Cloning Of Arabidopsis Hydroxynitrile Lyase Gene Promoter And Its Inducible Expression Patterns Analysis Treated With Hormone And Abiotic Stress

Hydroxynitrile lyases(HNL,E.C.4.1.2.x)are the enzymes,which catalyze enantioselective cleavage and synthesis of cyanohydrins.They are the versatile group of enzymes,which are also proven that they might play a significant defensive role in plant system against microbial attack.In chemical industries,hydroxynitrile lyase is used as an important industrial biocatalyst for the synthesis of' chiral cyanohydrins by exploiting the reversible enzymatic reaction.The currently known HNLs can be divided into two groups:R-selective enzymes evolved from oxidoreductase ancestors,such as HNLs from various Rosaceae and from Linum usitatissimum,and S-selective enzymes derived from hydrolases with an ?/?-hydrolase fold;these encompassing the enzymes from Hevea brasiliensis(HbHNL),Manihot esculenta(MeHNL),and Sorghum bicolor(SbHNL).In nature,HNLs catalyze the cleavage of cyanohydrins,known as cyanogenesis.The phenomenon of cyanogenesis is widely observed in higher plants.It has been reported in more than 4000 species of vascular plant taxa comprising 105 families of flowering plants,pteridophytes(ferns)and gymnosperms.Biological activity of HCN(by-product of HNL)was mostly correlated with the in hibition of the terminal cytochrome oxidase in the mitochondrial respiratory pathway,and some experimental data reported a protective effect of cyanide in plants against predators such as herbivores.Some data also indicate that HCN,apart from being toxic,plays a regulatory(maybe signaling)function in many physiological processes,e.g.seed germination,nitrate as similation or in plant responses to some environmental stimuli.Recently,an ?/?-hydrolase fold from the noncyanogenic plant Arabidopsis thaliana has been identified which present the first exception to this accepted rule with the first R-selective HNL.GeneChIP data analysis also showed that the expression of Arabidopsis HNL gene is significantly induced by the treatment of drought,cold and NaCl stress.But its regulatory roles are also unknow.In this study,to investigate the previously unclearly characterization of the AtHNL promoter,an approximately 2.1 kb of HNL promoter(pHY1,-2023 to +67)and its deletion fragments(5D1,-933 to +67;5D2,-733 to +67;5D3,-533 to +67;-333 to+67,5D5,-133 to +67,5D6,-30 to +67),were fused to ?-glucuronidase(GUS)reporter gene and stably transformed into Arabidopsis.The transgenic plants were then subjected to several environmental stimuli(especially to salinity,drought,and freezing stress)and plant hormone treatment.The results showed that the GUS activites of pHNL overexpressiong transgenic plants could be induced by NaCl and freezing stress,and methyl jasmonate(MeJA).This might be correlated with the stress induced cis-element appeared in the sequence.Additionally,AtHNL promoter is also differently inhibited by drought,abscisic acid(ABA),gibberellin(GA)and salicylic acid(SA)treatment,which might be correlated with the CGTCA-motif,TATC-box,GARE-motif,and TCA-element appeared in the sequence.Additionally,by analyzing the GUS staining and activities of different AtHNL promoter fragment overexpressing Arabidopsis,the possible important cis-element appeared in 5D1,5D2,5D3,5D4 and 5D5 were compared with pHNL.It showed that the possibility of negative regulatory elements located between-2023 to-933,and-533 to-333 could not be easily ruled out.Moreover,combined with the treatment with plant hormone and by the different stress conditions,the expression patterens of different fragment of AtHNL promoter were also analyzed.It showed that similar expression patterens were obsereved in some kind of treatment(such as GA and ABA).This implied that there is a complicated regulation pathway of AtHNL promoter under different treatment.In summary,all above results might provide some important theory basis for exploring the inducible expression pattern and new cis-element of AtHNL promoter.