Researches Of RNAi Vectors Constructing And Genetic Transformation About EST Of MYB Transcriptional Factors Family

MYB transcriptional factor is one of the largest families in plants, which plays important role in plants in regulation of secondary metabolism, responding to hormones and environmental factors, also regulation of cell differentiation, cell cycles and the formation of leaves and others organs. Because of its widely physiological functions, the research of the structure and function of plant transcription factor have been researched in the molecular biology field.According to controlling the expression of target gene in transcriptional level by transcription factors, it is an important way for plants to control their growth development and physiological metabolism. Most of MYB transcription factors take part in the metabolism of flavonoids in plants. In recent years, MYB transcription factors are confirmed in flavonoids metabolic pathways. Recent research has found that MYB transcription factors play a role in regulation of secondary metabolism of phenypropanoids in plants. Among of them, the R2R3-MYB transcription factors are widely involved in the metabolic pathways in phenypropanoids, then control the color of plants. Until now, almost all MYB transcription factors reported which are related to synthesis of anthocyanidins are R2R3-MYB proteins. But we have newly founded that CPC which is single MYB-type gene can also control plant color. These evidences help us deeply understand MYB transcription factors, also provide new strategy for us to control the anthocyanidins conduction.In this dissertation, several steps were taken to realize this idea:1,Construct MYB transcription factor- EB175067 RNAi vectorAccording to the EST sequences (EB 175067) in NCBI, which is thought to be relevant to plant color comparing to Arabidopsis, is also a MYB transcription factor. We cloned this gene in petunia, and constructed an ihp-RNA vector through homologous recombination method (BP reaction). The target gene is inserted in the Phellsgate2 vector through traditional cloning way combing with Gateway method.2,Transform CPC over-expression vector and EB 175067 RNAi vector into petunia and tobacco.We have transformed the EB175067 RNAi vector into early-flower tobacco through the leaf disc method mediated by agrobaeterium tumefaciens. PCR analysis of the transformed plants shows that we have obtained 60 positive transformed plants, which confirmed that the EB175067 gene was already transformed into early-flower tobacco.The CPC transcription factors which contain single MYB structure originally participate in the forming of trichome and root hair in plants, but we found that it can also adjust the gene forming of late flavonoids. In the high excressing CPC transfer-tobacco, with the changing of trichome and root hair distribution, its anthocyanidins is affected, its flowers turning lighter and whiter. So in the experiment, we also transfer over-express CPC to petunias V26, in order to prove the mechanism and function of CPC transcription factors. According to the PCR detection of transfer plants, we got 35 positive transfer plants, it shows that the exogenous gene had inserted into the genome of plants.3,Optimize the transformation system of petunia species V26According to several main factors influenced the transformation efficiency by agrobaeterium tumefaciens, we explored the time of agrobaeterium infection, co-cultue time and delaying choosing time influencing to the efficiency of transformation. In this experiment, we have found that when the petunia leaves infected in 5min, co-cultured 3 days on co-culture medium without experiencing delay selection, and then transformed into selection and differention medium, the transformation efficiency is obviously improved.