叶酸代谢基因缺失对ALS线虫运动能力影响

Effects of folate metabolism-related gene deletion on motor ability in ALS C. elegans

目的了解5-甲酰四氢叶酸环连接酶(Y106G6E.4)、蛋氨酸合成酶(R03D7.1)和四氢叶酸脱氢酶(Dao-3)基因突变对肌萎缩侧索硬化症(ALS)秀丽隐杆线虫运动能力的影响。方法通过与N2株系杂交获得单拷贝G85R:YFP雄虫,分别与VC2322(Y106G6E.4)、RB755(R03D7.1)和VC1197(Dao-3)突变株系回交,单线虫PCR法鉴定后代三突变基因纯合性,荧光鉴定超氧化物歧化酶Sod1(G85R)基因纯合的双突变体,光镜下观察双突变体在NGM平板上的爬行距离及在M9培养液中的摆动次数。结果成功构建了G85R/VC2322、G85R/RB755和G85R/VC1197三个双突变体,经PCR和荧光鉴定表明其相应的基因型均为纯合型;与ALS模型线虫比较,双突变体线虫G85R/VC2322、G85R/RB755和G85R/VC1197每分钟摆动次数[(14.6±2.39)、(17.4±2.23)、(10.2±1.64)]和相对爬行距离[(22.25±3.59)、(23.25±3.30)、(21.5±2.07)]均减小,且差异均有统计学意义(均P<0.05)。结论体内叶酸代谢相关基因改变将进一步损伤ALS线虫的运动能力,为深入探讨叶酸代谢途径在ALS发生发展中的作用奠定基础。

Objective To construct a double mutants of 5-formyltetrahydrofolate cyclo-ligase （ Y106G6E. 4 ）, methi-nine synthase（ R03DT. 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 （ R03DT. 1 ）, and VC1197 （ Dao-3 ）, respectively. Y106G6E. 4, R03DT. 1 and Dao-3 homozygous genotypes of the progenies were identified with single worm PCR. Then the hermaphrodites withthe homozygous genotypes were self-fertilized. Superoxide dismutase 1 （ Sodl ） 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 sta- tistically significant（ P 〈 0. 05）. Conclusion Double mutants could be obtained by hydridization in C. elegans. Folaterelated 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.