Mutation Of OsGA20ox2 Gene Based On CRISPR/Cas9 Editing System In Rice (Oryza Sativa L.)

As an important agronomic trait of rice(Oryza sativa L.), rice height had the close relationship to the yield.The lodging of rice was easier to occur for the taller ones.The ability of lodging resistance and fertilizer tolerance and close planting could be improve by reducing the height of rice.OsGA20ox2 gene encoded GA20 ox oxidase whose mutation could lead to the decline of rice height.The application of the mutation of OsGA20ox2 gene have caused the green revolution on agricultural production in the 20 th century, increasing the production of rice significantly.The OsGA20ox2 gene mutation of rice would be obtained by the CRISPR/Cas9 technology which could edit the OsGA20ox2 gene. Because of separation from T-DNA inserted loci, the mutated OsGA20ox2 gene could be left alone on the genome of rice. After self-crossing or back-crossing, the rice without any transgenic component whose height had declined could be gained.With the rice mature seeds as the material, the factors that affect the genetic transformation of rice shoot tips of Agrobacterium-mediated were studied and optimized in this research. According to published rice genome sequence from the NCBI database, we analyzed the OsGA20ox2 gene bioinformatically and designed the target sequence for CRISPR/Cas9.And the plant expression vector was construsted containing CRISPR/Cas9 system and bar gene. T0 transgenic rice was gained by Agrobacterium-mediated genetic transformation, which was identified by herbicide resistance and polymerase chain reaction(PCR). Its main agronomic traits were determined and analyzed in the grain filling period.The main results as follows:(1)The efficiency of Agrobacterium-mediated shoot tip genetic transformation of rice was affected by many factors which were measured in this research by the GUS gene momentary expression rate. Agrobacterium concentration, vacuum processing time and vacuum osmotic pressure were direct influence factors of rice shoot tips genetic transformation. The result showed that the optimized transgenic condition was bacterial concentration OD600 being 0.8, vacuum processing time being 8 min, vacuum osmotic pressure being 20 kPa. The condition in the genetictransformation laid a foundation for transforming OsGA20ox2 gene target specific CRISPR/Cas9 plant expression vector into rice by Agrobacterium.(2) According to published rice genome sequence from the NCBI database, we analyzed the OsGA20ox2 gene bioinformatically and designed the target sequence for CRISPR/Cas9 by E-crisp online tools.We constructed OsGA20ox2 gene target specificity CRISPR/Cas9 plant expression vector with the herbicide resistance selectable marker gene bar. By the Agrobacterium-mediated shoot tips genetic transformation, 12 transgenic rice Nipponbare plants and 4 transgenic Lailong rice were obtained which were verified by glufosinate-ammonium herbicide selection and PCR detection.(3)The main agronomic characters of the transgenic rice plants were investigated in the grain filling period. The results showed that the height of transgenic Nipponbare rice(71.3 cm) was diminished 16.9 percent than the wild type Nipponbare rice(85.8 cm), the height of transgenic Lailong rice(159.0 cm) was diminished 11.58 percent lower than wild-type Lailong rice(P < 0.05).Compared to the wild type, there were no significant difference between transgenic rice and wild-type rice in agronomic traits, such as effective tiller number, flag leaf length, flag leaf width,ear length, effective grain number, etc. The studies indicated that using the CRISPR/Cas9 technology on rice OsGA20ox2 gene fixed-point editing could cause reduction of plant height and have little effect on other agronomic traits.It was proved that the CRISPR/Cas9 gene editing technology could create new rice germplasms with site-directed mutagenesis which had important application value.