Application of osmkk6 protein and its coding gene in regulating plant seed development
A plant seed and protein technology, applied in the field of genetic engineering, can solve problems such as seed chalking and embryo development delay, and achieve the effect of improving expression efficiency
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0070] Example 1, Bioinformatics analysis of OsMKK6
[0071] OsMKK6 is a member of group A in the rice OsMKK family, and the gene is located on chromosome 1. 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 Os01g0510100 and LOC_Os01g32660, respectively. The gene has 8 exons. The present invention designs the target sequence of the sgRNA in the CRISPR / Cas9 system on the third exon of the gene. In order to knock out OsMKK6.
Embodiment 2
[0072] Example 2, Design of OsMKK6 Target Site and Construction of Related Knockout Vectors
[0073] Design the sgRNA sequence in the third exon region of the rice genome, as follows:
[0074] 5'-GTGGTATCGTCCAactagtT CGG -3' (sequence 4);
[0075] The lowercase letters in it are the recognition sequence of the endonuclease SpeI site, the underlined sequence is the PAM sequence, which is named C-OsMKK6.
[0076] C-OsMKK6 was introduced into the 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-OsMKK6-F: 5'- GTTT GTGGTATCGTCCAactagtT-3';
[0080] C-OsMKK6-R: 5'- AAAC AactagtTGGACGATACCAC-3'.
[0081] (3) Anneal C-OsMKK6-F and C-OsMKK6-R to form a doubl...
Embodiment 3
[0083] Example 3, Activity screening of OsMKK6 target site sgRNA
[0084] After a large amount of the recombinant vector pCAMBIA2300-C-OsMKK6 constructed in Example 2 was extracted, it was transferred into the protoplasts of the rice variety Nipponbare in a PEG-mediated manner, cultured at 25°C in the dark for 48 hours, and then the genomic DNA of the protoplasts was extracted and used a specific The primers are used to amplify the OsMKK6 gene containing the target fragment C-OsMKK6 by PCR, and then the PCR amplification product containing the target fragment C-OsMKK6 is digested with Spe I (if some bands of the PCR amplification product cannot be cut, it is indicated that the implementation The target site designed in Example 1 is active), and the PCR amplification product that cannot be cut by the restriction endonuclease Spe I is sequenced.
[0085] The primer sequence used to amplify the target fragment C-OsMKK6 is as follows: upstream primer OsMKK6-iden-F: GCAATGCATGGGATC...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More