| Ethylene is an important plant hormone that plays an important role in promoting fruit ripening and regulating many growth and development processes such as cell division and expansion,tissue differentiation,seed germination,root hair formation and flowering.In this study,large grain ordinary maize inbred line Dan 232 and small grain burst maize inbred line N04 were used as materials to clone maize ethylene signal transduction factors ZmCTR1 and ZmEIN2 homologously and conduct sequence analysis.The promoter sequence of ZmCTR1 was cloned and the cis acting element was predicted.Subcellular localization vectors were constructed for subcellular localization analysis.Overexpressed and CRISPR vectors were constructed,transformed into maize and Arabidopsis thaliana,and genetic transformation analysis was carried out.Overexpression vectors of maize ethylene receptors ZmERS14 and ZmERS25 were constructed and transfected into Arabidopsis thaliana for genetic transformation analysis.Subcellular localization vectors of ZmERS14 and ZmERS25 genes were constructed for subcellular localization analysis.Grains of schizotypic maize mutant ZMERS25 14 days after pollination were selected for RNA-SEP analysis.The main results are as follows:1.Using maize inbred lines N04 and Dan 232 as materials,RNA was extracted.According to the sequence of B73,primers were designed to clone the ZmCTR1 gene.A 1200 bp fragment was amplified,and the open reading frame(ORF)was 1074 bp.Compared with the sequences in B73 and Dan 232,five bases were changed,resulting in the difference of the two encoded amino acids.The sequence of ZmEIN2 gene in the materials B73 and N04 is consistent,and the ORF length of B73 is 1431 bp,while the ORF of Dan 232 is 1738 bp compared with that of B73,which is 7 bp longer than that of B73.2.The promoter sequence of ZmCTR1 gene in the maize inbred line Dan 232 and N04 was cloned homologously.Compared with the promoter sequence of ZmCTR1 gene in B73,the promoter sequence of N04 and Dan 232 was 254 bp missing at 977 bp,and there were 17 base differences and 3 base deletions between Dan 232 and N04.The cis-acting elements of ZmCTR1 promoter were predicted,including plant hormone abscisic acid response elements,light response elements GT1 and auxin response elements,etc.Two segments of ZmCTR1 promoter were selected with about100 bp each sequence,and successfully connected to the Pabai yeast single heterovector.The working concentration of Ab A was selected as 300 ng/ m L and 400ng/ m L.3.Overexpression vectors of ZmCTR1(Dan 232 and N04),ZmERS14 and ZmERS25 genes were constructed and successfully transformed into Arabidopsis thaliana plants,and T2 generation seeds of ZmCTR1(Dan 232 and N04)and ZmERS14 genes were obtained,and T0 generation seeds of ZmERS25 gene were obtained.The transgenic maize plant expression vector p BCXUN-ZmCTR1(Dan 232 and N04),the overexpression vector of ZmERS14 gene and the transgenic maize embryo of B104 with CRISPR-ZmCTR1 were constructed,and the CRISPR positive plants of ZmCTR1 gene were obtained.4.Subcellular localization vectors for ZmCTR1(Dan 232 and N04),ZmERS14 and ZmERS25 genes were constructed.The results of subcellular localization showed that ZmCTR1(Dan 232 and N04)genes were localized on the cell membrane,and the prediction results of subcellular localization showed that ZmERS14 and ZmERS25 genes were localized on the endoplasmic reticulum.5.The maize homozygous mutant of ZmERS25 gene showed schizotypic phenotype.Transcriptome sequencing was performed on schizotypic and normal wild-type seeds 14 days after pollination,and 967 differentially expressed genes,involving 103 pathways,were identified.812 genes were up-regulated and 155 genes were down-regulated.GO enrichment of differential genes is mainly involved in ribosome,photosynthesis,pyruvate metabolism,citrate cycle,biosynthesis of valine,leucine and isoleucine,and regulation of plant hormones. |
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