Cloning And Functional Analysis Of GW5L,a Gene Regulating Grain Width And Weight In Rice(Oryza Sativa L.)

Rice(Oryza sativa L.)is one of the world’s most important food crops,supporting nearly 60% of the world’s population.In recent years,as environmental issues have intensified and the area of cultivated land has decreased,how to increase yield has become a major challenge in rice production.Grain shape not only determines grain weight,affects rice yield,but also determines the quality and market value of rice.With the development of molecular biological technology and the completion of rice genome sequencing,more and more genes that regulate grain size have been cloned in rice.However,because the researchers know little of the molecular mechanisms underlying of the key regulatory genes,coupled with problems with the linkage of undesirable traits in the actual breeding process,the utility of the cloned grain shape regulatory genes is limited.Therefore,to keep cloning of key genes that regulate grain shape and to analysis the molecular mechanism of grain shape regulation are of great significance for the cultivation of new varieties of high-yield and high-quality rice.In this study,we identified a novel gene controllinggrain width and weight,grain width 5 like(GW5L)by means of the strategy of homologous cloning of GW5 in rice.GW5 L interacts with GSK2 protein kinase and regulates BR signaling by inhibiting its phosphorylation,thus affecting grain shape.In addition,GW5 L interacts with the calmodulin protein,OsCaM1-1,which contributes to salt resistance reaction.In this study,the structure,function,transgenic phenotype and regulatory pattern of GW5 L gene were studied and analyzed.The main results are as follows.1.Homologous cloning was adopted to clone the GW5 L gene in the rice genome with the GW5-encoded protein sequence as a reference.Due to the highly consistent protein structure,GW5 L and GW5 are assumed to have similar biological functions.Transgenic experiments showed that GW5 L overexpressing plants exhibited typical BR overdose phenotypes,including shorter plant height,larger angle of leaves,and slender seeds;GW5L knock-out lines also showed phenotypes of widening and shortening seeds.Therefore,GW5 L is a negative regulator of grain width.2.BR is an important plant hormone that plays an important role in rice.The results of the lamina inclination experiment proved that the phenotype of GW5 L overexpression lines were due to the enhancement of BR signal.The results of qRT-PCR on GW5 L overexpression lines showed that the BR synthetic genes,which had feedback regulation effect,were reduced in GW5 L overexpression lines.Therefore,it is inferred that GW5 L influences the grain shape through the BR signaling pathway and plays a positive regulator role in the BR signaling pathway.3.To confirm the interaction of GW5 L and GSK2,a GW5-interacting partner,yeast two-hybrid,bimolecular fluorescence complementation(BiFC)and in vitroPull-down experiments were performed.The results confirmed that GW5 L indeed interactedwith GSK2.In vitro phosphorylation experiments further demonstrated that GW5 L can inhibit GSK2 autophosphorylation and phosphorylation of downstream BZR1 transcription factors.GW5 L regulates BR signaling by inhibiting the function of the downstream protein kinase GSK2,ultimately affecting grain size.4.Both yeast two-hybrid and BiFC experiments confirmed the interaction between GW5 L and the calmodulin protein OsCaM1-1.OsCam1-1 can be involved in ABA-mediated salt stress resistance,and it is speculated that GW5 L is related to salt resistance in rice.Salt stress experiments showed that the GW5 L overexpression lines were more sensitive to salt stress,and the results of qRT-PCR further showed that the increase in salt sensitivity of GW5 L overexpressing lines might due to changes in the expression level of OsCam1-1.These results showed that GW5 L may associate with OsCaM1-1 mediated salt stress resistance,which provides clues for revealing the relationship between IQ protein and salt stress.5.Although GW5 L and GW5 have similar protein structure and biochemical function,their expression patterns are somewhat different.Compared with the high expression of GW5 in spikes,GW5 L was expressed in all organs examined at a high level.These results suggest that GW5 L and GW5 are likely to regulate different metabolic pathways in different tissues,pending further study.