球等鞭金藻中Δ5去饱和酶基因的克隆与功能鉴定

ISOLATION AND FUNCTIONAL ANALYSIS OF A Δ5 DESATURASE GENE FROM ISOCHRYSIS GALBANA

球等鞭金藻(Isochrysis galbana)是一类单细胞海洋微藻,富含二十二碳六烯酸(DHA,22:6Δ4,7,10,13,16,19)。我们利用RACE的方法从球等鞭金藻cDNA文库中同源克隆到一个大小为1329 bp的cDNA片段,编码442个氨基酸的多肽,分子量约49.9 kD。生物信息学分析表明,其编码产物N端具有细胞色素b5结构域,以及与电子传递有关的三个富含组氨酸的结构域,与Pavlova salinaΔ5去饱和酶同源性最高,达56%,故将该基因命名为IgD5。酿酒酵母功能鉴定实验表明,其编码的蛋白质具有Δ5去饱和酶活性,能够将二高-γ-亚麻酸(DGLA,20:3Δ8,11,14)转化成花生四烯酸(AA,20:4Δ5,8,11,14),转化效率平均为34.6%,最高可达40.3%。

The very long chain （〉~C20） polyunsaturated fatty acids （VLCPUFA）, such as arachidonic acid （AA; 20：4036）, eicosapentaenoic acid （EPA; 20：5033） and docosapentaenoic acid （DHA; 22：6033） are essential for human health and nutrition. Dietary supplementation with these fatty acids is not only helpful for fetal neuronal, but also can reduce the risk of cardiovascular disease, hypertension, inflammatory and other diseases. Human beings can synthesize these fatty acids from the two essential fatty acid, linoleic acid （LA; 18：2036） and ct-linolenic acid （ALA; 18：3033）, which must be obtained from the diet. However, the synthetic efficiency is somewhat limited, and can not meet our daily requirement. AA can be obtained easily from meat, egg and milk, often in excessive amounts, EPA and DHA, however, can only be obtained from marine fish that are so often omitted from the modern diet, in much lower amounts than required. This shortage is further worsened by the fact that the natural marine fish resources have been depleting fast in recent years. In addition, the recent findings of toxic chemicals in fish oil has created fears for the consumption of fish products hence reduced the intake of EPA and DHA even further. Therefore, alternative source of these VLCPUFAs are therefore de- sirable. To obtain them from oil plants in commercial and sustainable quantities is particularly attractive. However, no oil-seed species produce such products naturally. The VLCPUFA biosynthesis pathways in organisms such as filamen- tous fungus and marine microalgae have been elaborated. Accordingly, genes encoding for elongases and desaturases involved in their metabolic pathway have been cloned from a variety of organisms including algae, mosses, fungi, nematodes and humans in the last 10 years. The reconstruction of the VLCPUFAs metabolic pathway into higher plants has been achieved in Arabidopsis, linseed, mustard and soya bean by introducing a set of 3--9 fatty acid desaturase and elongase genes. However, the production of VLCPUFAs, especially DHA, in these transgenic plants is somewhat low, much lower than that found in the EPA and DHA producing microorganisms from which these genes were originally isolated. This may be attributed to the usual ＇pick-and-mix＇ strategies to choose the gene set to produce these transgen- ics. We aim to mine the whole set of enzymes from the same organism and use them as a gene set for the production of EPA and DHA in oilseed plants. Isochrysis galbana, a marine microalga, rich in docosahexaenoic acid （DHA, 22：6n-3, △^4, 7,10,13,16,19）, was chosen in this study. The A8 desaturation pathway may be operating in the production of EPA/DHA in this organism, and this pathway was thought to be more efficient over the conventional A6 desaturation pathway. Previously, two of the five genes involved in the conversion of C 18 fatty acid substrates to the final product DHA have been isolated. The first one was the A9 elongase gene that catalyses the first step in the DHA biosynthesis pathway, the elongation of LA to EDA and A LA to ETrA. The second one was a A4 desaturase, which was involved in the final step of the biosynthesis of DHA. Both enzymes showed high catalytic activities and also had restricted substrate specificity. Here, we reported the isolation of the third gene, a A5 desaturase gene that was capable to convert DGLA to AA. That was achieved by the RACE strategy using different degenerate primers based on the conserved motifs of known desaturase sequences to isolate a partial eDNA from an/. galbana eDNA library. The full length eDNA was subsequently assembled and it consisted of 1329 nucleotides, encoding a protein of 442 amino acids with predicted molecular mass of 49.9 kD. Bioinformatics analysis showed that it shared homol- ogy with other functionally known front-end fatty acid desaturases and the highest homology of 56% was found with a A5 desaturase from Pavlova salina. As characterized by this family of desaturases, it contained an N-terminal cytochrome b5 domain, and three histidine rich motifs （his-boxes） related to electron transfer. Functional analysis by expression in Sac- charomyces cerevisiae revealed that it could convert DGLA （20：3△^8,11, 14） to AA （20：4△^5, 8,11,14） by introducing a double bond in the acyl chain at the A5 position, indicating that this newly isolated cDNA sequence encodes a protein that specifically catalyzes for the conversion of C20-A5-polyunsaturated fatty acid, AA, hence it was designated as IgD5.