Regulation Network Analysis Of Gene ZmCLA1 In Maize

Maize is an important crop in China,which plays a very important role in ensuring national food security.Increase the density is an important way to improve maize yield.Leaf angle is an important trait for density tolerance,therefore,analysis the genetic regulatory networks and molecular mechanisms of maize leaf angle development,not only has important scientific value,but also has important practical significance for the high density breeding with high yield.This study is based on the map based cloning maize leaf angle candidate gene Zm CLA1 and preliminary validation of the gene function.Screening of genes that interact with Zm CLA1 by Co-Immunoprecipitation technique,The function of Zm CLA1 gene will further verified by transgenetic Arabidopsis,and the interaction network between relationship gene in the expression level.Using yeast two hybrid technique,the genes that have been identified in the interaction network were further screened and their interactions were identified by Co-Immunoprecipitation.The aim is to systematically analyze the interaction network and its molecular regulation mechanism of Zm CLA1 in regulating the formation of maize leaf angle.The main results are as follows:1?According to the preliminary experimental results of yeast two hybrid,by Agrobacterium mediated method,the fusion vector Zm CLA1 respectively with KGD,IAA17 and CC4 fusion vectors into tobacco leaf transient expression protein.By Co-Immunoprecipitation analysis,it is found that the Zm CLA1 and gene KGD,iaa17,and CC4 interacting protein band was positive,indicating that Zm CLA1 and gene KGD,IAA1,CC4 with direct interaction.Proved that the key gene Zm CLA1 controlling maize leaf angle with the other 3 genes KGD,CC4,IAA17 interactions exist in cells.2 ? By Arabidopsis thaliana mediated by Agrobacterium mediated transformation,the Zm CLA1 overexpression vector into Arabidopsis homolog mutant cla1,T3 generation transgenic lines and non transgenic Sister Lines(control)leaf angle phenotypic analysis results show that overexpression of(average OX-CLA1)transgenic lines leaf angle was significant higher than the control,indicating that Zm CLA1 positive regulation of leaf angle.By Arabidopsis thaliana mediated by Agrobacterium mediated transformation,the Zm CLA1 overexpression vector into Arabidopsis homolog mutant cla1,T3 generation transgenic lines and non transgenic Sister Lines(control)leaf angle phenotypic analysis results show that overexpression of(average OX-CLA1)transgenic lines leaf angle was significantly higher than the control,indicating that Zm CLA1 positive regulation of leaf angle.Further quantitative PCR analysis showed that the transgenic lines of CLA1 mutant were compared with the wild type and the mutant lines,and the expression of CLA1 was comparable to that of the wild type.The gene expression of KGD and CC4 was significantly higher than that of the mutant,but the expression level of IAA17 was lower than that of the mutant.These results indicate that CLA1 is positively regulated by KGD,CC4 and negatively regulates the IAA17 gene,which influences the development of leaf angle.3?KGD and the interaction of 60 positive clones screened from Yu 82 yeast c DNA library using yeast two hybrid technique,which has the correct reading frame ORF was 12,and verified by yeast rotary.The result showed that KGD interacts with the mitogenic mitogen activated protein kinase MAPK(GRMZM2G017351).Co immunoprecipitation results showed that KGD interacts with MAPKt in the cell.In summary,in the regulation network of leaf angle development,Zm CLA1 positively regulates the expression of KGD and CC4 genes,and negatively regulates the expression of IAA17 gene,while KGD interact with downstream gene MAPK.Therefore,Zm CLA1 may regulate the cell differentiation and division of the occipital region by KGD and MAPK,which leads to the change of the angle of the leaf.