Identification of a novel Drosophila gene, beltless, using injectable embryonic and adult RNA interference (RNAi).
Identification of a novel Drosophila gene, beltless, using injectable embryonic and adult RNA interference (RNAi).
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DOI：
10.1186/1471-2164-4-33
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发表时间：
2003-08-12
期刊：
影响因子：
4.4
通讯作者：
Manev H
中科院分区：
文献类型：
--
作者：
Dzitoyeva S;Dimitrijevic N;Manev H
RNA干扰(RNAi)是一种由双链RNA触发的过程，可导致基因表达的靶向下调/沉默，可用于功能基因组学，即功能丧失研究。在这里，我们报道了使用RNAi来鉴定一个新的发育重要的果蝇(果蝇)基因(对应于一个假定的基因CG5652/GM06434)，我们基于胚胎功能丧失的表型将其命名为Belless。除0-6h胚胎外，无带基因在所有发育阶段均有表达。原位RT-PCR定位于晚期胚胎的腹侧脊髓和脑，以及成虫的神经系统、卵巢和附腺中的无带mRNA。RNAi是通过将短的(22bp)无带双链RNA注射到胚胎或成年果蝇体内而诱导的。胚胎RNAi改变了腹部A2-A4段从部分形成到缺失牙带(因此没有牙带)的表皮表型。胚胎无带RNAi是致命的。成年RNAi导致卵巢萎缩一半，产卵量减少。我们还研究了DF(1)RK4果蝇，在这些果蝇中，缺失会移除16个基因，包括无带基因。在一些胚胎中，我们观察到了与无带RNAi相似的角质层异常。在将DF(1)RK4胚胎区分为可见的牙带和缺失的牙带后，我们检测了无牙带的mRNA的存在；在缺失牙带的胚胎中没有检测到无带的mRNA。我们已经确定了一个在发育上具有重要意义的新的果蝇基因，无带，它已经在使用RNA干扰进行的功能丧失研究中得到了表征。推测的无带蛋白与线虫抗氟西汀(NRF)NRF-6基因以及一些未鉴定的线虫和果蝇的NRF-6基因有同源性，其中一些基因具有显著的酰基转移酶结构域。未来的研究应该阐明无带在果蝇发育和成虫中的作用和机制，包括在成虫神经系统中的作用。
RNA interference (RNAi) is a process triggered by a double-stranded RNA that leads to targeted down-regulation/silencing of gene expression and can be used for functional genomics; i.e. loss-of-function studies. Here we report on the use of RNAi in the identification of a developmentally important novel Drosophila (fruit fly) gene (corresponding to a putative gene CG5652/GM06434), that we named beltless based on an embryonic loss-of-function phenotype. Beltless mRNA is expressed in all developmental stages except in 0–6 h embryos. In situ RT-PCR localized beltless mRNA in the ventral cord and brain of late stage embryos and in the nervous system, ovaries, and the accessory glands of adult flies. RNAi was induced by injection of short (22 bp) beltless double-stranded RNAs into embryos or into adult flies. Embryonic RNAi altered cuticular phenotypes ranging from partially-formed to missing denticle belts (thus beltless) of the abdominal segments A2–A4. Embryonic beltless RNAi was lethal. Adult RNAi resulted in the shrinkage of the ovaries by half and reduced the number of eggs laid. We also examined Df(1)RK4 flies in which deletion removes 16 genes, including beltless. In some embryos, we observed cuticular abnormalities similar to our findings with beltless RNAi. After differentiating Df(1)RK4 embryos into those with visible denticle belts and those missing denticle belts, we assayed the presence of beltless mRNA; no beltless mRNA was detectable in embryos with missing denticle belts. We have identified a developmentally important novel Drosophila gene, beltless, which has been characterized in loss-of-function studies using RNA interference. The putative beltless protein shares homologies with the C. elegans nose resistant to fluoxetine (NRF) NRF-6 gene, as well as with several uncharacterized C. elegans and Drosophila melanogaster genes, some with prominent acyltransferase domains. Future studies should elucidate the role and mechanism of action of beltless during Drosophila development and in adults, including in the adult nervous system.
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