Cloning And Expression Of P53 - Like Gene In

Tribolium castaneum is not only an important pest in general, but also a model organism, and has been completed the whole genome sequencing, so it is a good material in study of phosphine molecular toxicology and phosphine resistance mechanism. Tribolium castaneum belongs to genus Tribolium （Coleoptera: Tenebrionidae）. Due to using the single phosphine fumigation for a long time, its phosphine resistance problem has been increasingly prominent and its resistance research has become one of hot topics in the study of insect resistance. In recent years, most researches, especially researches on the mechanism of phosphine resistance, and the phosphine resistance of T. castaneum have achieved gene level.So far, the p53gene is the most closely associated with human tumor gene. Under normal circumstances, low level of the expression of p53protein in the cell, when the p53gene is activated, the expression level of p53gene would increase, the activation of p53gene is required for certain conditions, such as cells under the stimulation of specific cytokines or DNA damage, lack of oxygen, metabolic resistance change, and the activated p53gene would be involved in the repair of DNA damage, cell cycle regulation, apoptosis induced process. Recent studies show that the p53gene as an important intermediary factor in the process of cell metabolism, not only related to tumor suppressor, but also important to the regulation of mitochondrial respiration. p53can adjust the effectively balance of aerobic respiration and glycolysis, and has significantly correlation with the generation of reactive oxygen species, oxidative stress and cell death process in the process of breathing.Our laboratory in the previous studies had selected28pairs primers of microsatellite （Short Tandem Repeats, STR） polymorphism by building part of T. castaneum genomic library and using bioinformatics methods, which comes from the T. castaneum whole genome and EST library. It was found that STR polymorphism loci DT802192appears obvious lack of polymorphism phenomenon which located in third chromosome in the resistant strain, this reslut prompt the molecular markers associated with phosphine resistance genes. Depth analysis found that the similar to p53gene exist in DT802192upstream, so study on the similar to p53gene mRNA expression level would provide a theoretical basis to the T. castaneum phosphine resistance mechanism.This study put T. castaneum as the research object, using RT-PCR technology to clone the whole coding area of similar to p53gene from T. castaneum; using bioinformatics methods to analyze its nucleotide sequence and protein sequence; using qRT-PCR technology to detect the similar to p53gene of mRNA expression under phosphine stress in different T. castaneum strains of developmental stages. The main results are as follows:1. The the cDNA whole coding area of T. castaneum similar to p53gene is825bp, the cDNA accession number of the similar to p53gene is XM₉61543.2. Through the bioinformatics analsis, the result shows that the similar to p53prtotein’s relative molecular weight is30894.0Da and pI is5.55. The similar to p53protein shares low similarity with other insects. There is no signal peptide of this protein, which belongs to the hydrophilic nonsecreted protein. The similar to p53protein does not exist transmembrane area, so the protein is not a transmembrane protein. The similar to p53protein include three protein kinase C phosphorylation site. The secondary structure of similar to p53was determined by bioinformatics software and the result is a kind of mix protein.2. The blast between different strains of T. castaneum shows that there were6different loci, which are respectively located in21,102,108,116,229,251loci. Motif scan online to predict the function of the protein motif, eventually find that compared with the submitted sequence of NCBI, one of monoclonal sequences in sensitive strains has one more protein kinase C phosphorylation site （SRR）, which located in the252-254loci; one of monoclonal sequences in resistent strains has one less protein kinase C phosphorylation site （SCK）.3. This research was conducted to determine stable referece gene（s） for normalization in future expression studies on the red flour beetle （Tribolium castaneum）. The expressions of candidate reference genes β-actin、 GAPDH、α-Tubulin、SYN1、 SYN6、RPS3、RPS18、RPL13a in Tribolium castaneum after exposure to the phosphine were detected by qRT-PCR using relative quantification. According to the qRT-PCR reaction result, these eight candidate reference genes were all specifically amplified by the primers pairs with high efficiency. Then the expression stability of these genes was analysed by geNorm and NormFinder software. Based on the results of geNorm analysis, the stability of the eight candidate reference genes from high to low was RPS18=RPL13a （0.007）> SYN6（0.015）> RPS3（0.038）> β-actin （0.044）> a-Tubulin （0.105）> SYN1（0.154）> GAPDH （0.205）, and V2/3of pairwise variances analysis of eight reference genes was0.006. The results show that RPS18and RPL13a are the most suitable reference genes in Tribolium castaneum after exposure to the phosphine and the appropriate number of reference genes is2, whereas GAPDH, α-Tubulin and β-actin which have a wide range of application were poorly ranked. On the other hand, based on the results of the NormFinder analysis, the stability of eight candidate reference genes from high to low is RPS18（0.002）> RPL13a （0.016）> β-actin （0.042）>RPS3（0.044）> SYN6（0.055）> a-Tubulin （0.066）> SYN1（0.077）> GAPDH （0.126）. The results show that RPS18is the most suitable reference gene, and the best combinations of two genes are RPS18and RPL13a. All above results suggest that RPS18and RPL13a are suitable reference genes and could be used to normalize mRNA levels in different strains of Tribolium castaneum after exposure to phosphine.4. This study use qRT-PCR technology to detect the similar to p53gene of mRNA expression under phosphine stress in different T. castaneum strains of developmental stages （embryo,4instar of larva, pupa, and adult）. The results are as follows:a. The similar to p53gene are expressed in embryo of different T. castaneum strains, but the response degree of expression level are different; secondly, the gene expression changes after phosphine induction respectively in sensitive strain and resistent strain. Under normal circumstances, the similar to p53gene has no significant difference between sensitive strain and resistent strain （p>0.05）; the gene expression rises significantly in sensitive strain phosphine induction; the gene expression shows a gradually rising trend in resistant strain under phosphine induction. b. The similar to p53gene are expressed in4instar larva of different T. castaneum strains, but the response degree of expression level are different; secondly, the gene expression has no significant change after phosphine induction respectively in sensitive strain and resistent strain. Under normal circumstances, the similar to p53gene has no significant difference between sensitive strain and resistent strain （p>0.05）; the gene expression rises significantly in sensitive strain phosphine induction; in resistant strain, the gene expression shows a sharp drop after20h phosphine induction, then appeared a gradually rising trend, c. The similar to p53gene are expressed in pupa of different T. castaneum strains, but the response degree of expression level are different; secondly, the gene expression has no significant change after phosphine induction respectively in sensitive strain and resistent strain. Under normal circumstances, the similar to p53gene has no significant difference between sensitive strain and resistent strain （p>0.05）; the gene expression has no significant change in sensitive strain phosphine induction; the gene expression also has no significant change trend in resistant strain under phosphine induction. d. The similar to p53gene are expressed in adult of different T. castaneum strains, but the response degree of expression level are different; secondly, the gene expression has no significant change after phosphine induction respectively in sensitive strain and resistent strain. Under normal circumstances, the similar to p53gene has outstanding statistical significance between sensitive strain and resistent strain （p<0.01）; the gene expression rises significantly in sensitive strain phosphine induction; in resistant strain, the gene expression has no significant change after20h and60h phosphine induction, then appeared a sharp drop after100h phosphine induction. In conclusion, there could be concluded that the similar to p53gene may participate in the Tribolium castaneum resistence mechanisms; and the down-regulation of similar to p53gene expression may be associated to the generation of Tribolium castaneum resistence, the up-regulation of similar to p53gene expression is a lethal factor.