Study On Molecular Mechanism Of Rice OsMPK15 Gene Editing Leading To Decrease Of Leaf Angle

Mitogen-activated protein kinase（MAPK）is highly conserved in eukaryotes and exists in the form of the MKKK-MKK-MAPK cascade.The existing studies have shown that rice MAPK functions involve a variety of biological processes such as growth and development,response to abiotic and biotic stress,and hormone response.Our laboratory cloned the MAPK gene OsMPK15（LOC＿Os11g17080）from Nipponbare rice in the early stage.The expression pattern analysis showed that this gene is predominantly expressed in leaves and leaf sheaths,and hormones such as SA,JA and ABA can induce its up-regulation.In order to identify the function of the gene,the laboratory used CRISPR/Cas9 technology to construct OsMPK15 knockout rice,and screened two homozygous knockout lines mpk15-7 and mpk15-8.Phenotypic analysis showed that the leaf angle of knock-out rice was significantly reduced.Based on this research,this study screened and identified the T3 generation of OsMPK15 gene knock-out rice.The sequencing results of the edited target sequence PCR product showed that the mutation sites of OsMPK15 gene remained stable in the T3 generation of mpk15-7 and mpk15-8 strains.In knock-out rice,a base deletion occurred in the OsMPK15 sequence,leading to frameshift mutations and nonsense mutations in the gene,which was expected to produce a truncated protein that loses the MAPK conserved domain.The statistical results of agronomic characteristics of the T3 generation of OsMPK15 knock-out rice showed that compared with wild-type rice,the leaf angles of inverted first leaf,inverted second leaf,inverted third leaf and inverted fourth leaf of knocked out plants mpk15-7 and mpk15-8 were all reduced.Among them,the leaf angle of the first and second leaves decreased more significantly,with an average decrease of 29.53% and 32.44%,respectively,and reached a statistically significant level.In addition,the grain type of OsMPK15 gene knocked out rice changed.In order to explore the reason for the decrease in leaf angle of rice knocked out by OsMPK15 gene,paraffin section technology was used to conduct cytological detection on the leaf occipital of the first leaf.The results showed that compared with the wild type,the number of thick-walled cells per unit area at the abaxial and paraxial axis of knock-out rice leaves significantly increased.For this reason,q RT-PCR was employed to detect the expression of cell wall synthesis-related genes in the leaf occiput,and the results showed that compared with wild-type rice,the expression of marker genes related to cellulose and hemicellulose synthesis in the leaf occipital of knock-out rice very significant up-regulation,indicating that OsMPK15 knock-out rice may increase the cellulose and hemicellulose content in the cell wall of the leaf occipital and improve the supporting ability of the cell wall.Since the thickening of the cell wall was related to the formation of the secondary wall,the phloroglucinol staining method was used to qualitatively analyze the deposition of lignin in the cell wall of the occipital tissue.The results showed that lignin was more accumulated in the knock-out rice than in the wild-type rice.At the same time,the detection of the expression level of lignin marker genes in the leaf pillows also confirmed that compared with wild-type rice,the expression of lignin-related genes was extremely significantly up-regulated in knocked out rice.This indicates that the phenotype of reduced leaf angle in OsMPK15 knockout rice is related to the increase in the number of thick-walled cells in the leaf occipital tissue,and the increase in the accumulation of lignin in the secondary wall of the leaf occipital,thereby improving the mechanical support capacity of the leaf occipital.Since the leaf angle phenotype is regulated by a variety of plant hormones,and brassinolide（BR）is the dominant one,this study further explored whether OsMPK15 knock-out rice leaf angle reduction is related to BR hormone regulation.The method of gradient BR treatment was used to take 2cm rice tissue at the angle between leaf nodes and coleoptile as materials,the BR sensitivity test of OsMPK15 knock-out rice was carried out,and the results showed that the sensitivity of OsMPK15 knock-out rice to BR was reduced.Using q RT-PCR technology to detect the expression characteristics of OsMPK15 gene in wild-type rice leaf occiputs after BR treatment,the results showed that the expression of OsMPK15 gene was significantly increased after BR treatment for 10 h and 12 h,indicating that the expression of OsMPK15 in the occipital region was positively regulated by BR hormone.The expression of BR biosynthesis and signaling pathway related genes in leaf pillow tissues of OsMPK15 knock-out rice and wild-type rice were compared by q RT-PCR.The results showed that,compared with the wild type,the expression of BR biosynthesis related gene D2,D11 and BRD1 was extremely significantly up-regulated in the knocked out rice leaf pillow,while the expression of BR signaling pathway related gene Os BRI1 and Os BZR1 was extremely significantly down-regulated.These results indicate that OsMPK15 gene knock-out may affect BR homeostasis and signal transmission by affecting the expression of BR marker genes,thus leading to decreased leaf angle.Because MAPK exists in the form of a cascade reaction in the regulation of biological processes,and Os MKKs located upstream of OsMPK15 have not been reported yet.To this end,this study screened the Os MKKs upstream of OsMPK15 in leaf angle regulation.Firstly,q RT-PCR was used to analyze the correlation of gene expression,and the expression of Os MKKs in rice leaf occipitals was detected.Bioinformatics prediction of binding protein interaction was conducted,and four Os MKKs that may be located in the upstream of OsMPK15 were selected preliminarily.The four Os MKKs are: Os MKK4（LOC＿Os02g54600）,Os MKK9/5（LOC＿Os06g09190）,Os MKK6（LOC＿Os01g32660）and Os MKK10-2（LOC＿Os03g12390）.In order to identify whether OsMPK15 interacts with these four Os MKKs,the BD recombinant vector was constructed using OsMPK15 genes,and these four Os MKKs genes were cloned from leaf occipital tissues,and a yeast two-hybrid AD recombinant vector was constructed.The BD vector and the four AD vectors were co-transformed into yeast.According to the growth of the co-transformants on the defective medium,OsMPK15 not only binds to Os MKK9/5,but also binds to Os MKK10-2,suggesting that in leaf angle regulation,The upstream kinase candidate molecules of OsMPK15 may be Os MKK9/5 and Os MKK10-2.The agronomic traits showed that the grain length and grain thickness of OsMPK15 knocked out rice became longer and thinner,but the thousand-grain weight remained unchanged.In order to explore the reasons for the change of grain type,the inner and outer glumes of the grains were observed by scanning electron microscopy.The results showed that the number of cells that knocked out the inner and outer glumes of rice per unit area decreased,while the length of the inner and outer glumes increased.The q RT-PCR technique was used to detect the expression of grain type-related genes in young ears.The results showed that genes regulating cell number,such as GW8,GS3 and TGW6,were extremely down-regulated,while genes affecting cell size,such as GS2,were extremely up-regulated.It indicates that OsMPK15 gene knockout may inhibit the proliferation of inner and outer caryopsis cells by changing the expression level of granulotype-related genes,but promote the elongation of inner and outer caryopsis cells,resulting in changes in granule shape.It is inferred that the thousand-grain weight does not change,which may be the result of the grain length becoming longer and the grain thickness becoming thinner.In this study,we used OsMPK15 knockout rice constructed earlier in the laboratory to study the molecular mechanism of leaf angle reduction.It was found that OsMPK15 gene knockout increased the number of thick-walled cells and lignin deposition in the leaf occipital tissue of rice,which enhanced the mechanical support capacity of the leaf occipital tissue,and finally resulted in the decrease of leaf angle.This study also demonstrated that the reduction of leaf angle caused by OsMPK15 gene knockout was related to BR hormone.In addition,the Os MKKs located upstream of OsMPK15 in leaf angle regulation were screened and the yeast two-hybrid system protein interaction test was performed,and two candidate Os MKK genes were locked.It lays the foundation for the subsequent analysis of the molecular network of OsMPK15 in leaf angle regulation,and is also expected to provide candidate genes for the molecular design and breeding of ideal rice plant types.