nicastrin基因沉默的斑马鱼模型构建及其色素异常机制的初步研究

目的:探讨nicastrin（nct）基因对斑马鱼黑素细胞生物学功能的影响。方法:利用吗啉代寡核苷酸（MO）技术针对斑马鱼nct基因mRNA设计nct-MO序列，同时设计对照MO序列（ctrl-MO），并构建5′端为MO靶序列的增强绿色荧光蛋白（EGFP）mRNA，通过显微注射的方式将nct-MO或ctrl-MO与EG...

Construction of a nicastrin gene-silenced zebrafish model and a primary study on the mechanism of abnormal pigmentation

【Abstract】 Objective To evaluate the effect of nicastrin （nct） gene on the biological functions of melanocytes in zebrafish. Methods By using a morpholino oligonucleotide （MO） technology, a nct-MO sequence targeting the zebrafish nct mRNA was designed, so was a MO control （ctrl-MO） sequence. Then, the enhanced green fluorescent protein （EGFP） mRNA with MO target sequence at its 5′ end was synthesized, and co-microinjected with the nct-MO or ctrl-MO sequence into the zebrafish embryos to verify the silencing efficiency of nct-MO and observe changes in developmental phenotypes in zebrafish. With wild-type zebrafish as a blank control group, real-time fluorescence-based quantitative PCR （RT-PCR） was conducted to determine the mRNA expression of melanin synthesis- and notch signaling pathway-related genes, including mitfa, tyr, tyrp1a, tyrp1b, dct, pmela, notch1a, notch1b and hey1 genes. One-way analysis of variance was used for the comparison of means among multiple groups, and least significant difference（LSD）-t test for multiple comparisons. Results Eight hours after zebrafish fertilization, green fluorescence was observed in the zebrafish embryos in the ctrl-MO + EGFP mRNA group, but not in the nct-MO + EGFP mRNA group or blank control group. Forty-eight hours after fertilization, the proportion of pigmented area among the whole area of the tail of zebrafish larvae was significantly lower in the nct-MO group （0.169 ± 0.083） than in the ctrl-MO group （0.258 ± 0.042, t = 3.202, P = 0.005）, and disorderly pigment distribution in the tails was observed in the nct-MO group. RT-PCR revealed significant differences in the mRNA expression of pmela, tyrp1a and hey1 genes among the nct-MO group, ctrl-MO group and blank control group （all P < 0.05）, but no significant difference was observed in the mRNA expression of mitfa, tyr, tyrp1b, dct, notch1a or notch1b genes among the 3 groups （all P＞0.05）; the relative expression levels of pmela and tyrp1a mRNAs were significantly lower in the nct-MO group （0.708 ± 0.028, 0.558 ± 0.136, respectively） than in the ctrl-MO group （1.023 ± 0.142, 1.016 ± 0.134, respectively, both P < 0.05）. Conclusion The nct gene may affect biological functions of melanocytes by regulating melanin synthesis in zebrafish.