Cloning Of Chicken Delta-6 Desaturase And Its Expression In Different Tissues

LC-PUFA play an important role in the body.Δ~6-desaturase is the limited enzyme in the formation of PUFA.Because of the important role in the formation of polyunsaturated fatty acids.In this experiment we carried out chickenΔ~6-desaturase research. A 2141bp cDNA was obtained by two RT-PCR and 3,RACE.Sequence analysis showed this cDNA sequence han an open reading frame(ORF) of 1335bp coding 444 amino acids .This is the first successful cloning of chickenΔ~6-desaturase.Intron and exon boundaries of coding sequence was obtained by sequence analysis.This gene includs 12 exons and 11 introns.Through the semi-quantitative PCR, we compared fatty acid dehydrogenase in differ -ent tissues, we found expression ofΔ~6-desaturase in the liver is the most.This experiment lay the foundation for further study of the gene in the formation of polyunsaturated fatty acids.This study is divided into two parts :一,The cloning of chickenΔ~6-desaturase1,The cloning of partial code ofΔ~6-desaturase A 1323 bp DNA fragment was amplified from Gushi chicken based on the sequence information for predicted delta-6 fatty acid desaturase of gallus(XM421053) by RT-PCR (GenBank Accession: EF636888). The base sequence comparability was beyond 74%.2,The cloning of 5,end ofΔ~6-desaturaseUsing the nucleotide sequence of partial CDS,Blast on the NCBI,we find a EST(BI390105).This EST is partial lapped with partial CDS in the 5,end. According to the EST designing gene-specific primers for 5,end of the amplification,We gained a 283bp nucleotide(D6D2). The fragment is overlapped 99bp with partial CDS. It received the gene fragment was part of the same sequence.3,The cloning of 3,UTR ofΔ~6-desaturaseIn order to obtain the 3'untranslated region ofΔ~6-desaturase,we designed gene-specific primers for the 3,untranslated region amplifacation by using 3, RACE kit.We obtained a 705bp nucleotide(D6D3) finally. The fragment is overlapped 36bp with partial CDS.A 2153bp cDNA was obtained through DNAMAN linking.二,The detection for expression of chickenΔ~6-desaturase gene in different tissues by semi-quantitative PCRIn order to detect the expression of chickenΔ~6-desaturase gene in different tissues,we detected the mRNA relative content ofΔ~6-desaturase in liver,heart and lung etc.The result showed that the defference of theΔ~6-desaturase mRNA relative content of liver ,spleen, pancreas and lungs was not significant;liver was higher significantly than breast;the difference of breast,cardiac and abdominal fat was not significant; the lung was significantly higher than that of the bodiminal fat .we didn,t detectΔ~6-desaturase in the stomach muscle.