The Network Analysis Of ZmCLA5-1 Gene Regulating The Angle In Maize

Corn is an important food crop in China and plays an important role in the national food security strategy.At this stage,with the optimization of the planting structure of food crops,the planting area of corn is decreasing.To increase the total corn yield and increase the yield,it becomes one of the major breeding objectives at this stage.Appropriate increase of planting density is a way to increase the yield.In the plant type traits,the angle of stem and leaf is the key factor for limiting the planting density.Therefore,it is of great scientific value and practical significance to study the genes and functions of leaf angle traits.In this study group,we used compact inbred maize Yu 82 and loose inbred maize Shen 137 as materials to study the corn leaf angle.Through preliminary mapping and fine mapping,we obtained the candidate gene Zm CLA5-1 that regulates the angle of corn leaf,through the identification of the transgene and phenotype preliminarily proved that the gene has the function of regulating the angle of leaves.On this basis,this study used yeast two-hybrid technology to screen for the Zm CLA5-1 interaction protein,using yeast two-hybrid and bimolecular fluorescence complementation(Bi FC)to test the potential interaction protein,at the same time,q RT-PCR analysiswas carried out to define the regulation mechanism of the gene and interaction genes on the angle of the leaf in maize.Provide the basis for further analysis the regulation of the angle development.1.Zm CLA5-1 gene was screened by yeast two-hybrid,and 75 positive clones were screened from Yu-82 yeast two-hybrid library with 53 correct reading frames.Among them,three genes were involved in the regulation of the development of leaf angles,namely GRMZM2G336858,GRMZM2G133624 and GRMZM2G003096 genes.It is involved in the regulation of leaf angle by the BR synthesis pathway.Confirmation of the yeast two-hybrid technique was used to verify that these three genes interact with Zm CLA5-1.At the same time,using the Bi FC to verify GRMZM2G133624 and GRMZM2G003096,it was proved that both genes interact with Zm CLA5-1 and emit a yellow fluorescence signal.This shows that these three genes are involved in the regulation of leaf angle development.2.The results of q RT-PCR showed that the overexpression of Zm CLA5-1 in maize plants was significantly higher than that in the wild type,and the expression of Zm CLA5-1 was significantly increased in the 4 leaf stage and 5 leaf stage,and the expression of GRMZM2G003096 was significantly decreased,indicating that the negative regulation of GRMZM2G003096 by Zm CLA5-1 is located in the downstream of Zm CLA5-1;The expression level of GRMZM2G336858 and.GRMZM2G133624 was not significantly different in the 4?5 and 6 leaf stage,indicating that the two genes were not affected by the overexpression of Zm CALA5-1,probably located in the upstream of Zm CLA5-1.3.The yeast two-hybrid technique was used to screen the GRMZM2G336858,and 100 positive clones were screened from the Yu-82 yeast two-hybrid library with 40 correct reading frames.Through bioinformatics analysis,it was found that two genes are involved in the regulation of the development of leaf angles.The gene GRMZM2G123886 has the activity of activating MAPK.The MAPK protein can participate in the development of leaf angles through the auxin pathway.It is validated by yeast two-hybrid,and it is proved that this gene interacts with GRMZM2G336858.In summary,this experiment explored the genes related to the formation of leaf angles around the Zm CLA5-1 gene.These genes can regulate the development of leaf angles through the pathway of brassinosteroids and auxin.