Editing Badh2 To Improve Fragrance In Japonica Rice Via CRISPR/cas9 Technology

Rice is one of the most important food crops in the world.About half of the world's population is rice-based.For the purpose of pursuing the quality of life,the fragrant rice breeding has attracted interest from researchers in the rice breeding industry.The rice fragrance trait is determined by the recessive and negative regulation gene,Badh2.It is on the chromosome 8,and its loss-of-function mutants produce fragrance.Compared with traditional breeding,the CRISPR/Cas9 gene editing technology can be used to modify and engineer genes with short cycle and high efficiency as a new method of rice breeding.In this study,non-fragrance japonica rice variety,Dongnong 425,was used as experimental materials and we constructed two CRISPR/Cas9 knock-out vectors for site-specific editing in the second and third exons of Badh2.We used the mature genetic transformation system in japonica rice and the mature transgenic detection to obtain homozygous mutant materials,which have different degrees of fragrance,no T-DNA components and no significant changes in main agronomic traits.This study provides theoretical basis and material basis for accelerating the cultivation of fragrant japonica rice varieties.The specific research contents and results are as follows:(1): CRISPR/Cas9 knock-out vectors,pYLCRISPR/Cas9-B1-g RNA(B1 vector)and pYLCRISPR/Cas9-B2-g RNA(B2 vector)were constructed,and the two targets of B1 vector were located in the second and third exon,respectively.Both targets of the B2 vector were located in the second exon.The Agrobacterium mediated transformation was carried out to transform Dongnong 425,B1 vector and B2 vector obtained 18 and 19 regenerated plants,respectively.After positive transgenic detection,15 and 17 positive plants with B1 and B2 were obtained in T0 generation,respectly.(2): At the peak of the tillering stage,whole genome DNA of the T0 generation plants was extracted by CTAB method,and sequence analysis was performed near the target.The results showed that the mutation types of the positive plants were mainly homozygous mutations and biallelic mutations.There were 5 homozygous transgenic plants with B1 vector.The homozygous rate was 33.33%,and the genotypes were mostly 1-2 bp small fragment insertions or deletions.There were 9 homozygous plants transgenic with B2 vector.The homozygous rate was 52.94%,and the genotype partly showed 67 bp and 76 bp large fragment base deletion.Finally,a total of 8 homozygous lines with different genotypes were obtained in T0 generation.The amino acid sequences of homozygous mutant lines and wild type lines were analyzed.We found that all homozygous mutant lines had frameshift mutations,and finally truncated protein lacking a large number of amino acids was obtained.(3): 30 individual plants were selected from each line in T1 generation to extract leaf DNA,and Cas9-F/R primers were used for detection.A total of 26 homozygous plants without T-DNA components were screened from the 3 lines of B1.A total of 39 homozygous plants without T-DNA components were screened from the 5 lines of B2.At the same time,fragrance detection of the rice seeds of the 8 homozygous mutant lines was carried out by chewing method and potassium hydroxide immersion method.The results showed that all the 8 lines showed different degrees of fragrant,and the 3 lines edited by the B1 vector have more fragrance.(4): Furthermore,the main agronomic traits of the 8 homozygous mutant lines were investigated.As a result,there were no significant difference from the wild type in the number of panicles per plant,number of grains per panicle,seed setting rate and 1000-grain weight except the height of plants.The main agronomic traits of the mutants were not affected by the editing of Badh2.