Cloning And Functional Analysis Of Potato Gene StR2R3-MYB1

Plant R2R3-MYB transcription factor involved in a wide range of stress responses, development and differentiation, regulation of secondary metabolism and other vital activities. Firstly, I apply modern bioinformatics technology to compare, analyse and forecast potato StR2R3-MYB1gene sequence, function domain features to speculate its cellular localization and biological function, and then using quantitative PCR to analyse its response of the light, temperature stress and the relevance with anthocyanin synthesis. And Finally by CDS sequence for homology cloning potato StR2R3MYB1gene to do cellular localization analysis, organization express specificity and light temperature response pattern analysis system. I research potato StR2R3-MYB transcription factor answer mode under different environmental conditions of light, and by constructing gene over-expression and interference lines for further systematic research on potato StR2R3-MYB1molecular mechanism of stress responses laid an important foundation. The main results of this paper are as follows:(1)The use of bioinformatics prediction of potato R2R3-MYB transcription factor function, and other species such as Arabidopsis R2R3-MYB amino acid sequence analysis showed that the evolutionary tree, potato and tomato R2R3-MYB S1CMYB1, eggplant SmMYB the evolutionary relationships close homology of up to99%homology and with the Arabidopsis MYB113also more than70%.(2)Homology cloning method using potato R2R3-MYB transcription factor cloned. Find potato StR2R3-MYB1gene homologous CDS sequences in NCBI website to design cloning primers and to do RT-PCR. Sequence analysis showed that the CDS full-length is798bp, encoding265amino acids, amino acid sequence analysis showed that, StR2R3-MYB1contains two DNA-bingding domain, two tandem myb-domain, belonging to the class R2R3MYB transcription factors. Bioinformatics methods to analyze the nucleotide and amino acid sequences showed that:a lot of light elements, such as the presence of associated regulatory regions StR2R3-MYB1of:G-Box, Box4, Box Ⅱ, I-Box, MNF1etc. While found that ABA induced related components ABRE; circadian related components circadian; jasmonate-related components CGTCA-motif, StR2R3-MYB1likely to induce the transcription of the light signal, and environmental stress. StR2R3-MYB1do not have transmembrane signaling and coiled coil, the presence of the N-terminal signal peptide has a clear hydrophilic regions. By subcellular localization analysis StR2R3-MYB1may be located within the nucleus. The article also constructed three-level structure of the model StR2R3-MYB1.(3)By building StR2R3-MYB1of YFP fusion expression vector, using the method of Arabidopsis protoplasts transformed subcellular localization analysis, confocal laser scanning microscopy visible StR2R3-MYB1positioned on the nucleus. Real-time PCR results showed that:StR2R3-MYB1in turn increases on the roots, stems and leaves. Plants from the darkness into the light environment relative expression StR2R3-MYB1upward trend, and expression of StR2R3-MYB1was induced by light. After processing experiment shows monochromatic far-red light, blue light on the expression of StR2R3-MYB1has induced the expression of the corresponding amount reaches a certain degree feedback mechanism is activated. But the red light entrainment for StR2R3-MYB1is not obvious within24hours, consistent expression levels compared to controls.(4) Through the use of stepwise regression analysis method to know the influence of R2R3-MYB transcription factor for CHS, DFR and PAL gene transcription levels under different conditions of light and temperature. Potato R2R3-MYB transcription factor gene expression of CHS and DFR influence coefficient exceeded0.5, with a significant positive impact on the expression of CHS and DFR, indicating that potato R2R3-MYB transcription factors may be involved in regulation of anthocyanin synthesis in response to the outside light environment, temperature Stress.(5) Over-expression vector and interference vector were constructed, and we using Agrobacterium-mediated transformation of potato to transform wild-type E3. The sliced tubes potato, infection with Agrobacterium bacteria carried in the co-culture medium after two days transferred to culture differentiation medium, placed in the light incubator and using Bialaphos sodium salt as selection pressure induced buds to get basta resistance positive plants when differentiated shoot rooting out.