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

Abstract

The invention discloses a plant expression vector pBI121-LTPCRBKT and pBI121-BHYBKT and a method for efficiently producing astaxanthin by transforming a host cell by using the vector and culturing a transgenic plant. According to the method, the astaxanthin is efficiently produced at low cost by using transgenic plant cells, tissues or organs as reactors. The astaxanthin transgenic plant which is obtained by the method can be used as a raw material for extracting the astaxanthin or directly used as an additive to be used in industry of health care food, cosmetics, animal husbandry and the like.

Description

Transgenic plant are utilized to produce the method for astaxanthin

Technical field:

The invention belongs to plant genetic engineering field, relate to the method utilizing transgenic plant to produce object.In particular to structure plant vector, obtain recombinant host cell by it, and obtain the method for the transgenic plant producing astaxanthin.

Background technology:

Astaxanthin (3,3 '-dihydroxyl 4,4 '-diketo β-carotene) is keto-acid carotenoid, and be the antioxidant that occurring in nature is the strongest, its anti-oxidant activity is 500 times of vitamin-E, also far above other conventional antioxidants.Medical research proves that astaxanthin has protection skin and eyes, improves immunizing power, anti-cardiovascular disease, anti-inflammatory, antitumor, anti-ageing biological function of waiting for a long time.Astaxanthin is extensively present in hydrobiont, as micro-algae and hydrocoles (animal itself can not be synthesized, and must be obtained by food chain).The mankind obtain astaxanthin mainly through edible aquatic products.Its astaxanthin of the fishery products propagated artificially derives from the chemosynthesis astaxanthin making an addition to its feed for nursing.Artificial astaxanthin contains multiple harmful material, and its use causes the pollution of food chain undoubtedly.Along with various countries are to the strict restriction of Chemicals for aquaculture, artificial astaxanthin will be withdrawn from the market sooner or later.

The biosynthesizing of astaxanthin only betides minority biology and its output is usually very low.Haematocoocus Pluvialls is the biology that occurring in nature content astaxanthin is the highest, is unique single celled eukaryotic green alga (ancestors of plant) for commercially producing natural astaxanthin at present.But haematococcus pluvialis growing is slow, responsive to growing environment, can not resemble spirulina and carry out large-scale low-cost cultivation.Utilize Haematocoocus Pluvialls to commercially produce thing that natural astaxanthin is nearly ten years.Because having high input, technical requirements is high, is limited by the hereditary property of Haematocoocus Pluvialls self simultaneously, and current Haematocoocus Pluvialls breeding enterprise can only produce astaxanthin with little industrial scale and higher production cost.This causes the major cause that natural astaxanthin price is high He supply falls short of demand.

All the time, people attempt by optimizing Haematocoocus Pluvialls culture condition or screening the production problem that mutant solves astaxanthin, but do not obtain breakthrough so far.Constantly have nearly ten years and attempt by microorganism chemical activators pathway key enzyme gene expressions such as Haematocoocus Pluvialls in model animals, as intestinal bacteria, yeast and model plant also obtain certain success.The highest (the Hasunuma T of tobacco content astaxanthin of the monooxygenase gene of bacterial origin is wherein expressed with Chloroplast Genome Transformation, et al. Plant Journal 2008,55:857-868), but its content only reaches less than 1/10th of Haematocoocus Pluvialls, reach far away low cost industrialization production level.The major cause of the synthesis of restriction plant efficient and accumulation astaxanthin is that plant endogenous highly active β-carotene hydroxylase (BHY) makes hydroxylation β-carotene have comparative advantage and typical β-carotene oxygenase (BKT) is difficult to catalysis hydroxylation β-carotene and becomes astaxanthin.Be separated and functional analysis novel B KT by its high expression in the plant specific tissue containing a large amount of carotenoid and organ, as the fruit of tomato, the block root etc. of Radix Dauci Sativae is the key point of this difficult problem of solution.

Summary of the invention:

The object of the invention is the problems referred to above existed for prior art, provide a kind of genetic engineering technique that utilizes plant efficient is synthesized and accumulates the method for astaxanthin.

For achieving the above object, this invention takes following technical scheme:

Plant expression vector pBI121-LTPCRBKT and pBI121-BHYBKT, be connected to the plant chloroplast signal peptide sequence of guiding gene product location by the β-carotene oxygenase BKT from chlamydomonas and the β-carotene hydroxylase BHY gene from Haematocoocus Pluvialls, be inserted into after correctly engaging with signal peptide sequence reading frame on plant expression vector pBI121 and obtain.

Plant expression vector pBI121-LTPCRBKT and pBI121-BHYBKT of the present invention builds by the following method and obtain:

Tomato ribulose carboxylase small subunit chloroplast(id) is led the bacterial expression vector pCRBKT that peptide sequence LTP is inserted into containing CRBKT and obtain carrier pLTPCRKBT, then LTP+CRBKT nucleotide fragments enzyme is scaled off to be inserted on plant expression vector pBI121 obtain expression vector pBI121-LTPCRBKT;

PBI121-LTPCRBKT HPBHY is substituted CRBKT fragment and obtains pBI121-LTPHPBHY, then the CaMV35S::SlTpHpBHY::nos sequence on this carrier is cut by pcr amplification and enzyme on the ClaI site on rear insertion vector pBI121-LTPCRBKT and obtain carrier pBI121-BHYBKT.

Apply plant expression vector pBI121-LTPCRBKT and pBI121-BHYBKT of the present invention and set up the method for producing astaxanthin transgenic plant, comprise plant expression vector pBI121-LTPCRBKT and pBI121-BHYBKT built containing β-carotene oxygenase BKT and β-carotene hydroxylase BHY, then obtain BKT and BHY 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.

The gene β-carotene oxygenase BKT that the present invention is above-mentioned and β-carotene hydroxylase BHY and recombinant vectors pBI121-LTPCRBKT and pBI121-BHYBKT is preparing the application in recombinant host cell, and described recombinant host cell is obtained obtained by described recombinant vectors through heat shock method by Agrobacterium.

The gene β-carotene oxygenase BKT that the present invention is above-mentioned and β-carotene hydroxylase BHY and recombinant vectors pBI121-LTPCRBKT and pBI121-BHYBKT is preparing the application in transgenic plant cells and transgenic plant, described transgenic plant cells and transgenic plant are, by agriculture bacillus mediated, CRBKT or BHYBKT in recombinant vectors pBI121-LTPCRBKT and pBI121-BHYBKT in recombinant host cell according to claim 4 is imported to vegetable cell, then under microbiotic kantlex selective pressure, obtain high-yield astaxanthin transfer-gen plant by plant tissue and adventitious organogenesis.

The gene β-carotene oxygenase BKT that the present invention is above-mentioned and β-carotene hydroxylase BHY and recombinant vectors pBI121-LTPCRBKT and pBI121-BHYBKT is utilizing the application in transgenic plant production astaxanthin, comprise plant expression vector pBI121-LTPCRBKT and pBI121-BHYBKT built containing β-carotene oxygenase BKT and β-carotene hydroxylase BHY, then transfer-gen plant is obtained by agriculture bacillus mediated method, BKT and BHY is expressed in transfer-gen plant and the carotenoid of catalysis plant generation astaxanthin step itself.

The preparation-obtained recombinant host cell of method of the plant expression vector pBI121-LTPCRBKT above-mentioned with the present invention and pBI121-BHYBKT transformed host cell.

The plant expression vector pBI121-LTPCRBKT above-mentioned with the present invention and pBI121-BHYBKT transformed host cell, cultivate the transgenic plant of the prepared product astaxanthin obtained of method of transgenic plant.

Recombinant host cell of the present invention is preparing the application in transgenic plant cells and transgenic plant.

Present invention also offers application plant expression vector pBI121-LTPCRBKT and pBI121-BHYBKT of the present invention and set up transgenic plant that the method for producing astaxanthin transgenic plant obtains as the starting material extracting astaxanthin, and preparing in protective foods, prepare in makeup, the application prepared in animal cultivation product.

Accompanying drawing explanation

Fig. 1 bacterial expression vector pCRBKT physical map;

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

The physical map of Fig. 3 plant expression vector pBI121-BHYBKT;

Fig. 4 contrast and product astaxanthin tomato plant, flower and fruit;

Fig. 5 produces the Molecular Detection that astaxanthin tomato CRBKT and HPBHY expresses.

Embodiment:

Hereafter by reference to the accompanying drawings, introduce the present invention in more detailed manner with the following example of the present invention, but do not limit technical scheme of the present invention with this.

Embodiment 1:

The clone of chlamydomonas β-carotene oxygenase, transformation and in bacterial expression system anlysis:

(1) clone of chlamydomonas carotene oxygenase:

Chlamydomonas Chlamydomonas reinhardtii cc-124 is provided by Chamydomonas Center (Duke University, USA).Chlamydomonas TAP nutrient solution/base is cultivated.Total RNAs extraction with TRI extracting solution (Molecular research center, Cincinnati, OH, USA), with about 10 ⁸reinhardtii cell extract total serum IgE, method is with reference to the step of specification sheets.The synthesis of cDNA adopts SuperScript ^tMiII One-Step RT-PCR system (Invitrogen, Carlsbad, CA, USA), 15 minutes synthesis cDNA at 50 DEG C.With chlamydomonas cDNA for template, pcr amplification is carried out in order to lower pair of primers, obtain carotene oxygenase gene, determine without after sudden change through order-checking, cut with HindIII and XbaI enzyme and obtain fragment to be connected to the support products that the corresponding restriction enzyme site of pBluescript II KS obtains be pCRBKTL.

PCR primer following (respectively upper, downstream primer design HindIII and XbaI site):

Forward primer: 5'-GAGAAGCTTCATGGGCCCTGGGGATACA-3'

Reverse primer: 5'-GCGTCTAGATCAAGCCATCACGCCAAC-3'

(2) transformation of chlamydomonas β-carotene oxygenase:

A first the chlamydomonas β-carotene oxygenase be cloned into is removed the C-end i.e. oxygenase redundance (115 amino acid of encoding) of other homologies relative by (), same is template with chlamydomonas cDNA, pcr amplification is carried out with following primer, obtain the beta carotene monooxygenase gene of brachymemma, determine without sudden change through order-checking, cut with HindIII and XbaI enzyme and obtain fragment to be connected to the support products that the corresponding restriction enzyme site of pBluescriptII KS obtains be pCRBKTS.

PCR primer following (respectively upper, downstream primer design HindIII and XbaI site):

Forward primer: 5'-GAGAAGCTTCATGGGCCCTGGGGATACA-3'

Reverse primer: 5'-GCGTCTAGATCAGGCCAGGGCTGCGCCGCG-3'

(b) with carrier pCRBKTS for template, fallibility PCR(error prone PCR is carried out) with GeneMorph II Random Mutagenesis kit (Stratagene, La Jolla, CA), method steps is with reference to specification sheets, and primer is with described in (a).The condition of reaction can produce 0-4 base mutation on the DNA of every kb.Product HindIII and XbaI enzyme are cut and are obtained fragment and be connected to the corresponding restriction enzyme site of pBluescript II KS.

(3) chlamydomonas carotene adds ketolase in bacterial expression system anlysis:

The carrier carrier pCRBKTL that (1) and (2) obtains, pCRBKTS and random mutation obtained is respectively by the electroporated E. coli DH5 α competent cell containing pACCAR25 △ crtX carrier.Bacterium containing pACCAR25 △ crtX carrier can accumulate the substrate of zeaxanthin (Misawa et al., Journal of Bacteriology, 1990,177:6575-6584) as oxygenase.Microbial culture condition is with reference to the method (1990) of Misawa etc.Pigment composition analysis adopts high performance liquid chromatography (HPLC), and plant and instrument adopts Waters HPLC system (Waters, Milford, MA, USA) to equip a Waters Spherisorb 5 ì m ODS2 4.6 250 mm analytical column.Method adopts Baroli(Plant Cell 15:992-1008,2003) described method, as follows after improving: from 100% solution A [acetonitrile/methanol/0.1 M Tris-HCl (pH 8.0), 84:2:14, v/v/v] follow linear gradient to 100% solution B (methanol/ethyl acetate, 68:32, v/v), flow velocity is 1.2mL min-1, maintains 15 minutes, then 10 minutes solution B.Pigment standard pigment sample is determined by optical absorption peak and time of occurrence, and pigment quantitatively also adopts standard pigment sample amounts, and all standard pigment samples are purchased from Sigma and Wako.

Embodiment 2:

Plant expression vector construction containing chlamydomonas carotene oxygenase:

(1) one of carrier pBI121-LTPCRBKT builds and derives from pBI121-CRBKT (zhonget al, Journal of Experimental Botany 62:3659-69,2011).The RBCS1 signal peptide deriving from Arabidopsis in pBI121-CRBKT is replaced with the RBCS signal peptide (NCBI number of registration: M15236) of tomato.With tomato STb gene for template amplification tomato RBCS signal peptide, pcr amplification is carried out in order to lower pair of primers, obtain tomato RBCS signal peptide (LTP), determine without sudden change through order-checking, cut with SmaI and HindIII enzyme and obtain fragment to be connected to the support products that the corresponding restriction enzyme site of pBI121-CRBKT obtains be pBI121-LTPCRBKT.

PCR primer is as follows: (respectively upper, downstream primer design SalI-SmaI and HindIII site)

Forward primer: 5'-GCGTCGACCCGGGGAACCAAAAAAAGAGAGAAG-3'

Reverse primer: 5'-CCCAAGCTTGGCATGCAGCTAACTCTTCCAC-3'

(2) two pBI121-BHYBKT of carrier build and derive from pBI121-LTPCRBKT.First the CRBKT HPBHY of pBI121-LTPCRBKT is replaced.With the cDNA of Haematocoocus Pluvialls for its BHY encoding sequence of template amplification, pcr amplification is carried out in order to lower pair of primers, obtaining BHY determines without sudden change through order-checking, cuts obtain fragment to be connected to the support products that the corresponding restriction enzyme site of pBI121-LTPCRBKT obtains be pBI121-LTPHPBHY with HindIII and XbaI enzyme.

PCR primer following (respectively upper, downstream primer design HindIII and XbaI site):

Forward primer: 5'-GAGAAGCTTAATTACCACGATGCTG-3'

Reverse primer: 5'-GCGTCTAGACATCTAGTAACATAGA-3'

Secondly, with pBI121-LTPHPBHY for template amplification CaMV35S::SlTpHpBHY::nos fragment, pcr amplification is carried out in order to lower pair of primers, obtaining CaMV35S::SlTpHpBHY::nos fragment determines without sudden change through order-checking, cuts obtain fragment to be connected to the support products that the corresponding restriction enzyme site of pBI121-LTPCRBKT obtains be pBI121-BHYBKT with ClaI enzyme.

PCR primer following (respectively upper, downstream primer design ClaI-HindIII and ClaI-EcoRI site):

Forward primer: 5'-CCATCGATAAGCTTGCATGCCTGC-3'

Reverse primer: 5'-CCATCGATGAATTCCATCTAGTAACATAGA-3'

Embodiment 3:

The structure of transgenic plant:

(1) Agrobacterium of preparation containing plant expression vector:

Respectively pBI121-CRBKT obtained for the embodiment 2 of 2 microlitres and pBI121-BHYBKT plasmid are added to the competent cell that 200 microliter Examples 1 obtain, mix gently, place 30 minutes on ice.Then by bacterial suspension heat shock 60 seconds in 42 DEG C of water-baths, move to rapidly and place cooling in 5 minutes on ice.800 microlitre LB liquid nutrient mediums are added respectively, in 28 DEG C of recoveries 3 hours in bacterial suspension.Get on LB plate that appropriate bacterial suspension coats containing 50 mg/L Streptomycin sulphates and 50 mg/L kantlex, plate is placed in 28 DEG C of temperature baths 2 days.Picking list bacterium colony from flat board, through PCR qualification and enlarged culturing, extracts plasmid enzyme restriction qualification.Agrobacterium containing corresponding plasmid is carried out Plant Transformation.First 100 microlitre Agrobacterium nutrient solutions are accessed 3 ml LB liquid mediums and contain 50mg/L Streptomycin sulphate and 50mg/L kantlex, cultivate 48 hours.By centrifugal for culture recovery, abandon supernatant liquor, bacterial sediment thing is resuspended in 3 ml MS substratum (MS substratum adds 1% sucrose, 0.2 mg/L 2,4-D, 0.1 mg/L IAA, 100mg/L AS, pH 5.8) and obtains agrobacterium suspension.

(2) tomato cotyledon explant preparation:

Tomato (Solanum lycopersicum) seed (B-type LA0316) is provided by TomatoGenetics Resource Center (TGRC).Cultivate 14 days on MS solid medium after surface sterilization, treat that cotyledon grows.Cotyledon is cut and cultivates 1 day at MS preculture substrate (MS substratum adds 0.8% agar, 1% sucrose, 2 mg/L zeatin, 0.1 mg/ LIAA, 100 mg/L AS, pH 5.8).Cotyledon explant after preculture to be soaked in the agrobacterium suspension that (1) step obtains 10 minutes, explant after immersion is put in MS Dual culture plate, and (MS substratum adds 0.8% agar, 1% sucrose, 2 mg/L zeatin, 0.1 mg/L IAA, 100 mg/L AS, 300mg/L Pyocianil, 100 mg/L Streptomycin sulphates and 50mg/L kantlex pH 5.8) to move to light after one day without illumination cultivation in 28 DEG C under cultivate one day in 24 DEG C.Explant after Dual culture is after water cleaning, and (MS substratum adds 0.8% agar, 1% sucrose to be transferred to Callus formation substratum, 2 mg/L zeatin, 0.05mg/L IAA, 300mg/L Pyocianil and 100mg/L kantlex, pH 5.8) in 24 DEG C of cultivations.After one month, callus is transferred to the substratum that sprouts (MS substratum adds 0.8% agar, 1% sucrose, 1 mg/L zeatin, 0.03mg/L IAA, 200mg/L Pyocianil and 100mg/L kantlex, pH 5.8) to growing henna bud.Treat that bud grows two panels leaf, bud is cut and transfers to root media (MS substratum adds 0.8% agar, 1% sucrose, 0.1 mg/LIBA, and 100mg/L kantlex, pH 5.8), after about 1-2 week, grow adventive root.Taken out by plant after well developed root system, move in cultivating soil, in greenhouse, (24 DEG C, 16 h light/8 h dark) are cultivated.

Embodiment 4:

The qualification of transgenic plant and the mensuration of content astaxanthin:

(1) transgenic plant qualification:

Due to the pigment that astaxanthin is a kind of redness.It synthesizes the color by changing original plant organ in plant organ.Therefore the screening of transfer-gen plant starts from bud that callus grows, selects have the bud of colour-change to carry out root culture.About each vector plant produces 20 strains and turns plant, and its organ (comprising bud, blade, stem, flower and fruit) color is variant with non-transgenic plant.

(2) content astaxanthin of transgenic plant measures:

Plant usually can not synthesizing astaxanthin.HPLC measures the content astaxanthin of transgenic plant to obtain the transgenic plant of efficient synthesizing astaxanthin, and method is with bacterium pigment detection.Each vector families of plant obtain the highest transgenic plant of a strain content astaxanthin after measured, and the transgenic plant called after TB-bb8 of the transgenic plant called after TB-b10 of pBI121-LTPCRBKT, pBI121-BHYBKT, Fig. 4 is shown in plant trait analysis.The pigment composition analysis in table 1 of mature fruit.

Table 1 carotenoid content of tomato and per-cent

(3) determination of gene expression of transgenic plant:

Extract the total serum IgE of the mature fruit of transgenic plant TB-b10 and TB-bb8, synthesis cDNA, use the primer amplification 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。Pcr amplification the results are shown in Figure 5.

The method utilizing transgenic plant to produce astaxanthin of the present invention, by the screening of intestinal bacteria function reasonableness method and improvement BKT gene, build containing the plant expression vector through the BKT gene of improvement and the BHY gene with its efficient collaboration, both, use agrobacterium mediation converted plant, obtain transfer-gen plant, make BKT and BHY high expression in transfer-gen plant obtain the astaxanthin of high yield.

BKT gene source of the present invention is in chlamydomonas (Chlamydomonas reinhardtii cc-124, NCBI number of registration: AY860820, referred to as CrBKT); BHY derives from Haematocoocus Pluvialls (Haematococcus pluvialis, NCBI number of registration: AY187011; Referred to as HpBHY).

First transform CRBKT gene, CRBKT gene has more coding 115 amino acid whose 1 relative to other homologous genes ' end sequence, this sequence is removed, and obtains the enzyme gene (CRBKTQ164N) zeaxanthin to stronger ketonize ability by random mutation.Secondly build be used for Plant Transformation containing CRBKT muton expression vector.One of: the nucleotide sequence of coding CRBKT muton is placed under CaMV35S promoters driven, adds that guiding gene product enters the signal peptide of chloroplast(id).Two: by coding BHY nucleotide sequence be placed under CaMV35S promoters driven, add that guiding gene product enters the signal peptide of chloroplast(id), the location of controlling gene product.With being connected forming expression vector two containing CRBKT muton before.

Described plant expression vector completes by the following method:

(1) tomato ribulose carboxylase small subunit chloroplast(id) is led peptide sequence (LTP) bacterial expression be inserted into containing CRBKT and carries pCRBKT(Fig. 1) obtain carrier pLTPCRKBT, LTP+CRBKT nucleotide fragments enzyme is scaled off be inserted on plant expression vector pBI121 obtain expression vector pBI121-LTPCRBKT(Fig. 2).

(2) pBI121-LTPCRBKT HPBHY is substituted CRBKT fragment and obtains pBI121-LTPHPBHY, the CaMV35S::SlTpHpBHY::nos sequence enzyme on this carrier is scaled off on insertion vector pBI121-LTPCRBKT and obtains carrier pBI121-BHYBKT(Fig. 3).

After described expression vector pBI121-LTPCRBKT and pBI121-BHYBKT proceeds to Agrobacterium LBA4404 respectively, infect tomato cotyledon explant, obtain higher transformation efficiency with kantlex and leaf dithering.

Utilize method of the present invention, can the β-carotene oxygenase deriving from green alga be transferred in plant, and at each plant organ (blade, flower, fruit) middle high expression, cultivate the plant variety containing β-carotene oxygenase, its content astaxanthin up to 16 milligrams/gram, close to the content of Haematocoocus Pluvialls.Reach the requirement that astaxanthin is produced in industrialization.The biosynthesizing of astaxanthin only betides that minority is biological and output is usually very low, and the transgenic plant of being rich in astaxanthin can low cost industrialization production high value astaxanthin, meets the demand to this high anti-oxidation material on market.The astaxanthin in Haematocoocus Pluvialls source, owing to yielding poorly, is mainly used in high-end protective foods and makeup.Astaxanthin market is still by artificial sintetics is monopolized so far, is mainly used in animal feedstuff additive.High-yield astaxanthin transgenic plant can directly apply to animal-feed, and from safety, economy, nutritive value angle, genetically modified crops are more superior.Its advantage of the present invention is as follows: 1. the gene that the present invention is used is from the ancestors green alga of plant but not the gene of normally used bacterial origin.The more important thing is that the content of the plant astaxanthin of expressing this gene is far above the genes involved of expressing other sources; 2. the low production cost that obtained by transgenic plant mode of natural astaxanthin, producing astaxanthin transgenic plant can directly as fodder additives or the functional food of aquaculture; 3., relative to algal culture, crop plantation cost is low, operates easy less investment; 4. easier the expansion rapidly of the industrial scale of economic plants is used widely.

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.