CN104250650A

CN104250650A - Delta-6 fatty acid dehydrogenase gene and application thereof

Info

Publication number: CN104250650A
Application number: CN201410360120.2A
Authority: CN
Inventors: 张琦; 杨晓霞; 魏云林; 林连兵; 季秀玲
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2014-07-28
Filing date: 2014-07-28
Publication date: 2014-12-31

Abstract

The invention discloses a nucleotide sequence isolated from Mortierella isabellina M6-22 and encoding delta-6 fatty acid dehydrogenase, the nucleotide sequence is shown as SEQIDNO:1, and the amino acid sequence encoding by the gene is shown as SEQIDNO:2. Through the construction of recombinant vector and expression in Saccharomyces cerevisiae, the expression product has the functions of delta 6-fatty acid dehydrogenase, and can catalyze the linoleic acid to convert into gamma-linolenic acid.

Description

A kind of Δ 6-fatty acid dehydrogenase gene and application thereof

Technical field

The invention belongs to biological technical field and field of genetic engineering, relate to from a kind of filamentous fungus---the Mortierella isabellina of Mucor ( mortierella isabellina) Δ of cloning in M6-22 ⁶-fatty acid dehydrogenase gene, and this gene is directly connected with different carriers, proceed in bacterium, yeast, plant or animal, utilize its coded delta ⁶-fatty acid dehydrogenase produces and comprises the polyunsaturated fatty acids such as gamma-linolenic acid.

Background technology

Gamma-linolenic acid (γ-linolenic acid) is a kind of Multifunctional grease composition, it is the polyunsaturated fatty acid of needed by human, there is multiple important physiological activity, the physiological regulating control and substance metabolism of body play an important role, as reducing blood-fat, antithrombotic cardiovascular and cerebrovascular, prevention and therapy hypertension, atherosclerosis, strengthen the immunologic function of human body, also have antitumor, anti peroxidation of lipid, suppress the effect [Dong Jieming etc. of ulcer and the enhancing aspect such as Regular Insulin and fat-reducing, 2003, health research, 32 (3): 299-301].Especially the medical value in diabetes, cardiovascular disorder and cancer, gamma-linolenic acid has become the focus of academia's research.

Δ ⁶-fatty acid dehydrogenase energy specific catalytic linolic acid (linoleic acid, C18:2 Δ ^9,12) and alpha-linolenic acid (α-linolenic acid, C18:3 Δ ^9,12,15) carboxyl terminal the 6th and the 7th carbon atom between insert a new double bond, dehydrogenation forms gamma-linolenic acid (γ-linolenic acid, C18:3 Δ respectively ^6,9,12) and therapic acid (stearidonic acid, C18:4 Δ ^6,9,12,15), they form the polyunsaturated fatty acid (long-chain polyunsaturated fatty acid, LC-PUFAs) of other long-chain more further by carbochain extension and dehydrogenation, therefore Δ ⁶-fatty acid dehydrogenase is the key enzyme of synthesis of long-chain polyunsaturated fatty acids in people and mammalian body.These long chain polyunsaturated fatty acidss are body tissue microbial film moietys, play effect [the Napier JA et al maintaining cell normal function and increase body resistance, 1999, Curr.Opin.Plant Biol, 2:123-127], be also precursor [Horrobinn DF, 1992 that prostaglandin(PG), prostacyclin and leukotrienes etc. have the self-regulation thing of strong physiologically active simultaneously, Prog Lipid Res., 31:163-194].Δ ⁶the reduction of-fatty acid enzymic activity reduces causing the resultant quantity of gamma-linolenic acid, causes the various disorders of human body, and then causes various disease.Therefore, from different organism, Δ is cloned ⁶-fatty acid dehydrogenase gene, then proceed to different host cells after connecting from different expression vector and express, utilizes host to produce gamma-linolenic acid and other long chain polyunsaturated fatty acidss have very big application prospect.

Because people more and more recognize the importance of gamma-linolenic acid and other long chain polyunsaturated fatty acidss, therefore attention is turned to again Δ ⁶in the research of-fatty acid dehydrogenase.Δ ⁶-fatty acid dehydrogenase belongs to a class film intergrase, the difficulty due to separation andpreconcentration embrane-associated protein very large [MaKeon, 1981, Enzymology method, 12141-12147; Wang etc., 1988, plant physiology biological chemistry, 26:777-792], therefore conventional biochemical method is difficult to obtain comprising Δ ⁶-fatty acid dehydrogenase, and can only by carrying out preliminary study at interior various embrane-associated protein character desaturase superior bodies to the nucleotide sequence of enzyme, for Δ ⁶the biological function verification of-fatty acid dehydrogenase, many laboratories have employed clones relevant gene and proceeds in various recipient cell from various organism, in recipient cell, then observe the method that its lipid acid changes.The result of alignment shows, coded delta ⁶the nucleotide sequence of-fatty acid dehydrogenase has common mechanism characteristics: there are three Histidine conserved regions, forms the structure [Kyte et al, 1982, J.Mol.Biol.157:105-132] of tetratransmembrane, in addition, except cyanobacteria, and Eukaryotic Δ ⁶the aminoterminal of-fatty acid dehydrogenase is all containing being similar to cytochrome b5 (Cytb5) haem bonding pad (HPGG) [Napier JA, Sayanova O, Stobart AK, et al., 1997, Biochem.J., 157:105-132], these are all that maintenance dehydrogenase activity is necessary.

At present, from the different sourcess such as animal, plant, microorganism, many Δs are separated to by different kinds of molecules biological method ⁶the homologous sequence of-fatty acid dehydrogenase gene, and prove that there is corresponding biological function, but, although obtain a lot of Δ ⁶the gene order of-fatty acid dehydrogenase, but the technical difficulty existed in the purifying of enzyme, still not do not disclose its biochemical characteristic physical environment from enzyme level at present.Therefore, can only by means of the Δ excavating more different sources ⁶-fatty acid dehydrogenase gene sequence, carrys out more deep announcement Δ by gene sequencing ⁶the biological function of-fatty acid dehydrogenase, and for the in the future purifying of enzyme and the announcement of biochemical characteristic and further application lay the first stone.In addition, the dehydrogenase gene catalytic activity that these different sourcess are separated to is lower and great majority do not have strict specificity to fatty acid substrate chain length, double bond number and position of double bond, except target product, also can produce some non-specific lipid acid.Therefore, need to continue the Δ that screening has high substrate specificity ⁶-fatty acid dehydrogenase gene, and using gene engineering technique builds the transgenic rape tannin plant that specificity produces polyunsaturated fatty acid, directly improves oil quality, more has actual application value.This research excavates efficient, the specific gene of polyunsaturated fatty acid synthesis, for the quality-improving of transgenosis fatty acid product of new generation lays the foundation by multiple means.

Summary of the invention

An object of the present invention is to provide a kind of from Mortierella isabellina ( mortierella isabellina) be separated the Δ obtained in M6-22 ⁶the amino acid of-fatty acid dehydrogenase gene and this genes encoding, the fragment of this gene nucleotide series or nucleotide sequence is as shown in SEQ ID NO:1, or with the nucleotide sequence of SEQ ID NO:1 complementation, this gene order is long is 1515bp(base), wherein 1-1515 is coded delta ⁶the open reading frame of-fatty acid dehydrogenase mature polypeptide; Polypeptide as shown in SEQ ID NO:2 of the aminoacid sequence of this genes encoding or its fragment.

Another object of the present invention is to provide a kind of containing Δ ⁶the recombinant expression vector of-fatty acid dehydrogenase gene, be gene shown in SEQ ID NO:1 is directly connected from different expression vector (plasmid, virus or vehicle) constructed by recombinant vectors.

The Δ deriving from Mortierella isabellina M6-22 containing coding can be built by method well-known to those having ordinary skill in the art ⁶the expression vector of the nucleotide sequence of-fatty acid dehydrogenase and suitable to transcribe/translational control element.These methods comprise [Sambroook, et al, the Molecular Cloning such as recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology, a Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1989].Described coding Mortierella isabellina M6-22 Δ ⁶the nucleotide sequence of-fatty acid dehydrogenase can be effectively connected in the appropriate promotor of expression vector, synthesizes to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; The PL promotor of lambda particles phage; Eukaryotic promoter comprise CMV early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, the LTRs of retrovirus and some other known can the promotor expressed in prokaryotic cell prokaryocyte or eukaryotic cell or its virus of controlling gene.Expression vector also comprises the ribosome bind site and transcription terminator etc. of translation initiation.Inserting enhancer sequence in the carrier will make its transcribing in higher eucaryotic cells be enhanced.Enhanser is the cis-acting factors that DNA expresses, and usual nearly 10-300bp, acts on promotor transcribing with enhancing gene, as adenovirus cancers.

Another object of the present invention is to provide a kind of containing Δ ⁶the host cell of-fatty acid dehydrogenase gene or above-mentioned recombinant expression vector, this cell is bacterial cell, fungal cell, vegetable cell or zooblast, or the offspring of these host cells, and the representative example of host cell has: intestinal bacteria; Fungal cell or yeast; Vegetable cell is as rape, tobacco, soybean; Insect cell is as fruit bat S2 or Sf9; Zooblast is as CHO, COS or Bowes melanoma cells etc.

Optimize host cell with nucleotide sequence of the present invention or the recombinant vectors containing nucleotide sequence to be undertaken by method well-known to those having ordinary skill in the art.When host be prokaryotic organism as intestinal bacteria time, the competent cell that can absorb DNA can collect thalline in exponential phase of growth, uses CaCl ₂, the method such as electroporation carries out.When host is eukaryote, DNA infection protocol can be selected.The methods such as microinjection, electroporation, liposome packaging.

Nucleotide sequence of the present invention is DNA form, DNA form comprises the DNA of cDNA, genomic dna or synthetic, DNA can be strand or double-strand, and the coding region sequence of encoding mature polypeptide can or the varient of degeneracy identical with the coding region sequence shown in SEQ ID NO:2.

The present invention also comprises the Δ of encoding and obtaining from Mortierella isabellina M6-22 ⁶the fragment of the nucleotide sequence of-fatty acid dehydrogenase.As used in the present invention, term " fragment " refers to the nucleotide sequence of the polypeptide of identical biological function or the activity can encoded and substantially keep the present invention natural.By elaboration herein, such fragment, to be considered within the ken of those skilled in the art.

Polypeptide in the present invention and nucleotide sequence preferably provide with the form be separated, and are more preferably purified to homogeneous.

In the present invention, specific nucleotide sequence can obtain by multiple method.Such as, with hybridization technique isolated nucleic acid sequence well known in the art.These technology including, but not limited to: (1) hybridizes the nucleotide sequence to detect homology with probe and genome or cDNA library; (2) resistance screening of expression library is to detect the nucleotide sequence fragment of the clone with structural features.

Sequence dna fragment of the present invention also can obtain by following method: (1) is separated double chain DNA sequence from genomic dna; (2) chemical synthesising DNA sequence is to obtain the double-stranded DNA of described polypeptide.

In the above-mentioned method mentioned, isolation of genomic DNA is least conventional.The direct chemical synthesis of DNA sequence dna is the method often selected.The method more often selected is the separation of DNA sequence dna.The standard method being separated interested cDNA is donorcells separating mRNA from this gene of high expression level and transcribes, and forms plasmid or phage cDNA library.The method extracting mRNA has multiple proven technique, also can obtain from commercial channels with test kit.And construction cDNA library is also usual method.When in conjunction with polymerase chain reaction technology (PCR), even if few expression product also can be cloned.

Nucleotide sequence provided by the invention is a kind of efficient specific fatty acid dehydrogenase, be converted in microorganism cells body after it can being connected with carrier and produce unsaturated fatty acids, have product specificities high, with short production cycle, produce by place, weather, the impact in season and utilize different bacterial classifications and substratum to be applicable to the advantages such as development function grease, may be used for commercially producing, directly improve oil quality, more there is actual application value.Using gene engineering technique of the present invention builds the transgenic yeast cell production unsaturated fatty acids that specificity produces polyunsaturated fatty acid, have simple to operate, cost is low, feasibility advantages of higher, for the quality-improving of transgenosis fatty acid product of new generation lays the foundation.

Accompanying drawing explanation

Fig. 1 is the Δ of Mortierella isabellina M6-22 of the present invention ⁶-fatty acid dehydrogenase and Zygosaccharomyces rouxii Δ ⁶the amino acid sequence homology comparison diagram of-fatty acid dehydrogenase; Wherein MID6-1 is Mortierella isabellina M6-22 Δ of the present invention ⁶-fatty acid dehydrogenase, MRD6 is Zygosaccharomyces rouxii Δ ⁶-fatty acid dehydrogenase, Consensus is aminoacid sequence identical between two sequences;

The saccharomyces cerevisiae expression pY3MID6-1 of Fig. 2 constructed by the present invention;

Fig. 3 is the gas chromatogram that the present invention contains the transgenic yeast of pYES3/CT empty carrier;

Fig. 4 is the gas chromatogram that the present invention contains the transgenic yeast of recombinant plasmid pY3MID6-1.

Embodiment

Below in conjunction with drawings and Examples, the present invention is described in further detail, but scope is not limited to described content, the reagent used in embodiment and method, if no special instructions, all adopts conventional reagent and use ordinary method.

Embodiment 1: be separated Δ from Mortierella isabellina M6-22 ⁶the nucleotide sequence of-fatty acid dehydrogenase

According to the RNAseq analytical results of Mortierella isabellina M6-22, find a coded delta ⁶the mRNA sequence of-fatty acid dehydrogenase, analyzes the encoding sequence of acquisition one section coding whole protein further, and according to encoding sequence design and synthesis Auele Specific Primer (primer 1 and primer 2).Mortierella isabellina M6-22 is inoculated in PDA liquid nutrient medium simultaneously, cultivate 48h for 28 DEG C, collect mycelium, hypha powder is become by liquid nitrogen grinding, then adopt eukaryotic cell total RNA extraction reagent box (Promega Products) to extract the total serum IgE of Mortierella isabellina M6-22, recycling Reverse Transcription box (fermentas Products) reverse transcription synthesis Article 1 cDNA, with this cDNA for template, carry out PCR reaction with primer 1 and primer 2, react component used and amplification condition as follows:

Primer 1:MID6-1F1:5`-ATGCCTCCTAACAATAGGGCTATCGA-3`

Primer 2: MID6-1R1:5`-TCACGCATGAGGTGCTACCATCTCT-3`;

PCR amplification system (25 μ L) is composed as follows:

10×Ex Taq Buffer 2.5 μL

dNTP（2.5 μmol/L） 2 μL

Template 3 μ L

MID6-1F1（10 μmol/L） 2 μL

MID6-1R1（10 μmol/L） 2 μL

Ex Taq DNA polymerase（5U/μL） 2 μL

Aseptic ddH ₂o complements to 25 μ L

Amplification condition: 94 DEG C of sex change 4min, then carry out 30 circulations with 94 DEG C of 1min, 58 DEG C of 1min, 72 DEG C of 2min, amplified production is connected in pMD18-T carrier according to method in partial sequence amplification, sends order-checking after checking is correct.Result display the clip size obtained that increases be 1515bp, by the aminoacid sequence of its coding in Genebank database with Blast(Basic Local Alignment Search Tool) program carries out Homology search, result for retrieval shows that the homologous fragment the most similar to this fragment is Δ ⁶-fatty acid dehydrogenase, but and incomplete same, prove that the fragment increased is the Partial Fragment of new potential dehydrogenase gene.

Embodiment 2: the homology search of institute's isolated nucleic acid sequence

By the Δ inferred ⁶the aminoacid sequence of-fatty acid dehydrogenase carries out homology search on Genbank, and gained homologous sequence major part is the sequence of coded delta 6-fatty acid dehydrogenase, and minority is the sequence of putative protein, selects the Δ having done functional verification ⁶-fatty acid dehydrogenase does tetraploid rice, wherein with Zygosaccharomyces rouxii ( mucor rouxii) Δ ⁶-fatty acid dehydrogenase (AF290983_1) similarity is the highest, is 56%.(Fig. 1, MID6-1 are Mortierella isabellina M6-22 Δ of the present invention ⁶-fatty acid dehydrogenase, MRD6 is Zygosaccharomyces rouxii Δ ⁶-fatty acid dehydrogenase, Consensus is aminoacid sequence identical between two sequences, and this illustrates that the enzyme coded by nucleotide sequence provided by the invention is potential new Δ ⁶-fatty acid dehydrogenase.

Embodiment 3: the structure of yeast saccharomyces cerevisiae recombinant expression vector

Coding region sequence according to SEQ ID NO:1, design one pair of genes specificity amplification primer (primer 3 and primer 4):

Primer 3:MID6-1F2:5`-CG gGATCCaTGCCTCCTAACAATAGGGCTA-3`

Primer 4:MID6-1R2:5`-AT gCGGCCGCtCACGCATGAGGTGCTACCATC-3`

5 ‵ of these two primers hold underscore part to contain respectively bamh I He noti restriction enzyme site, amplification condition used and reactive component and with cDNA be template PCR reacting phase with, the sequencing result display of amplified production is consistent with the sequence of 1-1515bp shown in SEQ ID NO:1; Then 25ul PCR primer and 10ul pYES3/CT(Invitrogen company is got) carry out double digestion respectively, electrophoresis reclaims enzyme and cuts large fragment, and connect 4h with T4 ligase enzyme at 16 DEG C, connect product conversion intestinal bacteria HD5 α, by plasmid extraction and PCR screening positive clone, and carrying out order-checking qualification, plasmid construction the results are shown in Figure 2, and constructed contains Δ ⁶the expression vector called after pY3MID6-1 of-fatty acid dehydrogenase gene.

Embodiment 4: yeast saccharomyces cerevisiae recombinant expression vector and empty carrier transformed saccharomyces cerevisiae cell

The mono-bacterium colony of picking Wine brewing yeast strain INVSc1 is in 5ml YEPD liquid nutrient medium, and 30 DEG C of incubator overnight are cultivated, and the inoculum size by 1% is transferred in the YEPD liquid nutrient medium of 100ml, and 30 DEG C of shaking tables shake to OD600 between 1.3-1.5, 4 DEG C, 4000g centrifugal 5min collecting precipitation cell, with the aseptic ddH of 100ml precooling ₂o washed cell, 4 DEG C, 4000g centrifugal 5min collecting precipitation cell, then use the aseptic ddH of 50ml precooling ₂o washed cell, 4 DEG C, 4000g centrifugal 5min collecting precipitation cell, with the 1M sorbitol washes cell of 20ml precooling, 4 DEG C, 4000g centrifugal 5min collecting precipitation cell, the 1M sorbyl alcohol of the precooling of cell 0.5ml suspends, every 80ul is sub-packed in the centrifuge tube of 1.5ml precooling, get 1ug linearizing recombinant expression vector pY3MID6-1 and empty carrier pYES3/CT to join respectively in the competent cell of 80ul, go in the electric revolving cup of 0.2cm after mixing, hatch 5min on ice, proceed in electroporation (the electric shock condition: voltage 1.5kV that shocks by electricity, electric capacity 25uF, resistance 200 Ω, the electric shock time is 4-10ms), add the sorbyl alcohol of 1ml 1M precooling after electric shock immediately and blow even, 30 DEG C hatch 2h after coat SC-Trp(respectively without tryptophane) on Selective agar medium, put 30 DEG C and cultivate 3-4 days.

Embodiment 5: the abduction delivering of Yeast engineering bacteria and the gas chromatographic analysis of fatty acid methyl ester

Picking has transformed recombinant expression vector pY3MID6-1 and empty carrier pYES3/CT and the positive transformant occurred on SC-Trp Selective agar medium (tryptophane synthesis defective type substratum) flat board respectively, be inoculated in 15ml liquid SC-Trp Selective agar medium (containing 2% glucose) respectively, 30 DEG C are shaken bacterium and cultivate 24h, transfer in the SC-Trp Selective agar medium (containing the semi-lactosi of 2% and the raffinose of 1%) of 100ml, its initial OD 600 is made to be 0.4, add the xenobiotic substrates linolic acid of 0.5mmol/L, 30 DEG C are continued shaking culture 72h, 4000g collected by centrifugation thalline, with deionized water wash three times, thalline is in 50 DEG C of oven dry, grind, get the KOH-CH that 100mg dry mycelium powder adds 5ml 5% ₃in OH solution, 70 DEG C of reaction 3h, are cooled to room temperature after reaction terminates, by the pH value of the hydrochloric acid conditioning solution of 6M to 2.0, add 4ml 14%BF ₃-CH ₃oH, 70 DEG C of reaction 1.5h, synthesizing fatty acid methyl ester, add the NaCl solution that 10ml is saturated again, concuss mixes, be transferred in separating funnel, chloroform with 8ml 1:4: hexanaphthene extracting twice, merge the extracting solution of twice, solvent nitrogen containing fatty acid methyl ester is dried up, with the normal hexane back dissolving sample of 200ul, finally with 0.45um filtering with microporous membrane removing large granular impurity.

Embodiment 6: lipid acid gas chromatographic analysis

Gas chromatographicanalyzer device is adopted to analyze the abduction delivering situation of transgenic yeast engineering bacteria, gas chromatographicanalyzer device is Shimadzu GC-7A, pillar: fused-silica capillary column, 0.32 × 30m, solid support: Dienthyeneglycol succinate plated film thing: polyimide.Carrier gas: N2, linear speed 10cm/s.Splitting ratio: 100:1, vaporizer temperature: 250 DEG C, column temperature: 180 DEG C, tail blows: 50ml/min, detector: flame ionization ditector.The methyl esterification of fatty acid sample of above-mentioned preparation is carried out GC analysis, and applied sample amount is 1ul; Analysis software: Anstar.

Stratographic analysis the results are shown in Figure the transgenic yeast of 3,4, Fig. 3 displays containing pYES3/CT empty carrier, and Fig. 4 display is containing the transgenic yeast of recombinant plasmid pY3MID6-1; Compared with Fig. 3, in Fig. 4, retention time is that the peak that 15.05min is corresponding is consistent with gamma-linolenic acid methacrylate calibration, is Mortierella isabellina M6-22 Δ ⁶the gamma-linolenic acid that-fatty acid enzyme catalysis linolic acid produces.

Sequence table

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Asn Ile Leu His MET Pro Lys Thr Gln Ala Pro Leu Ala Ser His

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Ala Pro Thr Thr Ser Asn Asn Gln Ile Asp Val Asp Lys Ser Gln

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Ser Ile Ile Thr Arg Leu Tyr Asn Asp His MET Lys Val Ile Gln

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Glu Gln Lys Ala Arg Pro Gln Ala Asp Ile Gln Thr Val Arg Phe

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Lys Cys Asn Tyr Trp Lys Tyr Gly Lys Glu Cys Cys Arg Tyr Val

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Leu Phe Ile Tyr Leu Ala Val Tyr Leu Ile Leu Lys Gly Thr Gln

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Thr Trp His Tyr MET Leu Ser Ala Val Phe MET Ala Cys Phe Trp

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His Gln Leu Thr Phe Thr Ala His Asp Ala Gly His Asn Gly Ile

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Thr Gly Lys Tyr His Ile Asp His Leu Ile Gly Ile Phe Ile Ala

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Asp Phe Leu Gly Gly Leu Ser Ile Gly Trp Trp Lys Asn Asn His

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Asn Val His His Ile Val Thr Asn Asp Pro Ala His Asp Pro Asp

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Ile Gln His MET Pro Phe Phe Ala Ile Thr Thr Arg Ile Phe Asn

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Thr Tyr Ser Ser Tyr Tyr His Arg Val MET Lys Leu Asp Ala Ala

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Ala Lys Phe Phe Val Arg His Gln His Lys Leu Tyr Tyr Ile Ile

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Leu Ser Leu Gly Arg Phe Asn Leu His Asn Leu Ser Phe Val Phe

320 330

Leu Phe Thr Ala Pro Arg Val Lys Tyr Arg Trp Leu Glu Ile Phe

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Gly Ile Cys Val Phe Phe Thr Trp Tyr Ser Tyr Leu Leu Ser Phe

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370

Thr Leu Thr Val Pro Leu His Ile Gln Ile Thr Leu Ser His Phe

380 390

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Claims

1. a Δ ⁶-fatty acid dehydrogenase gene, its nucleotide sequence as shown in SEQ ID NO:1, the aminoacid sequence of encoding as shown in SEQ ID NO:2.

2. a host cell, described host cell contains Mortierella isabellina Δ according to claim 1 ⁶-fatty acid dehydrogenase gene.

3. Δ according to claim 1 ⁶-fatty acid dehydrogenase gene is producing the application in polyunsaturated fatty acid.