Cloning And Bioinformatic Analysis Of The Gene Encoding An O-methyltransferase In Maize,and Construction Of Its Prokaryotic Expression Vector

Resistance to pests in maize is highly related with the secondary metabolites of benzoxazinoids.In maize,the benzoxazinoids include DIBOA,DIMBOA,HDMBOA,DIM2BOA and HMBOA;among these,DIMBOA and HDMBOA are transformed into poisonous MBOA for pests due to pest feeding,and the transformation of HDMBOA into MBOA is faster than DIMBOA.DIMBOA and HDMBOA are stored as DIMBOA-Glc and HDMBOA-Glc,respectively,whereas HDMBOA-Glc is enzymatically synthesized from DIMBOA-Glc by O-methyltransferase.In maize,O-methyltransferase is encoded by the gene bx12(also called bx10c).When the mutation occurs within the bxl2 locus,DIMBOA-Glc could not be enzymatically transformed into HDMBOA-Glc,which results in divergent pest-resistances.However,the mechanism of expression and regulation of the gene bx12 is still unclear.Here,the target DNA fragment contained the gene bx12,named as Zm3325-ZaC546,was isolated from the inbred ZaC546,and its prokaryotic expression vector pGEX4T1-Zm3325(bx12)was constructed.The result of sequencing revealed that the target DNA fragment was accurately inserted into pGEX-4T-1;the 5'-end of the DNA fragment linked to the 3'-end of GST tag exactly.Consequently,the target gene and GST tag made a fusion gene GST-Zm3325(bx12),which could be read continuously and correctly.The target DNA fragment cloned was 1010bp long with a full ORF.Compared with the reference sequence GRMZM2G023325,the target DNA fragment had a single base mutation from the 204th G to A within its ORF,leading a change of the codon AAG to AAA,but no amino acid residue was altinated.Although the 883rd base G occured a deletion and the 905th base T was substituted by C,both mutations went after the ORF and did not influenced on its encoding function.The identity between the target DNA Zm3325-ZaC546 and reference DNA was 99.70%with 99.87%identity beween their ORFs,and 100%identity between proteins encoded by both ORFs.The protein encoded by the gene cloned was belonging to O-methyltransferase superfamily 2,which was a multifunctional enzyme with two large domains.One domain existed in N-terminal with methyltransferase dimerization,another one existed in C-terminal with,methyltransterase and O-methyltransferase.Nosignal peptide in the amino acid sequence of this enzyme was found,indicating that no transportation occurred after translation.