Improving the aroma quality of high-quality restorer line Guihui 852 by editing OsBADH2 gene with CRISPR-Cas9 technology

【Objective】CRISPR-Cas9 technology was used to edit the betaine aldehyde dehydrogenase gene（OsBADH2）of Guihui 852，in order to quickly improve the aroma quality of the high-quality restorer line Guihui 852 in Guangxi，and to provide parental resources and technical references for the creation of new fragrant rice germplasm in Guangxi.【Method】The sequence characteristics，conserved domains，conserved motifs and phylogenetic evolution of the OsBADH2 protein of Guihui 852，a high-quality non-fragrant rice in Guangxi，were analyzed. According to the information on the CRISPR-GE website，appropriate target sites were selected to construct the double mutation vector pLM62 for the first and second exons of the OsBADH2 gene and the single mutation vector pLM63 for the seventh exon. The OsBADH2 gene of Guihui 852 was targetedly knocked out using CRISPR-Cas9 technology and rice genetic transformation technology to obtain different types of osbadh2 mutants. Guihui 852 was used as a control，and the chewing method was used to identify whether the mutant osbadh2 had aroma. Finally，agronomic traits were determined and one-way ANOVA was performed to determine whether the mutation of OsBADH2 gene affected agronomic traits.【Result】The full length of OsBADH2 gene sequence was 6268 bp，including 15 exons and 14 introns. Its coding region（CDS）was 1512 bp，encoding 503 amino acid residues. It had a typical Aldedh domain（amino acids positions 16-485），belonging to aldehyde dehydrogenase，and its protein sequence and structure were relatively conservative. Through BLAST analysis of OsBADH2 amino acid sequence，three highly similar proteins in rice and four highly similar proteins in Arabidopsis were obtained. OsBADH1，OsBADH2，OsALDH2B2，OsALDH2C1 of rice，BADH1，BADH2 and AL2C4 of Arabidopsis thaliana all belonged to the ALDH-SF（Aldehyde dehydrogenase superfamily）members. OsBADH2 and OsBADH1 were paralogous genes，and were orthologous genes to BADH1 and BADH2 in A. thaliana. Four homozygous mutant lines（osbadh2-1-osbadh2-4）without T-DNA were successfully obtained under the genetic background of Guihui 852. All four types of mutations could cause frameshift mutations in the OsBADH2 gene，resulting in premature termination of the translation process，destroying the original structure and function of the OsBADH2 protein，and causing the rice of the four osbadh2 mutant lines to produce aroma. There were no significant differences in plant height，effective panicle number，panicle length，seed setting rate，1000-grain weight，and length-to-width ratio between the four osbadh2 mutant lines and Guihui 852 wild type（P>0.05），but there was extremely significant difference in seed setting rate between the osbadh2-2 mutant and the osbadh2-4 mutant（P<0.01）.【Conclusion】The four osbadh2 mutant lines of Guihui 852 all have aroma quality， and the comprehensive agronomic traits are not significantly different from those of the wild type，indicating that the CRISPR-Cas9 gene editing technology can quickly and directionally change the target traits of rice varieties，greatly shortening the breeding period.