Study On Drought Resistance Function Of Biological Clock Gene OsPRR37 And Application Of Gene Editing In Rice Breeding

In order to adapt to the white night and four seasons rhythm changes brought about by the rotation and rotation of the earth,plant circadian clock genes play an important role in maintaining the healthy growth and development of plants and coping with the changes of natural environment.Biological clock genes precisely regulate plant growth and development and stress resistance through negative feedback regulation mechanism.As the main component of biological clock core oscillator,PRRs family genes play an important role in plant photoperiod control flowering pathway,and affect plant stress resistance and yield.However,the role of this family gene in stress resistance is not clear enough.In this study,functional deletion mutants of OsPRR family genes（OsPRR1,OsPRR37,OsPRR73,OsPRR59,OsPRR95）produced by CRISPER/Cas9 technique were used to identify drought resistance and main agronomic characters.It was found that OsPRR37 played an important role in drought resistance of rice.Taking maintainer line Huhan 2B as an improved object,the gene was further edited by multi-target gene editing,and mutants with obvious improvement in growth period and yield were screened.The main results are as follows:1.In order to study the function of OsPRR family genes in stress resistance,the functional deletion mutants of OsPRR1,OsPRR37,OsPRR73,OsPRR59 and OsPRR95produced by CRISPER/Cas9 technology were collected,and the PCR fragments of the target sites were sequenced.it was found that each gene produced at least one base insertion or deletion mutant.Using these mutants as materials,these mutants were treated with PEG6000（20%）at germination stage and seedling stage,soil drought stress treatment at seedling stage,salt stress treatment at seedling stage,3μM/L ABA and mannitol treatment at seedling stage,and leaf water loss rate,senescence rate and agronomic characters at adult stage.2.OsPRR1 positively regulate the drought tolerance of rice,and can affect panicle development and control the number of tillers.Under PEG6000 stress,the seed germination rate of osprr1 was much lower than that of the control,the seedling survival rate decreased significantly,and the fresh weight and dry weight also decreased significantly.Field investigation showed that under normal conditions,the panicle length of osprr1 was similar to that of the control,but the number of primary and secondary branches decreased significantly,the plant height became shorter and the tiller increased.3.OsPRR59 did not respond to drought stress and salt stress at germination and seedling stage,but controlled the plant height of rice.The drought tolerance and salt tolerance of osprr59 mutants were identified at germination stage and seedling stage,and it was found that there was no significant difference in phenotype between the mutant and the control.Field investigation showed that the plant height of osprr59 under normal conditions was significantly lower than that of the control.4.OsPRR95 control the plant height and tiller number of rice at mature stage,regulate rice drought tolerance through ABA signal transduction pathway at germination stage,and positively regulate rice drought tolerance and salt tolerance at seedling stage.The germination osprr95 was treated with 150m M mannitol,and it was found that the bud length of the mutant was significantly higher than that of the control,but the root length of the mutant was significantly lower than that of the control,while the root length and bud length of the osprr95 treated with 3μmol/L ABA were not significantly different from those of the control.Whether under PEG6000 stress or 150m MNacl stress,the seedling survival rate,fresh weight and dry weight of osprr95 were significantly lower than those of the control.Field investigation showed that under normal conditions,the plant height and tiller number of osprr95 at adult stage were significantly lower than those of the control.5.OsPRR73 regulates drought tolerance and salt tolerance of rice,and affects plant height.Under PEG6000 stress,the seed germination rate of osprr73 was significantly lower than that of the control,and the seedling survival rate,fresh weight and dry weight were also significantly lower than those of the control.Under 150m MNacl stress,there was no difference in root length and bud length between osprr73 and the control at germination stage,but the survival rate,fresh weight and dry weight of mutants at seedling stage were significantly lower than those of the control.Field investigation showed that the plant height of osprr73 under normal conditions was significantly lower than that of the control.6.OsPRR37 affect the flowering time,plant height and yield-related agronomic characters of rice.Under normal conditions,the water loss rate and senescence rate of osprr37 in vitro were monitored,and it was found that the water loss rate and senescence rate of mutants were significantly lower than those of the control.For osprr37 growing under normal conditions,the flowering time was delayed by about half a month,and the1000-grain weight was significantly lower than that of the control,while the plant height,ear length,grain length,number of primary and secondary branches were significantly higher than those of the control.At the same time,OsPRR37 is regulating the drought tolerance of rice,and proper salt concentration will promote the growth of roots and buds during osprr37 germination.Under PEG6000 stress,the relative germination rate of osprr37 seeds was significantly lower than that of the control,and the seedling survival rate,fresh weight and dry weight were significantly lower than those of the control.When osprr37 was treated with 150m M mannitol and 3μmol/L ABA,the bud length was significantly lower than that of the control and the root length was significantly higher than that of the control under both stresses.The root length and bud length of osprr37 at germination stage treated with 150m MNacl were significantly higher than those of the control.7.There is a REC protein domain at the N-terminal of OsPRR37.In order to study the function of this domain,the drought tolerance of OsPRR37REC overexpressed plants was studied.It was found that the transgenic plants overexpressing REC domain could inhibit the expression of OsPRR37 gene,and the drought resistance of their plants decreased at seedling stage,indicating that OsPRR37REC domain could inhibit OsPRR37function,similar to osprr37.The drought tolerance of OsPRR37^REC overexpressed plants was significantly higher than that of the control at mature stage,and the yield was significantly increased,but the function of REC domain needed to be further studied.8.From the above and previous studies,it can be found that OsPRR37 has important development and research value in the study of stress resistance and high yield of rice.With Huhan 2B as the background,eight editing sites in pairs and single target editing on exon 10 of OsPRR37 were designed by genome editing technology,and four double target and one single target CRISPR/Cas9 gene editing vectors were constructed.About 150transgenic seedlings were obtained by Agrobacterium tumefaciens-mediated transformation of Shanghai Han 2B callus.Based on the preliminary observation of the field phenotype of the transgenic plants,it was found that there were great differences among different genotypes in heading time,plant height and the number of tillers,from which the plants with shorter growth period and larger panicle type were selected.as the basis for further research.To sum up,OsPRR family genes play an important role in rice growth and development,including controlling heading time,panicle development,plant height and tillering.OsPRR family genes are also widely involved in the response of rice to abiotic stress,regulating the drought and salt tolerance of rice to some extent.Because OsPRR37plays a variety of regulatory roles in all stages of rice growth and development and stress resistance,combined with CRISPR/Cas9 technology to edit its gene,it is expected to cultivate new abiotic stress resistant materials with growth stage change and yield increase,which will lay a foundation for rice molecular precision breeding.