Research On Blueberry Transformation With The LEA Gene And Cold Tolerance Analysis

With improving quality and standards of people's lives, people gradually increase health awareness. Many people favor the subject of foreign fruit, in which blueberry is much more welcome. As the blueberry fruit is expensive, processing technology constantly improving, and the fruit benefit to the physical health, planted area of blueberry is increased year by year around the world. In recent years, Chinese blueberry industry is gradually being taken seriously. In North China, due to the winter cold and dry, blueberry industriy has been limited. Therefore, in northern China, cultivation new variety of blueberry with high nutritional value and low temperature tolerance ability is imminent. In this study, the nutritional value of mainly blueberry cultivars in northern China was analysed. Meanwhile, the stems and leaves were used as explants to establish an efficient system for tissue regeneration, respectively. By Agrobacterium-mediated method, the LEA gene from cDNA library of Tamarix androssowii was introduced into Northland genome, which is a new way to genetic transformation to blueberry. The results are as follows:1. The content of amino acid, total sugar, protein, vitamin and mineral element were carried out in the tested seven blueberry varieties. The results showed that the test targets of Northland were outstanding, except Vb2 and calcium content in second place, the other test indicators were all listed in the first place;2. In different varieties of blueberry, the stems and leaves were used as explants, selected the type of basic medium, while identifying the types and the appropriate concentration of plant growth regulators. The results indicated that the modified WPM medium containing zeatin (Zt) was benefitted to differentiation, but the suitable concentration of Zt is different in different genotypes;3. Cefotaxime sodium is the optimized antibacterial antibiotics, it's effective concentration is 250 mg-L-1; Kanamycin is the optimized antibiotics for the transformant selection, it's effective concentration is 30 mg·L-1. The resistance LEA gene was transformed into blueberry by Agrobacterium-mediated method. The optimal genetic transformation system for Northland as follows:Leaves from 8- to 10-week-old adventitious shoot were used as explants for pre-culturing for 48 h in the dark at 25±1℃. The pre-cultured explants were then infected with A. tumefaciens diluted by liquid MWPMZ to OD600=0.5 for 1 h at 28℃. The infected explants were co-cultivated on solid MWPMZ in the dark for 4 days. Then the explants were transferred to MWPMZ+250 mg l-1 cefotaxime for 48 h in the dark at 25℃. The explants were then transferred to selection medium (solid MWPMZ+30 mg l-1 Km+250 mg l-1 cefotaxime) for 7 d in the dark at 25℃, followed by subculturing on fresh selection medium at 5-7 d intervals under a 16 h photoperiod with a photosynthetic photon flux density of 50μmol m-2 s-1.4.13 kanamycin resistant shoots were initially gained using Agrobacterium-mediated genetic transformation of blueberry. Five false-positive clones were removed by increasing the selection pressure. The remaining eight positive clones were carried out PCR, Southern blotting and RT-PCR detection. The results involved that, LEA gene was integrated into the genome of Northland, but different transformants with different copy numbers. The results of the one-step RT-PCR indicated that two lines were transcription silence, while the other transgenic lines were normal transcription;5. The 8 transgenic lines were tested for cold tolerance by measuring the activities of peroxidase (POD) and superoxide dismutase (SOD), malondialdehyde (MDA) content and relative electrolyte leakage (REL). The results showed that the MDA content and REL were lower than the control, SOD activity and POD activity was higher than the control after stress in the six transgenic lines which normal transcription, and there were significant differences. The results above illuminate that the exogenous LEA genes in Northland could normal expression, and increased transgenic Northland cold tolerance ability.