Cloning And Genetic Transformation Of BtACO Gene In Balsas Teosinte

As one of the main food crops and economic crops in the world,maize is widely used in industry and animal husbandry.In the 21 st century,the maize production is facing great challenges due to the rapid growth of the global population,the significant planting area reduction and the changing global climate environment.In addition,the sharp reduction of maize yield caused by pests is also the focus issue concerned.Improving the resistance of maize to insects is a long-term and fundamental measure to reduce the pest threat,but there is no good resistance gene source in maize.Balsas teosinte(Zea.mays ssp.parviglumis)is the wild ancestor of maize.Studies have shown that teosinte presented higher insect resistance than cultivated modern maize,and could be used as a natural gene pool for maize genetic improvement.In the present study,based on the transcriptome data analysis of teosinte that responding to armyworm feeding,a candidate insect-resistant gene was screened and cloned,and was transformed into Arabidopsis thaliana.The main results are as follows.1.Based on the previous transcriptome data,including statistical analysis of differentially-expressed genes number,and enrichment assessment of GO function and KEGG pathways,we identified plant hormone signaling pathways were significantly up-regulated,where ACO gene was highly induced in ethylene signaling pathway.Consider ethylene signal pathway is actively involved in plant diseases and insect defense,and ACO gene regulates the biosynthesis of ethylene and is the last key enzyme gene in ethylene signaling pathway,we selected BtACO gene as a candidate insect-resistant gene.At the same time,fluorescence quantitative PCR was adopted to analyze the relative gene expression of BtACO.The results showed 12 h after feeding,the BtACO gene was significantly up regulated compared with the control Therefore,we proposed BtACO gene was highly induced by armyworm feeding,and might be involved in the insect resistance response,thus could be used as an insect-resistant candidate gene;2.The partial coding sequence of BtACO protein was obtained from the transcriptome database of Balsas teosinte.The full-length CDS of BtACO gene was obtained by 3' RACE technology.The bioinformatics analysis of BtACO gene was carried out.The full-length CDS of the BtACO gene is 1071 bp in length,encoding 356 amino acids,the p I is 5.59,and contains 33 phosphorylation sites.BtACO has no signal peptide,and is mainly distributed in the cytoplasm.The protein belongs to the hydrophilic protein.The secondary structure analysis shows that alpha helix accounts for 28.09%,?-sheet is 15.17%,random coil accounts for 56.74%,and ?-turn is not included.Phylogenetic analysis showed that the amino acid sequence of BtACO is closely related to that of maize B73,which was consistent with the result that BtACO was a wild relative of cultivated maize.The homology comparison of amino acid sequence showed that BtACO protein has DIOX?N domain and 2OG-Fe II?Oxy domain,and leucine zipper structure.The protein sequence was highly conserved,which is a typical 2-ketoglutarate-dependent dioxygenase(2OGD)non-heme containing iron superfamily protein.3.The gene cloning vector PUC57-BtACO was constructed,also the gene expression vector p CAMBIA1300-BtACO / p CAMBIA1300-BtACO-GFP.The constructs were verified by enzyme digestion and gene sequencing.BtACO was transformed into Arabidopsis thaliana and Nicotiana benthamiana via Agrobacterium-mediated transformation method.T1 seeds from Arabidopsis were collected,and the positive plants were verified by antibiotic selection and PCR amplification.The transient expression of BtACO was observed with tobacco leaf by vacuum injection method.Most of the proteins were located in the cytoplasm of tobacco.