柴达木盆地黑果枸杞F3′5′H基因启动子克隆与活性分析

发布时间：2017-12-27 14:29

本文关键词：柴达木盆地黑果枸杞F3′5′H基因启动子克隆与活性分析　出处：《青海大学》2017年硕士论文　论文类型：学位论文

【摘要】：黑果枸杞主要分布于我国青海、宁夏、新疆等西部偏远地区,是我国西北地区特有的保持水土和缓解土壤盐碱化的理想植物。其黑色的果实中所含的大量花青素具有重要的医疗保健作用。类黄酮-3′,5′-羟化酶合成基因(F3′5′H基因)是合成使黑果枸杞果实呈黑色的矮牵牛色素的关键节点基因。为阐明F3′5′H基因的表达机理,明晰F3′5′H基因在黑果枸杞呈色中的作用,确定黑果枸杞及其白化果实中F3′5′H基因启动子的活性,最终揭示黑果枸杞及其白化果实呈色差异的原因。本研究以黑果枸杞果实为研究对象,以其白化果实为对照,首先确定了两者中F3′5′H基因的表达模式,继而克隆黑果枸杞果实F3′5′H基因全长cDNA序列,以该序列为模板,设计引物克隆两者中F3′5′H基因的启动子。将启动子与GUS基因融合构建植物瞬时表达载体,利用农杆菌介导的瞬时转化法转化烟草叶片,烟草培养后进行组织化学染色以确定启动子的启动活性。结果如下:1.利用实时荧光定量PCR(Real-time PCR)技术检测了F3′5′H基因在黑果枸杞及其白化果实中不同时期的表达模式,发现在S2-S5时期,其在黑果枸杞果实中的表达量明显高于白化果实中的表达量。2.利用RACE技术克隆得到长为1689bp的黑果枸杞F3′5′H基因的全长cDNA序列,其中包含长度为1527bp可编码508个氨基酸的编码区。对该全长cDNA序列及其编码的氨基酸以及蛋白质进行了生物信息学分析,发现黑果枸杞F3′5′H基因的保守性较高,F3′5′H蛋白为非分泌蛋白,多肽链内部含有57个磷酸化位点,且蛋白为亲水性蛋白。3.利用Genome Walking Kit(TaKaRa)试剂盒通过3次染色体步移克隆出黑果枸杞及其白化果实F3′5′H基因的启动子片段并测序。利用DNA MAN软件分析发现2个启动子序列的同源性高达90.3%,通过在线分析网站Plant Care对启动子序列进行预测,发现两者中均具有多个普遍存在于真核生物启动子中的基本元件如TATA-Box及CAAT-Box。除此之外,还有防御和胁迫应答元件TC-rich repeats、创伤响应元件WUN-motif以及多个与光响应相关的元件Sp1、Box I和G-box等。还存在一些其他类型的元件,如与胚乳表达相关的元件skin-1 motif和厌氧诱导元件ARE。另外在黑果枸杞启动子中预测到与茉莉酸甲酯响应相关的元件TGACG-motif,而在黑果枸杞白化果实启动子中没有预测到。4.分别将2个启动子与GUS报告基因融合以构建植物瞬时表达载体,进而利用农杆菌介导的瞬时转化法转化烟草叶片,烟草培养后,通过组织化学染色来确定启动子的启动活性。结果表明黑果枸杞及其白化果实的F3′5′H基因启动子均具有启动活性,进而利用荧光定量对2个启动子所驱动的GUS基因的表达量进行了分析,结果表明黑果枸杞所驱动的GUS基因的表达量是黑果枸杞白化果实的3.09倍。
[Abstract]:Lycium ruthenium is mainly distributed in remote areas of Western China such as Qinghai, Ningxia and Xinjiang. It is an ideal plant for preserving soil and water and alleviating soil salinization in Northwest China. The large amount of anthocyanins contained in its black fruit has an important health care role. The flavonoid -3 ', 5' - hydroxylase synthesis gene (F3 '5' H gene) is the key node gene to synthesize the black Petunia pigment of the fruit of the black fruit Lycium barbarum. In order to clarify the expression mechanism of F3 '5' H gene, clarify the role of F3 '5' H gene in the coloration of Lycium barbarum L., we determined the activity of F3 '5' H promoter in Lycium barbarum and its albino fruits, and finally revealed the reason of color difference of Lycium barbarum and its albino fruits. In this study, Lycium ruthenicum fruit as the research object, with its whitening fruit as the control, first determine the expression pattern of the F3 '5' H gene, and then cloned Lycium ruthenicum fruit F3 'cDNA 5' full-length H gene sequence to the sequence as a template, primers were designed to clone the F3 '5' H gene promoter. The promoter was fused with GUS gene to construct plant transient expression vector. The tobacco leaves were transformed by Agrobacterium mediated transformation, and histochemical staining was done after tobacco culture to determine promoter activity. The results are as follows: 1. using real-time fluorescence quantitative PCR (Real-time PCR) technique to detect F3 '5' H gene in Lycium ruthenicum fruit in different periods and albino expression patterns found in the S2-S5 period, its expression in Lycium ruthenicum fruit in the fruit was significantly higher than that in the albino scale up. 2., using RACE technology, we cloned the full-length cDNA sequence of 1689bp 'F3' 5 'H gene with a length of 1527bp, encoding 508 amino acids. The full-length cDNA sequence and its encoding amino acids and proteins were analyzed by bioinformatics, conserved found that Lycium ruthenicum F3 '5' H F3 '5' gene, H protein is a non secreted protein, polypeptide chain contains 57 phosphorylation sites, and the protein is a hydrophilic protein. 3., we used Genome Walking Kit (TaKaRa) kit to clone the promoter fragment of Lycium barbarum and its albino F3 '5' H gene and sequenced by 3 chromosome walking. Using DNA MAN software analysis, we found that the homology of 2 promoter sequences was as high as 90.3%. We predicted the promoter sequence by online analysis website Plant Care. It is found that both of them have many basic components which are ubiquitous in eukaryotic promoters, such as TATA-Box and CAAT-Box. In addition, there are defense and stress response components TC-rich repeats, trauma response element WUN-motif and multiple light response components Sp1, Box I and G-box. There are also some other types of components, such as the element skin-1 motif associated with the endosperm expression and the anaerobic inducer, ARE. In addition, TGACG-motif in response to methyl jasmonate was predicted in the promoter of Lycium barbarum, but not in the promoter of Lycium barbarum fruit. 4., the 2 promoters were fused with GUS reporter gene to construct plant transient expression vector, and then Agrobacterium mediated transformation was applied to transform tobacco leaves. After tobacco culture, the promoter activity of promoter was determined by histochemical staining. The results show that Lycium ruthenicum fruit whitening and F3 '5' H gene promoter with promoter activity, and expression of GUS gene by fluorescent quantitative driven on 2 promoter was analyzed. The results show that the expression of GUS gene driven by Lycium ruthenicum is 3.09 times the black fruit wolfberry whitening fruit.
【学位授予单位】：青海大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S567.19

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