淇河鲫MSTN基因相关microRNA的筛选与功能研究

发布时间：2018-05-14 03:39

本文选题：淇河鲫 + MSTN　；参考：《河南师范大学》2016年硕士论文

【摘要】：淇河鲫(Carassius auratus in Qihe River)属于银鲫的一种,天然雌核发育,脊背宽厚是区别于其它银鲫鱼最主要的特征。目前在淇河鲫育种、疾病免疫、饲料营养等方面已开展较多研究,但是淇河鲫生长性状相关的基础研究还相对薄弱,其脊背宽厚的生长机制尚不清楚。肌肉生长抑制素基因(myostatin,MSTN)是一种负调控因子,它最主要功能是调节动物的骨骼肌肉生长发育。microRNAs(miRNAs)是一类非编码单链小RNAs,近年来它的作用和功能越来越多地被发掘。miRNAs技术的发展为探索淇河鲫脊背宽厚这一问题开辟了一条新的途径。提取淇河鲫总RNA,克隆,得到淇河鲫MSTN3’UTR序列。根据MSTN 3’UTR序列通过TargetScans等软件预测出相关miRNAs,再利用RNAhybrid软件进行验证,最终选取miR-181a和miR-181b作为候选mi RNAs。以肌肉总DNA为模板,获得pri-miR-181a和pri-miR-181b全长。构建真核表达载体和miRNA慢病毒载体,通过双荧光检测和荧光原位杂交技术分别从分子水平和组织细胞定位方面确定MSTN和miR-181a、miR-181b之间的相互关系。采用qRT-PCR分析miR-181a、miR-181b和MSTN在淇河鲫脑、肌肉和心脏等8个组织的表达情况以及miR-181a、miR-181b和MSTN在胚胎发育及其在前期生长不同阶段的表达情况。由此进一步揭示淇河鲫肌肉生长相关基因间的多层次网络调控模式,探索淇河鲫肌肉生长机制。1、淇河鲫MSTN基因3’UTR的克隆提取总RNA,克隆得到MSTN基因3’UTR,拼接后获得3’UTR长度是865bp。在NCBI中,经过Blast比对发现,得到片段与鲤形目鱼类相似度较高,尤其与南亚野鲮(Labeo rohita)(91%,KR052242.1)和鲤鱼(Cyprinus carpio)(92%,LN590705.1),淇河鲫(Carassius auratus in Qihe river)(88%,KC851952.1)同源性最高。2、淇河鲫MSTN基因相关miRNAs的预测、验证及克隆根据MSTN基因3’UTR序列结果,利用RegRNA、Pictar、TargetScan、miRWalk、miRDB等软件,,预测相关miRNAs,进一步用RNAhybrid软件验证,选取可能与MSTN基因相关的miR-181a和miR-181b作为候选miRNAs。从淇河鲫肌肉中提取总DNA,克隆pri-miR-181a和pri-miR-181b。3、真核载体的构建及双荧光检测本实验中把MSTN 3’UTR序列和加突变位点的3’UTR序列双酶切,连接至pmirGLO载体;pri-miR-181a/b双酶切,连接至Lenti H1载体,分别构建真核表达载体和miRNAs慢病毒载体。构建的两个载体共转染至293T细胞,双荧光检测发现,miR-181a/b对MSTN基因均可能有一定的抑制作用,此外还发现miR-181a/b与MSTN还可能存在其他的结合位点共同作用调节MSTN基因的表达。4、MSTN和miR-181a在肌肉组织中的表达定位取淇河鲫背部和腹部肌肉,制成石蜡切片,合成探针,检测MSTN基因和mi R-181a在肌肉组织中的表达位置。结果表明,MSTN在肌纤维中表达量较高;mi R-181a在肌浆膜中表达量较高,并且在结缔组织中也有表达。此外还发现miR-181a在背部肌肉的表达量低于腹部,在腹部肌纤维中MSTN表达量显著高于背部,因此推测在背部肌肉中miR-181a对MSTN的抑制作用可能没有腹部明显。5、淇河鲫MSTN基因及相关miR-181a/b特异性表达qRT-PCR结果显示,淇河鲫MSTN基因在不同组织表达量存在显著性差异,其中心肌和脑中最高,肌肉其次,肾脏最低;胚胎发育及前期生长不同阶段,MSTN基因在原肠胚以前,出生20d表达量显著高于其他时期。miR-181a在肌肉和鳃的表达丰度显著高于其他组织,在原肠胚以前和孵化10d后也具有较高的含量。miR-181b在脑、肌肉和肠有较高的表达量,原肠胚以前和孵化10d后表达量显著高于其他时期。
[Abstract]:The Carassius auratus (Carassius auratus in Qihe River) is one of the silver crucian carp (Carassius auratus). The natural female nucleus is developed, and the back width is the main feature different from other silver carp. At present, much research has been carried out on the breeding, disease immunity and feed nutrition of the Carassius auratus in Qihe River, but the basic research on the long traits of the Carassius auratus in Qi He is still relatively weak and its backbone is wide. Myostatin (MSTN) is a negative regulatory factor. The most important function of the gene is to regulate the growth and development of skeletal muscle in animals..microRNAs (miRNAs) is a kind of non coding single strand small RNAs. In recent years, its function and function have been more and more exploited to explore the development of.MiRNAs technology to explore Qi. The general RNA of Carassius carassius carp opened a new way. The total RNA of Qihe crucian carp was extracted, and the MSTN3 'UTR sequence of Qihe crucian carp was cloned. According to the MSTN 3' UTR sequence, the related miRNAs was predicted by TargetScans software and then the miR-181a and miR-181b were selected as the candidate mi RNAs.. The whole length of pri-miR-181a and pri-miR-181b was obtained. The eukaryotic expression vector and the miRNA lentivirus carrier were constructed. The relationship between MSTN and miR-181a and miR-181b was determined by double fluorescence detection and fluorescence in situ hybridization. QRT-PCR analysis miR-181a, miR-181b and MSTN in Qihe crucian carp were used. The expression of 8 tissues, such as brain, muscle and heart, as well as the expression of miR-181a, miR-181b and MSTN in the development of embryo and the different stages of early growth, thus further reveal the multi-level network regulation mode between the muscle growth related genes of Qi He carp, explore the mechanism of muscle growth of Qi River Carassius auratus,.1, and the MSTN gene of Qi He crucian carp, 3 'UTR. The total RNA was extracted, and the MSTN gene was cloned to 3 'UTR, and the length of 3' UTR after splicing was 865bp. in NCBI. After Blast comparison, it was found that the similarity between the fragment and the carp shaped fish was higher, especially with the South Asian wild carp (Labeo rohita) (91%, KR052242.1) and the carp (Cyprinus carpio). (88%, KC851952.1) the highest homologous.2, the prediction, verification and cloning of the miRNAs related to the MSTN gene of Qi River Carassius auratus, verification and cloning based on the results of the MSTN gene 3 'UTR sequence, using RegRNA, Pictar, TargetScan, miRWalk, miRDB and other software to predict the related miRNAs. The total DNA, pri-miR-181a and pri-miR-181b.3 were extracted from the muscle of Qi He crucian carp, pri-miR-181a and pri-miR-181b.3 were cloned. In the construction of eukaryotic vector and double fluorescence detection, the MSTN 3 'UTR sequence and the 3' UTR sequence of the mutation site were cut into the pmirGLO carrier; pri-miR-181a/b double enzyme cut, connected to Lenti H1 vector, constructed the eukaryotic expression vector. The two vectors constructed with miRNAs were transfected into 293T cells. Double fluorescence detection found that miR-181a/b could inhibit the MSTN gene. Furthermore, it was found that miR-181a/b and MSTN may also have other binding sites to regulate the expression of the MSTN gene, and the expression of MSTN and miR-181a in the muscle tissue. Locating the muscles of the back and abdomen of the crucian carp, the paraffin section was made into a paraffin section and a probe was synthesized to detect the expression of the MSTN gene and MI R-181a in the muscle tissue. The results showed that the expression of MSTN in the muscle fibers was higher; the expression of MI R-181a in the serous membrane was higher, and the expression of MI was also expressed in the connective tissue. Moreover, miR-181a was also found in the back muscles. The expression of MSTN in the abdominal muscle was significantly higher than that in the back. Therefore, it is presumed that the inhibitory effect of miR-181a on MSTN in the back muscles may not be obvious in the abdominal.5. The qRT-PCR results of the MSTN gene and related miR-181a/b specific expression of the Qi He crucian carp found that the MSTN gene in Qi He crucian carp has a significant difference in the expression of different tissues. Among them, the highest in the heart and the brain, the second of the muscle and the lowest in the kidney; the MSTN gene was significantly higher in the expression of 20d in the muscles and gills than in other tissues before the embryo of the gastrula and at the different stages of the early growth of the embryo. The expression of the MSTN gene was significantly higher than that of the other tissues before and after the incubation of the proembryo and after the incubation of the 10d. In the brain, muscle and intestine, the expression level was higher, and the expression level of gastrula before and after hatching 10d was significantly higher than that in other periods.

【学位授予单位】：河南师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S917.4

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