| 【Objective】Rice Fatty Acid Desaturase 2 gene（FAD2）encodes the fatty acid desaturase and converts oleic acid（C18∶1）into linoleic acid（C18∶2）, the mutant of which is capable of improving the content of oleic acid in rice. To obtain the high oleic acid rice mutant and facilitate the development of rice bran oil, site-specific editing of OsFAD2 is conducted by using the CRISPR/Cas9 technology.【Method】 Nipponbare callus was used as explants to generate the transgenic plants by agrobacterium-mediated transformation protocol with CRISPR/Cas9-FAD2-sgRNA vector. Sanger sequencing and fluorescent quantitative PCR were used to identify the positive transgenic plants.Finally, gas chromatography-mass spectrometer（GC-MS）was employed to detect the content variation of fatty acids. 【Result】Gene target sequencing suggested that three OsFAD2 knockout plants contained the genome bases variation in T0 generation, but only one positive transgenic line did not present off-target phenotype. Further, positive transgenic plant seeds were reproduced to T1 generation, as a result, OsFAD2 mutation generated genetic homozygosis and stable sequence, and qRT-PCR detection indicated that the relative expression of OsFAD2 decreased significantly compared with that of wild-type plant. Fatty acid composition detection suggested that the oleic acid increased by 30% approximately in the T1 seeds of positive knockout lines.【Conclusion】High oleic acid rice mutants were obtained by targeted knockout of the coding sequence of OsFAD2 via CRISPR/Cas9 technology, providing germplasm resources for subsequent high-oleic rice bran oil breeding. |
