| Panthera Tigris has been widely distributed in Northeast China,the Russian Far East and the Korean Peninsula.It is the flagship species of biodiversity conservation and the top predator of the food chain in the ecosystem.But in recent decades,they have experienced severe population decline and geographical shrinkage because of habitat loss,poaching,prey exhaustion and disease.At present,less than 600 of the individual estimates are maintained in the Russian Sn Holt mountains(95%)and the Changbai Mountains(5%)on the Sino Russian border.2016 the Chinese government has launched a tiger leopard National Park(TLNP)to expand the scope of the Panthera tigers and leopards in China.In the macro ecological protection of the Amur tiger(such as wild populations,habitat restoration,along with the increase and decrease the interference of prey etc.)at the same time,the use of modern molecular biology,cell biology,bioinformatics technology and methods to carry out the tiger genetic resources collection,preservation,analysis and research,and to achieve the purpose of the protection in vitro,has become the focus of scientific research of the Amur tiger.In this study,we studied the miRNA expression profiles and transcriptome of Amur tiger,and the endogenous activation of miR-302/367 and iPSCs induced by fibroblasts.The main results are as follows:1 We used RNA-seq technology to analyze the small RNA sequencing of the lung tissue of the Amur tiger.The clean reads were mapped to the genome of the Amur tiger.And then we annotated the clean reads and analyzed the expression profiles which including 225 known miRNAs and 44 novel miRNAs.We also predicted these miRNAs target genes.After GO analysis,they were divided into 402 GO terms.KEGG pathway analysis of the target genes showed that it was mainly involved in cell proliferation,cell differentiation,foreign substance metabolism and cancer related signaling pathways.At the same time,some mature sequences and precursors of miR-302/367 sequences were obtained,which lay a theoretical foundation for reprogramming iPSCs from the Amur tiger fibroblasts by using miR-302/367.2 Next,we carried out bioinformatics analysis on the transcriptome of the Amur tiger lung,including Gene Ontology(GO)and Clusters of Orthologous of proteins(COG).9176 of the Amur tiger genes were annotated to 58 GO terms in GO analysis,and 58828 genes were annotated and classified in COG.We use Targetscan and miRanda prediction tools to predict the target gene in the Amur Tiger lung tissue transcriptome.We obtained 1861 miRNAs that regulated target genes from tiger lung tissue,described the miRNA biological regulation of target genes in lung function,and draped the miRNA regulate target gene function realization of lung tissue molecular network and miRNA regulates cell proliferation regulation network by regulating PI3K/Akt signal pathway.3 In order to predict and analysis of the Amur tiger miR-302/367 mature sequence and its promoter region,we obtained the remaining miR-302/367 mature sequence by using bioinformatics analysis.We got 4 sgRNAs targeting to miR-302/367 promoter region by bioinformatics software analysis.After connecting the artificial synthesis 4 sgRNAs to lentivirus expression vector,we construct 4 endogenous activation of lentivirus expression vector.The active region of dCas9 endonuclease was removed to reduce its size,and at the same time,the VPR transcriptional activation domain with strong gene activation was fused to the carboxyl terminus of dCas9N to construct dCas9N-VPR lentivirus expression vector to activate the expression of endogenous miR-302/367.293T cells were transfected with Liposome 3000,sgRNAs and dCas9N-VPR for lentivirus packaging,concentration and infection of Amur tiger fibroblasts,72h processing,qRT-PCR was used to detect the expression of miR-302/367.We found that 4 miRNA expression increased 10 times,reaching the purpose of endogenous activation,prepare for iPSCs reprogramming from Amur tiger fibroblasts.4 Finally,we induced iPSCs by using the endogenous expression of miR-302/367 in Amur tiger fibroblasts through the method of lentivirus infection.The clones were appeared after 5d induction.The expression of the surface markers of the stem cells was verified by the cell immunization of the obtained cells.Karyotype analysis showed no genetic variation in reprogramming of Amur tiger cells,and analyzed the expression of related genes in reprogramming of Amur tiger cells.Alkaline phosphatase staining analysis confirmed the high expression of alkaline phosphatase in Amur tiger cells.The orientated differentiation of the three germ layers in vitro showed that the iPSCs had the ability to differentiate in vitro.In summary,this study predicted the Amur tiger miR-302/367 mature sequence by high-throughput sequencing,constructed sgRNA Lentivirus expression which targeted into the promoter of miR-302/367,expressed stability in the Amur Tiger fibroblast by using activation of endogenous miR-302/367,and reprogrammed Amur tiger fibroblasts ino iPSCs.Amur tiger iPSCs have multilineage differentiation potential.It provides a cell research model for studying the early embryonic development of Amur tiger,and the tiger iPSCs can be used as a long-term preservation of genetic resources,providing a good material for the protection and research of Amur tiger.So that our country has achieved three protection ways in situ protection,ex situ conservation and in vitro protection on the protection of endangered species such as Amur tigers,Southern China tigers and other tigers. |
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