Genetic Transformation Mediated By Agrobacterium Tumefaciens And Physiological Analysis Of AtNHX1 Transformants Of Kiwifruit (Actinidia Deliciosa); The Function Of At4G12490 Of Arabidopsis Thaliana To Adversities

The dissertation contains two parts:1) AtNHXl from Arabidopsis thaliana was transformed into kiwifruit genome with Agrobacterium tumefaciens, putative transgenic plants were confirmed by PCR and identified by molecular and physiological analysis.2) The function of At4G12490 from Arabidopsis thaliana against adversity were identified through RNA gel blot analysis under different stress factors.1. Genetic transformation of kiwifruit mediated by Agrobacterium tumefaciens and analysis of salt tolerance of AtNHX1 transgenic plants.Kiwifruit is a kind of well-known fruits originate from China. Because of its distinctive flavors, high nutrition value and healthy components, kiwifruit attract more attention in recent years. Along with the improvement of living standard and changes of consumption structure, the requirement to high quality kiwifruit is increasing year after year. Abundant kiwifruit varieties distributed in China can provide resources for its breeding. However, because kiwifruit is a dioecious species with a complicated genetic background, traditional seedling propagation of kiwifruit could not meet the requirements, due to time-consuming, low survival rate and unstable properties. Modern biotechnology creates a new path for kiwifruit breeding. With the development of tissue culture system, genetic transformation has become the main method in kiwifruit breeding gradually. In our experiments, Actinidia deliciosa, with the highest yield of kiwifruit in China, was selected as the target material, and its high frequency regeneration system was established. With this system, AtNHXl from Arabidopsis thaliana was transformed into kiwifruit and transgenic plantlets were regenerated. The details are summarized as follows:Genetic transformation of kiwifruit with Agrobacterium tumefaciens. The calli, stem segments and leaf fragments were infected by LBA4404 containing pHZXl, and transformants were regenerated on the differentiation MS medium (2mg/L 6-BA,1mg/L NAA) supplemented with 600mg/L casin hydrolysate,20 mg/L kanamycin and 500mg/L cefotaxime, and rooted on MS medium containing 1.0mg/L IBA.Optimizing genetic transformation system of kiwifruit. We studied effects of preculture, infection and coculture time, Agrobacterium density and AS on transformation frequency of kiwifruit. Stem was much more suitable for transformation by Agrobacterium tumefaciens than leaf. Transformation frequency of leaf explants significantly increased if they were precultured for 1-2 days. On the contrary, preculture had no effect on stem transformation. However, the transformation frequencies decreased after 4 days preculture.Time of Agrobacterium infection and coculture were crucial to transformation. The proper infection time was 2 min for leaf and 15min for stem. Two days coculture with Agrobacterium was better for both stem and leaf. Suitable densities of Agrobacterium not only facilitated the infection process of Agrobacterium, but also reduced its detrimental effect to explants. The leaves required low density of Agrobacterium (optimal OD600=0.2) compared to stems (optimal OD600=0.6). However, high concentration of AS is toxic to explants. The effect of AS on transformation frequency was slight.Molecular identification of transgenic plants. Four transgenic lines of kiwifruit were identified by PCR amplification of a 560 bp fragment of AtNHXl gene. Three independent transgenic lines (TL1-TL3) were selected for Southern blotting analysis. The results showed TL1 and TL2 possessed a single copy of AtNHXl, while TL3 has two copies, and AtNHXl was expressed effectively in TLl and TL2.Physiological analysis of transgenic kiwifruits. Physiological analysis was carried out in two transgenic lines, TL1 and TL2. Without salt stress, Na+ and K+ contents were nearly the same in both transgenic and wild-type plants. Na+ and K+ contents in leaves and roots of transgenic lines were higher than that of the wild-type plants, especially at 200 mmol/1 NaCl concentration. When plants suffer from adversity, they usually produce abundant free proline to adjust osmotic-stress, and results in membrane lipid peroxidation is related to the oxidative degradation of polyunsaturated fatty acids and involves free radicals and produces malondialdehyde (MDA). After treated by NaCl, contents of free proline in leaves of transgenic lines were significantly higher than that of the wild-type plants, MDA contents of transgenic lines were less than that of the wild-type plants. This result further confirmed the function of the Na+/H+ antiporter in opposing to salt stress. These data indicated that overexpression of Na+/H+ antiporter gene AtNHXl was correlated with the alleviation of the detrimental effect of Na+The growth status of the transgenic lines obviously excelled the control plants when stressed by salt. Obvious salt stress effect on the growth of the transgenic plants and control could be observed. The leaves of the wild-type plants gradually turned yellow and withered after two weeks when the concentration of NaCl reach 200 mmol/L, whereas transgenic lines still kept green and grown. A remarkable improvement in biomass production at the vegetative growth stage was observed between the transgenic kiwifruit lines and wild-type plants under salt stress.The fresh shoot weight of wild-type was 31% of the non-saline condition with the treatment of 150mmol/L NaCl for 20 days, in comparison with that of the transgenic line TL1 (77% of the non-saline control), which was two times higher than the wild-type plants. The growth reduction of kiwifruit in saline conditions is mainly contributed to the Na+-specific toxicity and nutrient imbalance. Improved growth of the transgenic plants might be the result of the transporter compartmentalizing Na+ into the vacuole and maintaining high level·K+ in cytosol.Flavonoids take part in prohibiting cell aging and antagonizing ROS. They are important secondary metabolites and nutrition components of kiwifruit. The flavonoids contents in leaves, stems and roots of transgenic lines were measured after treatment with 200 mmol/1 NaCl for three days. In different tissues of kiwifruit, the flavonoids contents were different, leaf>stem>root in general. The contents of flavonoids in transgenic lines were significantly higher than that of the control, especially in leaves of transgenic lines. The flavonoid contents in whole plants of transgenic lines TL1 and TL2 increased 71% and 40% respectively compared to the wild-type plants. The contents of flavonoids in leaves of transgenic lines TLl and TL2 increased 68% and 44% respectively compared to that of wild-type plants. All these results coincided with the change of MDA content and confirmed that the transgenic lines had higher ROS elimination activity.2. The function of At4G12490 of Arabidopsis thaliana to adversities. Plants are constantly bombarded with various environmental signals, some of which cause stress and restrict growth and development. In response to those adversities, plants have developed a number of strategies that increase tolerance or adaptation to stress conditions, e.g. by altering gene expression profiles, leading to adaptive responses at the cellular or systemic levels. Multiple reports indicated HyPRP gene expression were induced by variable stress factors, but the studies mostly focused on a single or a few genes. At4G12490 is a member of EARLI1 family (a HyPRP gene subfamily) in Arabidopsis thaliana. The aim of our study is addressing the function of At4G12490.At4G12490 was induced by many stress factors. At4G12490 mRNA levels slowly increased after 2 hours of cold treatment, the RNA accumulation of At4G12490 reached to roust activation after 16h treated by cold. This suggests that At4G12490 was not a direct target of the cold stimulus, but was rather a downstream target of a cold-responsive regulatory factor. Seedlings were grown in long-day photoperiods for 20 d at room temperature, and then incubated for 24h at 4℃. At4G12490 RNA abundance was highest immediately after 16 h of cold treatment and returned to low levels after 6h after transfer to room temperature, which fell to basal level completely after 24h cold treatment. Thus, a 16h cold treatment resulted in robust activation of At4G12490; however, steady-state mRNA levels were not maintained after transfer to room temperature. Long-day photoperiods enhanced At4G124900 expression in the absence of cold. The basal level of At4G12490 mRNA was slight higher in plants grown at room temperature in long-day photoperiods than in plants grown in short-day photoperiods. Moreover, At4G12490 expression signals in plants grown in long-days photoperiods after 24h cold-treated were visible stronger than that in plants grown in short-days photoperiods with the same treatment. This result demonstrated that the expression of At4G12490 was activated by light; probably the gene was light dependent and cold inducing the gene expression need light to facilitate this process. The response of the plant to salt stress is gradually increased in 3d, the obvious activation of At4G12490 after 24h salt stress, At4G12490 mRNA abundance reach to the top point after 72h stress. The result indicated that the response to salt of At4G12490 is very slow and slight; probably it is not the direct target and the main function gene conscribe to AtNHXl family gene or other genes conferred salt tolerance of Arabidopsis.At4G12490 could be activated by drought stress. At4G12490 mRNA level increased with the elongation of treatment time. After 12h of treatment, mRNA abundance reached to highest point. SA, as a defense signal, activated the RNA expression, which illuminated that At4G12490 is involved in plant pathogen defense and stress tolerance.Salicylic acid (SA) is considered to be an important signaling molecule which is involved in local and endemic disease resistance in plants in response to various pathogenic attacks. Besides providing disease resistance to the plants, SA can modulate plant responses to a wide range of oxidative stresses. Within 24h, the transcript of At4G12490 was increased slowly and slight after 16h-treatment, whereas it ascended to the highest point suddenly after 24h-treatment.Seed germination rate and root growth of At4G12490 overexpression line under salt and drought stress. To estimate the effect of the gene on the physiological aspect of plant, At4G12490 overexpression line was employed. Under both drought and salt stress, germination rate of overexpression seeds was lower than that of Col-0, the root growth of overexpression plants higher than that of Col-0. These result confirmed that over-expression plants had drought and salt-tolerance at certain degree, whereas Col-0 seed gemination ability excels overexpression plants, this phenomenon was distinct when seeds were sown on 1/2MS supplemented with NaCl it appeared that the gene had side effect on seed germination.