Mutation effects of OsCDF1 gene and its genomic variations in rice

Create mutants：The experiment was conducted in the Agricultural College of Shenyang Agricultural University from 2018 to 2021. The SN9816 japonica rice variety cultivated by the Rice Research Institute of Shenyang Agricultural University was selected as the experimental material. Through Japan RAP-DB website（ http://rapdb.dna.affrc.go.jp/ ）The genomic DNA sequence of OsCDF1 (Os03g0169600, LOC_Os03g07360) gene was obtained, and the guide RNA (gRNA) primer was designed using the E-CRISP Design of the German Cancer Research Center (www.e-crisp. org/E-CRISP/designcrispr). The specificity of primer was tested by BLAST on RAP-DB website in Japan. The adhesive terminal GGCA was added to the 5 'end of the forward primer, and the adhesive terminal AAAC was added to the 5' end of the reverse primer. The primer was double-stranded and connected with pRGEB32 vector digested by Bsa Ⅰ. The positive/reverse primer gRF/R amplified fragment was designed with the flanking sequence of Bsa I restriction site on pRGEB32 vector. After the sequencing was correct, the connected vector was transferred into Agrobacterium tumefaciens by liquid nitrogen freeze-thaw method. The plasmid of Agrobacterium tumefaciens was extracted and inverted into the receptive state of Escherichia coli. After sequencing again, the vector construction was completed. The constructed vector was transferred into SN9816 callus by agrobacterium-mediated genetic transformation of rice mature embryos. Extract the DNA from the leaves of T1 generation plants as a template, and amplify the Hyg element by PCR using the hygromycin (Hyg) primer of the transgenic plants. The plants with Hyg element can be amplified, that is, T1 generation positive plants. Then use the flanking sequence of OsCDF1 gRNA to design mutation site detection primers. PCR products were amplified by mutation site detection primer, sequenced and analyzed to detect whether there was mutation at the editing site. Plant the mutated plants into the T2 generation, extract the DNA from the leaves of plants.