CN102888425B - Method for producing astaxanthin by using transgenic plant - Google Patents

Abstract

The invention discloses plant expression vectors pBI121-LTPCRBKT and pBI121-BHYBKT, and a method for transforming host cells with the vectors and cultivating transgenic plants to efficiently produce astaxanthin. The method uses transgenic plant cells, tissues or organs as reactors to produce astaxanthin with high efficiency and low cost. The astaxanthin transgenic plant obtained by the method of the invention can be used as a raw material for extracting astaxanthin or directly as an additive in industries such as health care food, cosmetics and animal breeding.

Description

Method for producing astaxanthin by utilizing transgenic plants

Technical field:

The invention belongs to the field of plant genetic engineering and relates to a method for producing target objects by using transgenic plants. In particular, it relates to a method for constructing a plant vector, obtaining a recombinant host cell, and obtaining an astaxanthin-producing transgenic plant.

Background technique:

Astaxanthin (3,3'-dihydroxy4,4'-diketo-β-carotene) is a ketone carotenoid, the strongest antioxidant in nature, and its antioxidant activity is 500 times that of vitamin E , is also much higher than other commonly used antioxidants. Medical research has proved that astaxanthin has biological functions such as protecting skin and eyes, improving immunity, anti-cardiovascular disease, anti-inflammation, anti-tumor, and anti-aging. Astaxanthin widely exists in aquatic organisms, such as microalgae and aquatic animals (animals themselves cannot synthesize it and must be obtained through the food chain). Humans mainly obtain astaxanthin by eating aquatic products. The astaxanthin of artificially farmed aquatic products comes from the chemically synthesized astaxanthin added to their feed. Artificial astaxanthin contains a variety of substances harmful to the human body, and its use will undoubtedly cause pollution to the food chain. With the strict restrictions on the use of chemical products in aquaculture in various countries, artificial astaxanthin will withdraw from the market sooner or later.

Astaxanthin biosynthesis occurs in only a few organisms and its yield is usually low. Haematococcus pluvialis is the organism with the highest astaxanthin content in nature and is the only single-celled eukaryotic green algae (the ancestor of plants) currently used for commercial production of natural astaxanthin. However, Haematococcus pluvialis grows slowly and is sensitive to the growth environment, so it cannot be cultured on a large scale and at low cost like Spirulina. The commercial production of natural astaxanthin using Haematococcus pluvialis is a matter of nearly ten years. Due to the large investment, high technical requirements, and the constraints of the genetic characteristics of Haematococcus pluvialis, the current Haematococcus pluvialls farming enterprises can only produce astaxanthin with a small production scale and high production costs. This is the main reason for the high price and short supply of natural astaxanthin.

For a long time, people have tried to solve the production problem of astaxanthin by optimizing the culture conditions of Haematococcus pluvialis or screening mutants, but no breakthrough has been made so far. In the past ten years, attempts have been made to express the genes of key enzymes in the astaxanthin synthesis pathway of microorganisms such as Haematococcus pluvialis in model organisms such as Escherichia coli, yeast and model plants and have achieved some success. Among them, the content of astaxanthin in tobacco that expresses the oxygenase gene derived from bacteria by transforming the chloroplast genome is the highest (Hasunuma T, et al. Plant Journal 2008, 55:857-868), but its content is only ten times that of Haematococcus pluvialis. Less than one-third, far from reaching the level of low-cost industrial production. The main reason for limiting the high-efficiency synthesis and accumulation of astaxanthin in plants is that the endogenous highly active β-carotene hydroxylase (BHY) in plants makes the hydroxylation of β-carotene occupy an absolute advantage, while the typical β-carotene oxygenase ( BKT) is difficult to catalyze the hydroxylation of β-carotene to astaxanthin. The key to solving this problem is to isolate and functionally analyze the novel BKT and express it efficiently in plant-specific tissues and organs containing a large amount of carotenoids, such as tomato fruit and carrot root.

Invention content:

The purpose of the present invention is to provide a method for efficiently synthesizing and accumulating astaxanthin in plants by using genetic engineering technology to solve the above-mentioned problems in the prior art.

To achieve the above object, the present invention has taken the following technical solutions:

Plant expression vectors pBI121-LTPCRBKT and pBI121-BHYBKT, linked to plants with guide gene product localization via the β-carotene oxygenase BKT from Chlamydomonas and β-carotene hydroxylase BHY from Haematococcus pluvialis The chloroplast signal peptide sequence is obtained by inserting the signal peptide sequence into the plant expression vector pBI121 after correctly joining the reading frame of the signal peptide sequence.

The plant expression vectors pBI121-LTPCRBKT and pBI121-BHYBKT of the present invention are constructed by the following method:

Insert the tomato ribulose carboxylase small subunit chloroplast guide peptide sequence LTP into the bacterial expression vector pCRBKT containing CRBKT to obtain the vector pLTPCRKBT, and then cut out the LTP+CRBKT nucleotide fragment and insert it into the plant expression vector pBI121 for expression Vector pBI121-LTPCRBKT;

Replace the CRBKT fragment with HPBHY in pBI121-LTPCRBKT to obtain pBI121-LTPHPBHY, and then insert the CaMV35S::SlTpHpBHY::nos sequence on the vector into the ClaI site on the vector pBI121-LTPCRBKT to obtain the vector pBI121 after PCR amplification and digestion -BHYBKT.

The method for establishing astaxanthin-producing transgenic plants using the plant expression vectors pBI121-LTPCRBKT and pBI121-BHYBKT of the present invention includes constructing the plant expression vector pBI121- LTPCRBKT and pBI121-BHYBKT, and then use the Agrobacterium-mediated method to obtain the transgenic plant step of expressing BKT and BHY in the transgenic plant and catalyzing the production of astaxanthin from the carotenoid of the plant itself.

The application of the above-mentioned genes β-carotene oxygenase BKT and β-carotene hydroxylase BHY and the recombinant vectors pBI121-LTPCRBKT and pBI121-BHYBKT in the preparation of recombinant host cells of the present invention, the recombinant host cells are produced by agricultural Bacteria are prepared by obtaining the recombinant vector through a heat shock method.

The application of the above-mentioned genes β-carotene oxygenase BKT and β-carotene hydroxylase BHY and recombinant vectors pBI121-LTPCRBKT and pBI121-BHYBKT in the preparation of transgenic plant cells and transgenic plants of the present invention, the transgenic plant cells And the transgenic plant is mediated by Agrobacterium. CRBKT or BHYBKT in the recombinant vector pBI121-LTPCRBKT and pBI121-BHYBKT in the recombinant host cell described in claim 4 is introduced into the plant cell, and then through the plant tissue and organogenesis pathway in High-yielding astaxanthin transgenic plants were obtained under the selection pressure of antibiotic kanamycin.

The application of the above-mentioned genes β-carotene oxygenase BKT and β-carotene hydroxylase BHY and the recombinant vectors pBI121-LTPCRBKT and pBI121-BHYBKT in the production of astaxanthin by using transgenic plants includes constructing The plant expression vectors pBI121-LTPCRBKT and pBI121-BHYBKT of β-carotene oxygenase BKT and β-carotene hydroxylase BHY were obtained, and then the transgenic plants were obtained by the method mediated by Agrobacterium, so that BKT and BHY could be expressed in the transgenic plants and catalyze The carotenoid itself generates astaxanthin step.

The recombinant host cell prepared by using the method of transforming the host cell with the above-mentioned plant expression vectors pBI121-LTPCRBKT and pBI121-BHYBKT of the present invention.

Astaxanthin-producing transgenic plants are obtained by transforming host cells with the above-mentioned plant expression vectors pBI121-LTPCRBKT and pBI121-BHYBKT of the present invention, and cultivating transgenic plants.

The application of the recombinant host cell of the present invention in the preparation of transgenic plant cells and transgenic plants.

The present invention also provides transgenic plants obtained by using the plant expression vectors pBI121-LTPCRBKT and pBI121-BHYBKT of the present invention to establish astaxanthin-producing transgenic plants as raw materials for extracting astaxanthin, and their use in the preparation of health food and cosmetics Application in the preparation of animal breeding products.

Description of drawings

Figure 1 Physical map of bacterial expression vector pCRBKT;

Fig. 2 Physical map of plant expression vector pBI121-LTPCRBKT;

Figure 3 The physical map of the plant expression vector pBI121-BHYBKT;

Fig. 4 Control and astaxanthin-producing tomato plants, flowers and fruits;

Fig. 5 Molecular detection of CRBKT and HPBHY expression in astaxanthin-producing tomato.

Detailed ways:

The following embodiments of the present invention are used to describe the present invention in more detail in conjunction with the accompanying drawings, but the technical solutions of the present invention are not limited thereto.

Example 1:

Cloning, transformation and analysis of Chlamydomonas β-carotene oxygenase in bacterial expression system:

(1) Cloning of Chlamydomonas carotene oxygenase:

Chlamydomonas reinhardtii cc-124 was provided by Chamydomonas Center (Duke University, USA). Chlamydomonas was cultured with TAP medium/medium. For total RNA extraction, TRI extraction solution (Molecular research center, Cincinnati, OH, USA) was used to extract total RNA from approximately 10 ⁸ Chlamydomonas cells, and the method was referred to the steps in the manual. Synthesis of cDNA SuperScript ^TM III One-Step RT-PCR system (Invitrogen, Carlsbad, CA, USA) was used to synthesize cDNA at 50°C for 15 minutes. Using the Chlamydomonas cDNA as a template, use the following pair of primers for PCR amplification to obtain the carotene oxygenase gene. After sequencing to confirm that there is no mutation, the fragment obtained by digesting with HindIII and XbaI is connected to the corresponding restriction site of pBluescript II KS The obtained vector product is pCRBKTL.

The PCR primers are as follows (HindIII and XbaI sites are designed for the upstream and downstream primers respectively):

Forward primer: 5'-GAGAAGCTTCATGGGCCCTGGGGATACA-3'

Reverse primer: 5'-GCGTCTAGATCAAGCCATCACGCCAAC-3'

(2) Modification of Chlamydomonas β-carotene oxygenase:

(a) First remove the C-terminus of the cloned Chlamydomonas β-carotene oxygenase (encoding 115 amino acids) relative to other homologous oxygenases, and use the Chlamydomonas cDNA as a template and the following primers for The truncated β-carotene oxygenase gene was amplified by PCR. No mutation was confirmed by sequencing. The fragment was digested with HindIII and XbaI and connected to the corresponding restriction site of pBluescriptII KS. The vector product was pCRBKTS.

The PCR primers are as follows (HindIII and XbaI sites are designed for the upstream and downstream primers respectively):

Forward primer: 5'-GAGAAGCTTCATGGGCCCTGGGGATACA-3'

Reverse primer: 5'-GCGTCTAGATCAGGCCAGGGCTGCGCCGCG-3'

(b) Using the vector pCRBKTS as a template, use the GeneMorph II Random Mutagenesis kit (Stratagene, La Jolla, CA) to perform error prone PCR (error prone PCR). The method steps refer to the instruction manual, and the primers are as described in (a). The reaction conditions can produce 0-4 base mutations per kb of DNA. The product was digested with HindIII and XbaI to obtain a fragment connected to the corresponding restriction site of pBluescript II KS.

(3) Analysis of Chlamydomonas carotene ketonease in bacterial expression system:

The vectors pCRBKTL, pCRBKTS obtained in (1) and (2) and the vectors obtained by random mutation were respectively transformed by electroporation into E. coli DH5α competent cells containing pACCAR25△crtX vector. Bacteria harboring the pACCAR25ΔcrtX vector can accumulate zeaxanthin (Misawa et al., Journal of Bacteriology, 1990, 177:6575-6584) as a substrate for oxygenase. Bacterial culture conditions refer to the method of Misawa et al. (1990). Pigment components were analyzed by high-performance liquid chromatography (HPLC), and the equipment used was a Waters HPLC system (Waters, Milford, MA, USA) equipped with a Waters Spherisorb® 5 μm ODS2 4.6 250 mm analytical column. The method adopts the method described by Baroli (Plant Cell 15: 992-1008, 2003), which is modified as follows: from 100% solution A[acetonitrile/methanol/0.1 M Tris-HCl (pH 8.0), 84:2:14, v/ v/v] Follow a linear gradient to 100% solution B (methanol/ethyl acetate, 68:32, v/v) at a flow rate of 1.2 mL min-1 for 15 minutes, followed by solution B for 10 minutes. The pigments were determined by the light absorption peak and appearance time with standard pigment samples, and the quantification of pigments was also quantified with standard pigment samples. All standard pigment samples were purchased from Sigma and Wako.

Example 2:

Construction of plant expression vectors containing Chlamydomonas carotene oxygenase:

(1) One of the vectors, pBI121-LTPCRBKT, was constructed from pBI121-CRBKT (zhong et al, Journal of Experimental Botany 62:3659-69, 2011). The RBCS1 signal peptide derived from Arabidopsis in pBI121-CRBKT was replaced with the RBCS signal peptide of tomato (NCBI accession number: M15236). The tomato RBCS signal peptide was amplified using the total tomato DNA as a template, and the following pair of primers were used for PCR amplification to obtain the tomato RBCS signal peptide (LTP). After sequencing, it was confirmed that there was no mutation, and the fragment obtained by digestion with SmaI and HindIII was ligated into pBI121- The vector product obtained from the corresponding restriction site of CRBKT is pBI121-LTPCRBKT.

The PCR primers are as follows: (SalI-SmaI and HindIII sites are designed for the upstream and downstream primers respectively)

Forward primer: 5'-GCGTCGACCCGGGGAACCAAAAAAAGAGAGAAG-3'

Reverse primer: 5'-CCCAAGCTTGGCATGCAGCTAACTTCTTCCAC-3'

(2) The second vector, pBI121-BHYBKT, was constructed from pBI121-LTPCRBKT. First, the CRBKT of pBI121-LTPCRBKT was replaced with HPBHY. Use the cDNA of Haematococcus pluvialis as a template to amplify its BHY coding sequence, and use the following pair of primers to perform PCR amplification to obtain BHY. After sequencing, it is confirmed that there is no mutation, and the fragment obtained by digesting with HindIII and XbaI is connected to the corresponding enzyme of pBI121-LTPCRBKT The vector product obtained by cleavage site is pBI121-LTPHPBHY.

The PCR primers are as follows (HindIII and XbaI sites are designed for the upstream and downstream primers respectively):

Forward primer: 5'-GAGAAGCTTAATTACCACGATGCTG-3'

Reverse primer: 5'-GCGTCTAGACATCTAGTAACATAGA-3'

Next, amplify the CaMV35S::SlTpHpBHY::nos fragment using pBI121-LTPHPBHY as a template, and perform PCR amplification with the following pair of primers to obtain the CaMV35S::SlTpHpBHY::nos fragment. After sequencing, it is confirmed that there is no mutation, and the fragment is obtained by digestion with ClaI The vector product obtained by ligation to the corresponding restriction site of pBI121-LTPCRBKT is pBI121-BHYBKT.

The PCR primers are as follows (ClaI-HindIII and ClaI-EcoRI sites are designed for the upstream and downstream primers respectively):

Forward primer: 5'-CCATCGATAAGCTTGCATGCCTGC-3'

Reverse primer: 5'-CCATCGATGAATTCCATTCTAGTAACATAGA-3'

Example 3:

Construction of transgenic plants:

(1) Preparation of Agrobacterium containing plant expression vector:

Add 2 microliters of the pBI121-CRBKT and pBI121-BHYBKT plasmids prepared in Example 2 to 200 microliters of the competent cells obtained in Example 1, mix gently, and place on ice for 30 minutes. Then the bacterial suspension was heat-shocked in a water bath at 42°C for 60 seconds, and quickly moved to ice for 5 minutes to cool down. Add 800 microliters of LB liquid medium to the bacterial suspension, and recover at 28°C for 3 hours. Take an appropriate amount of bacterial suspension and spread it on an LB plate containing 50 mg/L streptomycin and 50 mg/L kanamycin, and place the plate in a warm bath at 28°C for 2 days. Pick a single colony from the plate, identify it by PCR and expand it, extract the plasmid and identify it by digestion. Agrobacteria containing the corresponding plasmids are used for plant transformation. First, 100 microliters of Agrobacterium culture solution was inserted into 3 ml of LB liquid culture solution containing 50 mg/L streptomycin and 50 mg/L kanamycin, and cultured for 48 hours. The culture was recovered by centrifugation, the supernatant was discarded, and the cell pellet was resuspended in 3 ml MS medium (MS medium supplemented with 1% sucrose, 0.2 mg/L 2,4-D, 0.1 mg/L IAA, 100mg/L AS, pH 5.8) to obtain the Agrobacterium suspension.

(2) Preparation of tomato cotyledon explants:

Tomato (Solanum lycopersicum) seeds (B-type LA0316) were provided by the TomatoGenetics Resource Center (TGRC). After surface disinfection, culture on MS solid medium for 14 days until the cotyledon grows. The cotyledons were excised and cultured on MS pre-culture plates (MS medium supplemented with 0.8% agar, 1% sucrose, 2 mg/L zeatin, 0.1 mg/LIAA, 100 mg/L AS, pH 5.8) for 1 day. Soak the pre-cultured cotyledon explants in the Agrobacterium suspension obtained in step (1) for 10 minutes, and place the soaked explants on MS co-culture plates (MS medium supplemented with 0.8% agar, 1 % sucrose, 2 mg/L zeatin, 0.1 mg/L IAA, 100 mg/L AS, 300 mg/L carbenicillin, 100 mg/L streptomycin and 50 mg/L kanamycin (pH 5.8) at 28°C After culturing for one day without light, move to light and incubate at 24°C for one day. After co-cultured explants were washed with water, transferred to callus formation medium (MS medium supplemented with 0.8% agar, 1% sucrose, 2 mg/L zeatin, 0.05mg/L IAA, 300mg/L carbenicillin and 100 mg/L kanamycin, pH 5.8) at 24°C. One month later, the callus was transferred to the germination medium (MS medium supplemented with 0.8% agar, 1% sucrose, 1 mg/L zeatin, 0.03mg/L IAA, 200mg/L carbenicillin and 100mg/L card Namycin, pH 5.8) to brown-red buds. When the bud grows two leaves, cut the bud and transfer it to the rooting medium (MS medium supplemented with 0.8% agar, 1% sucrose, 0.1 mg/LIBA, and 100mg/L kanamycin, pH 5.8), about Adventitious roots grow after 1-2 weeks. After the root system developed, the plants were taken out, moved into the cultivation soil, and cultivated in the greenhouse (24°C, 16 hours of light/8 hours of darkness).

Example 4:

Identification of transgenic plants and determination of astaxanthin content:

(1) Identification of transgenic plants:

Because astaxanthin is a red pigment. Its synthesis in plant organs will change the color of the original plant organs. Therefore, the selection of transgenic plants starts from the shoots grown on the callus, and the shoots with color changes are selected for rooting culture. Approximately 20 transgenic plants were produced per vector-transformed plant, whose organs (including buds, leaves, stems, flowers and fruits) were colored differently from non-transgenic plants.

(2) Determination of astaxanthin content in transgenic plants:

Plants generally cannot synthesize astaxanthin. The astaxanthin content of the transgenic plants is determined by HPLC to obtain the transgenic plants for efficiently synthesizing astaxanthin, and the method is the same as that of bacterial pigments. It was determined that each vector transformed the plant system to obtain a transgenic plant with the highest content of astaxanthin. The transgenic plant of pBI121-LTPCRBKT was named TB-b10, and the transgenic plant of pBI121-BHYBKT was named TB-bb8. The analysis of plant traits is shown in Figure 4 . The pigment composition analysis of ripe fruit is shown in Table 1.

Table 1 Content and percentage of carotenoids in tomato

(3) Gene expression measurement of transgenic plants:

Extract the total RNA from the mature fruits of transgenic plants TB-b10 and TB-bb8, synthesize cDNA, and amplify with primers of crbkt and hpbhy respectively, hpbhy forward primer:

5'-CGCAAACGGGAGCAGCTGTCATA-3'; hpbhy reverse primer:

5'-CGCGCCACCAACCACCAAGA-3'. Crbkt forward primer:

5'-CCGCCTTCCGCCTGTTCTACTA-3'; reverse primer:

5'-CGGGCAATCTGGCGGCACTT. The results of PCR amplification are shown in Figure 5.

The method for producing astaxanthin using transgenic plants of the present invention is to screen and improve the BKT gene through the Escherichia coli functional complementation analysis method, construct a plant expression vector containing the improved BKT gene and the BHY gene that cooperates efficiently with it, and use Agrobacterium-mediated Transformed plants were introduced to obtain transgenic plants, and BKT and BHY were efficiently expressed in the transgenic plants to obtain high-yield astaxanthin.

The BKT gene of the present invention is derived from Chlamydomonas reinhardtii cc-124 (NCBI registration number: AY860820, referred to as CrBKT); BHY is derived from Haematococcus pluvialis (NCBI registration number: AY187011; referred to as HpBHY).

Firstly, the CRBKT gene is transformed. Compared with other homologous genes, the CRBKT gene has an extra 3' end sequence encoding 115 amino acids. This sequence is removed, and the enzyme gene (CRBKTQ164N) with stronger ketonization ability to zeaxanthin is obtained through random mutation. ). Secondly, construct the expression vector containing CRBKT mutant for plant transformation. One: the nucleotide sequence encoding the CRBKT mutant is placed under the drive of the CaMV35S promoter, plus a signal peptide that guides the gene product into the chloroplast. The second: placing the nucleotide sequence encoding BHY under the drive of the CaMV35S promoter, and adding a signal peptide that guides the gene product into the chloroplast to control the positioning of the gene product. Combined with the previous CRBKT-containing mutant to form the second expression vector.

The plant expression vector is accomplished by the following methods:

(1) Insert the tomato ribulose carboxylase small subunit chloroplast guide peptide sequence (LTP) into the bacterial expression carrier pCRBKT containing CRBKT (Figure 1) to obtain the vector pLTPCRKBT, cut the LTP+CRBKT nucleotide fragment and insert it The expression vector pBI121-LTPCRBKT was obtained on the plant expression vector pBI121 (Figure 2).

(2) Replace the CRBKT fragment with HPBHY in pBI121-LTPCRBKT to obtain pBI121-LTHPBHY, cut the CaMV35S::SlTpHpBHY::nos sequence on the vector and insert it into the vector pBI121-LTPCRBKT to obtain the vector pBI121-BHYBKT (Figure 3).

After the expression vectors pBI121-LTPCRBKT and pBI121-BHYBKT are respectively transformed into Agrobacterium LBA4404, tomato cotyledon explants are infected, and a higher transformation rate is obtained by screening with kanamycin and leaf color.

By using the method of the present invention, the β-carotene oxygenase derived from green algae can be transferred to plants, and expressed efficiently in various plant organs (leaves, flowers, fruits), and the oxygenated enzymes containing β-carotene can be cultivated. Enzyme plant species, its astaxanthin content is as high as 16 mg/g, which is close to the content of Haematococcus pluvialis. It has reached the requirements of industrial production of astaxanthin. The biosynthesis of astaxanthin only occurs in a few organisms and the yield is usually very low, and astaxanthin-rich transgenic plants can produce high-value astaxanthin at low cost to meet the market demand for this high-antioxidant substance . Astaxanthin derived from Haematococcus pluvialis is mainly used in high-end health food and cosmetics due to its low yield. So far, the astaxanthin market is still monopolized by synthetic products, which are mainly used as animal feed additives. High-yielding astaxanthin transgenic plants can be directly applied to animal feed. From the perspective of safety, economy and nutritional value, transgenic crops are superior. The advantages of the present invention are as follows: 1. The gene used in the present invention comes from the ancestral green algae of plants rather than the commonly used gene of bacterial origin. More importantly, the content of astaxanthin in plants expressing this gene is much higher than that of related genes expressing other sources; 2. The production cost of natural astaxanthin obtained through transgenic plants is low, and transgenic plants producing astaxanthin can be directly used as water Feed additives or functional foods for product farming; 3. Compared with algae farming, crop planting costs are low, easy to operate and less investment; 4. The production scale of economic plants is easier to expand rapidly and be widely used.

Claims (10)

1. plant expression vector pBI 121-BHYBKT, it is characterized in that it is by being connected to the plant chloroplast signal peptide sequence of guiding gene product location from the β-carotene hydroxylase BHY gene of Haematocoocus Pluvialls, be inserted into after correctly engaging with signal peptide sequence reading frame on plant expression vector pBI 121 and obtain, build by the following method and obtain: pBI 121-LTPCRBKT HPBHY being substituted CRBKT fragment and obtains pBI 121-LTPHPBHY, again the CaMV35S::SlTpHpBHY::nos sequence on this carrier is cut by pcr amplification and enzyme on the ClaI site on rear insertion vector pBI 121-LTPCRBKT and obtain carrier pBI 121-BHYBKT.

2. application rights requires that the plant expression vector pBI 121-BHYBKT described in 1 sets up the method for producing astaxanthin transgenic plant, it is characterized in that the method comprises the plant expression vector pBI 121-BHYBKT built containing β-carotene hydroxylase BHY, then obtain BHY and BKT by agriculture bacillus mediated method and express in transfer-gen plant and the transfer-gen plant step of the carotenoid of catalysis plant generation astaxanthin itself.

3. plant expression vector pBI 121-BHYBKT according to claim 1 is preparing the application in recombinant host cell, it is characterized in that described recombinant host cell is obtained obtained by described recombinant vectors through heat shock method by Agrobacterium.

4. plant expression vector pBI 121-BHYBKT according to claim 1 is preparing the application in transgenic plant cells and transgenic plant, it is characterized in that described transgenic plant cells and transgenic plant are, by agriculture bacillus mediated, the BHYBKT in carrier pBI 121-BHYBKT is imported to vegetable cell, then under microbiotic kantlex selective pressure, obtain astaxanthin transfer-gen plant by plant tissue and adventitious organogenesis.

5. plant expression vector pBI 121-BHYBKT according to claim 1 is utilizing the application in transgenic plant production astaxanthin, it is characterized in that described application comprises the plant expression vector pBI 121-BHYBKT built containing β-carotene hydroxylase BHY, then obtain transfer-gen plant by agriculture bacillus mediated method, BHY is expressed in transfer-gen plant and the carotenoid of catalysis plant generation astaxanthin step itself.

6. the transgenic plant of the product astaxanthin prepared by the method for producing astaxanthin transgenic plant are set up with plant expression vector pBI 121-BHYBKT according to claim 2.

7. the transgenic plant obtained by the method for claim 2 are as the raw-material application of extraction astaxanthin.

8. the transgenic plant obtained by the method for claim 2 are preparing the application in protective foods.

9. the transgenic plant obtained by the method for claim 2 are preparing the application in makeup.

10. the transgenic plant obtained by the method for claim 2 are preparing the application in animal cultivation product.