Cloning And Functional Analysis Of MYB Transcription Factor In Malus Pumila Var.niedz Wetzkyana Schneid

For a long time, with traditional breeding methods, we met with many difficulties such as the long breeding period, leading to the breeding process become slowly. Genetic engineering technique has been becomeing the important potential breeding method.MYB, as a transcription factor which was almost involved in the development and metabolism of all aspects of the plants, was widely found in plants. In recent years, a number of MYB transcription factor are found in volving in the control of plant flavonoid pathway regulation in order to affect floral organ and the colour of fruits. Therefore, cloning of MYB genes and verifing its function has far-reaching significance.On the basis of the characteristics of Malus pumila var.niedzwetzkyana schneid., We have cloned three genes, MpMYB30, MpMYBPA1 and MpMYB5B by use of the RACE, PCR technology and so on. In order to provid the theory of genetic engineering modifieding and expression regulation, we have studied the functional properties of Malus pumila var.niedzwetzkyana schneid. and the model plants preliminaryly.The major findings are as follows:1. One of the MYB transcription factors, named MpMYBPA1 was cloned by PCR technology from the leaves of niedzwetzkyana schneid. The result showed that the total length of MpMYBPA1 gene was 1020bp. Bioinformatics analysis showed that the protein of MpMYBPAl gene encoded had two MYB HTH DNA-binding domains, which shared comparability with Vitis viniferna. Analysis of PCR positive lines by Real-time quantitative PCR revealed MpMYBPA1 expressed in all these lines, the expressions of the gene MpMYBPAl which is also influenced by different temperature and ABA in the fruit were much higher. In order to further study its function, a plant expression vector with a green fluorescent protein gene has been const ructed and was t ransformed into onion cell walls by gene gun method. The result of transient expression showed that was transformed into the cell wall of onion by gene gun method. The result of transient expression showed that MpMYBPA1 gene was located in the nucleus which is in line with the characteristics of transcription factors. Then we constructed a overexpression vector of and transferred it into tobacco, obtained 10 hyg positive tobacco plants. PCR analysis showed that four of these tobacco plants are PCR positive. Analysis tobacco plants by RT-PCR revealed MpMYBPAl expressed in two of these tobacco plants.2. One of the MYB transcription factors, named MpMYB5B was cloned by PCR technology from the leaves of niedzwetzkyana schneid. The result showed that the total length of MpMYB5B gene was 1557bp. Bioinformatics analysis showed that the protein of MpMYB5B gene encoded had two MYB HTH DNA-binding domains, which shared comparability with Vitis viniferna. Analysis of PCR positive lines by RT-PCR and Real-time quantitative PCR revealed MpMYB5B expressed in all these lines, the expressions of the gene MpMYB5B which is also influenced by different temperature and ABA in the fruit were much higher. In order to further study its function, a plant expression vector with a green fluorescent protein gene has been const ructed and was transformed into onion skin cells by gene gun method. The result of transient expression showed that was transformed into the cell wall of onion by gene gun method. The result of transient expression showed that MpMYB5B gene was located in the nucleus which is in line with the characteristics of transcription factors. Then we constructed a overexpression vector of and transferred it into tobacco, obtained 9 hyg positive tobacco plants. PCR analysis showed that six of these tobacco plants are PCR positive. Analysis tobacco plants by RT-PCR revealed MpMYB5B expressed in four of these tobacco plants.3. One of the MYB transcription factors, named MpMYB30 was cloned by RACE technology from the leaves of niedzwetzkyana schneid. The result showed that the total length of MpMYB30 gene was 1661bp. Bioinformatics analysis showed that the protein of MpMYB30 gene encoded had two MYB HTH DNA-binding domains, which shared comparability with Vitis viniferna and Ricinus communis; the protein’s molecular formula was C1790H2794N538S1 and the relative molecular weight was 41579.8Da; the isoelectric point was 6.3, and contained more a-helix in its secondary structure, but did not existed a signal peptide and transmembrane regions in its multi-peptide. The sequence of MpMYB30 gene was digested by Xbal I and Sac I, then linked into pCAMBIA-S1300+vector, after that it was confirmed by resistance selected and PCR analysis.