Cultivation method of green, safe and marker-free transgenic rice
A genetically modified rice, marker-free technology, applied in genetic engineering, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problem of not meeting the needs of food safety control of genetically modified rice, and solve the problem of food safety. Effect
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Embodiment 1
[0026] [Example 1] Construction of plant expression vector
[0027] pBlue2KS-PAb, pMD18T-LF-GLCMS and pDTM3 are all preserved in our laboratory (Key Laboratory of Agricultural Genetic Engineering, Fujian Academy of Agricultural Sciences). These plasmids were transformed into DH5α host bacteria by heat shock, 30% glycerol Store at low temperature. For a schematic diagram of its plasmid structure, see figure 1 , 2 , 3 shown.
[0028] The PsbR-cryIAb-Tnos expression cassette fragment was inserted into the plasmid pMD18T-LF-GLCMS that was digested with StuI and dephosphorylated by calf alkaline phosphatase to construct the intermediate vector pMG. -PAb. Plasmid pMG-PAb was digested with HindIII and SapI, and the end of the fragment was filled in with Klenow large fragment enzyme, and then inserted into the plasmid pDTM3 digested with HpaI and dephosphorylated by calf alkaline phosphatase to obtain the final plant expression Vector pDMG-PAb-LF. For a schematic diagram of its...
Embodiment 2
[0029] [Example 2] Obtaining of transgenic rice
[0030] 2.1 NB medium formula (basic medium)
[0031] Large amounts of N6: Potassium nitrate KNO 3 2830 mg.L -1 , ammonium sulfate (NH 4 ) 2 SO 4 463 mg.L -1 , potassium dihydrogen phosphate KH 2 PO 4 400 mg.L -1 , magnesium sulfate MgSO 4 ·7H 2 O 185 mg.L -1 , Calcium Chloride CaCl 2 2H 2 O 166 mg.L -1 ;
[0032] B5 trace: boric acid H 3 BO 4 3 mg.L -1 , manganese sulfate MnSO 4 ·H 2 O 7.58 mg.L -1 , Zinc Sulfate ZnSO 4 ·7H 2 O 2mg.L -1 , potassium iodide KI 0.75 mg.L -1 , Sodium Molybdate Na 2 MoO 4 2H 2 O 0.25 mg.L -1 , copper sulfate CuSO 4 ·5H 2 O0.025 mg.L -1 , cobalt chloride CoCl 2 ·6H 2 O 0.025 mg.L -1 ;
[0033] Iron salt: ferrous sulfate FeSO 4 ·7H 2 O 27.8 mg.L -1 , Disodium EDTA Na 2 EDTA 37.3 mg.L -1 ;
[0034] Inositol: Myo-inositol 100 mg.L -1 ;
[0035] Organic ingredients: ThiamineHCl 10 mg.L -1 , PyridoxineHCl 1 mg.L -1 , Niacin 1 mg.L -1 , Hydrolyzed Casein Cas...
Embodiment 3
[0060] [Example 3] Expression analysis of target gene in transgenic plants
[0061] Take about 0.1g of young leaves, young stems, young roots, and mature seeds of brown rice and polished rice, respectively, and put them in 1.5ml EP tubes, add 300ul sterilized water and a steel ball, and put them in the FASTPrep of MP company. - 24 samples shaker for 20 seconds, 4M / S. After standing for a few minutes, insert a CryIAb / Ac-specific colloidal gold test strip into it, wait for 5 minutes, and observe the results. If the test strip shows a single band, it is negative, indicating that the target gene in the sample is not expressed; if the test strip shows double bands, it is positive, indicating that the target gene in the sample is expressed. See the results Figure 6 .
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