Application of rice OsFD2 gene in regulation and control of seed size
A technology for rice seeds and rice, which is applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problem that the molecular mechanism regulating the size of rice seeds has not been fully elucidated.
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Embodiment 1
[0037] Embodiment 1 Contains the construction and genetic transformation of target gene overexpression vector
[0038] 1. Design primers according to the sequence and cDNA sequence of the OsFD2 gene provided by NCBI, and clone the OsFD2 gene from the rice variety Zhonghua 11 by conventional methods. The sequence of the OsFD2 gene is shown in SEQ ID No.1, and the coding region of the OsFD2 gene The sequence is shown in SEQ ID No.2, and the encoded protein sequence is shown in SEQ ID No.3. The gene is located on chromosome 6, and its gene locus number in rice is LOC_Os06g50600 (Os06g0720900).
[0039] SEQ ID No. 1:
[0040]
[0041]
[0042]
[0043] SEQ ID No.2:
[0044]
[0045]SEQ ID No. 3:
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[0048] 2. Select a suitable enzyme cutting site and digest the Pox vector. The cloned OsFD2 gene was connected to the Pox vector by homologous recombination. Subsequently, the ligation product was transformed into a competent DH5α and positive colo...
Embodiment 2
[0053] Construction and genetic transformation of embodiment 2 OsFD2 knockout vector
[0054] 1. Obtain the whole genome sequence (SEQ ID No.1) of OsFD2 through NCBI, through the target design in the website (CRISPR-GE(Genome Editing)-Liu YG Lab(http: / / skl.scau.edu.cn / ) Design and select targets with low off-target rate, and design corresponding target primers. The primer sequence is F: ggcaCCATCTGGTAGTACTCCTGG R: aaacCCAGGAGTACTACCAGATGG.
[0055] 2. Connect the target point to the U3 gRNA expression cassette to construct the CRISPR / gRNA vector ( figure 2 ).
[0056] 3. Clone the U3 gRNA expression cassette and connect it to the pYLCRISPR / Cas9-Mtmono binary vector ( image 3 ) to obtain the OsFD2 knockout vector.
[0057] 4. Using Agrobacterium tumefaciens-mediated genetic transformation to introduce the above-mentioned OsFD2 knockout vector into rice callus, and obtain plants fd2-1 and fd2-2 transformed from two different calli.
[0058] 5. The target position is amplif...
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