Application of OsMPK4 protein and coding gene in regulation and control of growth of plant seeds
A plant seed and protein technology, applied in the field of genetic engineering, can solve the problems of chalkiness of seeds, delayed embryo development, etc., and achieve the effect of improving expression efficiency
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Embodiment 1
[0070] Embodiment 1, the bioinformatics analysis of OsMPK4
[0071] OsMPK4 is a member of group B in the rice OsMPK family, and the gene is located on chromosome 10. Its accession numbers in the Rice Annotation Project Database (RAP-DB) of Japan and TheMSU Rice Genome Annotation Project Database (RGAP7) of the United States are Os10g0533600 and LOC_Os010g38950, respectively. The gene has 7 exons. The present invention designs the target sequence of the sgRNA in the CRISPR / Cas9 system on the fourth exon of the gene. In order to knock out OsMPK4.
Embodiment 2
[0072] Example 2, Design of OsMPK4 Target Site and Construction of Related Knockout Vectors
[0073] Design the sgRNA sequence in the fourth exon region of the rice genome, as follows:
[0074] 5'-AGGGCTAAAATATgtgcacT CGG -3' (sequence 4);
[0075] The lowercase letters are the recognition sequence of the endonuclease ApaLI site, the underlined sequence is the PAM sequence, which is named C-OsMPK4.
[0076] C-OsMPK4 was introduced into vector pCAMBIA2300-2x35S::Cas9-OsU3::sgRNA using type II endonuclease AarI. The specific operation is as follows:
[0077] (1) pCAMBIA2300-2x35S::Cas9-OsU3::sgRNA was digested with AarI and the large backbone fragment was recovered.
[0078] (2) According to the sgRNA sequence designed above, synthesize primers with the following cohesive ends (underlined):
[0079] C-OsMPK4-F: 5'- GTTT AGGGCTAAAATATgtgcacT-3';
[0080] C-OsMPK4-R: 5'- AAAC CgtgcacATATTTTAGCCCT-3'.
[0081] (3) Anneal C-OsMPK4-F and C-OsMPK4-R to form a double-strand...
Embodiment 3
[0083] Example 3, Activity screening of OsMPK4 target site sgRNA
[0084] After a large amount of the recombinant vector pCAMBIA2300-C-OsMPK4 constructed in Example 2 was extracted, it was transferred into the protoplasts of the rice variety Nipponbare in a PEG-mediated manner, cultured in the dark at 25°C for 48 hours, and then the genomic DNA of the protoplasts was extracted, and used a specific Primers are used to amplify the OsMPK4 gene containing the target fragment C-OsMPK4 by PCR, and then the PCR amplification product containing the target fragment C-OsMPK4 is digested with ApaLI (if the PCR amplification product has some bands that cannot be cut, explain the embodiment The target site designed in 1 is active), and the PCR amplification product that cannot be cut by the restriction endonuclease ApaLI is sequenced.
[0085] The primer sequences used to amplify the target fragment C-OsMPK4 are as follows: upstream primer OsMPK4-iden-F: GCACCTTGTTCCTTTGGTC (No. 2071-2089 ...
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