豌豆pBR-F3H基因RNA干扰表达载体的构建与遗传转化

发布时间：2018-04-05 14:30

本文选题：RNA干扰　切入点：豌豆　出处：《吉林大学》2016年硕士论文

【摘要】：类黄酮化合物是豆科植物中一类重要的次生代谢产物,其主要活性成分是由异黄酮化合物、黄酮化合物、查耳酮化合物以及花青素等组成,在豌豆的类黄酮代谢途径中有需要关键酶基因,包括黄酮合成酶基因(FNS)、黄烷酮-3-羟化酶基因(F3H)以及异黄酮合成酶基因(IFS)等等。异黄酮化合物在生物化学活性研究发现具有抗氧化活性、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预防和抑制多种癌症疾病,但是研究发现异黄酮类化合物只是在豆科某些植物中存在,使用常规而传统的育种方式获得异黄酮化合物时间和周期较长,使其成分的提高不显著。目前,关于异黄酮代谢途径的研究是当今异黄酮化合物研究的焦点问题,但是通过RNAi技术对豌豆中异黄酮生物合成途径的研究尚未见报道。本实验运用RNAi技术构建干扰黄烷酮-3-羟化酶基因(F3H)的载体pBR-F3H,通过农杆菌介导辅助抽真空的豌豆茎尖转化法和花粉管通道法将pBR-F3H载体整合到豌豆基因组中,从而使更多的前体(黄酮合成酶,黄烷酮3-羟化酶和异黄酮合成酶有共同的前体4,5,7-三羟黄烷酮)流向异黄酮合成的分支,从而达到定向提高豌豆异黄酮含量的作用。经过实验后,得到干扰黄烷酮-3-羟化酶基因(F3H)的pBR-F3H载体和转基因阳性植株4棵(两种转化方法分别获得2棵),并对转基因阳性植株异黄酮含量进行测定,发现辅助抽真空茎尖转化体系获得的2株转基因阳性豌豆植株的异黄酮含量相比非转基因对照豌豆植株分别提高了1076.01μg/g和968.86μg/g,异黄酮含量分别提升了53.01%和47.74%;然而花粉管通道转化体系获得的2株转基因阳性豌豆植株的异黄酮含量相比非转基因对照豌豆植株分别提高了1069.04μg/g和1204.15μg/g,异黄酮含量分别提升了38.37%和43.23%。在将来的实验中还要进行Southern blot,northern blot,western blot等分子检测,以完善检测体系。通过转基因手段得到的豌豆新品种,不仅异黄酮含量得到显著的提高,而且大大节约了种质改良所需的周期,为进一步对类黄酮类化合物进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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S643.3;Q943.2

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