007052071.1)相似度最高,與克萊門柚(XP_006445271.1)也有較高的同源性。2.GhPPR9亞細(xì)胞定位結(jié)果預(yù)測(cè)該基因定位在線粒體上；GhPPR9在根、莖、葉、花及纖維的不同發(fā)育時(shí)期均有表達(dá),在花和開花后5天的纖維中表達(dá)較高,且不受脅迫誘導(dǎo),可能與花和纖維發(fā)育有關(guān)。3.GhPPR11蛋白定位于細(xì)胞核與細(xì)胞質(zhì)中。GhPPR11在不同組織中表達(dá)量都很低;對(duì)生長(zhǎng)1周的棉花幼苗分別進(jìn)行ABA,NaCl,干旱和高溫處理,GhPPR11均有響應(yīng),并且在6小時(shí)、9小時(shí)對(duì)四種脅迫處理的響應(yīng)達(dá)到峰值,表明GhPPR11的表達(dá)可能與棉花耐逆性有關(guān)。4、利用病毒介導(dǎo)基因沉默(VIGS)的方法,構(gòu)建VIGS載體,對(duì)棉花中的GhPPR9及GhPPR11分別進(jìn)行了基因干涉,檢測(cè)了它們的表型特征,與WT和對(duì)照組相比,在植株高度上表型變化不大,棉桃數(shù)量相對(duì)較少,GhPPR9的葉綠素含量低。
[Abstract]:PPR(Pentatricopeptide repeat protein is a new class of protein superfamily found in plants in recent years. It widely exists in terrestrial plants and plays an important role in plant growth and development. Participate in the regulation of gene expression, including transcription, translation of RNA editing and RNA splicing. Studies have shown that the absence of PPR gene in plants will lead to plant growth retardation, leaf yellowing, seed abortion, late flowering and a series of bad phenotypes. In this study, Upland cotton Xuzhou 142 was used as experimental material and primers were designed using cotton EST library to clone the cDNA and DNA full length sequences of GhPPR9 and GhPPR11 gene by PCR technique, and the structural characteristics were analyzed by bioinformatics. Real-time fluorescence quantitative PCR was used to study the expression patterns of genes in different tissues and under different stress conditions, and mesophyll protoplasts were used to study the localization of two PPR proteins in the cells. On this basis, the VIGS vector was constructed and transformed into cotton. The expression of GhPPR9 and GhPPR11 genes were interfered to detect the physiological and biochemical indexes of transgenic cotton in order to study the function of the two genes in cotton. The main results are as follows: the cDNA length of 1: 1 GhPPR9 is 3072 BP, encoding 1023 amino acids, with no intron, molecular weight 114.22 KD. The isoelectric point 7.17.GhPPR9 belongs to the P subfamily of PPR gene family with 25 PPR motifs. There is a high homology of the cDNA of .GhPPR11, encoding 637 amino acids with no intron, molecular weight 71.53kD. the isoelectric point 7.79.GhPPR11 belongs to the PLS subfamily of the PPR gene family, and the isoelectric point 7.79.GhPPR11 belongs to the PLS subfamily of the PPR gene family with a molecular weight of 71.53kD. 13 PPR motifs, GhPPR11, have the highest similarity with PPR protein in cocoa (XP007052071.1), and have higher homology with Clemeno pummelo XP0045271.1). 2. The results of subcellular localization of GhPPR9 predict that the gene is located on mitochondria in root, stem and leaf. At different developmental stages of flower and fiber, the expression was higher in the fiber of flower and 5 days after anthesis, and was not induced by stress. It may be related to flower and fiber development. 3. GhPPR11 protein is located in nucleus and cytoplasm, and the expression of GhPPR11 in different tissues is very low. The response to four stress treatments reached a peak at 6 hours or 9 hours, indicating that the expression of GhPPR11 might be related to cotton stress tolerance. The VIGS vector was constructed by virus mediated gene silencing (VIGS). The phenotypic characteristics of GhPPR9 and GhPPR11 in cotton were detected by gene interference. Compared with WT and control, the phenotypic changes were not obvious, and the chlorophyll content of GhPPR9 was lower than that of cotton peach.
【學(xué)位授予單位】：陜西師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q943.2

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本文編號(hào)：1893804