木薯块根β-胡萝卜素积累的蛋白质组学研究

发布时间：2018-05-28 19:51

本文选题：木薯 + HPLC　；参考：《海南大学》2015年硕士论文

【摘要】：木薯(Manihot esculenta Crantz)属大戟科木薯属植物,是世界上近7亿人的主要口粮。普通白心木薯块根蛋白质和类胡萝卜素含量较低,长期食用会产生各种疾病。β-胡萝卜素是维生素A的前体,其含量是衡量木薯营养价值的重要指标。因此,对木薯β-胡萝卜素含量的测定、合成途径关键酶及差异蛋白质组学的研究显得尤为重要,对筛选高类胡萝卜素含量的木薯种质具有重要的意义。本研究选用食用黄心木薯品种SC9自然杂交F1代木薯为研究材料,利用HPLC测量其β-胡萝卜素含量,同时对块根颜色和含量进行统计分析。选取具有代表性的6个木薯品系,分别从基因和蛋白质水平检测β-胡萝卜素合成关键基因LYC-b的表达水平,结合木薯块根差异蛋白质组学分析,探讨木薯块根β-胡萝卜素积累的分子机制,为培育高类胡萝卜素的木薯品种提供理论依据。研究结果如下： 1、优化木薯β-胡萝卜素含量的HPLC方法,流动相为甲醇：叔丁基甲醚(70：30,v/v),流速为1.0ml/min,保留时间为12.7min左右。结果显示,该方法分离效果好,精密性和重复性好,分析速度快,能够满足批量检测要求。 2、木薯块根β-胡萝卜素含量随颜色(白色、乳白色、黄色、深黄色)的加深而升高。60份木薯块根样品中,黄色块根样品与白色块根样品的比例为1：1；其中深黄色样品占10%,黄色样品占40%,乳白色样品占13%,白色样品占37%。 3、β-胡萝卜素合成关键基因LYC-b的表达水平与木薯块根颜色和β-胡萝卜素含量没有正相关；但颜色最深且β-胡萝卜素含量最高木薯31-R块根LYC-B酶表达水平最高。 4、差异蛋白质组学分析得出直接或间接参与类胡萝卜素合成的蛋白有葡萄糖-6-磷酸异构酶、醛缩酶、烯醇化酶、电子传递黄素蛋白-泛醌氧化还原酶、琥珀酸脱氢、素细胞色P450、谷胱甘肽转移酶、抗坏血酸过氧化物酶、谷胱甘肽脱氢酶、蛋白质二硫键异构酶、醛酮还原酶、超氧化物歧化酶、脱水素蛋白和钙调蛋白等,它们均在木薯31-R及24-R木薯块根中上调表达。
[Abstract]:Cassava (Manihot esculenta Crantz) belongs to the family Euphorbiaceae cassava, which is the main food ration for nearly 700 million people in the world. The content of protein and carotenoid in the tuber root of common white cassava is low, and it can produce various diseases for a long time. 尾 -carotene is the precursor of vitamin A. the content of 尾 -carotene is an important index to measure the nutritional value of cassava. Therefore, the determination of 尾 -carotene content in cassava, the study of key enzymes and differential proteomics of synthesis pathway are particularly important, and it is of great significance to screen cassava germplasm with high carotenoid content. In this study, the natural hybrid F1 cassava (SC9) was used as the research material. The 尾 -carotene content was measured by HPLC, and the color and content of root were analyzed statistically. Six representative cassava strains were selected to detect the expression of 尾 -carotene biosynthesis key gene LYC-b at gene and protein levels, respectively, and combined with differential proteomics analysis of cassava tuber root. To explore the molecular mechanism of 尾 -carotene accumulation in cassava tubers and provide theoretical basis for the cultivation of cassava varieties with high carotenoid content. The findings are as follows: 1. The HPLC method of cassava 尾 -carotene content was optimized. The mobile phase was methanol: tertiary Ding Ji methyl ether 70: 30 v / v, flow rate was 1.0 ml / min and retention time was about 12.7min. The results show that the method has good separation effect, good precision and repeatability, fast analysis speed and can meet the requirements of batch detection. 2. The 尾 -carotene content of cassava tuber root increased with the deepening of color (white, milky white, yellow, dark yellow). The ratio of yellow root sample to white root sample is 1: 1, of which 10 are dark yellow sample, 40 yellow sample, 13 milky white sample and 37 white sample. 3. The expression of 尾 -carotene synthesis key gene LYC-b was not positively correlated with the color of cassava root and the content of 尾 -carotene, but the highest expression level of 尾 -carotene and 尾 -carotene was found in the root of cassava 31-R. 4, differential proteomics analysis showed that the proteins directly or indirectly involved in the synthesis of carotenoids were glucose-6-phosphate isomerase, aldolase, enolase, electron transfer protein-ubiquinone redox enzyme, and succinic acid dehydrogenation. Cytochrome P450, glutathione transferase, ascorbic acid peroxidase, glutathione dehydrogenase, protein disulfide isomerase, aldehyde-ketone reductase, superoxide dismutase, dehydrin protein and calmodulin, etc. They were upregulated in the tuber roots of cassava 31-R and 24-R.
【学位授予单位】：海南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S533

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