Effects Of OsMYBAS1 Gene Overexpression On Seed Germination Of Rice

Direct-seeding rice saves time and effort and greatly reduces costs.The low vigor and poor seedling emergence ability of existing rice varieties seriously restrict the popularization of direct seeding technology.Therefore,excavating the key genes that regulate the germination of rice seeds and cultivating new rice varieties with strong germination ability are of great significance for the promotion of direct seeding rice.The R2R3 MYB transcription factor plays various roles in plant growth and development,but it is rarely reported whether it regulates rice seed germination.Our previous research used CRISPR/Cas9 gene editing technology to obtain the osmybas1 mutants.The results of phenotypic identification indicated that this gene might play a positively regulatory role in the germination of rice seeds.In order to verify the results,this paper used Nipponbare（wild type）and OsMYBAS1overexpressing T₂ rice seeds as materials to determine the germination traits,physiological and biochemical indexes under different sowing depths as well as applying in exogenous hormone.Furthermore,OsMYBAS1 overexpressing rice was used for RNA-seq and real-time RT-PCR analysis.The object was to lay a foundation for clarifying the molecular mechanism of OsMYBAS1 transcription factor regulating rice seed germination,which would also provide the key genes or germplasm for breeding new rice varieties with strong germination ability.The conclusions were exhibited as follows:（1）Determination of phenotypic,physiological and biochemical indexes of overexpression lines under different sowing depths:The germination potential,germination rate,germination index,vigor index and seedling length among three OsMYBAS1 overexpression lines were significantly higher than those of wild type under 0 cm sowing depth,which increased by 29.3～46.9%,28.6～38.1%,36.8～50.0%,67.2～95.3%and 7.5～27.7%,respectively.There was no significant difference in SOD,POD,APX activity and MDA content.Under 4 cm sowing depth,the results showed that the germination potential,germination rate,germination index,vigor index and root length of three OsMYBAS1 overexpression lines were better than wild type,which increased by 12.6～37.2%,27.9～38.9%,2.6～83.7%,35.9～119.7%and 10.7～36.1%,respectively.MDA content was significantly lower than that of wild type,decreased by 28.6～58.7%and APX activity was significantly higher than that of wild type,increased by 2.9～6.6folds.Collectively,the results suggest that OsMYBAS1 may positively regulate seed germination and antioxidant capacity,and the regulation effects at 4 cm sowing depth are more obvious.（2）Determination of phenotypic,physiological and biochemical indexes of overexpression lines treated with gibberellin and its synthesis inhibitor at 4 cm seeding depth:After the application of 5×10^-5mol/L GA₃,the germination rate,germination index,root length and seedling length of three OsMYBAS1overexpression lines were significantly higher than those of wild type,which increased by 19.9～34.9%,8.9～23.5%,18.8～19.7%,22.3～52.4%,respectively.Compared to WT,SOD,POD,CAT and APX activity of OsMYBAS1 overexpression lines were increased,in which the increase of CAT and APX were significantly by24.6～126.1%,3.5～37.1%,respectively.MDA content was significantly lower than that of wild type by 27.7～46.9%.After the treatment with 5×10^-5 mol/L uniconazole（UCZ）,germination rate,germination index,root length and seedling length of three OsMYBAS1 overexpression lines were significantly higher than those of wild type,which increased by 10.6～26.4%,16.1～51.8%,20.1～40.1%,respectively.There was no significant difference in SOD,POD,CAT and APX activity,while MDA content of OsMYBAS1 overexpression lines was significantly lower than that of wild type,and decreased by 11.8～20.6%.Moreover,we found that the promotion of gibberellin on OsMYBAS1overexpression lines was more obvious,while the inhibition of UCZ on overexpression lines was less than that of wild type.The results show that OsMYBAS1 may affect germination ability by regulating gibberellin metabolism.（3）Transcriptome sequencing and fluorescence quantitative analysis of seedlings after 14 days’germination of overexpression lines at 4 cm sowing depth:We found that the signal pathway of the related genes,MYB（Os03g55590,Os05g51160）,WRKY（Os01g43650）,ABA（Os01g16980;PP2C）,ethylene（Os10g37920;ERF10）,was up-regulated in RNA-Seq analysis in OsMYBAS1 overexpressed lines.Moreover,real-time RT-PCR results showed that the expression of Os01G43650,Os03g55590,Os05g51160,Os01g16980 and Os10g37920 in overexpressed lines was increased by 1,3,3,3,and 3times,respectively,which was consistent with the RNA-seq results.The results suggest that MYB,WRKY,ABA and ethylene signal pathways are also involved in the regulation of OsMYBAS1 on seed germination ability.