Analysing Sequence Characters of ADSL and PurH Gene and Correlation between Genes Expression and IMP Content in Duck
|School||Nanjing Agricultural College|
|Course||Animal Genetic Breeding and Reproduction|
|Keywords||duck ADSL gene PurH gene Real-time PCR IMP High-performance liquid chromatography|
Inosine monophosphate (IMP, inosine monophosphate acid) is an important flavor substance in livestock and poultry meat, the IMP content of meat was regarded as an important indicator of meat quality in international. IMP de novo synthesis was involved in ten step reactions in animal. Adenylosuccinate lyase(ADSL), which is a key enzyme in the process of synthesis of IMP, catalyzed reaction of the eighth step. 5-Amino-4-imidazolecarboxamide ribonucleotide transformylase/IMP cyclohydrolase (ATIC), the product of the PurH gene, is a bifunctional enzyme that possesses the final two activities in the de novo purine biosynthesis pathway. ATIC has key regulate role in the synthesis of IMP.The CDS(coding sequences) of Liancheng White ducks, Jinling White ducks and Cherry Valley ducks’ADSL and PurH genes were cloned. The study was aimed to analyze genes sequences, protein structure, relative expression levels of ADSL and PurH gene in muscle and the correlation between genes expression and IMP content in three varieties. This research revealed the ADSL and PurH genes structure of excellent native ducks of China, established the foundation for further research with its biological function and relationship with flavor substances.1 cDNA cloning and sequencing of duck ADSL and PurH genesHomology comparison of ADSL and PurH genes in number of species and looking for highly conserved region, and designed a pair of primers in the highly conserved region. and then designed two primer sequences according to the fragment was amplified by previous primers. Duck ADSL and PurH genes cDNA sequence, which contains the complete coding region, were obtained by RT-PCR and chromosome walking technique. Their CDS length were 1380 bp and 1782 bp, respectively. 2 Bioinformatics analysis of ADSL and PurH proteinsThe three varieties ducks’ coding region sequences, protein structure and phylogenetic relationships were analyzed by bioinformatics software. ADSL and PurH were predicted encoding 459 and 593 amino acids, respectively. The physical and chemical properties of duck ADSL indicated that ADSL is a alkaline protein. And the protein has more strongly signal peptide and transmembrane segments in cells. The secondary structure of duck ADSL were mainly made by a helixes, and also contain fewβsheets,βturns and disorder coils. Duck’s ADSL protein contained the classical domain region, which was highly conserved in the PRK08937, and had the same domain regions as chicken ADSL proteins. the protein of PurH has no strongly signal peptide, but contained transmembrane segments. The secondary structure of PurH were mainly made by a helixes andβsheets, also contain fewβturns and disorder coils. MGS-like superfamily and AICARFT_IMPCHas superfamily typical domains in other animals were found in ducks’ PurH. Homology analysis showed that ducks and chickens relationship was closest, ADSL homology with other species are all more than 75%, ATIC homology with other species are all more than 79%, indicating that these two genes were relatively conservative in animals evolutionary development.3 RT-PCR analysis of mRNA expression of ADSL and PurH gene in muscleRT-PCR was employed to examine the relative expression levels of ADSL gene in muscle of three varieties. The results showed that expression levels of ADSL and PurH genes among different varieties were extremely remarkable(P<0.01), the level of Jinling White ducks was higher than Cherry Valley ducks, but lower than Liancheng White ducks. Female was higher than male in varieties(P<0.01). ADSL gene expression among different locations were remarkable (P<0.05) other than those in cherry valley ducks(P>0.05); PurH gene expression among different locations in Liancheng White ducks were remarkable (P<0.05) other than those in Jinling White ducks and cherry valley ducks(P>0.05).4 IMP content detection in muscle of ducksIMP content in breast and leg muscle of three varieties were detected by HPLC. The results showed that IMP content among different varieties were extremely remarkable(P<0.01), IMP content of Jinling White ducks was higher than Cherry Valley ducks, but lower than Liancheng White ducks. IMP content of Female was higher than male in the same variety (P<0.05). IMP content of breast was higher than thigh in the same individual (P<0.05). 5 Analysing correlation between genes expression and IMP content in duckThe correlation between genes expression and IMP content in ducks were analysed. The results showed that two genes relative expression and IMP content were postivly correlated. Correlation coefficient(R2) of ADSL mRNA expression and IMP content were 0.9592,0.9618,0.9577 in different varieties, sexes and locations(except Cherry Valley ducks), respectively. Correlation coefficient(R2) of PurH mRNA expression and IMP content were 0.9609,0.9584 in different varieties and sexes. Correlation coefficient(R2) of PurH mRNA expression and IMP content between breast and thigh in Liancheng White ducks was 0.8156, there were no correlation of PurH mRNA expression and IMP content between breast and thigh in Jinling White ducks and Cherry Valley ducks.