CRISPR/Cas9 System-based Editing Of OsMPK15 Gene In Rice

Mitogen-activated protein kinases?MAPKs?are ubiquitous in eukaryotes,including yeasts,plants and mammals,which highly conserved during evolution.Up to now,their functions involved in many biological process,such as cell division,cell differentiation,biotic and abiotic stress responses,hormonal responses.Previously,the cDNA coding region of rice OsMPK15 gene was cloned from Nipponbare roots,and it was found that the OsMPK15 gene showed differential expression in various tissues of rice,and the abundance in leaves and leaf sheaths is the highest.The expression of OsMPK15was responsive to both environmental stress signals and hormone signals,which was most sensitive to drought and ABA,suggest that the gene may be involved in the related functions of leaves,and is most likely involved in rice abiotic stress response.In order to identify the functions of the OsMPK15 gene,CRISPR/Cas9 genome editing technique was used to construct OsMPK15knockout rice in this study,aims to further study the role of the gene in the growth and development of rice.The OsMPK15 gene knockout carrier was constructed by CRISPR/Cas9 technology,and 3homozygous transgenic lines were obtained by Agrobacterium mediated transformation,which were named mpk15-1,mpk15-7 and mpk15-8,respectively.Target site-specific analysis showed that there was no off-target in the three transgenic lines.Rice phenotype observation showed that the germination of mpk15-7 and mpk15-8 seeds was delayed,and the germination rate was decreased,leaf erect and leaf angle was reduced,heading date was postponed,but there were no significant differences in other agronomic traits,such as pollen viability,plant height,tiller number,grain number per spike,grain number,1000-grain weight.Considering that the leaf angle of rice is closely related to the regulation of Brassinolide?BR?,qRT-PCR were applied to detect the expression of OsBRI1,OsBZR1,OsMDP1,OsLIC,BU1,ILI1,D1 and OsBAK1,the key genes in the BR signaling pathway.The results showed that the expression levels of OsBAK1 and OsBZR1,the transcriptional regulators of BR synthesis genes,were reduced by 70%and 40%,respectively.The expression of BR receptor encoding gene OsBRI1 and BR signaling genes BU1 and D1 also decreased.Among them,the expression of D1was down to 40%of wild type,reaching a very significant level.The expression level of OsILI1and OsLIC decreased and increased slightly,respectively.But the expression of OsMDP1decreased to 50%,and reached the extreme significant.These results demonstrated that knockout of OsMPK15 gene inhibited the synthesis and signaling of BR.It has been verified that decrease of leaf angle in rice tends to be accompanied by the enhancement of photosynthesis.Accordingly,qRT-PCR were performed to detect the effect of light on the expression of OsMPK15 gene,and it was found that the expression of the gene in the shoots and roots were inhibited by light.Compared with control,the net photosynthetic rate of mpk15-7 and mpk15-8 increased to 1.5 and 2 times,and the total content of chlorophyll a and b increased significantly,while the stomatal conductance and transpiration rate had no significant changes.It was proved that knockout of OsMPK15 gene resulted in the improvement of photosynthesis in rice.In order to explore the relationship between rice OsMPK15 gene and abiotic stress response,10%PEG6000 was used to mimic drought stress.It was found that the activities of antioxidant enzymes SOD,POD,CAT and APX in mpk15-7 and mpk15-8 was generally lower than that of wild type rice during 24 hours after treated,but contents of MDA and H₂O₂ were higher,and the soluble protein content was significantly lower than that of control,suggest that OsMPK15 gene is involved in the rice drought stress response.In order to explore the relationship between OsMPK15 gene and seed germination,the germination rates of control,mpk15-7 and mpk15-8 seeds were statistically analyzed.The germination time,germination rate and growth condition of rice seeds were further determined by ABA treatment at different concentrations.It was found that OsMPK15 knockout reduced the germination rate and delayed the germination time.In addition,ABA significantly inhibited the germination and growth of rice seeds,and the effect on mpk15-7 and mpk15-8 was more notable.Because the sensitivity to ABA was further increased after OsMPK15 knockout,it can be deduced that OsMPK15 gene is involved in the ABA signaling pathway.In this study,three homozygous lines of OsMPK15 knockout rice were constructed using CRISPR/Cas9 technology.It was found that the function of the gene was involved in rice seed germination,leaf angle regulation,photosynthesis and drought stress,and its expression was regulated by light and ABA.This study systematically analyzed the function of OsMPK15 gene in the growth and development of rice,facilitate the further study and provided a candidate gene for rice molecular breeding.