Studies On The Expression Of MiR5753 And MiR5251 And Their Regulation Of Flavonoids Biosynthesis In Safflower

Safflower(Cachamus tinctorius. L) is a member of compositae family. As the effective secondary metabolites extracted from safflower, flavonoids have important pharmacological research significance. Micro RNA is a kind of endogenous small single-stranded RNA molecule with 20-25 nucleotides and no open reading frame, has no function of encoding proteins.Micro RNA is a kind of negative regulatory factor, and plays an important regulatory role in the plant. Recent discovery shows that micro RNAs plays an important role in regulating secondary metabolism in plants, and these results encourage people to strengthen the study of medicinal plants of micrornas. This research analyzed the expression of mi R5251, mi R5753 and their target genes LDOX in different flowering through the Real-time PCR, and determined the flavonoid content in each flowering of safflower, built mi R5251 and mi R5753 overexpression vector to transformate arabidopsis, and finally to clarify mi R5251 and mi R5753 regulating the biosynthesis of flavonoids by targeting LDOX according to the difference between the flavonoids contents of transgenic and wild type arabidopsis.The results as following:1.By detecting flavonoids content in different florescence using ultraviolet spectrophotometer at 400 nm, we found that the level of flavone was the hignest in full-bloom stage, lower in initial bloom stage, and lowest in bud stage.2.The chapter aims at determining the change of expression of mi R5251, mi R5753 and their target genes LDOX in different varieties and flowering by q RT-PCR, the results showed that with the increase of the flowering extent, the expression of mi R5251 and mi R5753 and flavonoid content increased, however, the expression of LDOX decreased in full-bloom and initial bloom stage.3.By double enzyme digestion, we can successfully construct mi R5251 and cmi R5753 precursor to p BASTA expression vector, and then moved into arabidopsis thaliana by using agrobacterium-mediated transformation to obtain 20 T3 generation transgenic arabidopsis thaliana of mi R5251 and 14 T3 generation transgenic arabidopsis thaliana of mi R5753.4.The results of quantitative analysis of miR5753 and mi R5251 precursor sequence in transgenic arabidopsis thaliana showed that the expression of mi R5251 and mi R5753 increased. Analyzing the flavonoid content of high expression transgenic strain selected by quantitative screening, the results showed that mi R5251 and mi R5753 can improve the flavonoid level of arabidopsis leaves, petals, stem, and mi R5251 and mi R5753 can up-regulate the synthesis of flavonoid in the arabidopsis thaliana.