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  本文選題：RNA干擾　切入點：豌豆　出處：《吉林大學(xué)》2016年碩士論文

【摘要】：類黃酮化合物是豆科植物中一類重要的次生代謝產(chǎn)物,其主要活性成分是由異黃酮化合物、黃酮化合物、查耳酮化合物以及花青素等組成,在豌豆的類黃酮代謝途徑中有需要關(guān)鍵酶基因,包括黃酮合成酶基因(FNS)、黃烷酮-3-羥化酶基因(F3H)以及異黃酮合成酶基因(IFS)等等。異黃酮化合物在生物化學(xué)活性研究發(fā)現(xiàn)具有抗氧化活性、抗溶血活性以及抗真菌活性等方面具有重要作用,因此利用它的活性可以預(yù)防和抑制多種癌癥疾病,但是研究發(fā)現(xiàn)異黃酮類化合物只是在豆科某些植物中存在,使用常規(guī)而傳統(tǒng)的育種方式獲得異黃酮化合物時間和周期較長,使其成分的提高不顯著。目前,關(guān)于異黃酮代謝途徑的研究是當(dāng)今異黃酮化合物研究的焦點問題,但是通過RNAi技術(shù)對豌豆中異黃酮生物合成途徑的研究尚未見報道。本實驗運用RNAi技術(shù)構(gòu)建干擾黃烷酮-3-羥化酶基因(F3H)的載體pBR-F3H,通過農(nóng)桿菌介導(dǎo)輔助抽真空的豌豆莖尖轉(zhuǎn)化法和花粉管通道法將pBR-F3H載體整合到豌豆基因組中,從而使更多的前體(黃酮合成酶,黃烷酮3-羥化酶和異黃酮合成酶有共同的前體4,5,7-三羥黃烷酮)流向異黃酮合成的分支,從而達(dá)到定向提高豌豆異黃酮含量的作用。經(jīng)過實驗后,得到干擾黃烷酮-3-羥化酶基因(F3H)的pBR-F3H載體和轉(zhuǎn)基因陽性植株4棵(兩種轉(zhuǎn)化方法分別獲得2棵),并對轉(zhuǎn)基因陽性植株異黃酮含量進(jìn)行測定,發(fā)現(xiàn)輔助抽真空莖尖轉(zhuǎn)化體系獲得的2株轉(zhuǎn)基因陽性豌豆植株的異黃酮含量相比非轉(zhuǎn)基因?qū)φ胀愣怪仓攴謩e提高了1076.01μg/g和968.86μg/g,異黃酮含量分別提升了53.01%和47.74%;然而花粉管通道轉(zhuǎn)化體系獲得的2株轉(zhuǎn)基因陽性豌豆植株的異黃酮含量相比非轉(zhuǎn)基因?qū)φ胀愣怪仓攴謩e提高了1069.04μg/g和1204.15μg/g,異黃酮含量分別提升了38.37%和43.23%。在將來的實驗中還要進(jìn)行Southern blot,northern blot,western blot等分子檢測,以完善檢測體系。通過轉(zhuǎn)基因手段得到的豌豆新品種,不僅異黃酮含量得到顯著的提高,而且大大節(jié)約了種質(zhì)改良所需的周期,為進(jìn)一步對類黃酮類化合物進(jìn)行研究起到一定作用。
[Abstract]:Flavonoids are important secondary metabolites in legumes. Their main active components are isoflavones, flavonoids, chalcone compounds and anthocyanins.There is a need for key enzyme genes in the flavonoid metabolism pathway of pea, including the flavonoid synthase gene (FNSN), the flavanone-3-hydroxylase gene (F3H) and the isoflavone synthase gene (IFS), and so on.Isoflavones play an important role in biochemical activity studies, such as antioxidant activity, antihemolytic activity and antifungal activity. Therefore, the use of isoflavones can prevent and suppress many kinds of cancer diseases.However, it is found that isoflavones only exist in some plants of Leguminosae, and the time and cycle of obtaining isoflavones by conventional and traditional breeding methods are longer, so the improvement of the composition of isoflavones is not significant.At present, the study of isoflavone metabolic pathway is the focus of the study of isoflavones, but the biosynthesis of isoflavones in pea by RNAi technology has not been reported.In this study, RNAi technique was used to construct the vector pBR-F3H that interfered with the F3H gene of flavanone-3-hydroxylase gene. The pBR-F3H vector was integrated into the pea genome by Agrobacterium tumefaciens assisted vacuum extraction of pea stem tip transformation and pollen tube pathway.Therefore, more precursors (flavonoid synthase, flavanone 3-hydroxylase and isoflavone synthase with common precursor, 45,5-trihydroxyflavanone) flow to the branches of isoflavone synthesis, thus achieving the function of increasing the content of isoflavones in pea.After the experiment, the pBR-F3H vector and 4 transgenic plants were obtained, which interfered with the F3H gene of flavanone -3-hydroxylase gene. Two transgenic plants were obtained by two methods, and the isoflavone content of the transgenic plants was determined.It was found that the contents of isoflavones and isoflavones in the two transgenic positive pea plants obtained by assisted vacuum stem tip transformation system were increased by 1076.01 渭 g / g and 968.86 渭 g / g, respectively, and the contents of isoflavones were increased by 53.01% and 47.74%, respectively, compared with those of the non-transgenic control.The contents of isoflavones and isoflavones were increased by 1069.04 渭 g / g and 1204.15 渭 g / g, respectively, and the contents of isoflavones were increased by 38.37% and 43.23%, respectively.In order to perfect the detection system, Southern blotr Northern blotterwestern blot and other molecules will be detected in the future experiments.The new pea varieties obtained by transgenic method not only increased the content of isoflavones significantly, but also greatly saved the period needed for germplasm improvement, which played a certain role in the further study of flavonoids.
【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】：2016
【分類號】：S643.3;Q943.2

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