Title Identification and function analysis of genes involved in development of intermuscular bone of wuchang bream (Megalobrama amblycephala) Title (croatian) IDENTIFIKACIJA I FUNKCIJSKA ANALIZA GENA KOJI SU UKLJUČENI U RAZVOJ INTERMUSKULARNIH KOSTIJU WUCHANG DEVERIKE (MEGALOBRAMA AMBLYCEPHALA) Author Shiming Wan Mentor Tea Tomljanović (mentor)Committee member Tomislav Treer (predsjednik povjerenstva)Committee member Daniel Matulić (član povjerenstva)Granter University of Zagreb Defense date and country 2020-05-22, Croatia Scientific / art field, BIOTECHNICAL SCIENCES Universal decimal classificationUDC ) 638/639 - Apiculture. Keeping, breeding and management of insects and other arthropods. Hunting. Fishing. Fish breeding Abstract Intermuscular bones (IB), which occur only in the myosepta of the lower teleosts, are segmental, serially homologous ossifications from connective tissue in the myosepta. Most of the freshwater aquaculture fishes around the world, especially Cyprinidae species, possess a certain amount of IB. These IB have brought about many adverse impacts on the deep processing and food value of a fish species, because they are difficult to remove and make the fish unpleasant to eat. Meanwhile, IB reduces the economic value of a fish species, resulting in low price and exports. Main results and conclusions are as follows:
The results of tissue section observation revealed the microstructural features of IB, CT and muscle tissue, indicating that IB has a potential differentiation relationship with CT. The miRNA comparative analysis between IB and connective tissue finally obtained a total of 218 known miRNAs belong to 97 miRNA families. Of them, 188 miRNAs were expressed in both tissues, while 13 and 17 miRNAs were specifically expressed in IB and connective tissue, respectively.
On the basis of fthis key development stages of IB (S1: IB haven’t emerged; S2: a few IB with small length emerged in the tail; S3: more IB of greater length gradually emerged in the tail; S4: all of the IB in the tail have a mature morphology and length. IB have also emerged throughout the body of the fish from the tail to the head), it is constructed a reference transcriptome to analyze miRNA and mRNA expression profiles over fthis IB developmental stages. The wild-type construct GLO-runx2b was not reduced by miR-206-3p mimics, which showed the inaccuracy of software prediction and the necessity of experimental verification.
Considering significant enrichment of BMP signaling pathway in previous studies, it is verified the function of bmpr2a/2b receptors through CRISPR-Cas9 technology and focused on its role in intermuscular bone and skeleton development. Phylogenetic and genomic structure analysis indicated that the sequence characteristics of bmpr2a and bmpr2b genes in zebrafish were differentiated in the evolutionary process. Gene knockout results showed that the loss of bmpr2a and bmpr2b function did not cause significant changes in the IB phenotype, but bmpr2b-/- mutant showed different degrees and types of mineralized bone malformations. The significant changes in the expression levels of BMP-Smad signaling molecules and various bone development genes, and the phenotypic changes in bmpr2b-/- individuals revealed an important role for bmpr2b in bone mineralization of zebrafish.
Abstract (croatian) U ovom istraživanju otkrivena je mikrostruktura i potencijalni odnos diferencijacije intermuskularnih kostiju i okolnog vezivnog tkiva u miosepti kod vrste wuchang deverike. Na temelju razlika u strukturi tkiva i usporedne analize transkripta, otkrivena je specifična ekspresija miRNA u različitim tkivima i ciljanim genima. Analizom KEGG staze otkriven je veliki broj specifičnih gena značajnih za skeletni razvoj. Genetski podaci intermuskularnih kostiju identificirani su kroz diferencijaciju tkiva.
Korelacijska analiza ekspresije mRNA i miRNA u ključnim razvojnim fazama intermuskularnih kostiju otkrila je mRNA i miRNA koje su posebno eksprimirane u različitim razvojnim fazama, kao i različito eksprimirane gene i miRNA između različitih razvojnih stadija, uključujući gene za TGF-β putove čija se razina ekspresije nastavlja smanjivati / povećavati u različitim razvojnim fazama. Utvrđena je uska veza TGF-β sa signalnim putovima MAPK i ERK kojima se reguliraa diferencijacija osteoblasta. Istraživanje je otkrilo veliku količinu genetskih informacija o intermuskularnim kostima iz razvojne perspektive.
Analiza interakcije miRNA-mRNA otkrila je veliki broj miRNA i mRNA povezanih s razvojem kostiju. Validacijom odnosa između skeniranih razvojnih miRNA i mRNA na temelju dualnog luciferaznog gena otkrila je da miR-133b-3p značajno inhibira 3'UTR ekspresiju tgfbrla i runx2a te da miR-206-3p značajno inhibira ekspresiju 'UTR sekvence. Međutim, eksperimentalni rezultati također su pokazali da prekomjerna ekspresija miR-206-3p nije uzrokovala supresiju ekspresije plazmida GLO-runx2b, što sugerira nesigurnost ciljnih gena predviđenih softverom i nužnost daljnjih eksperimenta za validaciju.
Funkcionalna provjera gena kandidata na temelju CRISPR-Cas9 otkrila je ulogu bmpr2b u mineralizaciji kostiju model riba zebrica, ali nije pronađen izravan regulatorni učinak na fenotip intermuskularnih kostiju.
U ovom radu postignuto je više istraživačkih inovacija. Prvi put je promatrana mikrostruktura intermuskularnih kostiju i srodnih tkiva, istražena je njihova potencijalna diferencijacija i konstruirana je baza podataka mRNA i miRNA transkriptora intermuskularnih kostiju i potencijalno diferenciranog tkiva. Također je na temelju karakteristika razvoja intermuskularnih kostiju, konstruirama je transkriptna baza podataka o mRNA i miRNA, otkrivajući potencijalni regulatorni odnos između miRNA i mRNA u razvoju intermuskularnih kostiju. Prvi put je sustavno proučavan mehanizam molekularne regulacije intermuskularnih kostiju riba kombinirajući morfologiju, histologiju, transkriptomiju i tehnologiju uređivanja gena CRISPR-Cas9
Stoga je ostvaren i predviđeni znanstveni doprinos u sveobuhvatnoj izgradi genetičke baze intermuskularnih kostiju wuchang deverike i generalizacije važnih genetskih informacija vezanih uz nastanak i razvoj intermuskularnih kostiju koje se mogu koristiti za daljnje istraživanje molekularnog mehanizma razvoja intermuskularnih kostiju i molekularno genetskih tehnika kod uzgoja wuchang deverike.
Keywords
Megalobrama amblycephala
intermuscular bone
development
miRNA
gene expression
gene function
Keywords (croatian)
Megalobrama amblycephala
intermuskularne kosti
ravoj
miRNA
ekspresija gena
funkcija gena
Language english URN:NBN urn:nbn:hr:204:172763 Promotion 2020 Study programme Title: Agricultural Sciences Catalog URL http://161.53.95.232/cgi-bin/wero.cgi?q=580503001368 Type of resource Text File origin Born digital Access conditions Open access Terms of use Public note Mentor: Zexia Gao
Povjerenstvo: Kaijian Wei, Hong Liu, Jingou Tong Repository Repository Faculty of Agriculture University of Zagreb Created on 2020-06-16 10:13:20
