Photorespiration metabolism modified branch and application of photorespiration metabolism modified branch in C3 plant
A technology of metabolic transformation and photorespiration, applied in the field of plant genetic engineering, can solve metabolic problems and achieve the effects of reducing CO2 emissions, increasing biomass, and increasing photosynthetic rate
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Embodiment 1
[0049] Example 1 Obtaining of TPC-OsGLO3, TPC-OsOXO3, and TPC-OsCATC fusion protein expression genes
[0050] (1) Acquisition of TPC-OsGLO3 fusion protein expression gene
[0051] Primers were designed according to the cDNA sequences of OsGLO3 and TPC provided by NCBI (http: / / www.ncbi.nlm.nih.gov / ), and the primer sequences were as follows:
[0052] OsGLO3-F: 5′-catg agatct atggagctaatcacaaac-3' (the underline is the recognition site of the restriction endonuclease BglII).
[0053] OsGLO3-R: 5′-tta atggtgatggtgatgatg cctgtcgctgtcggtgat-3' (the underlined part is the HIS tag sequence).
[0054] TPC-1F: 5'-ATGGCCCCCTCCGTGATG-3';
[0055] TPC-1R: 5′-ctac agatct catgcacctgatcctgcc-3' (the underline is the recognition site of the restriction endonuclease BglII).
[0056] Using the cDNA of 2-week-old seedling leaves of the japonica rice variety Zhonghua 11 as a template, under the guidance of primers OsGLO3-F and OsGLO3-R, and primers TPC-1F and TPC-1R, the OsGLO3 and TPC g...
Embodiment 2
[0068] Example 2 Obtaining of TPC-OsGLO3, TPC-OsOXO3, and TPC-OsCATC Fusion Protein Expression Cassette Sequences
[0069] (1) Acquisition of TPC-OsGLO3 fusion protein expression cassette Pubi-TPC-OsGLO3-Tnos
[0070] Primers were designed according to the TPC-OsGLO3 fusion protein expression gene sequence (SEQ ID NO: 1), and the primer sequences were as follows:
[0071] TPC-OsGLO3-F: 5′-gact ctgcag atggccccctccgtgatg-3' (PstI recognition site is underlined).
[0072] TPC-OsGLO3-R:5′-cagt ggatcc ctaatggtgatggtgatgatg-3' (the BamHI recognition site is underlined).
[0073] Using the pMD18-TPC-OsGLO3 vector described in Example 1 as a template, under the guidance of primers TPC-OsGLO3-F and TPC-OsGLO3, the TPC-OsGLO3 gene was amplified by conventional methods, and the gene was cloned into recombinant Donor vector Pubi-DI (vector map see figure 2 ) Between the PstI and BamHI restriction sites of the multiple cloning site, the vector Pubi-DI-TPC-OsGLO3 was obtained.
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Embodiment 3
[0092] Example 3 Obtaining GOC-pYL1305 carrier for transformation of GOC photorespiratory metabolic branch
[0093] With Pubi-DI-TPC-OsGLO3, 2×P35s-DII-TPC-OsOXO3 and Pubi-DI-TPC-OsCATC as donor vectors, pYL1305 (for the vector map, see Figure 4 ) is the acceptor carrier, referring to the method of Lin et al. (10):5962-5967), through three rounds of recombination, the GOC photorespiratory metabolic transformation branch carrier GOC-pYL1305 was finally obtained, and the vector map is as follows Figure 5 shown. The specific reorganization method is as follows:
[0094] (1) Preparation of donor plasmid and recipient plasmid
[0095] Collect 10 mL of the overnight bacterial solution, extract the plasmid with a plasmid mini-extraction kit, detect and measure the plasmid concentration by electrophoresis, and control the plasmid concentration at 100-200 ng / μL.
[0096] (2) Co-transfection and recombination of donor plasmid and recipient plasmid
[0097] Mix 3-4 μL of plasmids ...
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