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Screening Proteins Interacting with GATA-5 and Functional Research of miR-142-3p in Haematopoiesis

Author: LiHaiTao
Tutor: JiaHaiBo
School: Huazhong University of Science and Technology
Course: Biomedical Engineering
Keywords: Yeast Two Hybridization GATA-5 SMART cDNA miR-142-3p zebrafish In-situ hybridization O-stainning
CLC: Q78
Type: Master's thesis
Year: 2011
Downloads: 28
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Abstract

(1) Objective: We screen the proteins interacting with GATA-5 through Yeast Two Hybridization, and lay foundations for the further research of the function and mechanism of GATA-5.Methods: We extracted the total RNA from the zebrafish eggs (day1 and day2) use theTrizol reagent, and used the total RNA as reverse transcriptive template, CDSIII and SMART III as primer to synthesize the first strand of cDNA , The products we got which possessed the same homologous arms through the long distance PCR (LD-PCR).At the base of this property, we have accomplished the homologous recombination through transforming the SMART cDNA,Sma1 linearized PGADT7 -Rec plasmid into the yeast strain of Y187. Meanwhile we transformed PGBKT7-Rec-GATA-5 recombinant plasmid into yeast strain of AH109 for test the transcriptional activation and toxicity, Then picked the yeast clonies which grew on plates via these two kinds of yeast strain mating,We made the preliminary research of genes than may be interacting with GATA-5 by a series of experimental methods(Colony-lift Filter Assay, yeast PCR , Basic Local Alignment Search Tool )Results:The SMART cDNA from the zebrafish eggs has been build finished, after agarose electrophoresis ,the coverage of cDNA segments were from 250bp to 3000bp. Now ,we have screened out five genes that may be interact with GATA-5. Conclusion: The diversity and capacity of the cDNA library fit the screening requirements and qualified enough for the reseach of the other heart development related genes, but the genes that have already been screened out remaining need to be further identified(2) MicroRNAs(miRNAs) in viruses and eucaryotes are endogenous small non-protein coding and single strand RNA which have 21-23 nucleotides.They can degrade targeted mRNAs or inhibit their translation by binding to 3‘UTR regions of their targeted mRNAs according to the principle of complementary base pairing. So far hundreds of miRNA genes have been identified in eucaryotes and many miRNAs are evolutionarily conserved. It is supposed that miRNAs regulate up to 30% of the genes in human genome.and play an important role in growth, development and occurrence of disease.In recent years, model organisms extensively used promote the foots of scientific research. In this paper,we used the zebrafish as a tool for research the function of miR-142-3p in haematopoiesis through technologies of microinjection, O-stainning,and in-situ hybridization.The results indicated that overexpression of miR-142-3p in zebrafish embryos reduced the production of red cells via O-stainning , and we used RNA probes (GATA-2,scl-1,pu-1 ) for whole in-situ hybridization , The overexpression of miR-142-3p in zebrafish embryo had no impact on differentiation in haemangioblast and haematopoietic stem cell, but the copies of pu-1 arised in myeloid progenitor.we assumed that overexpression of miR-142-3p in zebrafish embryo reduced the differentiation from haematopoietic stem cell to erythoid progenitor,and increased the differentiation from haematopoietic stem cell to myeloid progenitor.

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