本文選題：家蠶 + 微小RNA��； 參考：《蠶業(yè)科學(xué)》2017年06期

【摘要】：對家蠶中部絲腺小RNA高通量測序獲得若干新miRNA,生物信息學(xué)分析發(fā)現(xiàn)Bmo-miR-0047可能對家蠶絲膠蛋白基因Bm Ser-1有調(diào)控作用。設(shè)計特異性莖環(huán)引物,利用半定量RT-PCR檢測分析家蠶5齡3 d幼蟲絲腺(包括前、中、后部絲腺)、頭部、脂肪體、表皮、中腸、馬氏管、精巢/卵巢、血淋巴細(xì)胞和氣管等12個器官組織中Bmo-miR-0047的表達(dá)情況。結(jié)果顯示,中部絲腺中Bmo-miR-0047的相對表達(dá)量明顯高于其他組織,說明在家蠶5齡幼蟲期Bmo-miR-0047對Bm Ser-1基因的表達(dá)調(diào)控存在時空特異性。為了進(jìn)一步分析Bmo-miR-0047對Bm Ser-1基因表達(dá)的調(diào)控作用,構(gòu)建重組表達(dá)載體pc DNA3.0(ie1-egfp-pri-miR-0047-SV40)和p GL3(A3-luc-Bm Ser-1 3'UTR-SV40),并以海腎熒光素酶報告質(zhì)粒pRL-CMV為內(nèi)參,利用家蠶卵巢培養(yǎng)細(xì)胞Bm N進(jìn)行共轉(zhuǎn)染。轉(zhuǎn)染后檢測發(fā)現(xiàn)實(shí)驗組Bm N細(xì)胞中的熒光素酶活性與對照組相比顯著減弱,表明Bmo-miR-0047在體外培養(yǎng)細(xì)胞中對Bm Ser-1基因的表達(dá)具有負(fù)調(diào)控作用。
[Abstract]:A number of new miRNAs were obtained by high-throughput sequencing of the small silkgland RNA in the central silkworm. Bioinformatics analysis showed that Bmo-miR-0047 might regulate the silkworm fibroin gene BM Ser-1. Specific stem ring primers were designed and semi-quantitative RT-PCR was used to detect and analyze the silk glands (including anterior, middle and posterior silk glands, head, fat body, epidermis, midgut, Markov duct, spermary / ovary) of the 5th instar larvae of Bombyx mori (Bombyx mori). Expression of Bmo-miR-0047 in 12 organs such as blood lymphocytes and trachea. The results showed that the relative expression of Bmo-miR-0047 in the central silk gland was significantly higher than that in other tissues, indicating that the regulation of Bm Ser-1 gene expression by Bmo-miR-0047 in the 5th instar larvae of Bombyx mori was space-time specific. In order to further analyze the regulatory effect of Bmo-miR-0047 on the expression of BM Ser-1 gene, the recombinant expression vector pc DNA3.0 Ie1-egfp-pri-miR-0047-SV40) and p GL3(A3-luc-Bm Ser-1 3 UTR-SV40G were constructed. The eukaryotic expression vector pRL-CMV was used as the internal reference, and the BMN was cotransfected with silkworm ovary culture cells. After transfection, the luciferase activity of BmN cells in the experimental group was significantly decreased compared with that of the control group, indicating that Bmo-miR-0047 had a negative regulatory effect on the expression of Bm Ser-1 gene in cultured cells in vitro.
【作者單位】： 江蘇科技大學(xué)生物技術(shù)學(xué)院江蘇省蠶桑生物學(xué)與生物技術(shù)重點(diǎn)實(shí)驗室;中國農(nóng)業(yè)科學(xué)院蠶業(yè)研究所農(nóng)業(yè)部蠶桑遺傳改良重點(diǎn)開放實(shí)驗室;
【基金】：國家自然科學(xué)基金項目(No.31402143,31372376) 江蘇省自然科學(xué)基金項目(No.BK20141285) 江蘇科技大學(xué)博士科研啟動項目(No.1732931702)
【分類號】：S881.26

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