Cloning And Functional Analysis Of The AmDEFH28 Gene Of Antirrhinum Majus

Antirrhinum majus is a very special plant in the study of plant leaf order.It has different leaf order forms on the same plant,the basal leaf order is mainly opposite,and the upper and middle leaf order is mainly spiral.Leaf primordium originates from the apical rneristem of plants(SAM),and its location on the stem tip determines the arrangement of leaves on the stem(leaf order).Mads-box gene family is a very important transcriptional regulatory factor,which has an important influence on the apical meristem(SAM)of plants.In this study the goldfish grass varieties”summer of pan powder,as the experimental material,through the adoption of a single enzyme and homologous recombination excessive expression vector was constructed,and the method of agrobacterium mediated success will be excess expression vector into arabidopsis gene,got different strains of phenotype observation,supplemented by bioinformatics analysis,to explore by MADS-box genes AmDEFH28 function and influence on plant phyllotaxis.1.According to the data analysis of the antirrhinum sinensis leaf sequence transcriptome conducted by the research group before,the target gene AmDEFH28 was searched through Blast software,and the primers were designed to extract the total RNA of antirrhinum sinensis.After rt-pcr,the target gene AmDEFH28 was cloned.The CDS sequences with a total length of 759bp were obtained by bioinformatics analysis of the target gene AmDEFH28.The similarity of the target gene AmDEFH28 and PhFBP29 was the highest(85.77%).The protein encoded by AmDEFH28 gene is composed of 252 amino acids.The molecular formula is C127lH2072N3840394S8.Its instability index is 68.49.There is no protein signal peptide sequence and no splicing site.There were 5 potential phosphorylation sites.The secondary structure and tertiary structure of the target gene were predicted.2.The relative expression of the target gene AmDEFH28 in antirrhinum was analyzed by fluorescence quantitative PCR.AmDEFH28 was expressed in different organs of antirrhinum during the growth cycle.3.The test using pCAMIBA1301 plasmid vector,through single enzyme and homologous recombination constructed AmDEFH28 excess expression vector,putting the vehicle into e.coli,and the conductance method import GV1301 agrobacterium,will AmDEFH28 excess expression vector through the inflorescence impregnation method into arabidopsis thaliana,and use the kanamycin,gentamicin,rifampicin and hygromycin B such as antibiotics screening step by step,finally get the homozygous transgenic arabidopsis thaliana plants.4.The test through the images,and microscopic imaging phenotypes observed in the transgenic arabidopsis thaliana,using type Colombia(Columbia-0)wild arabidopsis thaliana as control group,found that the transgenic arabidopsis growth is slow,the plant small,blade,blade shape,size,branch,flower bud differentiation and inflorescence axis spare delay,phyllotaxis appeared opposite,change the phenotype of floral organ development.5.The target gene was obtained from the antirrhinum auriculata leaf sequence transcriptome,and the subsequent phenotypic observation found that the transgenic arabidopsis thaliana presented the opposite phenotype,indicating that the target gene AmDEFH28 had a certain effect on the leaf order of plants.