Abstract: Objective To construct a double mutants of 5-formyltetrahydrofolate cyclo-ligase(Y106G6E.4),methionine synthase(R03D7.1),and 5,10-methenyl-tetrahydrofolate synthetase(Dao-3) and G85R::YFP and to detect motor ability of amyotrophic lateral sclerosis(ALS) Caenorhabditis elegans(C.elegans) after folate metabolism-related gene deletion.Methods C.elegans was cultured in nematode growth medium(NGM) agar plates.Hermaphrodites of G85R::YFP strain were hybridized with N2 males to obtain the G85R mutant males; then G85R mutant males were backcrossed to VC2322(Y106G6E.4),RB755(R03D7.1),and VC1197(Dao-3),respectively.Y106G6E.4,R03D7.1 and Dao-3 homozygous genotypes of the progenies were identified with single worm PCR.Then the hermaphrodites with the homozygous genotypes were self-fertilized.Superoxide dismutase 1(Sod1) gene was identified with fluorescence intensity.Crawling distance on the plates and thrashing times in liquid media of the C.elegans with all double mutants were observed.Results Double mutants were successfully constructed.PCR and fluorescene test showed that the mutant worms had the corresponding homozygous genotypes.Compared with ALS C.elegans,both thrash times in liquid media(14.6±2.39,17.4±2.23,and 10.2±1.64) and crawling distance on the plates(22.25±3.59,23.25±3.30,and 21.5±5.07) were decreased in G85R/VC2322,G85R/RB755 and G85R/VC1197 double mutants.The differences were statistically significant(P<0.05).Conclusion Double mutants could be obtained by hydridization in C.elegans.Folate-related metabolic changes might cause damage to motor ability of ALS worms,suggesting that folate metabolism may play an important role during the initiation and progression of ALS.