Wang De-Pei, Sun Wei, Li Ming-Chun, Wei Dong-Sheng, Zhang Ying-Hui, Xing Lai-Jun
Tianjin Key Laboratory of Microbial Funitional Genomics, Department of Microbiology, NanKai University, Tianjin 300071, China.
Sheng Wu Gong Cheng Xue Bao. 2006 Jul;22(4):581-6.
Thamnidium elegans is a kind of phycomycete that produces essential unsaturated fatty acids, particularly y-linolenic acid. In this process, delta6-Fatty acid desaturase (D6D) plays a key role due to its enzymatic properties that catalyze the delta6 site dehydrogenation of precursor linoleic acid (18:2delta(9, 12) n-6) and a-linolenic acid (18:3delta(9, 12, 15) n-3). This reaction is the first and rate-limiting step of highly unsaturated fatty acids (HUFA) synthesis pathways. After we have isolated and cloned the gene coding delta6-fatty acid desaturase from Thamnidium elegans As3.2806 (GenBank accession number DQ099380), our interest focuses on the promotion and regulation of the gene transcription. To achieve this aim, we designed long primers and used nested inverse PCR to amplify DNA flanking sequences. First, genome of Thamnidium elegans was extracted and digested with restriction enzymes EcoR I and Kpn I , respectively. Then we ligated the digested DNA with T4 ligase at low concentration which is propitious for linear DNA to joint intromolecule. According to the sequence of delta6-fatty acid desaturase gene of Thamnidium elegans, we designed a couple of 35nt long inverse primers and two couples of shorter inverse primers for inverse PCR. Three rounds of PCR reactions were performed. In the primary reaction, the ligated DNA was used as a template, and the product was used as the template of the secondary reaction, the tertiary reaction was achieved in the same way. After all the three rounds of reactions, we got a nice product about 4 kb from the EcoR I digested sample, in which a 1.3kb 5' upstream sequence (GenBank accession number DQ309425) of delta6-fatty acid desaturase gene containing several putative regulatory elements including TATA. box, FSE-2, AP-1 sites, CCAAT cis-element site and STRE-binding site was derived after sequencing. All of these implied intensely that this 1.3kb fragment is a condition-regulated promoter. It is the first report about Thamnidium elegans detla6-fatty acid desaturase gene promoter. The procedure described here is a rapid and simple method and particularly useful to isolate flanking sequences from fungal genome. box, FSE-2, AP-1 sites, CCAAT cis-element site and STRE-binding site was derived after sequencing. All of these implied intensely that this 1.3 kb fragment is a condition-regulated promoter. It is the first report about Thamnidium elegans delta6-fatty acid desaturase gene promoter. The procedure described here is a rapid and simple method and particularly useful to isolate flanking sequences from fungal genome.
雅致枝霉是一种能产生必需不饱和脂肪酸，尤其是γ-亚麻酸的藻状菌。在此过程中，Δ6-脂肪酸去饱和酶（D6D）因其催化前体亚油酸（18:2Δ(9,12)n-6）和α-亚麻酸（18:3Δ(9,12,15)n-3）的Δ6位点脱氢的酶特性而发挥关键作用。该反应是高度不饱和脂肪酸（HUFA）合成途径的第一步且是限速步骤。我们从雅致枝霉As3.2806中分离并克隆了编码Δ6-脂肪酸去饱和酶的基因（GenBank登录号DQ099380）后，我们的兴趣集中在该基因转录的促进和调控上。为实现这一目标，我们设计了长引物并使用巢式反向PCR扩增侧翼DNA序列。首先，分别提取雅致枝霉的基因组并用限制性内切酶EcoR I和Kpn I进行消化。然后我们用低浓度的T4连接酶连接消化后的DNA，这有利于线性DNA分子内连接。根据雅致枝霉Δ6-脂肪酸去饱和酶基因的序列，我们设计了一对35nt长的反向引物和两对较短的反向引物用于反向PCR。进行了三轮PCR反应。在第一轮反应中，连接后的DNA用作模板，产物用作第二轮反应的模板，第三轮反应以同样的方式进行。经过所有三轮反应后，我们从EcoR I消化的样品中得到了一个约4kb的良好产物，测序后从中获得了Δ6-脂肪酸去饱和酶基因1.3kb的5'上游序列（GenBank登录号DQ309425），其中包含几个推定的调控元件，包括TATA盒、FSE-2、AP-1位点、CCAAT顺式元件位点和STRE结合位点。所有这些都强烈表明这个1.3kb的片段是一个条件调控启动子。这是关于雅致枝霉Δ6-脂肪酸去饱和酶基因启动子的首次报道。这里描述的方法是一种快速简单的方法，尤其适用于从真菌基因组中分离侧翼序列。盒、FSE-2、AP-1位点、CCAAT顺式元件位点和STRE结合位点在测序后获得。所有这些都强烈表明这个1.3kb的片段是一个条件调控启动子。这是关于雅致枝霉Δ6-脂肪酸去饱和酶基因启动子的首次报道。这里描述的方法是一种快速简单的方法，尤其适用于从真菌基因组中分离侧翼序列。
