【摘要】：miRNA是一類由內(nèi)源基因編碼的長(zhǎng)度約為21-24 nt的非編碼單鏈RNA分子,廣泛存在于真核生物中。研究表明,miRNA在基因的轉(zhuǎn)錄后調(diào)控中起著非常重要的作用,參與調(diào)控胚胎發(fā)育、細(xì)胞分化、增殖、神經(jīng)發(fā)生、代謝及細(xì)胞凋亡等幾乎所有的生物過程,動(dòng)物中約有50%以上編碼蛋白基因的表達(dá)受到mi RNA的調(diào)控。家蠶是鱗翅目昆蟲的重要代表,家蠶絲膠基因BmSer-1、BmSer-2是中部絲腺特異表達(dá)的基因,也是蠶絲蛋白的主要基因。研究顯示蠶絲蛋白基因的表達(dá)受到miRNA的調(diào)控,但未見miRNA對(duì)絲膠基因表達(dá)調(diào)控研究的報(bào)道。為了研究家蠶miRNA對(duì)絲膠基因表達(dá)的調(diào)控作用,我們利用Solexa技術(shù)對(duì)家蠶P50和裸蛹(Nd)5齡幼蟲中部絲腺組織RNA進(jìn)行了高通量測(cè)序,鑒定獲得若干保守miRNA和新mi RNA;通過生物信息學(xué)分析,預(yù)測(cè)到bmo-miR-275等可能參與BmSer-1、Bm Ser-2的轉(zhuǎn)錄后調(diào)控,并分別構(gòu)建miRNA和靶基因3′UTR重組表達(dá)載體,在細(xì)胞水平進(jìn)行了驗(yàn)證,取得如下主要研究結(jié)果。1.家蠶P50與裸蛹(Nd)突變品種5齡幼蟲中部絲腺miRNAs的鑒定利用Solexa技術(shù)對(duì)家蠶P50與裸蛹(Nd)突變品種5齡3d幼蟲中部絲腺進(jìn)行了高通量測(cè)序,檢測(cè)到272個(gè)保守miRNAs,并預(yù)測(cè)到333個(gè)新的miRNAs;其中86個(gè)miRNAs在P50與Nd中的表達(dá)存在顯著差異。2.新預(yù)測(cè)家蠶miRNAs的qRT-PCR鑒定為了驗(yàn)證預(yù)測(cè)結(jié)果的可信性,對(duì)其中10個(gè)新預(yù)測(cè)mi RNA進(jìn)行qRT-PCR鑒定,結(jié)果與測(cè)序結(jié)果相符;利用軟件進(jìn)行靶基因預(yù)測(cè),保守miRNA和新預(yù)測(cè)miRNA分別預(yù)測(cè)到2522和1376個(gè)靶基因。3.家蠶絲膠基因BmSer-1、BmSer-2及相關(guān)mi RNA表達(dá)特性分析通過靶基因預(yù)測(cè),發(fā)現(xiàn)bmo-miR-2771和bmo-miR-275分別是BmSer-1、BmSer-2的潛在靶基因,利用半定量PCR方法分析它們的表達(dá)特性,發(fā)現(xiàn)bmo-miR-2771只在中部絲腺中表達(dá)。4.Bmo-miR-2771對(duì)家蠶絲膠蛋白基因BmSer-1表達(dá)的調(diào)控作用構(gòu)建表達(dá)bmo-miR-2771的重組質(zhì)粒pcDNA3.0(ie1-egfp-pri-mir-2771-SV40)和表達(dá)BmSer-1 3′UTR的重組質(zhì)粒pGL3(A3-luc-BmSer-1-3′UTR-SV40),共轉(zhuǎn)染家蠶BmN細(xì)胞,結(jié)果顯示bmo-miR-2771對(duì)BmSer-1基因表達(dá)具有負(fù)調(diào)控作用。5.Bmo-miR-275對(duì)家蠶絲膠蛋白基因BmSer-2表達(dá)的調(diào)控作用構(gòu)建表達(dá)bmo-miR-275的重組質(zhì)粒pcDNA3.0(ie1-egfp-pri-mir-275-SV40)和表達(dá)BmSer-2 3′UTR的重組質(zhì)粒pGL3(A3-luc-BmSer-2-3′UTR-SV40),共轉(zhuǎn)染家蠶BmN細(xì)胞,結(jié)果顯示實(shí)驗(yàn)組熒光素酶活性降低,再轉(zhuǎn)染人工合成bmo-mi R-275inhibitor后活性略有回升,證實(shí)bmo-miR-275對(duì)BmSer-2基因表達(dá)具有負(fù)調(diào)控作用。上述研究結(jié)果為闡明miRNA對(duì)蠶絲蛋白基因表達(dá)的調(diào)控機(jī)制和miRNA的功能提供了新的實(shí)驗(yàn)數(shù)據(jù)。
[Abstract]:MiRNA is a class of non-coding single-stranded RNA molecules encoded by endogenous genes with a length of about 21-24 nt, which is widely present in eukaryotes. Studies have shown that miRNA plays a very important role in the posttranscriptional regulation of genes, and is involved in almost all biological processes, such as embryonic development, cell differentiation, proliferation, neurogenesis, metabolism and apoptosis. About 50% of the encoded protein genes in animals are regulated by mi RNA. Bombyx mori (Bombyx mori) is an important representative of Lepidoptera insects. The silkworm gum gene BmSer-1,BmSer-2 is a gene specifically expressed in the middle silk gland and is also the main gene of silk protein. The study showed that the expression of silk protein gene was regulated by miRNA, but there was no report on the regulation of sericin gene expression by miRNA. In order to study the regulatory effect of silkworm miRNA on sericin gene expression, high throughput sequencing of RNA in the middle silk gland of silkworm P50 and bare pupae (Nd) 5 instar larvae was carried out by using Solexa technique. Some conservative miRNA and new mi RNA; were obtained. By bioinformatics analysis, it was predicted that bmo-miR-275 might be involved in the post-transcriptional regulation of BmSer-1,Bm Ser-2, and the recombinant expression vectors of miRNA and target gene 3'UTR were constructed, which were verified at the cell level. The main results are as follows. 1. Identification of miRNAs in the central silk gland of the 5th instar larvae of silkworm P50 and naked pupae (Nd) mutants the high throughput sequencing of the central silk glands of the 5th instar larvae of silkworm P50 and bare pupae (Nd) mutants was carried out using Solexa technique. 272 conserved miRNAs, were detected. And predicted 333 new miRNAs; There were significant differences in the expression of 86 miRNAs between P50 and Nd. 2. 2. QRT-PCR Identification of newly predicted Silkworm miRNAs in order to verify the credibility of the predicted results, 10 new predicted mi RNA were identified by qRT-PCR, and the results were consistent with the sequencing results. Using software to predict target genes, conservative miRNA and new predictive miRNA were used to predict 2522 and 1376 target genes, respectively. Analysis of BmSer-1,BmSer-2 and related mi RNA expression characteristics of Silk Gum Gene in Silkworm, bmo-miR-2771 and bmo-miR-275 are potential target genes of BmSer-1,BmSer-2 by prediction of target genes. Their expression characteristics were analyzed by semi-quantitative PCR. It was found that bmo-miR-2771 was expressed only in the central silk gland. The regulation of 4.Bmo-miR-2771 on the BmSer-1 expression of silk glue protein gene was used to construct the recombinant plasmid pcDNA3.0 (ie1-egfp-pri-mir-2771) expressing bmo-miR-2771. -SV40) and the recombinant plasmid pGL3 (A3-luc-BmSer-1-3'UTR-SV40) expressing BmSer-1 3'UTR, Cotransfected silkworm BmN cells, The results showed that bmo-miR-2771 had a negative effect on the expression of BmSer-1 gene. Construction of bmo-miR-275 Recombinant plasmid pcDNA3.0 (ie1) by 5.Bmo-miR-275 on BmSer-2 expression of Silk Glue protein Gene -egfp-pri-mir-275-SV40) and the recombinant plasmid pGL3 (A3-luc-BmSer-2-3'UTR-SV40) expressing BmSer-2 3'UTR, The results of co-transfection of silkworm BmN cells showed that luciferase activity decreased in the experimental group and increased slightly after transfection of synthetic bmo-mi R-275inhibitor, which confirmed that bmo-miR-275 had a negative effect on the expression of BmSer-2 gene. These results provide new experimental data for elucidating the regulation mechanism of miRNA on silk protein gene expression and the function of miRNA.
【學(xué)位授予單位】：江蘇科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S881.2

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