A kind of gene of Thalassiosira vissonii twpepc1 and its application and method for cultivating high-yielding rice
A technology of seaweed and seaweed, applied in the field of plant genetic engineering, can solve the problems that the yield of PEPC transgenic plants is not well improved, cannot exert due C4 effect, etc., and achieves overcoming the dependence of rosette structure , the effect of increasing activity and increasing yield
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Embodiment 1
[0051] Cloning of TwPEPC1 Gene from Thalassiosira vissonii
[0052] Material preparation: Thalassiosira vissonii, the culture conditions are temperature 22°C, light intensity 3000lux, light-dark ratio 16h / 8h.
[0053] Nutrient solution composition: NaNO 3 74.8mg / L, NaH 2 PO 4 4.4mg / L, vitamin mother solution 1ml (vitamin B1 0.1g / L, vitamin B12 0.5 mg / L, vitamin H 0.5mg / L, trace element mother solution 1ml (EDTA-Na 2 4.360 g / L, FeCl 3 .6H 2 O 3.150g / L, CuSO 4 .5H 2 O 0.010g / L, ZnSO 4 .7H 2 O 0.022g / L, CoCl 2 .6H 2 O 0.010g / L, MnCl 2 .4H 2 O 0.180g / L, Na 2 MoO 4 .2H 2 O 0.006g / L), pH=8.0, the culture medium was prepared with seawater, sterilized at 121°C for 15 minutes before use.
[0054] step:
[0055] (1) Extraction of Thalassiosira vissonii RNA:
[0056] Thalassiosira wilsonii RNA was obtained by using the method of Trizol Reagent (Invitrogen, Carisbad, CA, USA). The Thalassiosira wilsonii RNA was detected by 0.8% agarose gel electrophoresis and concentra...
Embodiment 2
[0064] Construction of Transformation Vector of Thalassiosira vissonii TwPEPC1 Gene
[0065] (1) Restriction vector pC1300s
[0066] The pC1300s vector was transformed on the basis of pCAMBIA1300 (the vector publicly used by CAMBIA Company), and the 35s promoter and 35s polyA vector map and the information of the multiple cloning site were respectively introduced at both ends of the multiple cloning site. figure 1 . pC1300s was digested with Kpn I, and dephosphorylation reaction was carried out after electrophoresis detection that the digestion was linear. The reaction system and conditions were referred to the instructions of Promega. Service Co., Ltd.) recovery, electrophoresis to detect the integrity of its digestion, and stored in a -20 ° C refrigerator.
[0067] (2) Enzyme digestion of TwPEPC1 cloning plasmid
[0068] The correct TA cloning plasmid was sequenced with Kpn I single enzyme digestion, and the digested product was separated by 0.8% agarose electrophoresis, ...
Embodiment 3
[0072] Agrobacterium-mediated genetic transformation
[0073] The Agrobacterium-mediated genetic transformation method of the present invention mainly refers to the method shown in the "Agrobacterium-mediated Genetic Transformation Operation Manual" issued by the State Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University (Lin Yongjun, etc., Agrobacterium-mediated Mudanjiang 8 No. Establishment of high-efficiency transgenic system, Acta Crops Sinica, 2002, 28(3): 294-300). The recipient of the transformation was the embryogenic callus induced from the mature seeds of the rice variety "Zhonghua 11". The hygromycin-resistant callus is obtained through precultivation, infection, cocultivation and screening, and then through differentiation, rooting, seedling training and transplanting to obtain transgenic plants.
[0074] (1) Abbreviation of reagents and solutions
[0075] The title and the abbreviation of the main reagent involved in the medium preparati...
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