棉花中两个新的PPR基因的克隆及功能分析

发布时间：2018-05-15 20:20

本文选题：PPR蛋白 + GhPPR　；参考：《陕西师范大学》2016年硕士论文

【摘要】：PPR(Pentatricopeptide repeat)蛋白是近年来在植物中新发现的一类蛋白质超家族,广泛存在于陆生植物中,在植物生长发育过程中具有重要作用。PPR蛋白大多定位在质体或线粒体中,参与基因表达调控的各项进程,包括转录、翻译、RNA编辑及RNA剪接等。研究表明植物中PPR基因缺失后会导致植株生长迟缓、叶片黄化、种子败育、花期延迟等一系列不良表型。本研究以陆地棉徐州142为实验材料,利用棉花EST库设计引物,通过PCR技术克隆得到GhPPR9、GhPPR11基因的cDNA及DNA全长序列；应用生物信息学方法分析其结构特征；利用实时荧光定量PCR研究基因在不同组织部位及不同逆境环境下的表达模式；用叶肉细胞原生质体研究两个PPR蛋白在细胞中的定位,在此基础上,构建VIGS载体,转化棉花,分别干涉GhPPR9和GhPPR11基因的表达,检测转基因棉花的各项生理生化指标,以期深入研究这两个基因在棉花中的功能。取得的主要研究结果如下：1、GhPPR9的cDNA全长3072bp,编码1023个氨基酸,没有内含子,分子量114.22kD,等电点7.17。GhPPR9属于PPR基因家族的P亚族,有25个PPR基序；与可可(XP_007038120.1),蓖麻(XP 002511099.1)等有较高的同源性。GhPPR11的cDNA全长1914bp,编码637个氨基酸,没有内含子,分子量71.53kD,等电点7.79。GhPPR11属于PPR基因家族的PLS亚族,有13个PPR基序；GhPPR11与可可中的PPR蛋白(XP_007052071.1)相似度最高,与克莱门柚(XP_006445271.1)也有较高的同源性。2.GhPPR9亚细胞定位结果预测该基因定位在线粒体上；GhPPR9在根、茎、叶、花及纤维的不同发育时期均有表达,在花和开花后5天的纤维中表达较高,且不受胁迫诱导,可能与花和纤维发育有关。3.GhPPR11蛋白定位于细胞核与细胞质中。GhPPR11在不同组织中表达量都很低;对生长1周的棉花幼苗分别进行ABA,NaCl,干旱和高温处理,GhPPR11均有响应,并且在6小时、9小时对四种胁迫处理的响应达到峰值,表明GhPPR11的表达可能与棉花耐逆性有关。4、利用病毒介导基因沉默(VIGS)的方法,构建VIGS载体,对棉花中的GhPPR9及GhPPR11分别进行了基因干涉,检测了它们的表型特征,与WT和对照组相比,在植株高度上表型变化不大,棉桃数量相对较少,GhPPR9的叶绿素含量低。
[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.
【学位授予单位】：陕西师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2

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