| Gene expression regulation is currently the most cutting-edge molecular biology research areas. Investigating on interactions between protein and DNA can clarify the mechanism of gene expression and development of the regulatory network. The pork production and the number of livestock in our country has been increasing in resent years, how to improve pork meak growth geneticly is an important part of pig breeding research. Studying the regulatory mechanism of muscle development, which has important theoretical and practical significance, will help to solve this problem fundamentally.This research is the continuing of the preliminary research of our lab. Including the following two respects:①Our group has finished some research on the TEF-1 about its effects upon pigs, through which we found that the expression level of TEF-1 is the highest in heart, followed by liver, lung tissues in the Tongcheng adult pig; and TEF-1 showed a gradually upregulation in different embryonic development stages. All these research showed that the TEF-1 has a potential effect on pig muscle development. Because there are no commercial antibodies on pigs, we group chose to do research on mouse firstly. So Ph.D Qiu has done ChIP-on-chip of TEF-1 gene on mouse, obtaining 745 downstream genes. This study, inspired by classical methods of transcription factors: analysis of promoter activity, gel mobility shift analysis and super-shift, hope to screening and confirmed several downstream target genes of TEF-1 regulation.②we select a class of SET domain containing histone lysine transferase named SMYD family, studying their function on pig muscle development based on the previous work of Ph.D.Peng. The main results were listed as follows:(1) Predict promoter of chip genes through bioinformatics. Three genes (RPS6KA2,FOS,MAP2K1IP1) were found to take part in the MAPK cascade signal pathway and were predicted to have TEF-1 binding sites in there promoter.(2) Transfecting TEF-1 overexpression vector into C2C12 cells, we observed the target genes’ expression trends. The results showed that the expression level of FOS, MAP2KIP1 were slightly higher after transfecting and RPS6KA2 were silightly lower.(3) Construct promoter vector of mouse RPS6KA2,FOS gene. RPS6KA2,FOS promoter were cloned in the multiple cloning sites before luciferase reporter gene (luciferase) of PGL3-basic vector.(4) The constructed FOS, RPS6KA2 promoter vector, together with the TEF-1 overexpression vector were transfected into BHK21. The results showed that after transfecting TEF-1 overexpression vector, the activity of RPS6KA2 promoter was significantly increased.(5) Homological alignment among various species and phylogenetic analysis of SMYD1-3 genes. The phylogenetic tree revealed that each of the SMYDs (collectively terming the SMYD1, SMYD2, and SMYD3) from mammalian were neatly clustered into one sub-branch, followed by the joining of chicken and fish successively, which was in accordance with the evolution track of these species. Herein, it was reasonable to presume that SMYDs originated from aquatic animals, and gradually mutated, coinciding with the come out of new creatures. In addition, SMYD1 and SMYD3 were clustered as one group, indicating they two were evolutionally closer and perhaps they derived from the same precursor, while SMYD2 was from another one.(6)The tissue expression profiles of porcine SMYD1-3 were determined using RT-PCR in adult LargeWhite.tissues. The results showed that SMYD2 and SMYD3 have the same expression patterns. Both genes were highly expressed in all nine tissues examined, while the expression of SmyDl was highly only in heart and skeletal muscle tissues(7). The SNPs of SMYD1 and SMYD2 with economic traits were analyzed in the experimental population and a resource population. The results showed that SMYD2 SNP. was significantly associated with average daily gain and SMYD1 SNP was significantly associated with Percentage of leaf fat and Percentage of leaf and caul fat.This study tried to screen and confirm several downstream target genes of TEF-1 related to muscle growth, using comprehensive analysis of binding site prediction, pathway and function. We tested and verified the target genes through bioinformatics prediction, overexpression, promoter activity analysis and other methods to ensure an an accurate result. It is our try to establish systematic research method of transcription factors in our lab. In addition, we continued to research the function of SMYD family genes in pig muscle development. The results that SMYD1 and SMYD2 are highly expressed in muscle tissue provided strong evidence for the two as candidate genes of pig breeding. |
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