Cloning and expression analysis of TaARF20 gene in wheat (Triticum aestivum L.)

【Objective】 To provide theoretical basis for analyzing the regulation mechanism of Auxin response factor (ARF)genes family in wheat by cloing and analyzing expression characteristic of TaARF20 gene.【Method】 The full-length cDNA sequence of TaARF20 gene was obtained from common wheat by homologous cloning technique,and its sequence characteristics were analyzed by bioinformatics. The active site of TaARF20 protein was determined by subcellular localization experiment. The TaARF20 gene was ligated with the expression vector Pcold-TF and transformed into Escherichia coli for prokaryotic expression. The expression patterns of TaARF20 gene in different tissues of common wheat and immature spike of common wheat and multi-ovary wheat at different development stages were analyzed by real-time fluorescence quantitative PCR.【Result】 Sequence analysis indicated that TaARF20 gene contained one intron and two exons, the coding region(CDS)was 1116 bp and encoded 371 amino acid residues. The molecular weight of TaARF20 protein was about 40.38 kD,theoretical isoelectric point(pI)was 4.96,fat solubility coefficient was 19.80,hydrophobicity coefficient was 0.985,and instability coefficient was 50.51 which indicated TaARF20 was an unstable protein. TaARF20 was located in the nucleus and contained ARF family domain and B3 DNA binding domain. Secondary structure analysis showed that the protein was mainly composed of alpha helix (18.06%),extended strand (59.57%),random coil (22.10%). The analysis of the promoter region revealed multiple cis-regulatory element responses,including hormonal responses, light and temperature responses. Alignment analysis of amino acid sequences and phylogenetic tree analysis showed that TaARF20 protein of common wheat had the highest amino acid sequence similarity and closest homology with Triticum dicoccoides ARF20. The result of SDS-PAGE showed that TaARF20 fusion protein was successfully expressed in prokaryotic cells in vitro. Real-time fluorescence quantitative PCR analysis showed that TaARF20 gene was expressed differently in root,leaf,immature spike and seed of wheat,and the expression in immature spike was significantly higher than that in other tissues. The relative expression level of TaARF20 gene in common wheat was lower than that in multi-ovary wheat for immature spike of 3 and 4 cm,but higher than that in multi-ovary wheat for immature spike of 5,6,8 and 9 cm in length.【Conclusion】 TaARF20 gene plays an important regulatory role in immature spike of wheat,which is speculated that it may be a key gene regulating spike shape,spike size and grain number per spike.