發(fā)布時間：2018-06-06 02:25

  本文選題：家蠶 + 微小RNA��； 參考：《蠶業(yè)科學》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有調控作用。設計特異性莖環(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、血淋巴細胞和氣管等12個器官組織中Bmo-miR-0047的表達情況。結果顯示,中部絲腺中Bmo-miR-0047的相對表達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基因的表達調控存在時空特異性。為了進一步分析Bmo-miR-0047對Bm Ser-1基因表達的調控作用,構建重組表達載體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粒pRL-CMV為內參,利用家蠶卵巢培養(yǎng)細胞Bm N進行共轉染。轉染后檢測發(fā)現(xiàn)實驗組Bm N細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)細胞中對Bm Ser-1基因的表達具有負調控作用。
[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】： 江蘇科技大學生物技術學院江蘇省蠶桑生物學與生物技術重點實驗室;中國農業(yè)科學院蠶業(yè)研究所農業(yè)部蠶桑遺傳改良重點開放實驗室;
【基金】：國家自然科學基金項目(No.31402143,31372376) 江蘇省自然科學基金項目(No.BK20141285) 江蘇科技大學博士科研啟動項目(No.1732931702)
【分類號】：S881.26

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