Cloning And Functional Characterization Of AnFALDH And CcFALDH Gene In Formaldehyde Metabolism

Formaldehyde is an important chemical raw material, which is widely used invarious industrial products closely related to the human life. It was officially classifiedas human carcinogens in2004. Currently, there are few studies about the formaldehydemetabolism in organisms which can absorb and degrade formaldehyde efficiently. Inthe present study, we chose two organisms with efficiently ability of absorbing anddegrading formaldehyde, and cloned faldh gene which is a key enzyme in themetabolism of formaldehyde. The summary of the main results are as follows:1. Expression analysis of Aspergillus nomius SGFA1under formaldehydetreatment. qRT-PCR was used to analyze the expression characteristics of genes fromdifferent formaldehyde metabolism pathways under formaldehyde treatment. Theresults showed that aldh (Aldehyde dehydrogenases), faldh (formaldehydedehydrogenases), fgh (S-formylglutathione hydrolase), fdh (Formate dehydrogenase)and yap1-like gene were induced by formaldehyde. Aldehyde dehydrogenases andyap1-like were stress responsive genes, and were not involved in the formaldehydemetabolism directly. Formaldehyde dehydrogenases, S-formylglutathione hydrolaseand formate dehydrogenase are the key enzyme involved in formaldehyde oxidationmetabolic pathways in Aspergillus nomius SGFA1.2. Cloning and sequence analysis of AnFALDH. AnFALDH gene was cloned byPCR, which consisted of1146nucleotide and encoded a polypeptide of381amino acids.AnFALDH belongs to the MDR (medium-chain dehydrogenase/reductase) family,which contains an ADH_N domain and zinc binding protein domain with an estimatedmolecular mass of40.4kDa and a theoretical isoelectric point (pI) of7.07.3. AnFALDH improves yeast resistance to formaldehyde. The result of comparativeanalysis of physiological function between wild type and transformed yeast show thatAnFALDH gene could improve yeast resistance to formaldehyde, and the ability offormaldehyde degradation of yeast increased to about22.3%.4. Cloning and sequence analysis of CcFALDH. The CcFALDH gene consisted of1140nucleotide and encoded a polypeptide of379amino acids. CcFALDH belongs tothe MDR family with an estimated molecular mass of40.6kDa and a theoreticalisoelectric point (pI) of6.51. 5. Overexpression of CcFALDH gene and Molecular analysis of transgenic tobacco.To construct the plant expression vector pBI-35S-CcFALDH, the plant expressionvector pBI-35S-CcFALDH was transferred into Agrobacterium tumefaciens EHA105by electroporation using a Gene Pulser (BioRad).Tobacco (Nicotiana tabacum cv. SR-1) was transformed according to the leaf disk co-cultivation protocol of Horsch et al.We obtained a total of36kanamycin resistant lines, and14PCR positive lines. Thesetransgenic lines were selected for Southern blot and Northern blot. The results ofNorthern blot indicate that FALDH gene expressed in TR1, TR3, TR7, TR9and TR15transgenic tobacco lines.6. Functional analysis of transgenic tobacco with CcFALDH. Wild type tobaccogermination rate was only half of the transgenic tobacco in the medium containing150mg l-1formaldehyde, however, there were no different of germination in a controlmedium. In the medium containing formaldehyde, transgenic tobacco lines could grownormally, but wild type tobacco growth was inhibited. The total chlorophyll content ofwild type tobacco only reached half of transgenic tobacco after formaldehyde treatment.The FALDH specific enzyme activities in the extracts of WT, TR7and TR15lines were0.033,0.104and0.108U mg-1protein, respectively. There are3.3-fold higher FALDH-specific activities in the transgenic tobacco lines in comparison to WT. The uptake rateof the transgenic plants for aqueous formadlehyde was examined. The transgenic plantsshowed2.18and1.89-fold higher uptake rate than the WT plants. The uptake rate ofthe transgenic plants for gaseous formaldehyde was examined. TR7and TR15werewith2.18and1.8-fold higher ability to absorb formaldehyde.