高异黄酮转基因大豆新品系的创制与鉴定

发布时间：2018-07-25 09:34

【摘要】：大豆是自花授粉作物,同时也是我国重要的经济作物,其含有丰富的蛋白质及不饱和脂肪酸等都具有重要的营养价值。随着人们生活水平的提高,对大豆的品质提出了更高的要求。大豆异黄酮是大豆类黄酮代谢途径的次生代谢产物,对于延缓衰老,改善更年期症状,预防骨质疏松,防癌抗癌,降低心脏病,心脑血管疾病等都有重要作用,已广泛应用于医疗、保健等方面。目前市场供不应求,选育高异黄酮含量大豆品种是解决这一问题的有效途径。本研究在大豆传统杂交方法的基础上,采用不去雄直接授粉的方法进行杂交,将该方法与去雄直接授粉后杂交荚成活率和伪杂交种进行对比。结果表明,不去雄直接授粉的方法的杂交荚成活率显著高于大豆去雄授粉的,而伪杂交种产生率两者无显著差别。同时母本不去雄较去雄更加省时省力、简单易行,更适合大规模推广。利用高效液相色谱法测定T4代,T5代,T6代转异黄酮合酶基因(IFS)、查尔酮异构酶(CHI)、MYB12B2转录因子大豆籽粒异黄酮含量。结果表明,高世代转基因材料籽粒总异黄酮含量均比其转基因受体品种异黄酮含量显著提高且稳定遗传。为了进一步提高转基因大豆品系的异黄酮含量,本研究通过利用不去雄直接授粉大豆杂交方法,将IFS基因基因过表达植株分别与吉大豆5号进行杂交,MYB12B2转录因子过表达植株与IFS、CHI过表达株系分别进行杂交,获得F1代植株,并通过PCR检测技术,Basta叶片涂抹,Bar基因试纸条等方法,结合田间农艺性状,鉴定携带目标基因的F1植株。对IFS,CHI基因过表达植株与吉大豆5号的杂交种F2代进行异黄酮含量进行检测,结果表明,IFS,CHI基因过表达植株与吉大豆5号杂交材料可以提高大豆异黄酮含量。且CHI基因过表达植株对于改善吉大豆5号总异黄酮含量的效果好于IFS基因过表达植株。对IFS,CHI基因过表达植株与MYB12B2转基因过表达植株的杂交种F2代进行异黄酮含量进行检测。结果表明,MYB12B2过表达植株与IFS,CHI基因过表达植株杂交F2代植株的异黄酮含量均比双亲有所提高,且杂交组合T(MYB12B2)x T(IFS)的杂交种异黄酮含量提高幅度高于杂交组合T(MYB12B2)x T(CHI)的杂交种异黄酮含量。
[Abstract]:Soybean is not only a self-pollinating crop but also an important cash crop in China. It contains abundant protein and unsaturated fatty acids and has important nutritional value. With the improvement of people's living standard, the quality of soybean is required higher. Soybean isoflavone is a secondary metabolite of soybean flavonoid metabolism pathway, which plays an important role in delaying senescence, improving menopausal symptoms, preventing osteoporosis, preventing cancer and anticancer, reducing heart disease, cardiovascular and cerebrovascular diseases, etc. Has been widely used in medical care, health care and other aspects. At present, the market is in short supply, and breeding soybean varieties with high isoflavone content is an effective way to solve this problem. On the basis of traditional soybean hybridization method, the method of direct pollination without male removal was used to carry out hybridization, and the survival rate of hybrid pods and pseudo-hybrids after direct pollination were compared. The results showed that the survival rate of hybrid pods without direct pollination was significantly higher than that of soybean without direct pollination, but there was no significant difference in the production rate of pseudo hybrids. At the same time, the mother did not go to male than to save time and effort, simple and easy, more suitable for large-scale promotion. The content of isoflavone synthase gene (IFS), chalcone isomerase (CHI) and MYB12B2 transcription factor in soybean grain was determined by high performance liquid chromatography (HPLC). The results showed that the contents of total isoflavones in the seeds of the transgenic materials in the higher generation were significantly higher than those in the transgenic recipient varieties and the inheritance of isoflavones was stable. In order to further improve the content of isoflavones in transgenic soybean lines, a direct pollination method was used to improve the content of isoflavones in transgenic soybean lines. The overexpression plants of IFS gene were hybridized with the over expression of MYB12B2 transcriptional factors and the overexpression lines of IFSX Chi, respectively. The F1 plants were obtained, and the leaves of Basta were smeared with the bar gene test strip by PCR detection technique. The F1 plants carrying the target gene were identified by combining the agronomic characters in the field. The content of isoflavones in F _ 2 generation of F _ 2 hybrids with the over-expression of IFSI Chi gene and Ji soybean 5 was determined. The results showed that the content of isoflavones could be increased between the over-expressed plants and the hybrids of Ji Soybean 5. The effect of CHI gene overexpression on improving the total isoflavone content of Ji soybean 5 was better than that of IFS gene overexpression. The content of isoflavones in F _ 2 hybrids of MYB12B2 transgenic plants and IFSS-CHI overexpression plants were detected. The results showed that the content of isoflavones in F2 generation of the over-expressed plants of MYB12B2 and IFSG-CHI gene was higher than that of their parents. The content of isoflavones in hybrid T (MYB12B2) x T (IFS) was higher than that in MYB12B2) x T (CHI) (hybrid T), and the content of isoflavone in hybrid T (MYB12B2) x T (CHI) was higher than that in hybrid T (MYB12B2) x T (CHI).
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S565.1

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