家蚕5龄幼虫中部丝腺miRNA的鉴定及对丝胶基因表达的调控作用
发布时间：2019-02-14 14:26
【摘要】：miRNA是一类由内源基因编码的长度约为21-24 nt的非编码单链RNA分子,广泛存在于真核生物中。研究表明,miRNA在基因的转录后调控中起着非常重要的作用,参与调控胚胎发育、细胞分化、增殖、神经发生、代谢及细胞凋亡等几乎所有的生物过程,动物中约有50%以上编码蛋白基因的表达受到mi RNA的调控。家蚕是鳞翅目昆虫的重要代表,家蚕丝胶基因BmSer-1、BmSer-2是中部丝腺特异表达的基因,也是蚕丝蛋白的主要基因。研究显示蚕丝蛋白基因的表达受到miRNA的调控,但未见miRNA对丝胶基因表达调控研究的报道。为了研究家蚕miRNA对丝胶基因表达的调控作用,我们利用Solexa技术对家蚕P50和裸蛹(Nd)5龄幼虫中部丝腺组织RNA进行了高通量测序,鉴定获得若干保守miRNA和新mi RNA;通过生物信息学分析,预测到bmo-miR-275等可能参与BmSer-1、Bm Ser-2的转录后调控,并分别构建miRNA和靶基因3′UTR重组表达载体,在细胞水平进行了验证,取得如下主要研究结果。1.家蚕P50与裸蛹(Nd)突变品种5龄幼虫中部丝腺miRNAs的鉴定利用Solexa技术对家蚕P50与裸蛹(Nd)突变品种5龄3d幼虫中部丝腺进行了高通量测序,检测到272个保守miRNAs,并预测到333个新的miRNAs;其中86个miRNAs在P50与Nd中的表达存在显著差异。2.新预测家蚕miRNAs的qRT-PCR鉴定为了验证预测结果的可信性,对其中10个新预测mi RNA进行qRT-PCR鉴定,结果与测序结果相符;利用软件进行靶基因预测,保守miRNA和新预测miRNA分别预测到2522和1376个靶基因。3.家蚕丝胶基因BmSer-1、BmSer-2及相关mi RNA表达特性分析通过靶基因预测,发现bmo-miR-2771和bmo-miR-275分别是BmSer-1、BmSer-2的潜在靶基因,利用半定量PCR方法分析它们的表达特性,发现bmo-miR-2771只在中部丝腺中表达。4.Bmo-miR-2771对家蚕丝胶蛋白基因BmSer-1表达的调控作用构建表达bmo-miR-2771的重组质粒pcDNA3.0(ie1-egfp-pri-mir-2771-SV40)和表达BmSer-1 3′UTR的重组质粒pGL3(A3-luc-BmSer-1-3′UTR-SV40),共转染家蚕BmN细胞,结果显示bmo-miR-2771对BmSer-1基因表达具有负调控作用。5.Bmo-miR-275对家蚕丝胶蛋白基因BmSer-2表达的调控作用构建表达bmo-miR-275的重组质粒pcDNA3.0(ie1-egfp-pri-mir-275-SV40)和表达BmSer-2 3′UTR的重组质粒pGL3(A3-luc-BmSer-2-3′UTR-SV40),共转染家蚕BmN细胞,结果显示实验组荧光素酶活性降低,再转染人工合成bmo-mi R-275inhibitor后活性略有回升,证实bmo-miR-275对BmSer-2基因表达具有负调控作用。上述研究结果为阐明miRNA对蚕丝蛋白基因表达的调控机制和miRNA的功能提供了新的实验数据。
[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.
【学位授予单位】：江苏科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S881.2
[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.
【学位授予单位】：江苏科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S881.2
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