【摘要】：大豆是自花授粉作物,同時也是我國重要的經(jīng)濟(jì)作物,其含有豐富的蛋白質(zhì)及不飽和脂肪酸等都具有重要的營養(yǎng)價(jià)值。隨著人們生活水平的提高,對大豆的品質(zhì)提出了更高的要求。大豆異黃酮是大豆類黃酮代謝途徑的次生代謝產(chǎn)物,對于延緩衰老,改善更年期癥狀,預(yù)防骨質(zhì)疏松,防癌抗癌,降低心臟病,心腦血管疾病等都有重要作用,已廣泛應(yīng)用于醫(yī)療、保健等方面。目前市場供不應(yīng)求,選育高異黃酮含量大豆品種是解決這一問題的有效途徑。本研究在大豆傳統(tǒng)雜交方法的基礎(chǔ)上,采用不去雄直接授粉的方法進(jìn)行雜交,將該方法與去雄直接授粉后雜交莢成活率和偽雜交種進(jìn)行對比。結(jié)果表明,不去雄直接授粉的方法的雜交莢成活率顯著高于大豆去雄授粉的,而偽雜交種產(chǎn)生率兩者無顯著差別。同時母本不去雄較去雄更加省時省力、簡單易行,更適合大規(guī)模推廣。利用高效液相色譜法測定T4代,T5代,T6代轉(zhuǎn)異黃酮合酶基因(IFS)、查爾酮異構(gòu)酶(CHI)、MYB12B2轉(zhuǎn)錄因子大豆籽粒異黃酮含量。結(jié)果表明,高世代轉(zhuǎn)基因材料籽�？偖慄S酮含量均比其轉(zhuǎn)基因受體品種異黃酮含量顯著提高且穩(wěn)定遺傳。為了進(jìn)一步提高轉(zhuǎn)基因大豆品系的異黃酮含量,本研究通過利用不去雄直接授粉大豆雜交方法,將IFS基因基因過表達(dá)植株分別與吉大豆5號進(jìn)行雜交,MYB12B2轉(zhuǎn)錄因子過表達(dá)植株與IFS、CHI過表達(dá)株系分別進(jìn)行雜交,獲得F1代植株,并通過PCR檢測技術(shù),Basta葉片涂抹,Bar基因試紙條等方法,結(jié)合田間農(nóng)藝性狀,鑒定攜帶目標(biāo)基因的F1植株。對IFS,CHI基因過表達(dá)植株與吉大豆5號的雜交種F2代進(jìn)行異黃酮含量進(jìn)行檢測,結(jié)果表明,IFS,CHI基因過表達(dá)植株與吉大豆5號雜交材料可以提高大豆異黃酮含量。且CHI基因過表達(dá)植株對于改善吉大豆5號總異黃酮含量的效果好于IFS基因過表達(dá)植株。對IFS,CHI基因過表達(dá)植株與MYB12B2轉(zhuǎn)基因過表達(dá)植株的雜交種F2代進(jìn)行異黃酮含量進(jìn)行檢測。結(jié)果表明,MYB12B2過表達(dá)植株與IFS,CHI基因過表達(dá)植株雜交F2代植株的異黃酮含量均比雙親有所提高,且雜交組合T(MYB12B2)x T(IFS)的雜交種異黃酮含量提高幅度高于雜交組合T(MYB12B2)x T(CHI)的雜交種異黃酮含量。
[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).
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S565.1

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