Genetic transformation method for neochloris oleoabundans
A genetic transformation method and technology of green algae, applied in the field of microalgae genetic engineering, can solve problems such as genetic transformation of oil-rich new green algae that is not suitable
Active Publication Date: 2016-06-01
GUANGDONG OCEAN UNIVERSITY
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Problems solved by technology
However, the inventor's previous research found that the existing methods in the literature are not suitable for the genetic transformation of oil-rich neochloroalgae
Method used
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Embodiment 1
[0043] Embodiment 1 liquid medium co-cultivation method transforms
[0044] Agrobacterium tumefaciens EHA105 was selected. The medium formula is (YEB medium): beef extract 5g / L, yeast 1g / L, peptone 5g / L, sucrose 5g / L, MgSO 4 ·7H 2 O0.4g / 100mL, pH=7.4, rifampicin 50mg / mL. Cultivate in shake flasks at 28°C to logarithmic phase (OD 600 =0.5-1.0). Take 50mL of Agrobacterium bacteria liquid, culture at 28°C at 200rpm for 48 hours, centrifuge at 5000rpm for 5min, discard the supernatant, add 40mL of 10% glycerol to resuspend the bacteria, ice-bath for 30min, then centrifuge at 4°C at 5000rpm for 5min, remove the supernatant. Add 30mL of 10% glycerol to resuspend, continue to centrifuge at 5000rpm at 4°C for 5min, discard the supernatant, add 30mL of 10%glycerol to resuspend the bacteria, centrifuge again at 4°C at 5000rpm for 5min, remove the supernatant and add 2mL of 10% glycerol to obtain the bacterial suspension , so as to produce electroporation-competent Agrobacterium.
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The invention discloses a genetic transformation method for neochloris oleoabundans. The method comprises the steps that 1, agrobacteria carrying binary expression vectors are prepared; 2, neochloris oleoabundans cells are prepared; 3, co-culture is carried out, wherein the agrobacteria stored in the step 1 are transferred into a liquid culture medium to be cultured for a logarithmic phase, and the obtained agrobacterium cells and the neochloris oleoabundans cells obtained in the step 2 are mixed to be cultured; 4, converter screening is carried out, wherein cells co-cultured in the step 3 are collected, the neochloris oleoabundans cells are prepared into cell suspension through the liquid culture medium, a screening flat plate with hygromycin is coated with the cell suspension, continuous illumination culture is carried out till neochloris oleoabundans emerges, the number of the neochloris oleoabundans emerging on the flat plate is calculated, and the emergent neochloris oleoabundans is picked, transferred, stored and detected. Transgene operation is carried out for the novel host neochloris oleoabundans, parameters in all the steps are optimized and limited, and a way is opened for the genetic engineering operation of the neochloris oleoabundans.
Description
technical field [0001] The invention belongs to the technical field of microalgae genetic engineering, and in particular relates to a method for genetic transformation of oil-rich neogreen algae. Background technique [0002] Neochlorisoleoabundans is a freshwater alga isolated from the desert in 1962. It has been identified as Chlorophyta, Chlorophyceae, Chlorophyceae, Chlorisoleaceae, and a member of the genus Neochloris. single-celled eukaryotic green algae. This algae has a high oil-yielding ability, and the lipid content can reach 17.5-54% of the dry weight, and the tri-fatty acylglyceride accounts for 80% of the total lipid content, and most of its fatty acid composition is 16-18 carbon Saturated or monounsaturated fatty acids. Therefore, it is very suitable as a resource for biodiesel. This microalgae is high in oleic acid, making up about 35 percent of the total fatty acids. In foreign countries, some people have found that this kind of microalgae has high nutrit...
Claims
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IPC IPC(8): C12N15/79
CPCC12N15/79
Inventor 李雁群张茜朱宏波谭瑜C·Q·兰卢虹玉吉宏武洪鹏志胡雪琼苏伟明
Owner GUANGDONG OCEAN UNIVERSITY