Cloning And Characterization Of Two Key Genes EcACS And EcACO Involved In Ethylene Biosynthesis

Ethylene is a kind of plant hormmones which play an important regulatory role of plant growth and abiotic stress.The researchers have found that the ethylene biosynthesis mainly include adenosylmethionine synthetase(S-Adenosyl-L-Methionine Synthetase,SAMS),1-aminocyclopropane-l-carboxylate synthase(ACS)and 1-aminocyclopropane-1-carboxylate oxidase(ACO).The ACS and ACO have been found that are two key en-zymes of ethylene biosynthesis pathway.Brassica napus L.is the main source of oil,but the weeds such as barnyardgrass impact on the yield and quality seriously.We studied ACS and ACO gene from resistant and susceptible to quinclorac of Echinochloa crus-galli(EcACS-R and EcACS-S,EcACO-R and EcACO-S),including the gene cloning,bioinfor-matics analysis,expression analysis,expression in prokeryotic and the enzymatic activity analysis.The main results were follows:1)1-aminocyclopropane-l-carboxylate synthase gene(ACS):The sequence analy-sis result showed that the EcACS have two different variants:the resistant was named EcACS-R;the susceptible was named EcAS-S.? The EcACS-R is 1630 bp in length with 1452 bp in the encoding region,and the EcACS-R encoding a protein of 483 amino-acids,pI 53.59,Mw 53 kD.The EcACS-S is 1670 bp in length with 1470 bp in the encoding re-gion,and the EcACS-S encoding a protein of 489 amino-acids,pI 54.02,Mw 54 kD.The phylogenetic tree analysis of EcACS genes found that the EcACS has high homology with the ACS genes of the monocot plants,Setaria italic and Zea mays et al.There are four non-synonymous nucleotide substitutions between EcACS-R and EcACS-S.The four non-synonymous SNPs' resulting amino acid substitutions were V230?G,D295?A,A370?V,K442?R,respectively.? The expression of EcACS-R and EcACS-S of barnyard grass was analyzed by qRT-PCR.?-Actin was used as the reference gene.The result showed that EcACS-R and EcACS-S have no significant difference.? Using the prokaryotic expression vector with MBP label pMAL-c5x,construct the expression vector MBP::EcACS,respec-tively,and conversion to E.coli BL21 and expression under the induction with 0.4 mmol·L-1IPTG 16 h.The 12%SDS-PAGE analysis show that the fusion protein expression stripe size is the same as the theoretical value.2)1-aminocyclopropane-l-carboxylate oxidase gene(ACO):The sequence analysis result showed that the EcACO have two different variants:the resistant was named EcACO-R;the susceptible was named EcACO-S.? The EcACO is 1000 bp in length with 936 bp in the encoding region.And the EcACO encoding a protein of 311 amino-acids,pI 5.4;Mw 35 kD.The phylogenetic tree analysis of EcACO genes found that the EcACO has high homology with the ACO genes of the monocot plants,Setaria italic,Zea mays and Sorghum bicolor et al.There are five non-synonymous nucleotide substitutions between EcACO-R and EcACO-S.The four non-synonymous SNPs' resulting amino acid substitu-tions were F98?Y,G101?D,Q206?R,G270?V,A272?G,respectively.? The expres-sion of EcACO-R and EcACO-S of barnyard grass was analyzed by real-time quantitative PCR.?-Actin was used as the reference gene.The result showed that EcACO-R and EcACO-S have no significant difference.? The fusion expression vector of MBP::EcACO was successfully constructed,and conversion to E.coli BL21 and expression under the in-duction with 0.4 mmol·L-1IPTG 16 h.The 12%SDS-PAGE analysis show that the fusion protein expression stripe size is the same as the theoretical value.? The supernatant was collected,and the soluble fusion protein was purified and assayed of Ethylene Production,the study showed that theamount of 5 ?g MBP::EcACO-S released ethylene was 2.15 times higher than the amount of 5 ?g MBP::EcACO-R release.The MBP::EcACO-S activity was higher than MBP::EcACO-R.