CN1854298A - Chimeric gene with delta 6 fatty acid dehydrogenase function and its use - Google Patents

Abstract

The present invention utilizes a fragment RnD6C (1139bp) derived from the DNA of black currant (Ribes nigrum L.) and two other approximately 208bp fragments RnD6A and RnD6B to construct two chimeric genes RnD6CA and RnD6CA with complete reading frames respectively. RnD6CB was expressed in yeast expression system. GC-MS analysis showed that the proteins encoded by these two chimeric genes could catalyze the production of γ-linolenic acid by adding substrate linoleic acid in yeast, and had the function of ^Δ6 fatty acid dehydrogenase. The invention can be applied to produce gamma-linolenic acid with yeast fermentation system and plant expression system by adopting genetic engineering technology.

Description

Chimeric gene with Δ6 fatty acid dehydrogenase function and its application

technical field

The present invention relates to plant coding gene and its application. Specifically, it involves two chimeric genes RnD6CA and RnD6CB with complete reading frames, which are respectively constructed from three segments of DNA sequences derived from black currant (Ribesnigrum L.). The present invention also relates to the polypeptide encoded by the chimeric gene with the function of ^Δ6 fatty acid dehydrogenase, as well as yeast expression vectors and plant expression vectors and host cells containing the DNA sequence, as well as using the gene to transform yeast and plants to produce γ - Application of linolenic acid.

Background technique

In the process of fatty acid metabolism, Δ ⁶ fatty acid dehydrogenase (Δ ⁶ -fatty acid desaturase, D6D) catalyzes the dehydrogenation of the sixth carbon atom of linoleic acid (linoleic acid, LA, 18:2 Δ ^9,12 ) to form γ-linseed Acid (γ-linolenic acid, GLA, 18:3Δ6, 9, 12) [1]. γ-linolenic acid can further form arachidonic acid, prostaglandins and leukotriene physiologically active substances through carbon chain extension and dehydrogenation. have important effects on a series of physiological functions.

As an essential unsaturated fatty acid for the human body, γ-linolenic acid has a series of biological functions on the human body, such as lowering blood fat, resisting lipid peroxidation, reducing weight, inhibiting ulcers, enhancing insulin, and antithrombotic cardiovascular disease [1, 2]. At present, γ-linolenic acid has been studied and utilized as a new vitamin-vitamin F in the world [3]. Therefore, as a key enzyme in the production of γ-linolenic acid—Δ6 fatty acid dehydrogenase has also been more and more researched and utilized. So far, Δ6 fatty acid dehydrogenase genes have been cloned from different organisms such as animals[4], plants[5], fungi[6] and nematodes[7]. ], tobacco [5, 10], rapeseed [6, 8] and soybean [11] etc. were successfully expressed.

In plants, only a few plants such as evening primrose (Oenothera sp.), borage (Borago officinalis L.) and black currant contain GLA[12], borage[5] and evening primrose[13]. The Δ6 fatty acid dehydrogenase gene has been reported abroad, and the two fusion genes involved in this patent are the first report of the Δ6 fatty acid dehydrogenase gene derived from black currant.

Since this type of gene encodes a protein, it can be expressed in vitro using the expression system of Saccharomyces cerevisiae. However, since the protein is located in the chloroplast or endoplasmic reticulum and is a membrane protein, even if it is expressed in the in vitro expression system, it will not be expressed. 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 bacterium is uracil-deficient INVSc I, which itself does not contain LA (linoleic acid) and GLA (γ-linolenic acid). Therefore, when the substrate linoleic acid is added Next, if the catalytic product γ-linolenic acid is detected in the yeast engineering bacteria, it can be confirmed that the protein encoded by the exogenous gene has been expressed in the yeast expression system and has catalyzed the external substrate. 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 [5, 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.

In plants, GLA only exists in a few plants such as evening primrose (Oenothera sp.), borage (Borago officinalis L.) and black currant [12], and the production of seed oil of these plants has become the world's largest The main commercial source of GLA. GLA can also be extracted by fermentation of oleaginous fungi, but the yields of GLA obtained by 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 γ-linolenic acid has important research significance and economic value.

Contents of the invention

The present invention provides two chimeric genes RnD6CA and RnD6CB, the nucleotide sequences of which are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively. These two chimeric genes are derived from black currant (Ribes nigrum L.) DNA fragments RnD6C and RnD6A, and RnD6C and RnD6B were constructed, and the encoded polypeptides are shown in SEQ ID NO: 6 and SEQ ID NO: 7, respectively (Figures 6 and 7).

The present invention provides three DNA fragments RnD6C, RnD6A and RnD6B derived from Black Currant (Ribes nigrum L.), the nucleotide sequences of which are respectively as SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 shown.

Another object of the present invention is to provide yeast expression vectors containing chimeric genes RnD6CA and RnD6CB, and yeast cells transformed with the expression vectors. The polypeptide expressed in the transformed yeast cell has the function of ^Δ6 fatty acid dehydrogenase, and can catalyze the production of γ-linolenic acid by adding the substrate linoleic acid.

Another object of the present invention is to provide a plant expression vector containing chimeric genes RnD6CA and RnD6CB. 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, inducible promoters containing DRE and/or ABRE elements, and plant expression vectors for microprojectile bombardment of monocotyledonous plants.

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, etc., and plant tissue site-specific expression promoters, etc. 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 plant hosts that can be used for transformation in the present invention are dicotyledonous plants selected from the group consisting of tobacco, rapeseed, sunflower, soybean, tomato, castor, sesame and peanut.

The invention also relates to the application of chimeric genes RnD6CA and RnD6CB in the production of gamma-linolenic acid.

Description of drawings:

Figure 1. The nucleotide sequence of the chimeric gene RnD6CA, SEQ ID NO:1.

Figure 2. The nucleotide sequence of the chimeric gene RnD6CB, SEQ ID NO:2.

Figure 3. Nucleotide sequence of RnD6A fragment, SEQ ID NO:3.

Figure 4. Nucleotide sequence of RnD6B fragment, SEQ ID NO:4.

Figure 5. Nucleotide sequence of RnD6C fragment, SEQ ID NO:5.

Figure 6. The polypeptide encoded by the chimeric gene RnD6CA, SEQ ID NO: 6, where the underlined parts are the Cytb5 functional domain and three His Box functional domains.

Figure 7. The polypeptide encoded by the chimeric gene RnD6CB, SEQ ID NO: 7, where the underlined parts are the Cytb5 functional domain and three His Box functional domains.

Fig. 8. Yeast expression vector diagram of chimeric gene RnD6CA/B.

Figure 9. The plant expression vector map of the chimeric gene RnD6CB/B.

Figure 10. GC-MS analysis of total fatty acids expressed by the chimeric gene RnD6CA/B in Saccharomyces cerevisiae

a) GC-MS of fatty acid composition of INVSc I yeast strain;

b) GC-MS of fatty acid composition of INVSc I 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 Saccharomyces cerevisiae expressing RnD6CA gene induced by adding LA substrate, the red arrow indicates the product peak;

f) Partial enlarged view of the product peak (X10);

g) GC-MS of total fatty acids in Saccharomyces cerevisiae expressing RnD6CB gene induced by adding LA substrate, the arrow points to the product peak.

Figure 11. MS spectrum in GC-MS analysis of catalytic product of RnD6CA/B gene expression in S.

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.

Example 1. Construction of chimeric genes

1. Preparation of black currant genomic DNA

Take the living black tea currant (Ribes nigrum L.) plant grown in Beijing Botanical Garden as the material, take about 100 mg of its young leaves, add steel balls (diameter 5mm) into a 7mL Ep tube, and freeze in liquid nitrogen for 20-30min , high-speed crushing on the vortex, 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). 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 RnD6A, RnD6B, RnD6C DNA fragments

Primers were designed to clone RnD6A, RnD6B, and RnD6C DNA fragments from the previously extracted DNA (Fig. 3, 4 and 5). The reaction system used is as follows:

PCR reaction system (50ul):

PCR products were electrophoresed (1% gel concentration), and then the gel was cut to recover the target fragments (the sizes of the three target fragments of RnD6A, RnD6B, and RnD6C were 0.22kb, 0.22kb and 1.1kb, respectively), and were connected into the pMD 18-T vector (Takara Biotech .(Dalian) Co.Ltd) sequencing verification.

The primers used are as follows:

1).Primers used for cloning of RnD6A DNA fragment

Forward primer: 5′-TTGAGCATCATTTGTTCCCTCGATTGCCTCGATGCC-3′ (SEQ ID NO: 8)

Reverse primer: Xba I

5'-GCGG TCTAGA TCACCCCGGGCAGGTGACAGCTT-3' (SEQ ID NO: 9)

2).Primers used for cloning of RnD6B DNA fragment

Forward primer: 5′-TTGAGCATCATTTGTTCCCAAAGATTGCCTCGATGCC-3′ (SEQ ID NO: 10)

Reverse primer: Xba I

5'-GCGG TCTAGA TCAGCCATAGGTGTTGACAGCTTCCC-3' (SEQ ID NO: 11)

3). Primers used for cloning of RnD6C DNA fragment

Forward primer: Hind III

5'-CGCG AAGCTT ATGGCTAATGCAATCAAGAAGTACATG-3' (SEQ ID NO: 12)

Reverse primer: 5'-GGGAACAAATGATGCTCAATTTGG-3' (SEQ ID NO: 13)

3. Construction of chimeric genes RnD6CA and RnD6CB

Using the RnD6A, RnD6B, and RnD6C fragments cloned and verified by sequencing above as templates, the sequences of chimeric genes RnD6CA and RnD6CB were obtained by PCR technology, and HindIII and Xba I restriction sites were introduced at both ends, and connected into pMD18-T , verified by sequencing.

The specific operation is as follows:

1). Preparation of RnD6A, RnD6B, RnDD6C fragments for chimeric genes

Using the same PCR reaction system and PCR program as in Section 2 of Example 1 above, RnD6A, RnD6B, and RnD6C fragments were respectively obtained and recovered by gel electrophoresis (1% gel concentration). in:

The RnD6A fragment is obtained by PCR amplification using the primer sequences shown in SEQ ID NO: 8 and SEQ ID NO: 9;

The RnD6B fragment is obtained by PCR amplification using the primer sequences shown in SEQ ID NO: 10 and SEQ ID NO: 11;

The RnD6C fragment is obtained by PCR amplification using the primer sequences shown in SEQ ID NO: 12 and SEQ ID NO: 13.

2) Chimeric gene preparation

A) Preparation of RnD6CA chimeric gene

The RnD6A (fragment 1) and RnD6C (fragment 2) fragments were fused to form the RnD6CA chimeric gene (Figure 1). The primer sequences used are:

Forward primer: Hind III

5'-CGCG AAGCTT ATGGCTAATGCAATCAAGAAGTACATG-3' (SEQ ID NO: 12)

Reverse primer: Xba I

5'-GCGG TCTAGA TCACCCCGGGCAGGTGACAGCTT-3' (SEQ ID NO: 9)

B) Preparation of RnD6CB chimeric gene

The RnD6B (fragment 1) and RnD6C (fragment 2) fragments were fused to form the RnD6CB chimeric gene (Figure 2). The primer sequences used are:

Forward primer: Hind III

5'-CGCG AAGCTT ATGGCTAATGCAATCAAGAAGTACATG-3' (SEQ ID NO: 12)

Reverse primer: Xba I

5'-GCGG TCTAGA TCAGCCATAGGTGTTGACAGCTTCCC-3' (SEQ ID NO: 11)

Use the following PCR reaction system for PCR fusion to obtain chimeric genes:

Fusion PCR reaction system (20ul):

Full-length chimeric gene PCR reaction system (50ul):

After electrophoresis, the PCR products were recovered by gel cutting, and the full-length fragments (about 1.3Kb) of the RnD6CA and RnD6CB chimeric genes were obtained respectively, and connected into the pMD18-T vector (the left end has a HindIII restriction site, and the right end has a BamH I restriction site ), transformed into Escherichia coli DH-5α (please provide the specific source) for preservation, and at the same time for sequencing verification.

Embodiment 2. Construction of chimeric gene yeast vector

From the pMD18-T vector containing chimeric genes RnD6CA and RnD6CB, the RnD6CA and RnD6CB genes were obtained after double-enzyme digestion using the HindIII and Xba I restriction sites, and were directional cloned into the yeast expression vector pYES2.0 (purchased from Invitrogen Corporation ), obtained yeast expression plasmids pYRnD6CA and pYRnD6CB, transformed into Escherichia coli DH-5α for preservation. Its carrier map is shown in Figure 8.

Example 3. Expression of chimeric genes in yeast

1. Transformation of Yeast

Referring to the method described in Invitrogen’s pYES2 Kit (Cat#V285-20), the yeast expression plasmids pYRnD6CA and pYRnD6CB (see Figure 8 for the plasmid map) of the above-mentioned chimeric genes were transformed into Saccharomyces cerevisiae auxotrophic strain INVSc I using lithium acetate. (Refer to the transformation method described in the pYES2 Kit of Invitrogen Company) (purchased from Invitrogen Company), and use the empty vector pYES2.0 plasmid as a control to obtain yeast cells containing each expression plasmid.

2. Induced expression in yeast transformed cells

Take the yeast single colony transformed with the yeast expression plasmid containing the chimeric gene, inoculate it in 50 ml of SC-U culture medium containing 2% raffinose (refer to the formula described in the pYES2 Kit of Invitrogen Company), and cultivate it overnight at 250 rpm on a shaker at 28°C . Then add Nonad P-40 (NP-40, purchased from BBI Company) (final concentration 1%), exogenous linoleic acid substrate (purchased from Sigma Company) (final concentration is 0.003%), and yeast expression induction 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 4. GC-MS analysis of chimeric 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 0.1g 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 (purchased from Aldtich Company), and after 5h in water bath at 70°C, add HCl was acidified until its pH reached 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% NaCI solution, mix well and let stand for a while. Then add 2 ml 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 10a, 10b and 10c, it shows that under the condition of not adding linoleic acid substrate, no new product peaks appear in the yeast engineering strain used and the yeast containing the induced empty pYES2 expression plasmid, indicating that the yeast engineering strain used and The total fatty acids of the empty pYES2 expression plasmid yeast did not contain the additional substrate linoleic acid (LA) and the catalytic product γ-linolenic acid (GLA).

When the linoleic acid substrate was added and the expression was induced, the control (empty pYES2.0) also had no product peak γ-linolenic acid (GLA) (see Figure 10d). After the pYRnD6CA/B yeast expression strain is induced, the additional substrate linoleic acid (LA) can be catalyzed to generate γ-linolenic acid (GLA) (see Figure 10e and 10g)

Comparing Figures 10e, 10f and 10g, it can be found that a new substance is formed when the retention time is 7.6 minutes. , its peak time and mass spectrum are consistent with those of the standard γ-linolenic acid (purchased from Sigma Company) (see Figures 11a and 11b), and it is determined to be γ-linolenic acid.

The above results proved that the two chimeric genes RnD6CA/B derived from black currant were successfully expressed in Saccharomyces cerevisiae, and the expressed protein could catalyze the production of γ-linolenic acid (GLA) by adding the substrate linoleic acid (LA).

Embodiment 5. Construction of chimeric gene RnD6CA/B plant expression vector

The construction of the plant expression vector of RnD6CA/B was carried out by means of conventional molecular biology (see "Refined Molecular Biology Experiment Guide" 2001, Science Press, translated by Yan Ziying and Wang Hailin). With RnD6CA/B DNA as template, by introducing BamHI site (replacing Hind III with BamH I site) and reverse primer (SEQ ID NO: 9(RnD6CA) and Sac I site was introduced into SEQ ID NO: 11 (RnD6CB) (replace Xba I with Sac I site), and it was inserted behind the CaMV 35S promoter in the pBI121 binary expression vector [14] to obtain a binary The map of the expression vector pRnD6CA/B is shown in FIG. 9 . After digestion with BamH I and SacI and identification of the inserted fragment by PCR, it was transformed into Agrobacterium LBA4404[15], and the plasmid was extracted, digested with PCR and confirmed by PCR. This expression vector can be directly used for transformation of plants (see Figure 9).

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

4. Hyekyung P. Cho, Manabu T. Nakamura, and Steven D. Clarke (1999) Cloning, Expression, and Nutritional Regulation of the Mammalian Δ ⁶ -Desaturase. JBC 274(1): 471-477.

5. Sayanova O, Smith MA, Lspinskas P, Stobart AK, Dobson G, Christie WW, Shewry PR, 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 Obacco. PNAs 94:4211-4216.

6. Haiping Hong, Nagamani Datla, Darwin W.Reed, Patrick S.Covello, Samuel L.MacKenzie, and Xiao Qiu(2002) High-Level Production ofγ-Linolenic Acid in Brassica juncea Using a Δ ⁶ Desaturase from Pythium irregulare.Plant Physiology 129:354–362.

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

8. Qiu X, Hong HP, Datla N, MacKenzie SL, Tayler CD, Thomas LT (2002) Expression of borage Δ 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, Tucker G (2002) Modification of fatty acid composition in tomato (Lycopersicone sculentum) by expression of a borage Δ ⁶ -desaturase. Mol Biotechnol. 21 (2): 123-128.

10. Olga Sayanova, Mark A. Smith, Peter Lapinskas, A. Keith Stobart, Gary Dobson, William W. Christie, Peter R. Shewry, And Johnathan A. Napier (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. PNAs94: 4211-4216.

11. Shirley Sato, Aiqiu Xing, Xingguo Ye, Bruce Schweiger, Anthony Kinney, George Graef and Tom Clemente (2004) Production of γ-Linolenic Acid and Stearidonic Acid in Seeds of Marker-Free Transgenic Soybean. Genomics 4 Molecular- 4 Genetics 4 Genetics 652.

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

13. Emilio Mendoza de Gyves, Caroline A. Sparks, Olga Sayanova, Paul Lazzeri, Johnathan A. Napier and Huw D. Jones (2004) Genetic manipulation of γ-linolenic acid (GLA) synthesis in a commercial variety of evening primrose (Oenothera sp.) Plant Biotechnology 2(4): 351-357.

14. Chen PY, Wang CK, Soong SC, To KY (2003) Complete sequence of the binary vector pBI121 and its application in cloning T-DNA insertion from transgenic plants. Molecular Breeding 11: 287-293.

15. Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) Abinary plant vetor strategy based on separation of vir-and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303:179-180.

IB051489-Sequence Listing

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ttgagcatca tttgttccct cgattgcctc gatgccagct taggacggtc tctccttttg 60

cgatggagct atgcaagaag cacaatttgc cttataggag tttgtccttt ttggaggcca 120

atgtcgcgac cattatgacg ttgaggactg cagccctgca agcgcgcgac ttgtccaacc 180

cggttccgaa gaacttgctc tgggaagctg tcacctgccc ggggtga 227

<210>4

<211>227

<212>DNA

<213> Artificial sequence

<400>4

ttgagcatca tttgttccca agattgcctc gatgccagct taggaaggtc tctccttttg 60

tgaaagagct ttgcaagaaa cacaatttgc cttataggag tttatcattt tttgaggcca 120

atgttgcaac cattaagacg ttgaggactg ccgccttgca agctcgcgac ctatccaacc 180

ctatttcaaa gaatttgcta tgggaagctg tcaacaccta tggctga 227

<210>5

<211>1139

<212>DNA

<213> Artificial sequence

<400>5

atggctaatg caatcaagaa gtacatgaca gttgaggagc taaaagtgca taacaagcca 60

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

IB051489-Sequence Listing

cctactggga acgattggtt tgagaagcag acaagtggga ctttggacat ttcgtgcccg 1080

tcttggatgg attggtttca tggtgggctg caattccaaa ttgagcatca tttgttccc 1139

<210>6

<211>448

<212>PRT

<213> Artificial sequence

<400>6

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 Ata 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 Ash 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

IB051489-Sequence Listing

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 Thr Val Ser Pro Phe Ala Met Glu Leu Cys Lys Lys

385 390 395 400

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

405 410 415

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

420 425 430

Asn Pro Val Pro Lys Asn Leu Leu Trp Glu Ala Val Thr Cys Pro Gly

435 440 445

<210>7

<211>448

<212>PRT

<213> Artificial sequence

<400>7

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

IB051489-Sequence Listing

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

IB051489-Sequence Listing

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

Thr 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>8

<211>36

<212>DNA

<213> Artificial sequence

<400>8

ttgagcatca tttgttccct cgattgcctc gatgcc 36

<210>9

<211>33

<212>DNA

<213> Artificial sequence

<400>9

gcggtctaga tcaccccggg caggtgacag ctt 33

<210>10

<211>36

<212>DNA

<213> Artificial sequence

<400>10

ttgagcatca tttgttccca agattgcctc gatgcc 36

<210>11

<211>36

<212>DNA

IB051489-Sequence Listing

<213> Artificial sequence

<400>11

gcggtctaga tcagccatag gtgttgacag cttccc 36

<210>12

<211>37

<212>DNA

<213> Artificial sequence

<400>12

cgcgaagctt atggctaatg caatcaagaa gtacatg 37

<210>13

<211>24

<212>DNA

<213> Artificial sequence

<400>13

gggaacaaat gatgctcaat ttgg 24

Claims (13)

1. mosaic gene RnD6CA, its nucleotide sequence is shown in SEQ ID NO:1.

2. mosaic gene RnD6CB, its nucleotide sequence is shown in SEQ ID NO:2.

3. the mosaic gene RnD6CA encoded polypeptide of claim 1, its aminoacid sequence is shown in SEQ ID NO:6.

4. the mosaic gene RnD6CB encoded polypeptide of claim 2, its aminoacid sequence is shown in SEQ ID NO:7.

5. an expression vector is characterized in that comprising claim 1 or 2 described mosaic genes.

6. the expression vector of claim 5, it is Yeast expression carrier or plant expression vector.

7. the expression vector of claim 6, wherein said plant expression vector is selected from pBIN19, pBI121, pB221, or contain the evoked promoter of DRE and/or ABRE element and the plant expression vector that is used for the monocotyledons micropellet bombardment.

8. the expression vector of claim 5, it is characterized in that also comprising promotor, described promotor is selected from cauliflower mosaic virus CaMV 35S, Ubiqutin, Actin or plant tissue position specific expression promoter, and described promotor is used in combination separately or with other plant promoter.

9. host cell is characterized in that comprising the expression vector of claim 5.

10. the host cell of claim 9, wherein said expression vector are that the mode by Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection or electroporation imports.

11. the host cell of claim 9, it is yeast cell or vegetable cell.

12. the host cell of claim 11, wherein said yeast cell are brewing yeast cell, described vegetable cell is the cell of tobacco, rape, Sunflower Receptacle, soybean, tomato, castor-oil plant, sesame or peanut.

13. the mosaic gene RnD6CA of claim 1 or the mosaic gene RnD6CB of claim 2 application in producing gamma-linolenic acid.

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