| For a long time, with traditional breeding methods, we face many difficulties such as the slower breeding process caused longer juvenile period while the fruit trees were genetic improving. Genetic engineering technique became one of the importantest potential breeding methods. Abiotic stresses, such as drought, salinity, extreme temperatures, chemical toxicity, and oxidative stress, have enormous damage impact on agriculture and result in the deterioration of the environment. Plants respond and adapt to abiotic stresses by a variety of biochemical and physiological mechanisms, which are associated with the rapid activation of signal transduction pathways. Apple is one of the most important fruit trees in the world. Due to their ecological adaptability, high nutritional value, storability, longer supply cycle, apples are major consumer products and obtain strongly recommended in many countries. However, in the process of planting fruit trees, with different degrees of stress, and in recent years, global environmental have been changed increasingly and then frequent worse weather, which have bad impact on apple’s quality and yield. In order to improve the capacity of its anti-Stress, stress tolerance related genes in a variety of apples is imminent.According to the research hotspot of the model plant Arabidopsis, we studied the expression characterization of these genes, we expect that it will be used in the genetic engineering breeding or regulate gene expression. The main results are as follows:1. Mdmyb6 gene from Malus domestica Borkh. were successfully synthesized by PCR method. The results of Real-Time PCR indicated that the expression of MdMYB6 gene was induced by salt stress, sucrose and PEG, and the transcript level of MdMYB6 gene was the highest in Malus demestic Borkh roots. 2. We analysis the sequence of Mdmyb6 isolated from Malus domestica Borkh. The full-length cDNA of Mdmyb6 has a 939 bp open reading frame (ORF) and encode a MYB transcription factors consisting in 312 amino acids. The Mdmyb6 deduced amino acid sequence showed high identity to sucrose-responsive element binding protein family members. In the sugar watering experiment, almost all of the leaves of WT plants turned red when treated with 180 mM sucrose, while the transgenic line showed no color change. We then assayed anthocyanin accumulation in plants treated with 180 mM sucrose or sorbitol. The transgenic lines showed significantly lower anthocyanin accumulation than the wild type plants. The results of Real Time-PCR indicated that over-expression of MdMYB6 in Arabidopsis influences biosynthesis of anthocyanins, likely by suppressing enzymes involved in anthocyanin biosynthesis such as CHS、CHI、DFR、UF3GT、LDOX and FLS. Over-expression of MdMYB6 also altered the expression of sucrose synthesis (AtSUS1)and transporter genes(AtSUC1、AtSUC2、AtSUC3). At the same time, gene involved in sugar signal gene (UGPase, AtHXKl) also changed.3. Mdmyb10 gene from Malus domestica Borkh were successfully synthesized PCR-based two-step DNA synthesis (PTDS) method.Mdmyb10, a regulatory gene of anthocyanin biosynthesis in apple fruit, into Arabidopsis and analyzed its function to osmotic stress in transgenic plants. Under high osmotic stress, the Mdmyb10 over-expressing plants exhibited growth better than wild-type plants. The elevated tolerance of the transgenic plants to osmotic stress was confirmed by the changes of flavonoids、chlorophyll、malondialdehyde and proline contents. These results preliminarily showed that the MdmyblO can possibly be used to enhance the high osmotic-tolerant ability of plants. |
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