| Rice(Oryza sativa L.)is an important food crop,and increasing its yield is of great significance for ensuring food security.Grain size is one of the key factors affecting grain weight and field yield.Studies have shown that the plant hormone Brassinosteroids(BRs)is an important hormone that regulating grain size,but its network of regulation has not yet been fully elucidated.Previously,our laboratory found that Os GSK3 is involved in the BR signaling pathway,and Os GSK3 mutant affects grain length.This topic focuses on its family protein OsGSK4.We use transgenic methods to study the effect of OsGSK4 on grain shape,and use biological techniques to study the molecular mechanism of OsGSK4 regulating rice grain length.The main findings are as follows:OsGSK4 contains a functional domain of S＿TKc,which has a close homology relationship with At BIN2 and Os GSK3.OsGSK4 is expressed in the roots,stems,leaves,nodes,panicles,and leaf sheaths of Nipponbare,with the highest expression level in panicles and nodes.OsGSK4 protein is located in the cell membrane,cytoplasm,and nucleus.After BL treatment,the distribution of OsGSK4 in the cell body will be affected,and the expression of OsGSK4 protein will be weakened.OsGSK4 gene can be induced by exogenous BL.Using the information of more than 3,000 rice germplasms provided by the RFGB database,the analysis found that gene OsGSK4 has two SNP,forming three haplotypes,named OsGSK4~A,OsGSK4~B,OsGSK4~C,the corresponding representatives in rice varieties are Indica 93-11,Japonica Nanjing 9108,and Japonica Nipponbare.According to Indica and Japonica classification and comparative analysis of grain length statistics,the haplotype of OsGSK4~A almost be found out in Indica,and the average grain length is significantly longer;the haplotypeof OsGSK4~B almost be take place out in Japonica,and the average grain length is also longer;the haplotype of OsGSK4~C almost be found out in Japonica,and the average grain length is shorter.We construct a series of transgenic rice and make surveys of agronomic traits.The survey results showed that the grain length of the overexpression material OE-OsGSK4~A was significantly longer,the plant height was significantly shorter,and the number of tillers increased slightly;cr-osgsk4~B showed larger grains,the plant height was shorter,and the number of tillers increased.Compared with the wild type,the grain length and grain width of the over-expression material OE-OsGSK4~C had no significant difference,and the tillers decreased.cr-osgsk4~C significantly increases the grain length,shortens the plant height,and increases the number of effective tillers.The phenotype of mutant osgsk4 is consistent with cr-osgsk4~C.Knockout of OsGSK4 will increase the sensitivity of plants to BR,and overexpression of OsGSK4 will show insensitivity to BR.It can be seen that the gene OsGSK4 participates in the BR signaling pathway,Using yeast two-hybrid technology and Bi FC system,it is verified that OsGSK4 can interact with the key gene Os BZR1 of BR signal transduction pathway.At the same time,it was screened and verified that OsGSK4 can interact with Os Di19-5 and Os KTN80c.Both Os Di19-5 and Os KTN80c can be inhibited by BL.The grain length of the mutant osdi19-5is significantly shorter,while the grain length of the mutant osktn80c has no obvious change.Therefore,OsGSK4 may interact with Os BZR1 and Os Di19-5 to regulate rice grain length through the BR signaling pathway.Gene Os Di19-5 is induced by a variety of abiotic stresses and participates in the regulation network of rice salt tolerance.It was verified that gene OsGSK4 was induced by ABA,salt stress,drought,and cold damage.The transgenic materials of OsGSK4 were treated with simulated abiotic stress.Under salt stress treatment,knocking out OsGSK4 can significantly improve the tolerance of rice to salt stress,and overexpression of OsGSK4 can reduce the tolerance of rice to salt stress.Under drought treatment,although the mutant osgsk4 had no obvious response,the knockout material cr-osgsk4~C significantly improved the drought tolerance of rice,while overexpression of OsGSK4 would reduce the drought tolerance of rice.Therefore,OsGSK4 may coordinately regulate the BR signaling pathway and stress response pathway.We performed transcriptome sequencing of cr-osgsk4.The analysis results show that the differentially expressed genes of OsGSK4 are enriched in the pathways of sugar metabolism,phenylpropanoid biosynthesis,and thiamine metabolism,which are involved in plant growth and development and response to abiotic stress.Therefore,OsGSK4 participates in the process of rice sugar metabolism and abiotic stress response.The SNP2 of the gene OsGSK4 detected DNA methylation modification.It is speculated that the grain length differentiation of the gene OsGSK4 is related to methylation modification.In summary,OsGSK4,a negative regulator of brassinosteroid signaling in rice,regulates rice grain length and participates in rice abiotic stress response. |
