Cloning And Bioinformatics Analyses Of The Arabidopsis BGLU23 And BGLU28 Genes

In higher plants,?-glucosidases(BGLU)belonging to glycoside hydrolase(GH)Family 1 have been implicated in several fundamental processes including defense.Phylogenetic analysis shows that among the 47 BGLU proteins,BGLU18–BGLU39(which includes BGLU23),form monophyletic clades that are specific to Brassicales,suggesting their function as myrosinases.Also,BGLU28 has been hypothesized to function in the release of thiol groups by breaking down various glucosinolates in response to sulfur deficiency.Both findings have led to the hypothesis that both genes may be involved in breaking down glucosinolates in intact plant tissues.To test this hypothesis,we cloned CDS encoding BGLU23 and BGLU28 and their promoters and carried out bioinformatics analysis on their amino acid and promoter sequences.Our findings are as follows:(1)Both genes were isolated using homology-based cloning methods.The 1575 bp and 1749 bp coding sequences that encode peptide sequences of 524 and 582 respectively were isolated by using specific primers and the CDS of both genes were identified separately using RT-PCR technique.The BGLU23 gene has an ORF of 1575 base pairs(bp),encodes a peptide sequence of 524 amino acids and possesses a relative molecular mass of about 59 721 Da,with an instability index of 27.14 which indicates a stable protein and the BGLU28 gene has an ORF of 1749 base pairs(bp),encodes a peptide sequence of 582 amino acids and possesses a relative molecular mass of about 67 299 Da,with an instability index of 41.75 which indicates that the protein may be unstable.(2)Both proteins showed hydrophilic properties,BGLU23 was predicted to have signal peptide with cleavage site between positions 24 and 25 while BGLU28 was predicted to have signal peptide with cleavage site between positions 21 and 22.For both proteins,all phosphorylation site scores were above the threshold(0.500)thus indicating that the confidence for these sites being true phosphorylation sites is high.BGLU23 had 2 confidently predicted functional domains while BGLU28 had just 1.(3)Evolutionary analysis revealed that BGLU21,BGLU22 and BGLU24 genes were the closest paralogs to BGLU23 while BGLU29,BGLU31 and BGLU32 genes were the closest paralogs to BGLU28.(4)Promoter sequences of BGLU23 and BGLU28 were analyzed and basic expression pattern of the genes were predicted.(5)Plant vectors respectively overexpressing BGLU23 and BGLU28 were constructed.In all,our findings present a foundational and theoretical framework for further studies on the functions of BGLU23 and BGLU28 in plant defense and related processes as these genes may also be involved in other plant processes aside defense.