【作者】 白义春；

【导师】 康相涛；

【作者基本信息】 河南农业大学 ， 动物遗传育种与繁殖， 2008， 硕士

【摘要】 多不饱和脂肪酸(PUFA)在机体内具有一系列重要的生理功能。Δ~6-脂肪酸脱氢酶则是形成多不饱和脂肪酸的限速酶。基于Δ~6-脂肪酸脱氢酶在多不饱和脂肪酸形成过程中的重要作用,本实验开展了对固始鸡Δ~6-脂肪酸脱氢酶基因的研究工作。本实验通过两次RT-PCR和一次3,RACE,获得了全长为2141bp的cDNA,该cDNA具有一个长度为1335bp、编码443个氨基酸的开放阅读框。这是Δ~6-脂肪酸脱氢酶基因首次在鸡上的成功克隆。以得到的鸡的Δ~6-脂肪酸脱氢酶基因序列为搜索对象,通过Contig分析,获得了鸡Δ~6-脂肪酸脱氢酶基因编码序列的内含子和外显子边界,序列分析表明该基因含有12个外显子和11个内含子。通过半定量PCR,比较脂肪酸脱氢酶在不同组织中的表达,发现在肝脏中表达量最大。该试验为进一步研究该基因在脂肪酸形成过程中的作用奠定了基础。本研究共分两个部分:试验一Δ~6-脂肪酸脱氢酶基因的克隆1Δ~6-脂肪酸脱氢酶基因部分编码序列的克隆根据GenBank上预测的鸡的Δ~6-脂肪酸脱氢酶基因序列XM421053设计引物,进行RT-PCR,扩增出一个长度为1323bp的片段(D6D1)。该序列与人、大鼠、小鼠、牛等动物的核苷酸同源性达到74%以上。2Δ~6-脂肪酸脱氢酶基因5,端的克隆将已获得的部分编码序列作为搜索对象,在NCBI网站进行Blast,获得了一段鸡EST(BI390105)。该序列与上述克隆的部分编码序列的5,端有一部分重复序列。根据该EST设计基因特异性引物进行5,端的扩增。获得了一个283bp的片段(D6D2)。该片段与部分编码序列有99bp的重叠区,表明所得到的片段为同一基因的部分序列。3、Δ~6-脂肪酸脱氢酶基因的3,UTR的克隆为了获得固始鸡Δ~6-脂肪酸脱氢酶基因的3,非翻译区端,根据所获得的部分编码序列设计基因特异性引物,利用3,RACE试剂盒进行3,末端cDNA的扩增,得到一个706bp的片段(D6D3)。该片段与部分编码序列的3,端有36bp的重叠区。通过DNAMAN将三个克隆片段连接,得到了2141bp的cDNA全长。试验二、利用半定量PCR检测鸡△6-脂肪酸脱氢酶在不同组织中的表达为了检测△6-脂肪酸脱氢酶在不同组织中的表达,利用半定量PCR检测△6-脂肪酸脱氢酶在肝脏、心脏、肺脏等组织中的mRNA的相对含量。结果表明,肝脏、脾脏、胰脏、肺脏中的Δ~6-脂肪酸脱氢酶mRNA相对含量差异不显著;肝脏显著高于胸肌;胸肌、心脏和腹脂的差异不显著;肺脏显著高于腹脂。而Δ~6-脂肪酸脱氢酶在肌胃中未见有表达。鸡Δ~6-脂肪酸脱氢酶基因的克隆及在不同组织中的表达为进一步研究Δ~6-脂肪酸脱氢酶在多不饱和脂肪酸的形成过程中的作用奠定了一定的基础。更多还原

【Abstract】 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.更多还原