Cloning And Functional Analysis Of Maize ZAP1 Gene On Flower Organ Development

The plant MADS-box genes play key roles in the regulation of floral organ development.The ABCDE model,based on the studies on floral mutants of dicot model plants,explains explicitly how five class genes regulate floral organs identity,among which A class genes not only regulate the organ identity of sepal and petal,but also are involved in the initiation of floral meristem.In addition to individual genes,the MADS-box gene contains almost all of these genes.A class genes have been studied in Arabidopsis and rice,but still remain to be elucidated in maize.Among which AP1-Like gene in mazie are replicated,resulting in ZAP1 and ZmMADS3.These two replicatied genes generate multiple transcripts through alternative splicing.To understand the biological function of AP1-Like genes in maize,we first performed bioinformatics analysis of each transcript of ZAP1 and ZmMADS3.Based on the results of AP1-Like genes bioinformatics analysis,we obtained functional ZAP1 transcripts from maize spikelets and constructed a plant expression vector.The wild type Arabidopsis inflorescence was invaded by Agrobacterium-mediated method.In order to further understand the function of ZAP1 gene during flower development and lay a foundation for the research of gene duplication and divergent function.In order to study the effects of alternative splicing on its function,we cloned and constructed another expression vector for the D class gene ZAG2 on the basis of previous studies of this group.The main results of the study are as follows:1.The nucleotide sequences and amino acid sequences of the ZAP1 and ZmMADS3genes were obtained from MaizeGDB and the Gramene website.We obtained preliminary results from bioinformatics analysis that ZAP1-T02 performed its AP1-Like gene function.The ZmMADS3-T03 gene has been verified to function as a transcript,which is consistent with our bioinformatics analysis.In addition,through alternative splicing,the individual conserved regions of transcripts change.We predict that they may also perform functions,but the effect of alternative splicing on function remains to be studied.2.We pass the Arabidopsis AP1 gene,the AP1-Like genes of maize,and its homologous protein sequences in Setaria italica,Sorghum bicolor and Brachypodium distachyon.The four proteins formed by alternative splicing of ZAP1 belong to the same branch.All ZAP1 proteins belong to the same branch with ZmMADS3 protein,and they share the highest homology with Sorghum bicolor.They belong to two major branches of Arabidopsis AP1 protein,respectively.3.The promoter elements of each gene were predicted by online tool PLANTCARE,including their transcripts generated by alternative splicing.The results showed that alternative splicing has no effect on the promoter elements owned by each transcript of each gene.Next,we analyzed the promoters of AP1-Like genes produced by maize in addition to the shared control of light,hormones and other regulatory elements.These results indicate that AP1-Like genes may be differences in functionality.4.We overexpressed the ZAP1-T02 gene of maize and cloned into the plant expression vector for further functional studies.The overexpression vector pCAMBIA 1300-ZAP1-T02was transformed into Arabidopsis via Agrobacterium-mediated method to get T₀ transgenic seeds.Hygromycin resistant transgenic Arabidopsis was screened and T₁ generation transgenic plants were obtained and their inflorescences were observed with obvious floral defects.This study lay the foundation for subsequent gain of homozygous transgenic Arabidopsis and further deep investigation of ZAP1 gene.5.In order to understand the function of ZAG2-T03 gene in flower development and the effect of alternative splicing on gene function,we cloned the ZAG2-T03 gene and constructed plant overexpression vector.