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Cloning, Expression of Δ-15 Fatty Acid Desaturase Gene and Activity Assay

Author: NiuLiHong
Tutor: ZhangJie
School: Chinese Academy of Agricultural Sciences
Course: Biochemistry and Molecular Biology
Keywords: Delta-15 fatty acid desaturase Chlamydomonas reinhardtii Alpha-linolenic acid Monodus subterraneus Transcriptome
CLC: Q78
Type: Master's thesis
Year: 2011
Downloads: 15
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Abstract


Polyunsaturate fatty acids (PUFAs) are vital to the health of human by playing important roles in prevention and treatment of several diseases, such as, cardiovascular disease. Alpha-linolenic acid (ALA) as the one of theω-3 PUFAs also plays important role in the health of human, the more important role is that ALA is the precursor of the long chain PUFAs, such as, eicosapentaenoic acid (EPA) and docosahexenoic acid (DHA). Two of the microalgaes Chlamydomonas reinhardtii and Monodus subterraneus are full of PUFAs, especiallyω-3 PUFAs. Delta-15 fatty acid desaturase is the key enzyme of the biosynthesis of ALA, which introduces a double bond between the existing double bond and carboxyl terminus at theΔ-15 position of fatty acyl chain of linoleic acid. In this study, the whole length gene of theΔ-15 fatty acid desaturase from C. reinhardtii and Arabidopsis thaliana were cloned with the method of RT-PCR, and the biological function of the genes were verified via the transformants of Pichia pastoris and tobacco. At the same time, the transcriptome of M. subterraneus was sequenced and analyzed with bioinformatics software to find the new genes of the biosynthesis of PUFAs. The main results are as follows:Total RNA of the exponential phases cell of C. reinhardtii and the fresh leaf of A. thaliana were extraceted and purified, the genes were cloned with the method of RT-PCR, and subcloned in the pMD19-T vector, then was sequenced. The nucleotide sequences were analyzed with Blast in the NCBI. The full length of the gene ofΔ-15 fatty acid desaturase from C. reinhardtii was 1,362 bp, encoding 419 amino acid recidues, and that from A. thaliana was 1,525 bp, encoding 447 amino acid recidues.Both of theΔ-15 fatty acid desaturase genes were inserted into Pichia pastoris expression vector pPIC3.5K to generate respectively the recombinant plasmid p3.5K-Ld15 and p3.5K-Nd15, which were subsequently transformed into Pichia pastoris strain GS115 for heterologous expression by electroporation method. After PCR detection, total fatty acids of Pichia pastoris were extracted from the induced cells and methylated. The resultant fatty acids methyl esters were analyzed with gas chromatography (GC), the results showed thatΔ-15 fatty acid desaturase gene were expressed in the Pichia pastoris. Compared with the transformant of pPIC3.5K, the contents of linoleic acid of the transformant of lyd15 were reduced from 44.3% to 26.9%, and that of ALA were elevated. The results of GC analysis of the total fatty acid methyl esters of the transformants of Pichia pastoris show that theΔ-15 fatty acid desaturase of C. reinhardtii introduces a double bond between the existing double bond and carboxyl terminus at theΔ-15 position of fatty acyl chain for linoleic acid to produce ALA. Meanwhile both of theΔ-15 fatty acid desaturase genes were inserted into the tobacco express vector pCAMBIA2300 to generate respectively recombinant plasmid p23-Ld15 and p23-Nd15, which were subsequently transformed into Agrobactrium tumefaciens strain LBA4404. Tobacco was transformed with co-cultivating leaf dics with the transformatants of Agrobactrium. The regenerated Kanamycin-resistant transformants were analyzed with PCR, the results show that both of theΔ-15 fatty acid desaturase genes had been inserted into the genome of tobacco. Subsequently, both of theΔ-15 fatty acid desaturase genes had expressed in the transformatants of tobacco with the method of RT-PCR.Monodus subterraneus is a microalga rich in polyunsaturated fatty acid (PUFA) especially eicosapentaenoic acid (EPA, 20:5ω-3). Since the biosynthesis of PUFA in M. subterraneus is different from other species, cDNA sequencing was processed in order to clone noval efficient genes of the biosynthesis of PUFAs. 26,903 expressed sequence tags (ESTs) were obtained. The number of the sequences ranging from 500 bp to 4,000 bp in length was 10,792. Among those unigenes, the 5,270 (30.6%) were hit with the clusters of Kyoto Encyclopedia of Genes and Genomes (KEGG). About 34 unigenes encoding PUFA biosynthesis enzymes were identified, included 6 desaturase genes and 4 elongase genes. These genes may facilitate the elucidation of EPA biosynthetic pathway in transgenic plant.

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