Identification Of The Aldehyde Dehydrogenase (ALDH) Gene Family From Silkworm And The Roles Of BmALDH1A2

Aldehyde dehydrogenase（ALDH）superfamily is a group of multifunctional enzyme proteins whose activity depends on NAD（P）⁺.They catalyze the irreversible oxidation reaction of endogenous and exogenous aldehydes to corresponding acids to protect the body of animals from the toxicity of aldehydes.Retinal dehydrogenase（RALDH）is one of the key enzymes in the carotenoid metabolic pathway that catalyze the irreversible oxidation of retinal to retinic acid,which is very important for the normal growth,differentiation and development of vertebrates and the maintenance of adult epithelial tissues.The function of insect ALDH gene family has only been reported in Drosophila melanogaster,especially its involvement in retinoic acid signaling pathway.To verify the function of ALDH gene family in carotenoid metabolism pathway of silkworm,we first analyzed,identified and classified ALDH homologous genes in silkworm genome,and then selected RALDH homologous genes（BmALDH1A1,BmALDH1A2,BmALDH1A3 and BmALDH1A4）for expression pattern detection,and further constructed expression vectors.The active enzyme proteins were obtained by inducing expression and protein purification.The enzymatic activity of BmALDH1A2 protein was detected,and the key amino acid sites affecting the enzymatic activity were determined by site-directed mutagenesis.Finally,the subcellular localization of BmALDH1A2 and BmALDH1A4 proteins was examined at the cellular level.The main research results are as follows:1.Identification of ALDH homologous genes in silkwormA total of 12 ALDH homologous genes were identified in silkworm genome by bioinformatics analysis,which were predicted to encode 18 proteins,all of which contain the Aldedh domain unique to the aldehyde dehydrogenase protein family.Among them,there are four genes homologous to human retinal dehydrogenase RALDH,named as BmALDH1A1,BmALDH1A2,BmALDH1A3 and BmALDH1A4,which contain 11 exons.The four RALDH homologous proteins shared more than 55%sequence identity,and their secondary and tertiary structures were also very similar.In the subsequent cloning sequencing,eight different m RNA splicing forms of BmALDH1A1 gene were identified,and eight different proteins were presumed to be encoded.2.Spatial and temporal expression patterns of RALDH homologous genes（BmALDH1A1,BmALDH1A2 and BmALDH1A4）in silkwormThe spatial and temporal expression patterns of RALDH homologous genes in the silkworm Dazao strain breed were examined by RT-PCR.The results showed that the expression level of BmALDH1A1 was lower than that of BmALDH1A2 and BmALDH1A4 during the whole developmental stage of silkworm.The expression levels of BmALDH1A1,BmALDH1A2 and BmALDH1A4 increased first and then decreased during the embryonic stage.The expression levels of BmALDH1A1 and BmALDH1A2 in sleeping silkworms were lower than those in corresponding rearing silkworms.The expression of BmALDH1A2 in female pupae and moths was generally higher than that in male pupae and moths,while there was no significant difference in the expression of BmALDH1A4 between male and female.The expression of BmALDH1A1,BmALDH1A2 and BmALDH1A4 was different in different tissues of the third-day larvae of the fifth instar of silkworm.BmALDH1A1 was highly expressed in the integument and spermary,BmALDH1A2 was the highest expressed in the midgut,and BmALDH1A4 was the highest expressed in the spermary.In conclusion,the expression of RALDH homologous gene in silkworm has tissue and time specificity.3.Subcellular immunofluorescence localization of BmALDH1A2 and BmALDH1A4 proteinsThe localization of BmALDH1A2 and BmALDH1A4 proteins in silkworm ovarian cells was studied by using the prepared polyclonal antibody.The results showed that BmALDH1A2 proteins were located in the mitochondria,while BmALDH1A4 proteins were located in the cytoplasm.4.Enzyme activity of BmALDH1A2 proteinAccording to the enzyme activity experiment,the enzymatic reaction time of BmALDH1A2 protein in vitro was determined to be 30 min,the reaction temperature was 30℃,the optimal pH was 7.5,and the protein content in the system was determined to be 0.125 mg.The reaction product of BmALDH1A2 protein was detected by HPLC after enzymatic reaction in vitro.The results showed that 0.125 mg of BmALDH1A2 protein could completely convert all-trans retinal（atRAL）of 10μM to all-trans retinic acid（atRA）in the presence of cofactor NAD⁺.5.Site-specific mutation of BmALDH1A2 gene and enzyme activity of mutant BmALDH1A2 proteinIn order to study the conserved amino acid sequence of BmALDH1A2 protein,three amino acid residues Glu182,Glu255 and Cys289 important in catalysis and coenzyme binding were mutated by site-directed mutagenesis,and the mutant BmALDH1A2 expression vectors were constructed and transformed into BL21 strain for induced expression and protein purification.Three mutant BmALDH1A2 proteins were successfully obtained,which were named as BmALDH1A2^E182S,BmALDH1A2^E255A and BmALDH1A2^C289S.The enzyme activity of the mutant protein BmALDH1A2^E182S was basically the same as that of wild-type protein BmALDH1A2.However,the mutant proteins BmALDH1A2^E255A and BmALDH1A2^C289S completely lost enzymatic activity.The reaction product of mutant BmALDH1A2 protein was detected by HPLC after enzymatic reaction in vitro.The results again showed that the mutant proteins BmALDH1A2^E255A and BmALDH1A2^C289S were completely devoid of function,while the mutant protein BmALDH1A2^E182S was as functional as the wild-type protein BmALDH1A2,which could convert atRAL to atRA in the presence of cofactor NAD⁺.