The Construction Of Zebrafish Wild Strain Indentification Criteria And The Discovery And Analysis Of Zebrafish Non-coding RNA

Zebrafish is a new model vertebrate, which has unique advantages in genetic & development research. The individual of zebrafish lines that commonly used at home and/or abroad are similar in the form which are difficult to distinguish, furthermore, there are lack of biological markers for detection. Therefore, the pollution between strains easily existed in daily experiment. However, in biomedical research, the quality of experimental animals has a significant impact on the experimental results of comparable quality, repeatability and accuracy. Therefore, genetic monitoring of laboratory animals is a routine and important work for assessing and ensuring the quality of laboratory animalsIn this study, we selected a large number of microsatellite markers, used own reared AB, TU, WIK strain and the strain purchased from Beijing fish market which inbred within 4 generations or more for the formation of the LF lines (in after referred as four different strains) as the experimental materials, through a number of polymerase chain reaction (PCR) experiments, detected by 3% or 4% agarose gel electrophoresis. According to the results of electrophoresis the different lanes including the number and the size of bands), we selected the three pairs of identification microsatellite markers primers for distinguishing these strains. The three pairs of primers were named z3782, z4299, z24244 respectively.z3782, z4299, z24244, and these three pairs of primers of microsatellite markers can distinguish between AB, TU, WIK, and LF four strains of zebrafish simultaneous. In experiments, we used three pairs of primers of microsatellite markers to identify the zebrafish of the same strains from different sources. And the experimental results are very stability and repeatability. Therefore, the three microsatellite DNA points are stable amplified in AB, TU, WIK, and LF. They showed significant polymorphisms among there four different strains. Therefore, the three microsatellite markers can be used in identifying of the four strains of experimental zebrafish AB, TU, WIK, and LF. The experimental results provides a simple reference for identification of the common strains of zebrafish. Non-coding RNA, ncRNA, which is a class of RNA molecules with unopened reading frame and do not code protein, including rRNA, tRNA, snRNA, snoRNA, microRNA which are known functional RNA, and also including some functional-unknown RNA. The common characters of these RNA are that they can be transcribed from the genome, but not translated into protein. They can act their biological function at the RNA level.More than 500 nt of mRNA-like non-coding RNA widely exist in various species. Most of them are tran-scripted by RNA polymerase and aslo be alternative splicing. They also have the structure of G cap and poly (A) tail. Some studies suggest that much of mRNA-like ncRNA be expression with spatial and temporal specificity. They also showed that they play a role in specific biological processes.This experiment focuses on the spatial and temporal expression specificity of mRNA-like ncRNA in zebrafish embryonic development. In this study, we have screened all of zebrafish mRNA-like ncRNA by various bioinformatics. Through summering and improving the screening conditions of mRNA-like ncRNA, the false positive rate of the experimental results was reduced. Through primer designing, Polymerase Chain Reaction (PCR), agarose gel electrophoresis and nucleotide sequencing, we successfully amplified two mRNA-like ncRNA genes. To further study the two amplified mRNA-like ncRNA genes, we firstly made semi-quantitative RT-PCR experiments to preliminary analyze time-specific of these two gene expression. Then we made molecular cloning, synthesis of RNA probes and the original in situ hybridization techniques, we analysised the spatial and temporal specificity of expression of two mRNA-like ncRNA genes in embryonic development of zebrafish.The results of semi-quantitative RT-PCR and in situ hybridization showed that:# 7, # 20, there two mRNA-like ncRNA genes in embryonic development of zebrafish are both space and time-specific expression. On the one hand, the results broadened the idea to study the embryonic development of the research. On the other hand, the results set the basis for the study of zebrafish mRNA-like ncRNA gene functional basis for the next step.