Specific promotor in rice embryo and application thereof
A promoter and rice technology, applied in the direction of using vectors to introduce foreign genetic material, cells modified by introducing foreign genetic material, DNA/RNA fragments, etc., can solve the problem of few miRNA promoters, difficult to find promoters, and miRNA matching The problem of multiple regions
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Embodiment 1
[0067] Example 1. Cloning of rice mesoembryo-specific promoter
[0068] Using the rice genomic DNA extracted by conventional methods as a template, the promoter P1 was amplified by conventional PCR using the following primer pair (SEQ ID NO.: 2 and 3):
[0069] Forward primer: GGCTGCAGCGGCTGCTGTTCTTATGGC (SEQ ID NO: 2)
[0070] Reverse primer: GGGATCCTGCACTGCAAATCCGGTTAG (SEQ ID NO: 3).
[0071] The inventors also used the same method to amplify two other promoters: P2 and P3, using the following different reverse primers:
[0072] Reverse primer: GGGATCCGCACTGCAAATCCGGTTAG (SEQ ID NO:4)
[0073] Reverse primer: GGGATCCACTGCAAATCCGGTTAG (SEQ ID NO: 5).
Embodiment 2
[0074] Example 2. Construction of GUS expression vector
[0075] The promoter sequences obtained in Example 1 were respectively connected to the commercially available vector pMD19-T vector (Takara Company), and verified by sequencing. Then use HindIII and BamHI to double digest the pMD19-T vector, reclaim the promoter fragment of about 2kb, and use the restriction endonuclease HindIII to this fragment and the binary vector pBI101.1 (Clontech Company, U.S.) containing the GUS reporter gene respectively Double enzyme digestion with BamHI, the digested product was recovered by gel, ligated with T4-DNA ligase, and transformed into Escherichia coli DH5α strain for preservation.
[0076] The final vectors obtained were named pBI101.1-P1-GUS, pBI101.1-P2-GUS and pBI101.1-P3-GUS. The recombinant vectors pBI101.1-P1-GUS, pBI101.1-P2-GUS and pBI101.1-P3-GUS were transformed into Agrobacterium strain GV3101 by heat shock transformation method, and verified by PCR amplification.
Embodiment 3
[0077] Embodiment 3. Obtaining of transgenic plants
[0078] The constructed vectors pBI101.1-P1-GUS, pBI101.1-P2-GUS and pBI101.1-P3-GUS were respectively transformed into commercially available Agrobacterium GV3101 strains, and then the wild-type rice was treated by flower dipping method. 11 for transformation.
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