Tandem gene for biosynthesizing astaxanthin in rape seeds, expression cassette and expression method

Abstract

The invention provides a tandem gene for biosynthesizing astaxanthin in rape seeds, an expression cassette and an expression method, and belongs to the technical field of gene engineering. The invention provides a tandem gene for biosynthesis of astaxanthin, and four carotenoid anabolism genes are firstly constructed into the tandem gene for a biosynthesis path of astaxanthin. The four carotenoid anabolism genes are driven based on the rape seed specific NAPIN promoter to construct an astaxanthin biosynthesis path in the rape seeds and synthesize the astaxanthin, so that the biosynthesis efficiency of the astaxanthin is improved; the expression of the astaxanthin biosynthetic gene and the accumulation of the astaxanthin in the seeds do not influence the growth, development and reproduction of the rape; in addition, fat-soluble carotenoid and astaxanthin are naturally integrated with grease in the rape seeds, and astaxanthin rapeseed oil is directly used as a product, so that the activity of astaxanthin is maintained, and the later astaxanthin extraction production cost can be reduced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a tandem gene for biosynthesis of astaxanthin in rapeseed, an expression cassette and an expression method. BACKGROUND

[0002] Astaxanthin is a natural pigment, also known as astacene and astaxanthin, which belongs to carotenoids. It exists in the shells of animals such as shrimps, crabs and fish, and in some algae, fungi and other organisms. Astaxanthin is one of the strongest natural antioxidants in nature, and has multiple biological activities such as antioxidant, anti-inflammatory, anti-tumor, immune enhancement and vision protection. It is widely used in medicine, health products, cosmetics, food and aquaculture industries. Among astaxanthin isomers, L-astaxanthin is mainly derived from Haematococcus pluvialis, and its antioxidant activity is the strongest among the three. D-astaxanthin is mainly derived from shrimp and crab shells, yeast and other organisms, and has partial antioxidant activity. Mesostaxanthin has no antioxidant activity and is mainly found in chemically synthesized astaxanthin.

[0003] Astaxanthin sources include natural extraction, chemical synthesis and biosynthesis. Natural astaxanthin exists in animals and microorganisms rich in carotenoids, such as Haematococcus pluvialis, Phaffia rhodozyma and Antarctic krill. Currently, commercial natural astaxanthin is mainly derived from Haematococcus pluvialis, but its biomass is low, and there are problems such as bacterial contamination, viral infection, excessive heavy metals and plasticizers, and low astaxanthin content during cultivation. In addition, the cost of astaxanthin extraction from Haematococcus pluvialis is high, and the required equipment is expensive. Chemical synthesis of astaxanthin includes total synthesis and semi-synthesis. Total synthesis of astaxanthin is to use chemical raw materials as raw materials to synthesize astaxanthin through chemical synthesis reaction. Semi-synthesis method is to use carotenoids such as canthaxanthin, lutein and zeaxanthin as raw materials to prepare astaxanthin. Chemical synthesis of astaxanthin requires multiple chemical reactions and biological catalytic reactions, and the synthesized astaxanthin is a mixture of multiple configurations and contains by-products, and the synthesis process has a high safety risk. Compared with natural astaxanthin, chemically synthesized astaxanthin has certain differences in structure, properties, applications and safety, and its stability, antioxidant activity and coloring property are significantly lower than those of natural astaxanthin.

[0004] In recent years, the production of astaxanthin in yeast, algae, bacteria and higher plants by synthetic biology method has become a new field of the industry. There are reports on astaxanthin biosynthesis in plants such as Arabidopsis, tomato, tobacco, rice, corn, potato and cotton as the chassis organisms. Most of them use the constitutive promoter of cauliflower mosaic virus CaMV35S (35S) to biosynthesize astaxanthin in plant vegetative tissues. There is also a report on the use of 35S promoter to drive astaxanthin biosynthesis genes to biosynthesize astaxanthin in the vegetative tissues of Brassica napus. However, the use of 35S constitutive promoter to drive genes to synthesize astaxanthin in plant vegetative tissues seriously inhibits photosynthesis of plants and affects the growth and development of plants. Therefore, how to use Brassica napus to synthesize astaxanthin without affecting the growth and development of Brassica napus has become a technical problem to be solved. SUMMARY

[0005] To solve the above technical problems, the application provides a tandem gene for biosynthesizing astaxanthin in Brassica napus seeds, an expression cassette containing the tandem gene and an expression method.

[0006] The application provides a tandem gene for biosynthesizing astaxanthin, which comprises, in sequence, a phytoene synthase gene, a phytoene desaturase gene, a beta-carotene hydroxylase gene and a beta-carotene ketolase gene.

[0007] In a preferred mode of the application, the phytoene synthase gene is derived from corn, the phytoene desaturase gene is derived from Pseudomonas syringae, the beta-carotene hydroxylase gene is derived from Haematococcus pluvialis, and the beta-carotene ketolase gene is derived from Chlamydomonas reinhardtii.

[0008] The application further provides a four-gene tandem expression frame, which comprises, in sequence, a phytoene synthase gene, a 2A peptide coding gene, a phytoene desaturase gene, a 2A peptide coding gene, a beta-carotene hydroxylase gene, a 2A peptide coding gene and a beta-carotene ketolase gene.

[0009] In a preferred mode of the application, the amino acid sequence of the protein encoded by the four-gene tandem expression frame is shown in SEQ ID No. 1.

[0010] In a preferred mode of the application, the nucleotide sequence of the four-gene tandem expression frame is shown in SEQ ID No. 2.

[0011] The application further provides a four-gene expression cassette, which comprises a seed-specific promoter and the above four-gene tandem expression frame.

[0012] In a preferred embodiment of the present invention, the seed-specific promoter includes those derived from rapeseed. NAPIN The promoter, the NAPIN The nucleotide sequence of the promoter is shown in SEQ ID No. 3.

[0013] The present invention also provides a vector or strain containing the above-mentioned four gene expression cassettes.

[0014] The present invention also provides a method for cultivating rapeseed that biosynthesizes astaxanthin, comprising the following steps: transforming rapeseed using the above-mentioned carrier or strain to obtain rapeseed that biosynthesizes astaxanthin in seeds.

[0015] The present invention also provides a method for biosynthesizing astaxanthin in rapeseed seeds, comprising the following steps: transforming rapeseed using the above-mentioned carrier or strain, culturing the successfully transformed rapeseed, and harvesting the seeds, wherein the seeds contain astaxanthin.

[0016] Beneficial effects: This invention provides a tandem gene for the biosynthesis of astaxanthin, and for the first time integrates four carotenoid biosynthetic genes: phytopene synthase. ZmPSY 8-Hydroxylycopene desaturase PaCrtl β-Carotene hydroxylase HpBHY and β-carotene ketolase CrBKT By introducing rapeseed, a tandem gene sequence for the astaxanthin biosynthesis pathway was constructed, and based on rapeseed seed specificity... NAPIN The promoter drives four carotenoid biosynthesis genes to construct an astaxanthin biosynthesis pathway and synthesize astaxanthin in rapeseed seeds, thereby improving the biosynthesis efficiency of astaxanthin; due to NAPIN The promoter-driven gene is highly expressed in the mid-to-late stages of rapeseed embryonic development. The expression of astaxanthin biosynthesis genes and the accumulation of astaxanthin in seeds do not affect the growth, development, or reproduction of rapeseed. Furthermore, as a fat-soluble carotenoid, astaxanthin is naturally integrated into rapeseed seeds and oils. Directly using astaxanthin-enriched rapeseed oil as a product preserves astaxanthin activity and reduces the cost of later astaxanthin extraction. This invention solves several long-standing technical problems encountered in astaxanthin production and the biosynthesis of astaxanthin in plants. Attached Figure Description

[0017] Figure 1 A schematic diagram illustrating the biosynthetic pathway of astaxanthin in rapeseed seeds; Figure 2 for 35S:PCBB and NAPINp:PCBB Schematic diagram of the construction of a four-gene tandem expression vector. In the diagram, A. 35S promoter drives the four-gene tandem expression vector; B. NAPIN promoter ( NAPINp()Driven four-gene tandem expression vector; P2A1-3, 2A peptide; TP, plasmid transport peptide; Figure 3 Recombinant plasmid 35S:PCBB Figure showing the verification results of transient expression of astaxanthin biosynthesis in Nicotiana benthamiana. Figure 4 for NAPINp:PCBB Phenotypic characteristics of astaxanthin color in transgenic rapeseed seeds. In the figure, A is a developing rapeseed seed; B is a mature rapeseed seed; C is a germinating rapeseed seed; WT is a wild-type rapeseed seed. NAPINp:PCBB Genetically modified rapeseed seeds; Figure 5 for NAPINp:PCBB Growth phenotype of transgenic rapeseed; WT in the figure represents wild-type rapeseed. NAPINp:PCBB It is genetically modified rapeseed; Figure 6 The image shows the results of astaxanthin content determination in genetically modified rapeseed seeds; WT in the image represents wild-type rapeseed seeds. NAPINp:PCBB 1-4 These are genetically modified rapeseed seeds; ND was not detected. Detailed Implementation

[0018] This invention provides a tandem gene for the biosynthesis of astaxanthin, comprising a phytoene synthase gene, a phytoene desaturase gene, a β-carotene hydroxylase gene, and a β-carotene ketolase gene connected in series.

[0019] This invention selected four carotenoid biosynthesis genes to construct the tandem gene, that is, to construct the astaxanthin biosynthesis pathway using the tandem gene, such as... Figure 1 As shown: phytoene synthase forms colorless phytoene by condensing two molecules of geranyl diphosphate (GGPP) → phytoene is catalyzed by phytoene desaturase to form lycopene, which is further metabolized into β-carotene → β-carotene is further catalyzed by β-carotene hydroxylase and β-carotene ketolase to form astaxanthin.

[0020] The present invention does not specifically limit the source of the four genes in the tandem gene; for example, in one embodiment, the phytoene synthase gene is derived from maize (…). ZmPSY The phytoene desaturase gene mentioned above is derived from Erwinia urediniosum (…). PaCrtl The β-carotene hydroxylase gene is derived from Haematococcus pluvialis (Haematococcus pluvialis). HpBHY The β-carotene ketolase gene is derived from Chlamydomonas reinhardtii ( CrBKT ).

[0021] This invention transforms the tandem gene into rapeseed and expresses the astaxanthin in rapeseed seeds, thus, based on the codon bias of rapeseed, the astaxanthin... ZmPSY , PaCrtl , HpBHY and CrBKT The gene sequences were codon-optimized, and the four genes were named as follows: sPSY , sCrtl , sBHY and sBKT .

[0022] The present invention also provides a four-gene tandem expression cassette, comprising, in sequence, a phytoene synthase gene, a 2A peptide encoding gene, a phytoene desaturase gene, a 2A peptide encoding gene, a β-carotene hydroxylase gene, a 2A peptide encoding gene, and a β-carotene ketolase gene.

[0023] In this invention, astaxanthin, as a carotenoid, is synthesized in the plastids of plant cells, therefore... sPSY The gene-encoded phytopene synthase already possesses a plassome transport peptide. In order to... sCrtl Encoded phytopene desaturase, sBHY Encoded β-carotene hydroxylase and sBKT The encoded β-carotene ketolase is located on the plastid, in sCrtl , sBHY as well as sBKT A plasmid localization transport peptide (TP) sequence is added to the 5' end of each gene.

[0024] The four genes described in this invention are expressed tandemly within the same open reading frame. Therefore, 2A peptide coding sequences (P2A1, P2A3, and P2A3) are inserted between the four genes, resulting in a single transcript during transcription. During protein translation, no peptide bond forms between the C-terminus and N-terminus of the 2A peptide, leading to protein self-cleavage. This allows for the independent expression of multiple proteins, ensuring the uniformity of the four synthases and guaranteeing the smooth operation of the astaxanthin biosynthetic pathway. The final four-gene tandem expression frame structure is shown below: sPSY -P2A1-TP- sCrtl -P2A2-TP- sBHY -P2A3-TP- sBKT The complete nucleotide sequence of the designed protein is shown in SEQ ID No. 2, which is 5283 bp in length, and the amino acid sequence encoding the protein is shown in SEQ ID No. 1, which encodes 1760 amino acids in length.

[0025] The present invention also provides a four-gene expression cassette, comprising a seed-specific promoter and the above-mentioned four-gene tandem expression cassette.

[0026] The seed-specific promoters described in this invention include those derived from rapeseed. NAPIN Promoters selected from the rapeseed genome database NAPIN The sequence 1603 bp upstream of the gene start codon ATG, and named... NAPINp The aforementioned NAPINp It can be obtained by amplification. During amplification, rapeseed genomic DNA is used as a template. The promoter fragment is cloned by PCR and constructed into a cloning plasmid for sequencing verification. Its nucleotide sequence is shown in SEQ ID No. 3.

[0027] This invention will identify clones. NAPINp Insert upstream of the four-gene tandem expression cascade to obtain NAPINp Seed-specific expression cassette driven by the driver NAPINp : sPSY -P2A1-TP- sCrtl -P2A2-TP- sBHY -P2A3-TP- sBKT (abbreviation) NAPINp : PCBB The four-gene expression cassette described in this invention is obtained through... Aat The hygromycin resistance gene is inserted at site II into the backbone of the MWV067 plant expression vector (Zhan J, Zhang X, Wang Y, Zhao H, Chu Y, Wang P, Chen Y, Wei X, Qin W, Liu M, Kong J, Li F, Ge X. The GhWL1-GhH1-GhGA2OX1 transcriptional module regulates Scottton leaf morphology. Advanced Science 2025 12: 2410783).

[0028] The present invention also provides a vector or strain containing the above-mentioned four gene expression cassettes.

[0029] By inserting the four-gene expression cassette of the present invention into the MWV067 plant expression vector, a recombinant vector expressing astaxanthin specifically for seeds can be obtained. In one embodiment, the recombinant vector was transformed into Agrobacterium GV3101 to obtain the strain.

[0030] The present invention also provides a method for cultivating rapeseed that biosynthesizes astaxanthin, comprising the following steps: transforming rapeseed using the above-mentioned carrier or strain to obtain rapeseed that biosynthesizes astaxanthin in seeds.

[0031] The present invention does not specifically limit the method of transformation. For example, in one embodiment, Agrobacterium-mediated hypocotyl infection of rapeseed (Huang KL, Wang H, Wei YL, Jia HX, Zha L, Zheng Y, Ren F, Li XB) is used to transform rapeseed (Huang KL, Wang H, Wei YL, Jia HX, Zha L, Zheng Y, Ren F, Li XB). The high-affinity transporter BnPHT1;4 is involved in phosphorus acquisition and mobilization for facilitating seed germination and early seedling growth of Brassica napus (BMC Plant Biology 2019 19:156.) After antibiotic screening, transgenic rapeseed lines were obtained. From the transgenic lines, rapeseed seeds with reddish-brown color in the later stages of development can be identified, and the cotyledons of mature rapeseed seeds also show a reddish-brown astaxanthin deposition phenotype.

[0032] The present invention also provides a method for biosynthesizing astaxanthin in rapeseed seeds, comprising the following steps: transforming rapeseed using the above-mentioned carrier or strain, culturing the successfully transformed rapeseed, and harvesting the seeds, wherein the seeds contain astaxanthin.

[0033] The method described in this invention is the same as that described above, and will not be repeated here.

[0034] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a tandem gene, expression cassette, and expression method for the biosynthesis of astaxanthin in rapeseed seeds provided by the present invention, should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1 rape NAPIN Promoter identification and cloning (1) According to rapeseed NAPIN Expression patterns of homologous genes in rapeseed seeds were screened to identify high-level expression specific to rapeseed. NAPIN Gene; (2) Selecting from the rapeseed genome NAPIN The promoter sequence 1600 bp upstream of the ATG start codon; (3) According to NAPIN Primer design based on promoter sequence: Upstream primer (SEQ ID No. 4): 5'-taataatcagttgaagcaattaagaatca-3'; Downstream primer (SEQ ID No. 5): 5'-ttgtgtatgttttgtagtgatgagttttg-3'; (4) Using the above primers, rapeseed genome was cloned by PCR. NAPIN Gene promoter DNA fragments; (5) PCR amplification NAPIN The gene promoter DNA fragment was ligated into the cloning plasmid pBluescript sk for sequencing analysis, and the sequence was found to be completely correct. NAPIN Gene promoter, named NAPINp The nucleotide sequence is shown in SEQ ID No. 3.

[0036] Example 2 sPSY , sCrtl , sBHY , sBKT Gene chemical synthesis (1) Retrieve from the database ZmPSY (GenBank: AAB60314) PaCrtl (GenBank: AHG94990) HpBHY (GenBank: AEA35045) and CrBKT Gene (GenBank: AEA35045) sequence; (2) Obtain P2A and TP sequences from the database; P2A sequence (SEQ ID No. 6): GSGATNFSLLKQAGDVEENPGP; TP sequence (SEQ ID No. 7): MASMISSSAVTTVSRASRGQSAAVAPFGGLKSMTGFPVKKVNTDITSITSNGGRVKC; (3) Remove ZmPSY , PaCrtl , HpBHY The stop codons of the three genes, then ZmPSY , PaCrtl , HpBHY as well as CrBKT Gene sequence tandem, in ZmPSY and PaCrtl , PaCrtl and HpBHY as well as HpBHY and CrBKT P2A and TP sequences are inserted between them respectively to form ZmPSY -P2A1-TP- PaCrtl -P2A2-TP- HpBHY -P2A3-TP-CrBKT Fusion expression sequence; (4) ZmPSY -P2A1-TP- PaCrtl -P2A2-TP- HpBHY -P2A3-TP- CrBKT Codon optimization of fused expression sequences for rapeseed yields results suitable for rapeseed. sPSY -P2A1-TP- sCrtl -P2A2-TP- sBHY -P2A3-TP- sBKT Fusion expression sequence; (5) Chemical synthesis on the plant expression vector WMV067 backbone sPSY -P2A1-TP- sCrtl -P2A2-TP- sBHY -P2A3-TP- sBKT The fusion expression sequence was verified by sequencing, and the nucleotide sequence is shown in SEQ ID No. 2.

[0037] Example 3 35S:PCBB Construction of constitutive plant overexpression vectors (1) Design upstream and downstream primers for cloning sequences based on the 35S promoter sequence in the plant expression vector pBI121; 35S upstream primer (SEQ ID No. 8): 5'-cactagtgcatgcctgcaggtccccag-3' 35S downstream primer (SEQ ID No. 9): 5'-tactagtgttctctccaaatgaaatgaac-3' (2) Using pBI121 plasmid as a template, the 35S promoter DNA fragment was amplified by PCR; (3) Insert the 35S promoter DNA fragment sPSY -P2A1-TP- sCrtl -P2A2-TP- sBHY -P2A3-TP- sBKT upstream formation of fusion expression sequence Figure 2 As shown in A 35S:sPSY -P2A1-TP- sCrtl -P2A2-TP- sBHY -P2A3-TP- sBKT ( 35S:PCBB Recombinant plasmid.

[0038] Example 4 Verification of astaxanthin biosynthesis in tobacco leaves 1. Agrobacterium-mediated transformation (1) Place Agrobacterium GV3101 competent cells stored at -80℃ on ice to thaw; (2) Use a pipette to take approximately 100 ng of 35S:PCBB The recombinant plasmid was added to GV3101 competent cells, mixed, and placed on ice for 10 min, then in liquid nitrogen for 1 min, and then quickly transferred to a 37°C water bath for 5 min. (3) Add about 1 mL of LB liquid culture medium, place in a shaker at 28℃, and shake at 200 rpm for 2-3 h; (4) Spread the bacterial culture onto LB solid medium containing 50 mg / L kanamycin (Kan) and incubate at 28°C for 48 h; (5) Select single colonies and perform PCR detection using the upstream primer of the 35S promoter sequence and the downstream primer of the sPSY gene. Positive single colonies are used for tobacco transformation.

[0039] Upstream primer (SEQ ID No. 10): 5'-gacgtaagggatgacgcaca-3'; Downstream primer (SEQ ID No. 11): 5'-ttgggccatcgaccaattca-3'; 2. With 35S:PCBB Preparation of Agrobacterium suspension of recombinant plasmid and injection infection of Tobacco Benzoenta leaf (1) Positive clones were cultured in LB liquid medium (containing 50 mg / L Kan) at 28°C with shaking at 200 rpm until OD. 600 The value is approximately 0.6.

[0040] (2) Place the bacterial culture in a high-speed centrifuge, centrifuge at 6000 rpm for 10 min, discard the supernatant and collect the bacterial cells; (3) Resuspend Agrobacterium in osmotic buffer (10 mM 2-morpholinoethanesulfonic acid, 10 mM MgCl2, 0.2 mM acetylsuccinone) and let stand at room temperature for 10 min; (4) Use a syringe to evenly inject the resuspended Agrobacterium tumefaciens solution onto the back of the tobacco leaves; (5) After the tobacco was injected, it was placed in the dark at 25℃ for 24 h, and then in the light at 25℃ for 2-3 days to observe the color formation of astaxanthin in the tobacco leaves.

[0041] The results are as follows Figure 3 As shown, two days after Agrobacterium injection, reddish-brown astaxanthin deposits appeared on tobacco leaves, and all four genes could be expressed in the leaves of Tobacco Benedict.

[0042] Example 5 NAPINp:PCBBConstruction of plant expression vectors for rapeseed genetic transformation 1. NAPINp:PCBB Construction and Agrobacterium-mediated transformation (1) The cloned NAPIN promoter DNA fragment insertion sPSY -P2A1-TP- sCrtl -P2A2-TP- sBHY -P2A3-TP- sBKT Upstream of the fusion expression sequence forms, such as Figure 2 As shown in B NAPINp:sPSY -P2A1-TP- sCrtl -P2A2-TP- sBHY -P2A3-TP- sBKT ( NAPINp:PCBB Recombinant plasmid.

[0043] (2) Place Agrobacterium GV3101 competent cells stored at -80℃ on ice to thaw; (3) Use a pipette to take approximately 100 ng of NAPINp:PCBB The recombinant plasmid was added to GV3101 competent cells, mixed, and placed on ice for 10 min, then in liquid nitrogen for 1 min, and then quickly transferred to a 37°C water bath for 5 min. (4) Add about 1 mL of LB liquid medium, place in a shaker at 28℃, and shake at 200 rpm for 2-3 h; (5) Spread the bacterial culture onto LB solid medium containing 50 mg / L kanamycin (Kan) and incubate at 28°C for 48 h; (6) Pick single colonies using the upstream NAPIN promoter primers and downstream sPSY Gene sequence primers were used for PCR detection, and positive monoclonal colonies were used for rapeseed transformation.

[0044] Upstream primer (SEQ ID No. 4): 5'-taataatcagttgaagcaattaagaatca-3'; Downstream primer (SEQ ID No. 11): 5'-ttgggccatcgaccaattca-3'; 2. With NAPINp:PCBB Preparation of Agrobacterium suspension of recombinant plasmids and genetic transformation of rapeseed (1) Positive clones were cultured in LB liquid medium (containing 50 mg / L Kan) at 28°C with shaking at 200 rpm until OD. 600 The value is approximately 0.6.

[0045] (2) Place the bacterial culture in a high-speed centrifuge, centrifuge at 6000 rpm for 10 min, discard the supernatant and collect the bacterial cells; (4) Resuspend Agrobacterium cells in a resuspension solution (5% sucrose and 0.02% surfactant) to OD. 600 The concentration was approximately 1.0, which was then used to prepare an Agrobacterium suspension for subsequent rapeseed conversion.

[0046] Example 6 Rapeseed genetic transformation 1. The specific steps for converting rapeseed using the hypocotyl infection method are as follows: (1) After soaking the rapeseed seeds in 70% ethanol solution for 1 min, add an appropriate amount of 84 disinfectant solution for 10-15 min, wash them with sterile water 5 times, spread them on MO (MS) medium, and place them in an artificial climate incubator at 24℃ for 4-5 days in the dark. (2) Cut the hypocotyl of the seedling and place the cut explant in the bacterial solution for 30 min. After discarding the bacterial solution, use filter paper to absorb the residual bacterial solution on the surface of the hypocotyl, and then transfer the explant to M1 medium (MS containing 2,4-dichlorophenoxyacetic acid (2,4-D), 0.3 mg / L kinetin (KT), 0.1 mM acetylsylgenone (AS)) and incubate in the dark at 24℃ for 2-3 days; (3) The explants were transferred into M2 medium (MS containing 1 mg / L 2,4-D, 0.3 mg / L KT, 200 mg / L termethin (TMT), 2 mg / L trans-zeatin (ZT) and 30 mg / L Kan) and cultured at 24℃ for 16 h light / 8 h dark. (4) After 3 weeks, the green and well-grown callus tissue was transferred to M3 medium (MS containing 0.1 mg / L IAA, 200 mg / L TMT, 2 mg / L ZT and 200 mg / L spectinomycin (Spe)) until regenerated green shoots differentiated; (5) When the regenerated shoots grow to 1 cm, cut off the small shoots and transfer them to M4 medium (MS containing 0.02 mg / L IAA, 1 mg / L IBA and 200 mg / L Spe) to induce rooting; (6) After the transformed seedlings have taken root and grown leaves, the DNA of the transformed seedlings is extracted and used for… NAPIN The upstream primer of the promoter (SEQ ID No. 4) and sPSY The gene was identified by PCR using the downstream primer (SEQ ID No. 11); (7) The positive seedlings identified by PCR were transplanted into nutrient pots in an artificial climate greenhouse for growth. After the seeds matured, individual plants were harvested to obtain T1 generation transgenic rapeseed seeds.

[0047] 2. Screening for homozygous positive transgenic rapeseed plants using spectinomycin, the specific steps are as follows: (1) The T1 generation transgenic rapeseed seeds were disinfected according to the above method for obtaining sterile rapeseed seedlings; (2) Spread the sterilized seeds evenly on MS solid medium containing 50 mg / L spectinomycin and culture them in the dark for about 5 days in an incubator, then switch to culture under normal light. (3) When two true leaves have grown, transplant the positive seedlings into the nutrient soil in the artificial climate greenhouse; (4) After the positive transgenic rapeseed seeds mature, each plant is harvested and numbered sequentially to obtain T2 generation transgenic rapeseed seeds. (5) The T2 generation transgenic rapeseed seeds were screened for spectinomycin resistance according to the above experimental steps. The lines with a segregation ratio of 3:1 were screened out, transplanted into an artificial climate greenhouse, harvested individually, and numbered and stored in sequence to obtain T3 generation transgenic rapeseed seeds. (6) The T3 generation transgenic rapeseed seeds were screened for spectinomycin according to the above experimental steps. All normally growing lines were screened out to obtain homozygous T3 generation transgenic rapeseed.

[0048] Example 7 Astaxanthin biosynthesis phenotype and biosynthesis gene expression verification in transgenic rapeseed seeds (1) During the cotyledon formation stage of transgenic rapeseed seeds, the seed color characteristics were observed. Negative seeds were green, while brownish-red seeds were positive lines. (2) Using green and brownish-red seeds as materials, total RNA was extracted and reverse transcribed into cDNA, and then used as follows: sPSY , sCrtl , sBHY , sBKT Four gene-specific primers were used to analyze the expression of astaxanthin biosynthesis genes by PCR.

[0049] sPSY Upstream primer for gene PCR (SEQ ID No. 12): 5'-tgaattggtcgatggcccaa-3'; sPSY Gene PCR downstream primer (SEQ ID No. 13): 5'-tcggtaacacccctttcagc-3'; sCrtl Upstream primer for gene PCR (SEQ ID No. 14): 5'-tccaatgccgatgtggttca-3'; sCrtlGene PCR downstream primer (SEQ ID No. 15): 5'-tgagtgagcacaggttcgac-3'; sBHY Upstream primer for gene PCR (SEQ ID No. 16): 5'-agggcgattgctgagagaag-3'; sBHY Gene PCR downstream primer (SEQ ID No. 17): 5'-ccgccgtatttgccagaatg-3'; sBKT Upstream primer for gene PCR (SEQ ID No. 18): 5'-gccgccgtattcttctcact-3'; sBKT Gene PCR downstream primer (SEQ ID No. 19): 5'-cacggagcataaggccatct-3'.

[0050] The results are as follows Figure 4 As shown, rapeseed seeds exhibiting a reddish-brown color in the later stages of development can be identified from the transgenic lines, and the cotyledons of mature rapeseed seeds also show a reddish-brown astaxanthin deposition phenotype. Furthermore, the growth traits of the transgenic lines of this invention are not affected. Figure 5 ).

[0051] Example 8 Astaxanthin test in genetically modified rapeseed seeds Take 50-100 mg of astaxanthin from rapeseed seeds, extract with acetone, collect the extract and dilute to 10 mL, filter, and determine the astaxanthin content using high-performance liquid chromatography (UPLC). Results are as follows: Figure 6 As shown in Table 1.

[0052] Table 1. Astaxanthin content in transgenic rapeseed seeds

[0053] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A tandem gene for biosynthesis of astaxanthin, characterized in that, The four-gene tandem expression cassette comprises, in series, a phytoene synthase gene, a phytoene desaturase gene, a β-carotene hydroxylase gene and a β-carotene ketolase gene.

2. The tandem gene according to claim 1, wherein The phytoene synthase gene is derived from Zea mays, the phytoene desaturase gene is derived from Xanthomonas campestris, the β-carotene hydroxylase gene is derived from Haematococcus pluvialis, and the β-carotene ketolase gene is derived from Chlamydomonas reinhardtii.

3. A quad-gene tandem expression cassette, characterized in that, The four-gene tandem expression cassette comprises, in series, a phytoene synthase gene, a 2A peptide coding gene, a phytoene desaturase gene, a 2A peptide coding gene, a β-carotene hydroxylase gene, a 2A peptide coding gene and a β-carotene ketolase gene.

4. The quad-gene tandem expression cassette of claim 3, wherein, The amino acid sequence of the protein encoded by the four-gene tandem expression cassette is shown as SEQ ID No.

1.

5. The quad-gene tandem expression cassette of claim 3, wherein, The nucleotide sequence of the four-gene tandem expression cassette is shown as SEQ ID No.

2.

6. A quad expression cassette comprising, The four-gene tandem expression cassette comprises, in series, a phytoene synthase gene, a phytoene desaturase gene, a β-carotene hydroxylase gene and a β-carotene ketolase gene.

7. The quad cassette of claim 6, wherein, The seed-specific promoter includes that from Brassica napus NAPIN The nucleotide sequence of the promoter is shown as SEQ ID No.

3. NAPIN The nucleotide sequence of the promoter is shown as SEQ ID No.

3.

8. A vector or strain comprising the four-gene expression cassette of claim 6 or 7.

9. A method for breeding Brassica napus to biosynthesize astaxanthin, characterized by, The method comprises the following steps: transforming Brassica napus with the vector or strain of claim 8 to obtain Brassica napus capable of biosynthesizing astaxanthin in seeds.

10. A method for biosynthesis of astaxanthin in Brassica napus seeds, characterized in that, The method comprises the following steps: transforming Brassica napus with the vector or strain of claim 8, culturing the transformed Brassica napus, harvesting seeds, and the seeds contain astaxanthin.

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