CN101921784B - Gene with Δ6 fatty acid dehydrogenase function and its application - Google Patents

Abstract

The invention utilizes two genes RnD6C and RnD6D with complete reading frames in DNA derived from black tea sugarcane (Ribes nigrum L.) to express the genes in a yeast expression system. GC-MS analysis shows that the proteins coded by the two genes in the yeast can respectively catalyze the additional substrates Linoleic Acid (LA) and alpha-linolenic acid (ALA) to respectively generate gamma-linolenic acid (GLA) and stearidonic acid (SDA) with delta⁶Fatty acid dehydrogenase function. The invention can be applied to the production of GLA and SDA by adopting a gene engineering technology and a lower eukaryotic cell expression system and a plant expression system.

Description

Gene with Δ6 fatty acid dehydrogenase function and its application

This application is a divisional application of the patent application whose application number is 2007101194771.

technical field

The present invention relates to plant coding gene and its application. Specifically, it involves two genes with complete reading frames, RnD6C and RnD6D, which are respectively derived from two DNA sequences of black currant (Ribes nigrum L.), and two genes with complete reading frames cloned on this basis. gene sequence. The present invention also relates to the polypeptide encoded by the gene with the function of ^Δ6 fatty acid dehydrogenase, as well as lower eukaryotic cell expression vectors and plant expression vectors containing the DNA sequence, host cells and transformation of lower eukaryotic organisms using the gene and plant production of GLA and SDA applications.

Background technique

Polyunsaturated fatty acids (PUFAs) refer to straight-chain fatty acids containing two or more double bonds and a carbon chain length of 18-22 carbon atoms, mainly including linoleic acid (LA, 18 :2Δ ^9,12 ), γ-linolenic acid (γ-linolenic acid, GLA, 18:3, Δ ^6,9,12 ), arachidonic acid (Arachidonic Acid, AA, 20:4Δ ^{5,8,11, 14.} Eicosapentaenoic Acid (EPA, 20: ^{5Δ5,8,11,14,17} ), Docosahexaenoic Acid (DHA, 20: ^{5Δ4,7,10, 11, 16, 19} ), etc. Among them, linoleic acid and linolenic acid are recognized as essential fatty acids (EFA), which can be further derivatized into highly unsaturated fatty acids with different functions, such as AA, EPA, DHA, etc. .

PUFAs have very important physiological functions. First, PUFAs play important roles in physiological functions such as cardiovascular exercise, brain, retinal and nervous tissue development. Second, DHA and EPA have good anti-cancer effects, such as preventing (especially postmenopausal women) breast cancer. Third, polyunsaturated fatty acids such as arachidonic acid, EPA, and DHA play an important role in immune regulation. Fourth, polyunsaturated fatty acids can also prevent skin aging, delay aging, and promote hair growth.

In the process of fatty acid metabolism, Δ ⁶ fatty acid desaturase (Δ ⁶ -fatty acid desaturase, D6D) catalyzes the dehydrogenation of the sixth carbon atom of the substrates linoleic acid (LA) and α-linolenic acid (ALA) respectively to form γ - Linolenic acid (GLA) [1] and stearidonic acid (SDA, 18: ^4Δ6,9,12,15 ).

γ-linolenic acid can further form AA, prostaglandins and leukotriene physiologically active substances through carbon chain extension and dehydrogenation. These products play a series of physiological functions in human brain development, vision, allergic reactions and cardiovascular exercise have an important impact. As an essential unsaturated fatty acid in the human body, γ-linolenic acid has a series of biological functions such as lowering blood fat, anti-lipid peroxidation, weight loss, inhibiting ulcer, enhancing insulin, and anti-thrombotic cardiovascular disease[1,2] At present, vitamin F has been studied and utilized as a new vitamin in the world [3]. SDA is the metabolic precursor of EPA and DHA. It is easily converted into EPA and DHA in the human body, and its utilization efficiency is 4 times that of ALA. It can effectively alleviate the physiological diseases related to the lack of EPA and DHA. Of course, the same effect can also be obtained by directly supplementing EPA or DHA.

However, the current commercial sources of PUFAs are mainly certain seed plants, marine fish and some animal tissues. Except for LA, existing sources cannot meet the growing market demand for PUFAs in terms of both total yield and quality. This is mainly based on the following reasons:

(1) The plant resources of PUFAs are limited by seasons and regions, and the yield and quality of PUFAs are unstable; moreover, most plant resources have low yields and are not suitable for large-scale planting. For example, the yield of evening primrose, borage and black currant containing γ-linolenic acid is only 300-600kg/ha, which is far lower than the 3t/ha yield of conventional oil crops such as rape [4].

(2) The limited natural marine fishery resources are lack of fishery resources due to overfishing, and the inherent fishy smell and oxidative instability of fish oil limit the further utilization of PUFAs;

(3) Due to the need to refine low-quality oils, the production cost of PUFAs is greatly increased. The resulting high market price of pure PUFAs hinders many of their potential uses.

In view of the fact that the supply of PUFAs will soon be unable to meet its demand and the insufficient supply of pure PUFAs required for application in biopharmaceuticals, it is necessary to seek alternative sources for commercial production.

Therefore, as the key enzyme in the production of GLA and SDA- ^Δ6 fatty acid dehydrogenase has been more and more researched and utilized. So far, ^Δ6 fatty acid dehydrogenase genes have been cloned from different organisms such as animals[5], plants[4], fungi[6] and nematodes[7], and have been cloned in Saccharomyces cerevisiae[7,8], tomato[ 9], tobacco [4, 10], rapeseed [6, 8] and soybean [11] were successfully expressed.

In plants, GLA and SDA only exist in a few plants such as evening primrose (Oenothera sp.), borage (Borago officinalis L.), and black currant [12], and the seed oil production of these plants is also Become the main commercial source of GLA and SDA in the world. GLA and SDA can also be extracted by fermentation of oleaginous fungi. However, the yields of GLA and SDA obtained through these existing production methods are very low, which is far from meeting the growing market demand [13]. Therefore, using Saccharomyces cerevisiae or transgenic oil crop plants to express exogenous ^Δ6 fatty acid dehydrogenase to produce GLA and SDA has important research significance and economic value. In addition, although borage [5] has a Δ ⁶ fatty acid dehydrogenase gene that has been reported abroad, the two genes of the present application are Δ ⁶ fatty acid dehydrogenase genes derived from black currant, which have so far been studied at home and abroad. None reported.

Contents of the invention

The present invention provides two genes RnD6C and RnD6D, the nucleotide sequences of which are respectively shown in SEQID NO: 1 and SEQID NO: 2, and these two genes are respectively derived from the DNA fragments of Black Currant (Ribesnigrum L.) , the encoded polypeptides are shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively.

Another object of the present invention is to provide lower eukaryotic cell expression vectors containing genes RnD6C and RnD6D, such as yeast expression vectors, and yeast cells transformed with the expression vectors. The polypeptide expressed in the transformed yeast cells has the function of ^Δ6 fatty acid dehydrogenase, which can respectively catalyze the addition of substrates linoleic acid and α-linolenic acid to generate γ-linolenic acid (GLA) and stearidonic acid ( SDA).

Since this type of gene encodes a protein, it can be expressed in vitro using the Saccharomyces cerevisiae expression system, but since the protein is a membrane protein, even if it is expressed in the in vitro expression system, it is difficult to separate and purify. Therefore, the method of investigating the catalytic activity of the expressed protein to the external substrate is generally used for functional identification.

In the Saccharomyces cerevisiae expression system adopted, the recipient strain is uracil-deficient INV ScI, which does not contain LA (linoleic acid), ALA (α-linolenic acid), GLA (γ-linolenic acid) and SDA, so , under the condition of adding the substrate linoleic acid, if the catalytic product GLA or SDA is detected in the yeast engineered bacteria, it can be confirmed that the protein encoded by the exogenous gene has been expressed in the Saccharomyces cerevisiae expression system, and the additional substrate produced Catalyzed. Therefore, Saccharomyces cerevisiae has been used as the most commonly used and effective expression system for the functional identification of exogenous Δ6 and Δ12-fatty acid dehydrogenase genes, and there have been many successful research reports [4, 6, 7]. The detection of fatty acids is mainly accomplished by GC-MS (gas chromatography-mass spectrometry). The principle is to separate different fatty acid components through GC (gas chromatography), and then conduct mass spectrometry qualitative analysis on each component through MS (mass spectrometry) detector, so as to determine the composition and content of each component. combined analysis techniques.

Another object of the present invention is to provide a plant expression vector containing genes RnD6C and RnD6D. Any expression vector that can direct expression of foreign genes in plants can be used. These plant expression vectors include, but are not limited to, binary Agrobacterium vectors, such as pBIN19, pBI121, pB221, pCambia 1300, pGreen and other plant expression vectors.

The vectors of the present invention may also contain an appropriate promoter. Any strong promoter can be used in the present invention. These promoters include, but are not limited to, cauliflower mosaic virus (CaMV 35S), Ubiqutin, Actin promoters. It can be used alone or in combination with other plant promoters.

The expression vector of the present invention can be introduced into plant cells by using Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation and the like.

The lower eukaryotic hosts that can be transformed using the method of the present invention are selected from the group consisting of yeast, algae and other lower eukaryotic organisms.

Plant hosts that can be transformed using the method of the present invention include plants such as tobacco, rapeseed, sunflower, soybean, tomato, castor, sesame and peanut.

The invention also relates to the application of genes RnD6C and RnD6D in the production of GLA and SDA.

Description of drawings

Figure 1. RnD6C fragment nucleotide sequence, SEQ ID NO:1.

Figure 2. RnD6D fragment nucleotide sequence, SEQ ID NO:2.

Figure 3. The polypeptide sequence encoded by the gene RnD6C, SEQ ID NO: 3, where the underlined parts are the Cytb5 functional domain and the three His Box functional domains.

Figure 4. The polypeptide sequence encoded by the gene RnD6D, SEQ ID NO: 4, where the underlined parts are the Cytb5 functional domain and three His Box functional domains.

Figure 5. Map of yeast expression vectors for genes RnD6C and RnD6D.

Figure 6. Map of plant expression vectors for genes RnD6C and RnD6D. a) Map of the plant expression vector of RnD6C/D with hygromycin (Hyg) as the selectable marker; b) Map of the plant expression vector of RnD6C/D without the selectable marker.

Figure 7. GC-MS analysis of total fatty acids expressed by genes RnD6C and RnD6D in Saccharomyces cerevisiae

a) GC-MS of fatty acid composition of INV ScI yeast strain;

b) GC-MS of fatty acid composition of INV ScI yeast strain and standard samples added with LA (linoleic acid) and GLA (γ-linolenic acid);

c) GC-MS of total fatty acids of yeast containing empty pYES2 expression plasmid;

d) Total fatty acid GC-MS of yeast containing empty pYES2 expression plasmid induced by adding LA substrate;

e) GC-MS of total fatty acids in yeast expressing RnD6C gene induced by adding LA substrate, the red arrow indicates the product peak;

f) GC-MS of total fatty acids in yeast expressing RnD6D gene induced by adding LA substrate, the red arrow indicates the product peak;

g) Total fatty acid GC-MS of yeast containing empty pYES2 expression plasmid induced by adding ALA substrate;

h) GC-MS of total fatty acids in yeast expressing RnD6C gene induced by adding ALA substrate, the red arrow indicates the product peak;

i) GC-MS of total fatty acids in yeast expressing RnD6D gene induced by adding ALA substrate, the red arrow indicates the product peak;

j) GC-MS of total fatty acids of transgenic rapeseed H165 seeds whose expression of RnD6D gene is activated by napin promoter, the red arrow indicates the product peak;

k) GC-MS of total fatty acids of control-untransformed rapeseed H165 seeds.

Figure 8. MS spectrum in GC-MS analysis of catalytic products of RnD6C and RnD6D gene expression in Saccharomyces cerevisiae, a: MS chart of GLA, the catalytic product expressed by RnD6C and RnD6D in yeast; b: MS chart of γ-linolenic acid standard.

Detailed ways

The present invention is described in detail below with reference to Examples and drawings. Those of ordinary skill in the art can understand that the following examples are for the purpose of illustration and should not be construed as limiting the present invention in any way. The protection scope of the present invention is defined by the appended claims.

Embodiment 1. Obtaining of RnD6C and RnD6D genes

1. Preparation of black currant genomic DNA

Using the live plant of black tea currant (Ribes nigrum L.) grown in Beijing Botanical Garden as the material, take its young leaves (about 100mg), add steel balls (diameter 5mm) into a 7ml Ep tube, and freeze in liquid nitrogen for 20 -30min, high-speed crushing on the vortex machine, repeat the operation 2-3 times until the material is completely crushed. Add 1-2ml of preheated CTAB extract solution (see "Refined Molecular Biology Experiment Guide" 2001, Science Press, translated by Yan Ziying and Wang Hailin), and mix well. 65°C water bath for 30min. Add an equal volume of chloroform, and gently extract for about 5 minutes. Centrifuge at 12000rpm room temperature for 10min. Take the supernatant, add 1/2 volume of isopropanol to mix well, and place at room temperature for 10 min to precipitate DNA. The precipitated DNA was picked out with a Tip, washed twice with 70% ethanol, and placed in 70% ethanol at room temperature for 30 min. Remove 70% ethanol and dry in air. Dissolve in appropriate amount of sterilized ddH ₂ O and store at -20°C.

2. Cloning of RnD6C and RnD6D DNA fragments

Design primers to clone the DNA fragments of RnD6C and RnD6D from the DNA extracted above. The reaction system used is as follows:

PCR reaction system (50μl):

After PCR product electrophoresis (1% agarose gel concentration), the target fragment (RnD6C, RnD6D, about 1.3 kb) was recovered by gel cutting, connected into pGEM-T vector (purchased from Promega Company), and sequenced for verification.

The primers used are as follows:

1). Primers used for cloning of RnD6C DNA fragment

forward primer

Kpn I

5'-CGCG GGTACC ATGGCTAATGCAATCAAG-3' (SEQ ID NO: 5)

reverse primer Sac I

5'-CGCC GAGCTC TCAGCCATAGGTGTTGAC-3' (SEQ ID NO: 6)

2). Primers used for cloning of RnD6D DNA fragment

Forward primer Kpn I

5'-CGCG GGTACC ATGGGTGAAAATGGAAGG-3' (SEQ ID NO: 7)

reverse primer Sac I

5'-CGCC GAGCTC TCAACCATAGGTGTTGAC-3' (SEQ ID NO: 8)

Embodiment two, the construction of RnD6C, RnD6D gene yeast vector

From the pGEM-T vector containing the genes RnD6C and RnD6D, use the Kpn I and Sac I restriction sites to obtain the RnD6C and RnD6D genes after double cutting, and directionally clone them into the yeast expression vector pYES2 (purchased from Ivitrogen Company) to obtain yeast expression Plasmids pYRnD6C and pYRnD6D, transformed into Escherichia coli DH-5α (preserved by our laboratory) were preserved. Its vector diagram is shown in Figure 5.

Example 3, Expression of RnD6C and RnD6D genes in yeast

1. Transformation of Yeast

Referring to the method described in Invitrogen's pYES2 Kit (Cat# V285-20), the yeast expression plasmids pYRnD6C and pYRnD6D of the above-mentioned chimeric genes were transformed into Saccharomyces cerevisiae auxotrophic strain INV Sc I (purchased from Invitrogen) using lithium acetate. , with the empty pYES2 plasmid as a control, yeast cells containing each expression plasmid were obtained.

2. Induced expression in transformed yeast cells

Get the yeast single colony transformed with the yeast expression plasmid containing the target gene, and inoculate it in 50ml SC-U culture fluid (with reference to the formula described in Invitrogen company pYES2 Kit) containing 2% raffinose SC-U culture fluid, 250rpm, 28°C, Cultivate overnight; add NP-40 (purchased from BBI Company) (final concentration 1%), exogenous linoleic acid and α-linolenic acid substrates (purchased from Sigma Company) (final concentration 0.003%), and induce yeast expression D-galactose (purchased from Amresco) (final concentration: 2%) was used at 250 rpm, and cultured at 22° C. for 48 hours to induce expression.

Embodiment four, RnD6C, the GC-MS analysis of RnD6D gene yeast expression product to Δ ⁶ fatty acid dehydrogenase substrate catalytic product

1. Extraction and methyl esterification of fatty acids.

The induced yeast cells were collected by centrifugation, washed with deionized water, and dried at 50°C. Take 40 mg of yeast powder (obtained by drying the yeast induced in Example 3.2 at 50°C) and grind thoroughly, add 5ml of 5% KOH-CH ₃ OH, bathe in water at 70°C for 5 hours, then add HCl to acidify until the pH reaches 2.0. Add 4ml of 14% BF ₃ -CH ₃ OH (purchased from Aldtich Company) solution, and bathe in water at 70°C for 1.5h. Add 2ml of 0.9% NaCl solution, mix well and let stand for a while. Add 2ml of chloroform:n-hexane (V/V1:4) for extraction, absorb the extract, and blow dry with _N2 . Finally dissolved in 100 μl ethyl acetate.

2. GC-MS detection and analysis experiment of the final product.

The GC-/MS instrument used is TurboMass (PerkinElmer Company), the column: BPX-70, 30m×0.25mm×0.25vm, the column temperature is 120°C, and the gasification chamber temperature is 230°C. Take 1 μl of the final product and load it with a split ratio of 10:1.

3. GC-MS result analysis.

Comparing Figures 7-1a, 7-1b and 7-1e, it shows that no new product peaks appear in the yeast engineered plants used and the yeast containing the induced empty pYES2 expression plasmid without the addition of linoleic acid substrate, indicating that The total fatty acids of the yeast engineering strains used and the empty pYES2 expression plasmid yeast do not contain additional substrates linoleic acid (LA), α-linolenic acid (ALA) and catalytic products γ-linolenic acid (GLA), steadetraene acid (SDA).

When the linoleic acid substrate was added and the expression was induced, the control (empty pYES2) also had no product peak γ-linolenic acid (GLA) (see Figure 7-1d). After the pYRnD6C/D yeast expression strain is induced, the additional substrate linoleic acid (LA) can be catalyzed to generate γ-linolenic acid (GLA) (see Figure 7-1e and 7-1f).

Comparing Figures 7-1d, 7-1e and 7-1f, it can be found that when the retention time is about 7.25min, a new substance is formed. Its peak time and mass spectrum are consistent with those of the standard γ-linolenic acid (purchased from Sigma Company) (see Figure 8a and Figure b), and it is determined to be γ-linolenic acid.

When the α-linolenic acid substrate was added and the expression was induced, the control (empty pYES2) also had no product peak SDA (see Figure 7-2g). After the pYRnD6C/D yeast expression strain is induced, the additional substrate α-linolenic acid can be catalyzed to generate SDA (see Figure 7-2h and 7-2i).

Comparing Figures 7-2g, 7-2h and 7-2i, it can be found that when the retention time is about 7.75min, a new substance is formed. Determined as SDA according to its peak time and mass spectrum.

The above results prove that the two genes RnD6C and RnD6D derived from black currant were successfully expressed in S. acid and SDA.

Embodiment five, the construction of RnD6C, RnD6D gene plant expression vector

1. Construction of napin promoter napin-T intermediate vector

The total DNA of rapeseed (mustard type) was extracted, (see Example 1.1 for the method). Primers used for cloning napin promoter fragments:

Forward primer: Hind III

5'-CGCG AAGCTT ACTACAATGTCGGAGAGACAAGG-3' (SEQ ID NO: 9)

Reverse primer: BamH I

5'-CGCG GGATCC TTGTGTATGTTCTGTAGTGATGAGTTTTG-3' (SEQ ID NO: 10)

Use Pyrobest DNA Polymerase PCR amplification (see Example 1.2 for the reaction system), PCR product electrophoresis (1% agarose gel concentration) and gel cutting to recover the target fragment (about 1.8kb), connected into the pGEM-T vector (purchased from Promega company), sequence verification.

2. Construction of the RnFD6C/D-T intermediate vector of RnD6C and RnD6D genes

According to the gene sequence of RnFD6C/D, design upstream and downstream specific primers containing restriction sites BamH I and Sac I respectively, use Pyrobest DNA Polymerase to amplify the target fragments RnFD6C and RnFD6D, recover the target fragments and clone them into the pGEM-T vector , picked a single colony, and verified by PCR, enzyme digestion and sequencing to obtain the RnFD6C/D-T intermediate vector.

3. Construction of plant expression vectors of RnD6C and RnD6D genes

1) Construction of pC1300-napin

Double digest Napin-T and pCambia 1300 with Hind III and BamH I, respectively, and recover the napin promoter fragment and pCambia 1300 vector fragment, connect and transform Escherichia coli, pick a single clone at random, and obtain the replacement 35S promoter by PCR and enzyme digestion verification The pC1300-napin plant expression vector is the napin promoter.

2) Construction of pC1300-nRnD6C/D plant expression vector

RnD6C/D-T and pC1300-napin were double-digested with BamH I and Sac I respectively, and the RnD6C/D target fragment and pC1300-napin vector fragment were recovered, connected and transformed into Escherichia coli, single clones were randomly selected, and obtained by PCR and enzyme digestion verification The plant expression vector of pC1300-n RnD6C/D. Its vector diagram is shown in Figure 6a.

3) Construction of pC1300-nRnD6C/D removing hygromycin (Hyg) selection marker

After the construction is complete, use Xho I to single-enzyme digest, then recover the large fragment, and after ligation and transformation, randomly pick a single clone for PCR and enzyme digestion verification to obtain the pC1300-nRnD6C/D plant expression that removes the hygromycin (Hyg) selection marker carrier. Its carrier map is shown in Figure 6b.

Embodiment 6, RnD6C, RnD6D genes and transformation in plants

1. Transformation of plant expression vector into Agrobacterium

Pick a single colony of Agrobacterium and inoculate it in 5ml of YEP liquid medium, culture at 28°C, 200rpm, shake overnight. Add 2ml of overnight cultured bacterial solution to 50ml of YEP medium, at 28°C, 220rpm, shake culture until OD ₆₀₀ is between 0.5-1.0. Centrifuge the bacterial solution at 5000rpm, discard the supernatant, suspend the pellet in 10ml of 0.5M NaCl, centrifuge at 5000rpm for 5min at 4°C, discard the supernatant, and resuspend the cells with _1ml of pre-cooled 20mM CaCl2. Take 0.2ml and add about 0.5-1μg plasmid DNA, mix gently, freeze in liquid nitrogen for 1min, heat shock at 37°C for 5min, add 1ml of YEP solution, and incubate at 28°C for 2-4h. Centrifuge the bacterial solution at 5000rpm, discard the supernatant, resuspend the cells in 0.2ml YEP medium, spread evenly on the YEP plate containing Kan, and incubate at 28°C for 48h. A single colony was randomly picked for PCR and enzyme digestion verification.

2. Transformation of rapeseed (H165) by Floral-dip method

Brassica napus plants at the initial flowering stage were selected for treatment, and the field was watered thoroughly for the first 1 day. The top flower buds of the main inflorescence and each branch inflorescence are removed, and the flowers that have opened are removed at the same time, and only the flower buds that are about to open are kept for transformation.

Use 10ml of fresh YEP+kan medium to cultivate Agrobacterium containing the plant expression vector of the target gene overnight at 28°C until 10:00 the next day, then transfer 5ml into 100ml fresh YEP+kan medium, and culture overnight for 22h. Centrifuge the Agrobacterium liquid at 2500rpm, discard the supernatant, resuspend with 100ml Transformation Bufer (MS basic medium + 5% sucrose + Silwet-77, pH 5.8) to OD ₆₀₀ =1.0, and then soak the rape inflorescence. 5 consecutive treatments at intervals of 1d within 10d. Put on a paper bag after soaking. After soaking for 24 hours, the paper bag was removed until the seeds were harvested.

Embodiment seven, the screening of T1 generation seed

The T1 generation seeds of rapeseed obtained after dipping treatment were surface sterilized with 75% alcohol for 30 seconds and 0.1% HgCl ₂ for 10 minutes, spread on the pre-poured screening medium, and were screened and cultivated for 7-15 days, and the green seedlings with normal growth were subjected to the second 2 times of resistance selection (7-15d), the resistant plants after the 2nd selection were subjected to the 3rd resistance selection (7-15d), the medium for the 2nd and 3rd selection was: Ms+Kan( 150mg/L). The green seedlings after the third screening are transferred to flowerpots and cultivated in the greenhouse until the seeds are harvested.

For the T1 generation rapeseed obtained after treatment with Agrobacterium containing pC1300-n RnD6C/D that removes the screening marker, they were directly sown in the field, leaf DNA was extracted, and positive plants were screened by PCR.

Embodiment eight, RnD6C, RnD6D gene plant expression thing is to the GC-MS analysis of Δ ⁶ fatty acid dehydrogenase substrate catalytic product

1. Extraction and methyl esterification of fatty acids.

Take 50-100 mg of mature control or transgenic rape seeds, grind them thoroughly in liquid nitrogen, add 5 ml of 5% KOH-CH ₃ OH, bathe in water at 70°C for 5 hours, then add HCl to acidify until the pH reaches 2.0. Add 4ml of 14% BF ₃ -CH ₃ OH (purchased from Aldrich) solution, and bathe in water at 70°C for 1.5h. Add 2ml of 0.9% NaCl solution, mix well and let stand for a while. Add 2ml of chloroform:n-hexane (V/V 1:4) for extraction, absorb the extract, and blow dry with _N2 . Finally dissolved in 100 μl ethyl acetate.

2. GC-MS detection and analysis experiment of the final product.

The GC/MS instrument used is TurboMass (PerkinElmer Company), the column: BPX-70, 30m×0.25mm×0.25vm, the column temperature is 120°C, and the gasification chamber temperature is 230°C. Take 1 μl of the final product and load it with a split ratio of 10:1.

3. GC-MS result analysis.

Comparing Figures 7-2j and 7-2k, compared with the non-transformed rapeseed control, two new product peaks appeared in the transformed rapeseed. Compared with the results of the standard and yeast GC-MS, it can be found that the retention time is 7.25 When the retention time is about 7.75 min, the product peak of GLA appears; when the retention time is about 7.75 min, the product peak of SDA appears. The above results proved that the gene RnD6D from black currant was successfully expressed in rapeseed, and the expressed protein could catalyze the production of GLA and SDA from the substrates linoleic acid (LA) and α-linolenic acid (ALA), respectively. This also shows that by transferring RnD6C/D into rapeseed, the transgenic rapeseed can be used as a bioreactor to produce GLA and SDA.

references:

1. Gunstone FD (1992) Gamma linolenic acid-occurrence and physical and chemical properties. Prog Lipid Res 31: 145-161.

2. Horrobin DF (1992) Nutritional and medical importance of gamma-linolenic acid. Prog Lipid Res 31: 163-194.

3. Huang YS and Milles DE(1996) Gamma-Linolenic Acid: Metabolism and Its Roles in Nutrition and Medicine. AOCS Press, Champaign, IL.

4. Sayanova O, Smith MA, Lapinskas P, Stobart AK, Dobson G, Christie WW, Shewry PR and Napier JA (1997) Expression of a borage desaturase cDNA containing an N-terminal cytochrome b5 domain results in the accumulation of high levels of Δ ⁶ - desaturated fatty acids in transgenic tobacco. Proc Natl Acad Sci USA 94: 4211-4216.

5. Hyekyung PC, Manabu TN and Steven DC (1999) Cloning, Expression, and Nutritional Regulation of the MammalianΔ ⁶ -Desaturase. J Biol Chem274 (1): 471-477.

6. Hong HP, Datla N, Reed DW., Covello PS, MacKenzie SL and Qiu X (2002) High-Level Production ofγ-Linolenic Acid in Brassica juncea Using aΔ ⁶ Desaturase from Pythium irregulare. Plant Physiol 129: 354-362.

7. Napier JA, Hey SJ, Lacey DJ and Shewry PR (1998) Identification of aCaenorhabditis elegans Δ ⁶ -fatty-acid-desaturse by heterologous expression in Saccharomyces cerevisiae. Biochem J 330: 611-614.

8. Qiu X, Hong HP, Datla N, MacKenzie SL, Tayler CD and Thomas LT (2002) Expression of boreageΔ ⁶ -desaturase in Saccharomyces cerevisiae and oilseed crops. Can J Bot 80: 42-49.

9. Cook D, Grierson D, Jones C, Wallace A, West G and Tucker G (2002) Modification of fatty acid composition in tomato (Lycopersicon esculentum) by expression of a borageΔ ⁶ -desaturase. Mol Biotechnol.21 (2): 123-128.

10. Sayanova O, Smith MA, Lapinskas P, Stobart AK, Dobson G, Christie WW, Shewry PR and Napier JA (1997) Expression of a borage desaturasecDNA containing an N-terminal cytochrome b5 domain results in the accumulation of high levels of Δ ⁶ -desaturated fatty acids in transgenic tobacco. Proc Natl Acad Sci USA 94: 4211-4216.

11. Sato S, Xing AQ, Ye XG, Schweiger B, Kinney A, Graef G and Clemente T(2004) Production ofγ-Linolenic Acid and Stearidonic Acid in Seeds of Marker-Free Transgenic Soybean. Crop Sci 44:646-652.

12. Ucciani E (1995) Nouveau Dictionnaire des Huiles Végétales-Composition en Acides Gras. Lavoisier, Paris pp 3-596.

13. Gyves EM, Sparks CA, Sayanova O, Lazzeri P, Napier JA and Jones HD (2004) Genetic manipulation of γ-linolenic acid (GLA) synthesis in commercial variety of evening primrose (Oenothera sp.) Plant Biotech2 (4): 351 -357.

sequence listing

<110> Institute of Genetics and Developmental Biology, Chinese Academy of Sciences

<120> Gene with Δ6 fatty acid dehydrogenase function and its application

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<160>8

<170>PatentIn version 3.1

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<212> DNA

<213>Ribes nigrum L.

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gaggatttgt ggatctctat tcagggtaag gtttacaatg tcactgattg gaaaaaggaa 120

cacccaggtg gggatagctc tctcctgaat ctaggtggtc aagatgtcac agatgcattc 180

attgcttatc atccaggtac tgcttggcaa tatcttgaca ggttctttac tgggtattat 240

ctcaaagatt tcaatgtctc agatgtatcc aaagattata gaaaacttgc atctgagttt 300

accaaaatgg gtctttttgc aaagaaaggg catggtgttt tctactcctt ttgtcttggg 360

gcattcttgt ttgctgtttg tgtttatggt gttttgtatt ctcaaagttt gtttgtgcat 420

ttgtgttgtg gtgggatttt ggggttttta tggatgcaaa gtggttatgc tggtcacgat 480

tctgggcatt atcagacaat gtctactcct ttttatacca atttggctca aattcttact 540

ggaaattgcc ttagtggtat tagtatggct tggtggaaat ggacccacaa tgctcaccac 600

gttgctgtta atagtattga ccatgaccct gatcttcagt acatgccatt ttttgtcctc 660

tctcccaaat tgttcaatgg cataacttct cgcttttatg ggaggaagtt ggagtttgat 720

gcttttgcta ggttcatggt aagttatcag cattggacat tttatccggt gatgattttt 780

gcacgatttt atatgtttgt gcccacgttt tttatgttgc tttcaaagaa aaaagtaccc 840

aacagacctt tgaatatagc gggaattatt gtgttctgga tttggtttcc tcttcttgtt 900

tcatgccttc ccaattggac tgagaggatg atgtttgtgc tagtgagctt tacagttact 960

tcaattcaac acgttacatt ttgtttgaat cactactcgg ctgataatta tatgggacat 1020

cctactggga acgattggtt tgagaagcag acaagtggga ctttggacat ttcgtgcccg 1080

tcttggatgg attggtttca tggtgggctg caattccaaa ttgagcatca tttgttccca 1140

agattgcctc gatgccagct taggaaggtc tctccttttg tgaaagagct ttgcaagaaa 1200

cacaatttgc cttataggag tttatcattt tttgaggcca atgttgcaat cattaagacg 1260

ttgaggactg ccgccttgca agctcgcgac ctatccaacc ctatttcaaa gaatttgcta 1320

tgggaagctg tcaacaccta tggctga 1347

<210>2

<211>1347

<212>DNA

<213>Ribes nigrum L.

<400>2

atgggtgaaa atggaaggaa gtacatgaca gttgaggagc taaaagtgca taataagcca 60

gaggatttgt ggatctctat tcagggtaag gtttacaatg tcactgattg gaaaaaggaa 120

cacccaggtg gggatagctc actcctgaat ctgggtggtc aagatgtcac agatgcattc 180

attgcttatc atccaggtac tgtttggcaa tatcttgaca ggttctttac tgggtattat 240

ctcaaagatt tcaaagtctc agatgtatcc aaagattaca gaaaacttgc atctgagttt 300

accaaaatgg gtctttttgc aaagaaaggg catggtgttt tctactcctt ttgtcttggg 360

gcattcttgt ttgctgtttg tgtttatggt gttttgtatt ctcaaagttt gtttgtgcat 420

ttatgttgtg gtgggatttt ggggttttta tggatgcaaa gtggttatgc tggtcatgat 480

tctgggcatt accagacaat gtctactcct ttttatacca atttggctca aattcttact 540

ggaaattgcc ttagtggtat tagtatggct tggtggaaat ggacccacaa tgctcaccac 600

attgctgtta atagtattga ccatgaccct gatcttcagt acatgccatt ttttgttctc 660

tctcccaaat tgttcaatag cataacttct cgcttttatg ggaggaagtt ggagtttgat 720

gcttttgcta ggttcatggt gagttatcag cattggacat tttatccggt gatgattttt 780

gcacgatttt atatgtttgt gcctacgttt tttttgttgc tttcaaagaa aaaagtaccc 840

aacagacttt tgaatatagc tggaattatt gtgttctgga tttggtttcc tcttcttgtt 900

tcatgcctac ccaattggac tgagaggatg atgtttgtgc tagtgagctt tacggttat 960

tcaattcaac acgttacatt ttgtttgaat cactactcgg ctgataatta tatgggacat 1020

cctactggga acgattggtt tgaaaagcag acaagtggga ctttggacat ttcgtgcccg 1080

tcttggatgg attggtttca tggtgggctg caattccaac ttgagcatca tttgttccct 1140

cgaatgcctc gatgtcagct taggaaggtc tctccttttg tgaaggaact ttgccagaag 1200

cataatttgc cttataggag tttgtcattt ttcgaggcca atgtcgcgac cattaagacg 1260

ttgaggactg cagccttgca agctcgtgac ctgtccaacc ctattacaaa gaatttgcta 1320

tgggaagctg tcaacaccta tggttga 1347

<210>3

<211>448

<212>PRT

<213>Ribes nigrum L.

<400>3

Met Ala Asn Ala Ile Lys Lys Tyr Met Thr Val Glu Glu Leu Lys Val

1 5 10 15

His Asn Lys Pro Glu Asp Leu Trp Ile Ser Ile Gln Gly Lys Val Tyr

20 25 30

Asn Val Thr Asp Trp Lys Lys Glu His Pro Gly Gly Asp Ser Ser Leu

35 40 45

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

50 55 60

Pro Gly Thr Ala Trp Gln Tyr Leu Asp Arg Phe Phe Thr Gly Tyr Tyr

65 70 75 80

Leu Lys Asp Phe Asn Val Ser Asp Val Ser Lys Asp Tyr Arg Lys Leu

85 90 95

Ala Ser Glu Phe Thr Lys Met Gly Leu Phe Ala Lys Lys Gly His Gly

100 105 110

Val Phe Tyr Ser Phe Cys Leu Gly Ala Phe Leu Phe Ala Val Cys Val

115 120 125

Tyr Gly Val Leu Tyr Ser Gln Ser Leu Phe Val His Leu Cys Cys Gly

130 135 140

Gly Ile Leu Gly Phe Leu Trp Met Gln Ser Gly Tyr Ala Gly His Asp

145 150 155 160

Ser Gly His Tyr Gln Thr Met Ser Thr Pro Phe Tyr Thr Asn Leu Ala

165 170 175

Gln Ile Leu Thr Gly Asn Cys Leu Ser Gly Ile Ser Met Ala Trp Trp

180 185 190

Lys Trp Thr His Asn Ala His His Val Ala Val Asn Ser Ile Asp His

195 200 205

Asp Pro Asp Leu Gln Tyr Met Pro Phe Phe Val Leu Ser Pro Lys Leu

210 215 220

Phe Asn Gly Ile Thr Ser Arg Phe Tyr Gly Arg Lys Leu Glu Phe Asp

225 230 235 240

Ala Phe Ala Arg Phe Met Val Ser Tyr Gln His Trp Thr Phe Tyr Pro

245 250 255

Val Met Ile Phe Ala Arg Phe Tyr Met Phe Val Pro Thr Phe Phe Met

260 265 270

Leu Leu Ser Lys Lys Lys Val Pro Asn Arg Pro Leu Asn Ile Ala Gly

275 280 285

Ile Ile Val Phe Trp Ile Trp Phe Pro Leu Leu Val Ser Cys Leu Pro

290 295 300

Asn Trp Thr Glu Arg Met Met Phe Val Leu Val Ser Phe Thr Val Thr

305 310 315 320

Ser Ile Gln His Val Thr Phe Cys Leu Asn His Tyr Ser Ala Asp Asn

325 330 335

Tyr Met Gly His Pro Thr Gly Asn Asp Trp Phe Glu Lys Gln Thr Ser

340 345 350

Gly Thr Leu Asp Ile Ser Cys Pro Ser Trp Met Asp Trp Phe His Gly

355 360 365

Gly Leu Gln Phe Gln Ile Glu His His Leu Phe Pro Arg Leu Pro Arg

370 375 380

Cys Gln Leu Arg Lys Val Ser Pro Phe Val Lys Glu Leu Cys Lys Lys

385 390 395 400

His Asn Leu Pro Tyr Arg Ser Leu Ser Phe Phe Glu Ala Asn Val Ala

405 410 415

Ile Ile Lys Thr Leu Arg Thr Ala Ala Leu Gln Ala Arg Asp Leu Ser

420 425 430

Asn Pro Ile Ser Lys Asn Leu Leu Trp Glu Ala Val Asn Thr Tyr Gly

435 440 445

<210>4

<211>448

<212>PRT

<213>Ribes nigrum L.

<400>4

Met Gly Glu Asn Gly Arg Lys Tyr Met Thr Val Glu Glu Leu Lys Val

1 5 10 15

His Asn Lys Pro Glu Asp Leu Trp Ile Ser Ile Gln Gly Lys Val Tyr

20 25 30

Asn Val Thr Asp Trp Lys Lys Glu His Pro Gly Gly Asp Ser Ser Leu

35 40 45

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

50 55 60

Pro Gly Thr Val Trp Gln Tyr Leu Asp Arg Phe Phe Thr Gly Tyr Tyr

65 70 75 80

Leu Lys Asp Phe Lys Val Ser Asp Val Ser Lys Asp Tyr Arg Lys Leu

85 90 95

Ala Ser Glu Phe Thr Lys Met Gly Leu Phe Ala Lys Lys Gly His Gly

100 105 110

Val Phe Tyr Ser Phe Cys Leu Gly Ala Phe Leu Phe Ala Val Cys Val

115 120 125

Tyr Gly Val Leu Tyr Ser Gln Ser Leu Phe Val His Leu Cys Cys Gly

130 135 140

Gly Ile Leu Gly Phe Leu Trp Met Gln Ser Gly Tyr Ala Gly His Asp

145 150 155 160

Ser Gly His Tyr Gln Thr Met Ser Thr Pro Phe Tyr Thr Asn Leu Ala

165 170 175

Gln Ile Leu Thr Gly Asn Cys Leu Ser Gly Ile Ser Met Ala Trp Trp

180 185 190

Lys Trp Thr His Asn Ala His His Ile Ala Val Asn Ser Ile Asp His

195 200 205

Asp Pro Asp Leu Gln Tyr Met Pro Phe Phe Val Leu Ser Pro Lys Leu

210 215 220

Phe Asn Ser Ile Thr Ser Arg Phe Tyr Gly Arg Lys Leu Glu Phe Asp

225 230 235 240

Ala Phe Ala Arg Phe Met Val Ser Tyr Gln His Trp Thr Phe Tyr Pro

245 250 255

Val Met Ile Phe Ala Arg Phe Tyr Met Phe Val Pro Thr Phe Phe Leu

260 265 270

Leu Leu Ser Lys Lys Lys Val Pro Asn Arg Leu Leu Asn Ile Ala Gly

275 280 285

Ile Ile Val Phe Trp Ile Trp Phe Pro Leu Leu Val Ser Cys Leu Pro

290 295 300

Asn Trp Thr Glu Arg Met Met Phe Val Leu Val Ser Phe Thr Val Thr

305 310 315 320

Ser Ile Gln His Val Thr Phe Cys Leu Asn His Tyr Ser Ala Asp Asn

325 330 335

Tyr Met Gly His Pro Thr Gly Asn Asp Trp Phe Glu Lys Gln Thr Ser

340 345 350

Gly Thr Leu Asp Ile Ser Cys Pro Ser Trp Met Asp Trp Phe His Gly

355 360 365

Gly Leu Gln Phe Gln Leu Glu His His Leu Phe Pro Arg Met Pro Arg

370 375 380

Cys Gln Leu Arg Lys Val Ser Pro Phe Val Lys Glu Leu Cys Gln Lys

385 390 395 400

sequence listing

His Asn Lcu Pro Tyr Arg Ser Leu Ser Phe Phe Glu Ala Asn Val Ala

405 410 415

Thr Ile Lys Thr Leu Arg Thr Ala Ala Leu Gln Ala Arg Asp Leu Ser

420 425 430

Asn Pro Ile Thr Lys Asn Leu Leu Trp Glu Ala Val Asn Thr Tyr Gly

435 440 445

<210>5

<211>28

<212>DNA

<213> Artificial sequence

<400>5

cgcgggtacc atggctaatg caatcaag 28

<210>6

<211>28

<212>DNA

<213> Artificial sequence

<400>6

cgccgagct c tcagccatag gtgttgac 28

<210>7

<211>28

<212>DNA

<213> Artificial sequence

<400>7

cgcgggtacc atgggtgaaa atggaagg 28

<210>8

<211>28

<212>DNA

<213> Artificial sequence

<400>8

cgccgagctc tcaaccatag gtgttgac 28

<210>9

<211>33

<212>DNA

<213> Artificial sequence

<400>9

cgcgaagctt actacaatgt cggagagaca agg 33

<210>10

<211>39

<212>DNA

<213> Artificial sequence

<400>10

cgcgggatcc ttgtgtatgt tctgtagtga tgagtttg 39

Claims (8)

1. gene RnD6C, its nucleotide sequence is shown in SEQ ID NO:1.

2. the gene RnD6C encoded polypeptide of claim 1, its aminoacid sequence is shown in SEQ ID NO:3.

3. an expression vector is characterized in that comprising the described gene of claim 1, and wherein said expression vector is pYES2.

4. an expression vector is characterized in that comprising the described gene of claim 1, and wherein said expression vector is pCambia 1300.

5. the expression vector of claim 4; It is characterized in that also comprising promotor; Said promotor is selected from cauliflower mosaic virus CaMV35S, Ubiqutin, Actin or plant tissue position specific expression promoter, and said promotor is used in combination separately or with other plant promoter.

6. rape host cell is characterized in that comprising the expression vector of claim 3 or 4.

7. the host cell of claim 6, wherein said expression vector are that the mode through Ti-plasmids, Ri plasmid, plant virus-based expression vector, directly DNA conversion, microinjection or electroporation imports.

8. the gene RnD6C of claim 1 produces the application among GLA and the SDA in the transgene rape bio-reactor.

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