CN105907780A - Transgenic breeding method producing astaxanthin in crop seed endosperm - Google Patents

Abstract

The invention discloses a transgenic breeding method for producing astaxanthin in the endosperm of crop seeds. The specific steps are S1. According to the codon preference of the breeding crop, optimize the sequences of the 4 genes of PSY , CrtI , BHY and BKT , and combine the optimized 4 genes with the endosperm expression promoter and plastid transit peptide of the breeding crop respectively fusion to construct gene expression cassettes respectively; construct the four gene expression cassettes in S1 into plant transformation vectors to obtain plant transformation vectors containing PSY , CrtI , BHY and BKT ; transform the plant transformation vectors described in S2 into breeding crops to obtain seeds GMO crops that synthesize astaxanthin with orange-red endosperm. The invention realizes the synthesis of astaxanthin in the seed endosperm of crops (especially rice), and its products can be directly used for food, and can also be used as raw materials for producing astaxanthin, which has important guiding significance for the breeding of new functional crops and production application value.

Description

A transgenic breeding method for producing astaxanthin in crop seed endosperm

technical field

The invention relates to the field of genetic engineering, in particular to a transgenic breeding method for producing astaxanthin in the endosperm of crop seeds.

Background technique

Astaxanthin, chemical name 3,3'-dihydroxy-4,4'-diketone-ketone, β'-carotene, also known as super vitamin E, is a kind of orange-red carotenoid pigment, in Organisms have super antioxidant properties, and their antioxidant activity is 550 times that of vitamin E. It has the functions of anti-radiation, preventing tumors, preventing cardiovascular diseases, preventing diabetes, delaying aging and improving immunity. It has broad application value in food, health care products, medical supplies, cosmetics, food additives, aquaculture and other fields. In nature, astaxanthin is mainly produced by some algae, bacteria, and phytoplankton, and higher plants cannot synthesize it due to the lack of key β-carotenoid ketolase (BKT). At present, the sources of astaxanthin are mainly extracted from crushed shrimp shells, fermented by Rhodotorula, cultured and extracted from Haematococcus pluvialis, and artificially synthesized. The method of natural product extraction has the problems of low yield and high cost, and the artificial chemical synthesis method is restricted due to safety risks. Therefore, using genetic engineering means to produce astaxanthin with plants as bioreactors can solve the above limiting factors. Because plants are rich in the precursor β-carotenoid of astaxanthin, the synthesis of astaxanthin has been realized in tobacco, tomato and potato by means of genetic engineering.

Rice is an important food crop and model plant, and its seed is also an excellent bioreactor. The protein and active nutrients of rice mainly exist in the seed coat and embryo, while the polished rice for daily consumption is removed from the seed coat and The endosperm of the embryo (the main component is starch), its nutritional value is greatly reduced. Therefore, it is the main goal of functional rice breeding to increase and strengthen the synthesis of specific nutritional components and functional substances in the endosperm by genetic engineering methods. For example, the "Golden Rice" that produces β-carotenoids in the endosperm, and the "Human Serum Protein Rice" that uses the endosperm as a bioreactor to produce biological activity. However, there is no report on the production of astaxanthin in rice endosperm, because rice lacks key enzymes for synthesizing astaxanthin, and it is also a very difficult problem to specifically need those enzymes to synthesize astaxanthin in endosperm. Subject research.

Contents of the invention

The present invention aims at the above shortcomings of the prior art, and provides a transgenic breeding method for producing astaxanthin in the endosperm of crop seeds.

In order to achieve the above object, the present invention is achieved through the following schemes:

The present invention discovers for the first time through research that PSY, CrtI, BHY and BKT are four essential key enzymes in order to synthesize astaxanthin in crop endosperm. Therefore, the present invention firstly protects the application of the four genes PSY, CrtI, BHY and BKT in transgenic breeding for producing astaxanthin from the endosperm of crop seeds.

In addition, on the basis of this study, the present invention provides a specific transgenic breeding method for producing astaxanthin in the endosperm of crop seeds, comprising the following steps:

S1. According to the codon bias of the breeding crop, optimize the sequences of the four genes PSY, CrtI, BHY, and BKT, and construct the genes with the endosperm expression gene promoter and plastid transit peptide of the breeding crop respectively with the optimized four genes expression cassette;

S2. assembling the four gene expression cassettes in S1 with the plant transformation vector plasmid in turn to obtain a plant transformation vector containing four genes PSY, CrtI, BHY and BKT;

S3. Transforming the embryonic callus of the breeding crop with the plant transformation vector in S2 to obtain a transgenic crop producing astaxanthin in the seed endosperm.

Astaxanthin, as an important functional substance, realizes its synthesis and accumulation in rice endosperm, which can greatly increase the nutritional quality and product added value of rice. Carotenoids cannot be synthesized in rice endosperm due to the lack of expression of genes involved in the carotenoid synthesis pathway (Fig. 1). The main component of "Golden Rice" is β-carotenoid, which is obtained through the specific expression of phytoene synthase (PSY) and bacterial phytoene dehydrogenase (Crt I) in the endosperm of the carotenoid synthesis pathway. produced. As the highest form of β-carotenoid, astaxanthin is specifically synthesized in rice endosperm, in addition to PSY and Crt I, β-carotenoid hydroxylase (BHY) and β-carotenoid Ketoketase (BKT). When these four genes are co-expressed in the endosperm, it is possible to achieve the goal of astaxanthin synthesis in the endosperm (Fig. 1). Therefore, compared with the production of β-carotenoids from Golden Rice, more genes are involved in the specific synthesis and accumulation of astaxanthin in rice endosperm, which is more difficult. At present, there is no official report on the relevant research at home and abroad.

Preferably, the breeding crops are rice, corn, wheat.

Preferably, the PSY gene is the PSY gene of maize, the CrtI gene is the CrtI gene of Erwinia, the BHY gene is the BHY gene of Haematococcus pluvialis, and the BKT gene is the BKT gene of Chlamydomonas reinhardtii.

Preferably, when the breeding crop is rice, optimize the PSY gene of maize, the CrtI gene of Erwinia, the BHY gene of Haematococcus pluvialis, and the BKT gene of Chlamydomonas reinhardtii according to the codon preference of monocots and rice The optimized genes are called PSY-r, CrtI-p, BHY-p and BKT-p, and their sequences are shown in SEQ ID NO:1-4.

Preferably, when the breeding crop is rice, the promoters used are the rice endosperm-specific storage protein gene promoters Pens1-Pens4, the sequences of which are shown in SEQ ID NO: 5-8; the plastid transit peptide is TP, which encodes the DNA sequence As shown in SEQ ID NO:9.

Preferably, when the breeding crop is rice, the breeding method comprises the steps of:

S1. According to the codon preference of monocots and rice, optimize the PSY gene of maize, the CrtI gene of Erwinia, the BHY gene of Haematococcus pluvialis, and the BKT gene sequence of Chlamydomonas reinhardtii. The optimized gene is called They are PSY-r, CrtI-p, BHY-p and BKT-p, and the sequences are shown in SEQ ID NO: 1-4; the four genes after sequence optimization are respectively linked to the rice endosperm-specific storage protein gene promoter Pens1- The coding sequences of Pens4 and the plastid transit peptide TP respectively construct gene expression cassettes; the rice endosperm-specific storage protein gene promoters Pens1-Pens4 and the plastid transit peptide TP coding sequences are respectively shown in SEQ ID NO: 5-9;

S2. constructing a plant transformation vector with the 4 gene expression cassettes in S1 to obtain a multigene vector containing PSY-r, CrtI-p, BHY-p and BKT-p gene expression cassettes;

S3. Transforming the multigene vector into Agrobacterium, transforming the callus of the breeding crop rice, and obtaining the transgenic rice.

SEQ ID NO:5-8 are the sequences of rice endosperm-specific storage protein gene promoters Pens1, Pens2, Pens3, Pens4 respectively, PSY-r and Pens1 are constructed as expression cassettes, CrtI-p and Pens2 are constructed as expression cassettes, BHY- p and Pens3 were constructed as an expression cassette, and BKT-p and Pens4 were constructed as an expression cassette.

More preferably, when the four gene expression cassettes in S2 construct the plant transformation vector, a multigene vector system is used to construct a T-DNA region containing PSY-r, CrtI-p, BHY-p and BKT-p four genes multigene carrier. Most preferably, the multigene carrier system is the multigene carrier system with patent number ZL02134869.3.

More preferably, the transformation method described in S3 is an Agrobacterium-mediated method.

The present invention also protects the carrier containing four genes PSY-r, CrtI-p, BHY-p and BKT-p shown in SEQ ID NO: 1-4. Preferably, the vector containing the four genes PSY-r, CrtI-p, BHY-p and BKT-p shown in SEQ ID NO: 1-4 is a multigene vector pYLTAC380-BBPC.

In order to show the effectiveness of the present invention, the present invention utilizes the genetic engineering carrier system (ZL02134869.3) that can be loaded with multiple genes to construct a T-DNA region that contains four key enzymes recombined in the synthesis of astaxanthin in the seed endosperm genes (PSY-r, CrtI-p, BHY-p and BKT-p, respectively fused with the TP coding sequence), and these four genes are respectively controlled by rice endosperm-specific promoters in multigene vectors (binary vectors). Through the method mediated by Agrobacterium, the multigene carrier that aggregates 4 target genes is transferred into rice to obtain a transgenic rice line. These 4 target genes are specifically expressed in the endosperm, and the transgenic rice with high specific synthesis and accumulation of astaxanthin (up to 3.68 μg/g) in the endosperm, the rice seeds can be directly eaten or used as raw materials for extraction and processing.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention realizes the synthesis of astaxanthin in the endosperm of crops (especially rice) for the first time, and its products can be directly used for food, and can also be used as raw materials for producing astaxanthin. The present invention has important guidance for the breeding of new functional crops Significance and production application value. In addition, the present invention can realize the synthesis and accumulation of useful active substances or nutrients (astaxanthin) in the endosperm of crop seeds, so the present invention provides a technical method for the multi-gene genetic engineering operation of important and complex biosynthetic pathways and important agronomic traits, It has important application value.

Description of drawings

Figure 1 shows the pathway of astaxanthin synthesis in the endosperm of transgenic crop seeds. Small black arrows represent reactions that may exist in rice endosperm; dotted arrows represent reactions that do not exist in rice endosperm; black thick and large arrows represent the introduction of exogenous transgenes The expressed enzyme catalyzes the reaction.

Fig. 2 is the structural representation (A) of the supply vector containing PSY-r, CrtI-p, BHY-p and BKT-p gene expression cassettes and the restriction endonuclease Not I restriction endonuclease Not I restriction endonuclease cut detection figure (B); Arrow represents The target gene expression cassette obtained after Not I digestion.

Figure 3 is a schematic diagram of the structure of the multigene vector pYLTAC380-BBPC containing four essential genes for astaxanthin (PSY-r, Crt I-p, BHY-p and BKT-p) (A) and the restriction endonucleases of different clones Restriction detection of Not I (B); HPT in the T-DNA region is hygromycin resistance gene.

Figure 4 is the PCR detection of 4 exogenous genes (ORF) in the genomic DNA of the rice transformant introduced into pYLTAC380-BBPC; CK+ is the multigene vector pYLTAC380-BBPC plasmid; WT is the wild-type control genomic DNA.

Fig. 5 is the expression detection of 4 exogenous genes in the developing seeds of the pYLTAC380-BBPC rice transformant 20 days after pollination by RT-PCR.

Figure 6 shows the phenotype of polished rice transformed with pYLTAC380-BBPC for the synthesis of astaxanthin; the wild type is the recipient variety Zhonghua 11.

Fig. 7 is HPLC detection of astaxanthin in rice seed endosperm transformed with pYLTAC380-BBPC.

detailed description

The present invention will be further elaborated below in combination with the accompanying drawings and specific embodiments. The embodiments are only used to explain the present invention, and are not intended to limit the scope of the present invention. The test methods used in the following examples are conventional molecular biology methods unless otherwise specified; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

Example 1

The present invention finds through research that PSY, CrtI, BHY and BKT are four essential key enzymes in order to synthesize astaxanthin in crop endosperm. Based on the above research basis, the present embodiment establishes a transgenic breeding method for producing astaxanthin in rice endosperm, which specifically includes the following steps:

S1. Construction of four essential gene expression cassettes:

S11. Synthesis of the coding regions of four essential genes (PSY-r, CrtI-p, BHY-p and BKT-p): Using the PSY gene (GenBank No.U32636.1) of maize as a template, according to monocotyledonous plants and rice Codon preference, using the Codon Optimization Tool program to optimize the codon, synthetically obtain the PSY-r of the DNA sequence shown in SEQ ID NO:1, clone it into a plasmid vector, and determine its sequence by sequencing; Erwinia Erwinia uredovoracrtI gene (GenBank No. D90087) as a template, according to the codon preference of monocotyledonous plants and rice, utilize the Codon Optimization Too program to optimize the codon, synthesize and obtain the DNA sequence CrtI-p shown in SEQ ID NO:2, clone it into a plasmid vector, and determine its sequence by sequencing; Haematococcus pluvialis BHY gene (GenBank No.BD250390.1) was used as a template, and according to the codon preference of monocots and rice, codons were optimized using the Codon Optimization Tool program, and the DNA sequence shown in SEQ ID NO:3 was synthesized BHY-p, cloned into a plasmid vector, sequenced to determine its sequence; using the Chlamydomonas reinhardtii BKT gene (GenBank No. JF304771.1) as a template, according to the codon bias of monocotyledonous plants and rice, the code was optimized using the Codon Optimization Tool program The progeny was synthesized to obtain the DNA sequence BKT-p shown in SEQ ID NO:4, cloned into a plasmid vector, and sequenced to determine its sequence.

S12. Synthesis of the plastid transit peptide coding sequence TP (Transit peptide): referring to the sequence of the pea RbcS small subunit gene (GenBank No. X00806), a sequence encoding the plastid transit peptide TP (SEQ ID NO: 9) was synthesized.

S13. Construction of four gene expression cassettes necessary for astaxanthin synthesis on the supply vector: Crt I-p gene expression cassette pYL322d1-Crt I-p construction: Reverse amplification of multi-gene supply vector I (Lin et al., 2003; ZL02134869. 3), obtaining the vector backbone fragment a; using rice genomic DNA as a template, amplifying the 1.5kb promoter Pens1 (SEQ ID NO: 5) of the rice endosperm-specific storage protein gene (GenBank No. BAC83236.1) as fragment b; Amplify the sequence TP (SEQ ID NO:9) of the plastid transit peptide as fragment c; amplify the Crt I-p gene as fragment d; amplify the CaMV 35S terminator 35sT from plasmid pCAMBIA1300 (GenBank No.AF234296.1) as Fragment e. On both sides of each fragment, there are 25bp homologous sequences, using the principle of Gibson assembly (Gibson, 2011), according to the method of one-step assembly of plasmid vector multi-fragments (Zhu et al., 2014), to obtain Crt I-p gene expression The vector pYL322d1-Crt I-p for the cassette (Figure 2). Construction of PSY-r gene expression cassette pYL322d2-PSY-r: Reverse amplification of multi-gene supply vector II (Lin et al., 2003; ZL02134869.3) to obtain vector backbone fragment a; using plasmid pSAT7 (GenBankNo.DQ5453) as a template, Amplify the agropine synthase terminator agsT as fragment b; amplify the reverse PSY-r gene as fragment c; use rice genomic DNA as a template to amplify the rice endosperm-specific storage protein gene (GenBank No.BAD61649.1) about 1kb Promoter Pens2 (SEQ ID NO: 6), as fragment d. There are 25 bp homologous sequences on both sides of each fragment. Using the principle of Gibson assembly, the vector pYL322d2-PSY-r containing the PSY-r gene expression cassette was obtained according to the method of one-step assembly of plasmid vector multi-fragments (Figure 2). Construction of BKT-p gene expression cassette pYL322d1-BKT-p: Reverse amplification of multi-gene supply vector I to obtain vector skeleton fragment a; use rice genomic DNA as template to amplify rice endosperm-specific storage protein gene (GenBank No.AAP50945. 1) Promoter Pens3 (SEQ ID NO:7) of about 2.4kb, as fragment b; amplify the sequence TP of plastid transit peptide (SEQ ID NO:5), as fragment c; amplify BKT-p gene, as fragment d; The manopine synthase terminator masT was amplified from plasmid pSAT3 (GenBank No. DQ005465) as fragment e. There are 25 bp homologous sequences on both sides of each fragment. Using the principle of Gibson assembly, according to the method of one-step assembly of plasmid vector multi-fragments, the supply vector pYL322d1-BKT-p containing the BKT-p gene expression cassette was obtained (Figure 2). Construction of BHY-p gene expression cassette pYL322d2-BHY-p: Reverse amplification of multigene supply vector II to obtain vector backbone fragment a; amplify manopine synthase terminator masT from plasmid pSAT3 (GenBank No. DQ005465) as fragment b; Increase the reverse BHY-p gene as fragment c; amplify the sequence TP (SEQ IDNO: 5) of the plastid transit peptide as fragment d; use rice genomic DNA as a template to amplify the rice endosperm specific storage protein gene (GenBank No. .BAC19997.1) about 2.4 kb promoter Pens4 (SEQ ID NO: 8), as fragment e. There are 25 bp homologous sequences on both sides of each fragment. Using the principle of Gibson assembly, the supply vector pYL322d2-BHY-p containing the BHY gene expression cassette was obtained according to the method of one-step assembly of plasmid vector multi-fragments (Figure 2).

S2. Assembly of the multigene vector pYLTAC380-BBPC for rice endosperm-specific synthesis of astaxanthin: the multigene vector system is composed of a transformable artificial chromosome (TAC)-based acceptor vector and two gene-loaded donor vectors, using Cre The /loxP site-specific recombination method allows different donor vectors to undergo more than two rounds of gene assembly with the acceptor vector alternately to construct a multigene vector (Lin et al., 2003).

Assembly of S21.CrtI-p gene expression cassette: Mix the donor vector (I) pYL322d1-CrtI-p plasmid with the acceptor vector pYLTAC380H2 plasmid, and electrically transform the competent cells of E. coli strain NS3529 expressing Cre enzyme. Transformants were screened on the chloramphenicol double-antibody plate, and the endogenously expressed Cre enzyme of NS3529 was used to realize the recombination of the donor vector plasmid and the deletion of the donor vector backbone. Wash the mixed colonies on the double-antibody plate and extract the plasmids, then digest with I-SceI to eliminate the unrecombined plasmids, transform Escherichia coli DH10B without the Cre gene, and obtain a gene containing 1 gene on the kalamycin plate. Vector pYLTAC380-C.

S22. Assembly of the PSY-r gene expression cassette: mix the supply vector (II) pYL322d2-PSY-p plasmid with the receiving vector pYLTAC380-C plasmid containing the Crt I-p gene expression cassette constructed in the step, and transform NS3529 competent cells by electric shock, The transformant was screened on the double-antibody plate of kalamycin and ampicillin, and the endogenously expressed Cre enzyme of NS3529 was used to realize the recombination of the donor vector plasmid and the deletion of the donor vector backbone. The mixed colonies on the double-antibody plate were washed and the plasmids were mixed, and then the unrecombined plasmid was cut with PI-SceI enzymes, and then E. coli DH10B was transformed, and the vector pYLTAC380-PC containing 2 genes was identified on the kalamycin plate.

S23. Assembly of BKT-p gene expression cassette: Mix the donor vector (III) pYL322d1-BKT-p plasmid with the receiving vector pYLTAC380-PC plasmid containing 2 target genes obtained in the step, and jointly transform the large intestine expressing Cre enzyme by electric shock The Bacillus strain NS3529 competent cells were screened for transformants on the double-antibody plate of kalamycin and chloramphenicol, and the endogenously expressed Cre enzyme of NS3529 was used to realize the recombination of the supply vector plasmid and the deletion of its backbone. Plasmids were extracted from the mixed colonies, and the non-recombined plasmids were eliminated by digestion with I-SceI, then transformed into DH10B, and the vector pYLTAC380-BPC containing 3 genes was obtained by identification on the kalamycin plate.

S24. Assembly of the BHY-p gene expression cassette: Mix the supply vector (IV) pYL322d2-BHY plasmid with the acceptor vector pYLTAC380-BPC plasmid containing 3 genes obtained in the step, and transform NS3529 competent cells by electroshock. Transformants were screened on a double-antibody plate against ampicillin, and the endogenously expressed Cre enzyme of NS3529 was used to realize the recombination of the donor vector plasmid and the deletion of its backbone. Wash the mixed colonies on the double-antibody plate and extract the plasmids, then digest with PI-SceI to eliminate the unrecombined plasmids, transform DH10B, and identify the polygenes containing 4 genes necessary for astaxanthin synthesis on the kalamycin plate Vector pYLTAC380-BBPC (see Figure 3).

S3. Rice transformation and detection of multigene vector pYLTAC380-BBPC

Genetic transformation of rice: the multigene carrier pYLTAC380-BBPC plasmid was transformed into Agrobacterium EHA105 for transforming rice embryo callus. Callus was induced from rice immature seeds or mature seeds at 25°C in the dark. Suspend an appropriate amount of Agrobacterium in the infection medium with 100 μmol/L acetosyringone, culture with shaking at 28°C (200rpm, 0.5h), adjust the OD550 value to 0.3-0.4 with a spectrophotometer, and then it can be used for healing. Infection of injured tissue. Select the granular embryogenic callus that is fresh in light yellow color and vigorously growing, mix it with the Agrobacterium bacterium liquid, soak for 20 minutes, absorb the bacterial liquid, transfer to the co-cultivation medium, and after 3 days of dark culture, transfer to the culture medium containing 50 mg/kg. On the selection medium of L hygromycin, subculture once every 2 weeks, and subculture twice. After resistance selection, the resistant calli with green spots were transferred to a differentiation medium to differentiate transformed seedlings and obtain transformed plants.

Genome PCR detection of transformed plants: Genomic DNA was extracted by SDS method as a template from the obtained leaves of T0 generation plants, and exogenous HPT, Crt I-p, PSY-r, BHY-p and BKT-p were detected by PCR amplification method . The primers used are as follows:

Primer F-HPT: 5'-GGACGCAACGCCTACGACTGGAC-3';

Primer R-HPT: 5'-TCATCGCAAGACC GGCAACAGGA-3';

A fragment of about 720bp was amplified.

Primer Forf-CrtIp: 5'-ATGAAGCCGACTACCGTGATAG-3;

Primer Rorf-CrtIp: 5'-CTAAATCAG GTC CTCCAACATC-3';

The coding frame of about 1.48 kb of Crt I-p gene was amplified.

Primer Forf-PSYp: 5'-ATGGCCATCATACTCGTACGAG-3';

Primer Rorf-PSYp: 5'-CTAGGTCTGG CC ATTTCTCAA-3';

The ORF of about 1.2 kb of PSY-r gene was amplified.

Primer Forf-BKTp: 5'-ATGGGTCCAGGCATCCAGCCTAC-3';

Primer Rorf-BKTp: 5'-CTAGGCGA GCGCAGCCCCGCGAG-3';

The ORF of about 1 kb of the BKT-p gene was amplified.

Primer Forf-BHYp: 5'-ATGGCCATCATACTCGTACGAG-3';

Primer Rorf-BHYp: 5'-CTAGGTCTGGCCATTTCTCAA-3';

The ORF of about 0.9 kb of BHY-p gene was amplified.

Amplification program used: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, extension at 72°C for 1.5 min, a total of 30 cycles; and finally extension at 72°C for 5 min. The results showed that none of the wild-type (WT) controls could amplify the exogenous genes, and the transgenic plants could amplify the above five genes ( FIG. 4 ).

RT-PCR detection of transgenic plant T1 seeds: Grind the orange-red developing seeds of the T1 generation of transgenic plants 20 days after pollination into powder under liquid nitrogen conditions, then extract the total RNA of the seeds by Trizol method, and use MMV reverse transcription Kit, oligDT primers reverse transcribe into cDNA, use the following primers and amplification conditions for RT-PCR detection of exogenous Crt I-p, PSY-r, BHY-p and BKT-p genes, rice endogenous Actin 1 gene as an internal reference . The primers used are as follows:

Primer F-RT-CrtIp282: 5'-TCGCATACCTCGAGCAGCAC-3';

Primer R-RT CrtI-p 282 (5'-TCAGG TCCTCCAACATCAGG-3';

A 282bp fragment of the Crt I-p gene was amplified.

Primer F-RT-PSYr381: 5'-GATGAGCTTGCACAGGCAGG-3';

Primer R-RT PSY-r 381: 5'-CAATGA ACATGGGAGCAGTAGC-3';

A 381bp fragment of PSY-r gene was amplified.

Primer F-RT-BKTp261: 5'-CTCCACTCGCTAACCAGCTG-3';

Primer R-RT-BKTp261: 5'-CGCGAG CTATCTGTCTGCAC-3';

A fragment of about 261bp of the BKT-p gene was amplified.

Primer F-RT-BHYp322: 5'-GGCTATGCTGCTCTGCACG-3';

Primer R-RT-BHYp322 (5'-CCTCTTA CTCCAGTCCAACTC-3';

A 322bp fragment of the BHY-p gene was amplified.

Amplification program used: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, and extension at 72°C for 30 sec, a total of 25 cycles; finally, extension at 72°C for 5 min. The results showed that the control wild-type seeds could not amplify the exogenous gene, and the orange-red transgenic seeds could amplify the specific correct band, and the exogenous gene could be expressed in the seeds (Fig. 5).

Example 2

Appearance and HPLC detection of astaxanthin in transgenic rice endosperm:

Observation of astaxanthin in the endosperm of transgenic rice: After the transgenic rice seeds introduced with the above four genes were processed into polished rice (removing the seed coat) and the rice grains were chopped and observed, it can be seen that the inside and outside of the whole polished rice were orange-red (Figure 6), indicating that Astaxanthin is synthesized in the endosperm.

Extraction and high-performance liquid chromatography (HPLC) identification of astaxanthin from transgenic rice seeds: Take 0.1 g of rice seeds and grind them into powder on ice, add 2 mL of methanol and continue to grind uniformly for 5 min; 100rpm) extraction for 10min; centrifuge at 4°C 8000rpm for 5min to collect the supernatant; repeat the extraction of the precipitate until the precipitate is white; centrifuge the supernatant at 4°C 8000rpm for 5min and then combine the supernatant, and concentrate and dry the collected supernatant at low temperature in the dark; finally Dried astaxanthin plus 600 μL methanol was used for the assay. Pass the above-mentioned sample to be tested through a 0.22 μm organic filter membrane, and then inject it into a 2 mL brown sampling bottle with an injection volume of 20 μL, and perform HPLC analysis with a C30 high performance liquid chromatography column. The mobile phase was methanol:water=95:5. The chromatographic conditions were as follows: the column temperature was room temperature, and the flow rate was 1 mL/min. The measurement wavelength is 480 nm. Astaxanthin HPLC pure standard was purchased from Sigma (CAS No. 472-61-7). Weigh 1 mg of astaxanthin standard sample, dissolve it in 10 mL of methanol, and then dilute with methanol to make 0 ppm, 5 ppm, and 10 ppm solutions respectively, and take 20 μL of samples according to the above chromatographic conditions. Linear regression analysis was performed with the peak area as the ordinate and the concentration of the standard as the abscissa. The results showed that the transgenic rice seeds had the same characteristic peaks as the astaxanthin standard sample, indicating that astaxanthin was successfully synthesized in the transgenic rice seeds, and the content could reach 2.68 μg/g (Figure 7).

SEQUENCE LISTING

<110> South China Agricultural University

<120> A transgenic breeding method for producing astaxanthin in the endosperm of crop seeds

<130>

<160> 9

<170> PatentIn version 3.3

<210> 1

<211> 1233

<212>DNA

<213> corn

<400> 1

atggccatca tactcgtacg agcagcgtcg ccggggctct ccgccgccga cagcat cagc 60

caccaggggga ctctccagtg cagtaccctc ctgaagacca aacgcccggc ggcaaggagg 120

tggatgcctt gttcacttct cggactgcac ccctgggagg ccggtagacc atcgccagcc 180

gtttatagtt ccttggctgt caatcccgcc ggggaagcag tcgtttcctc cgagcagaag 240

gtctacgacg tggtgctcaa gcaggccgca ctgctgaaaa gacaactgag aactcccgtc 300

ctggacgcaa ggccccagga catggacatg ccacgcaacg ggctcaagga agcctacgac 360

cgctgcggcg aaatctgtga ggagtatgcc aagacgttct atctcggtac aatgttgatg 420

acggaagaaa gacgccgggc tatttgggcg atatatgtgt ggtgccgcag aacagatgaa 480

ttggtggatg gaccgaacgc caactatatt accccctacag cactcgaccg gtgggaaaag 540

cgcttggagg acctgttcac tggacgcccg tatgatatgc tcgacgcggc tctgtccgat 600

accatcagca gatttccgat agacatccag ccctttcgcg atatgatcga aggcatgaga 660

tcggacctgc ggaaaacgag gtacaataat tttgacgagc tgtatatgta ctgctactat 720

gttgctggaa ctgtcgggtt aatgagcgta ccagtgatgg gcatagcggc ggagagcaag 780

gcaacgactg aatcagttta ctctgccgcg ctcgcattgg gcattgcaaa ccaacttacg 840

aacatactcc gggatgttgg agaggatgct cggaggggga ggatttatct gcctcaggac 900

gaacttgctc aggccggact gtcagacgaa gacattttta agggcgtggt cactaacagg 960

tggaggaatt ttatgaaaag acagataaaa cgcgccagga tgtttttcga ggaggccgag 1020

agaggcgtta ctgagctgtc tcaggcgtct cgctggccag tttgggcgtc gttgctgctg 1080

tataggcaaa tacttgacga gatcgaagcc aacgactaca acaacttcac gaagaggggcg 1140

tatgttggta aagggaagaa gttgctagca cttcctgtgg catatggaaa atcgctactg 1200

ctcccatgtt cattgagaaa tggccagacc tag 1233

<210> 2

<211> 1478

<212>DNA

<213> ErwiniauredovoracrtI

<400> 2

atgaagccga ctaccgtgat aggcgcgggg ttcgggggcc tcgctctcgc catcagactt 60

caagcagccg ggatcccagt gcttcttttg gagcaacgtg ataagcctgg cgggcgggcg 120

tacgtttatg aggaccagggg cttcaccttt gatgctggtc ccaccgtgat cacagacccg 180

tcggccattg aagaactttt cgctttggca ggcaaacaac ttaaggagta tgtggaggttg 240

cttccggtga cgcccttcta tagactgtgc tgggaaagcg ggaaagtctt caattatgac 300

aacgatcaga ccaggcttga ggctcaaata cagcagttca accccccgtga cgtggaaggc 360

tataggcagt ttcttgacta ctcccgggcc gtgtttaagg aaggatacct caaattgggg 420

actgtgccct tcctcagttt cagggacatg ttgcgggccg ctccgcagct tgctaagttg 480

caggcctggc ggtcagtgta cagcaaagtg gcttcctaca ttgaggatga gcacctcagg 540

caggccttca gcttccattc gcttctcgtg ggggggaacc cattcgcgac atcctctatc 600

tatactctca ttcacgcgtt ggagcgcgag tggggtgtgt ggttcccaag ggggggcact 660

ggcgcgctgg tccaggggat gatcaaactc tttcaggacc tcggcggcga agtggtgctg 720

aacgctaggg tatctcacat ggagacaaca ggcaacaaga ttgaagcagt ccatctcgaa 780

gacggtcgca ggtttctcac ccaggccgtg gcatcgaacg ctgatgttgt tcacacgtac 840

agggacttgc tctctcagca cccagcggcg gtgaagcagt cgaacaaact gcagaccaag 900

cgcatgagta actctctctt cgtcctctac ttcgggctca accaccatca cgaccagctg 960

gcgcatcaca ccgtttgctt tggcccccgg taccgtgagc tgatagacga aattttcaac 1020

cacgacggcc tggcggagga tttttcactc tatctgcacg ccccttgcgt tacagattct 1080

agtctcgccc ctgaaggctg tggttcgtat tatgtcctgg ctcccgtacc gcacttgggt 1140

actgcgaacc ttgactggac cgtggaaggc ccgaagctgc gtgaccgcat cttcgcatac 1200

ctcgagcagc actacatgcc ggggctccgt agtcagctcg tgactcacag gatgtttaca 1260

cctttcgact tccgcgatca actcaatgct taccacggat cagcgttttc ggttgagcca 1320

gttcttacgc agtccgcctg gtttcgtcct cacaatcggg acaagactat cactaacctg 1380

tatctcgtgg gagctggcac acaccccggg gccggcattc ccggtgtgat cggctcagcg 1440

aaagccactg caggccctgat gttggaggac ctgatta 1478

<210> 3

<211> 882

<212>DNA

<213> Haematococcus pluvialis

<400> 3

atgctgagca agttgcagtc aatctctgtg aaggcccgcc gcgtcgagct tgctcgggat 60

attacccggc cgaaggtgtg tctgcacgca cagaggtgtt ccttggtgcg gcttcgggtg 120

gctgcgcccc aaacagagga agcactcggc acggtgcagg ccgccggcgc gggagatgaa 180

cactccgcgg atgttgcgct gcagcagctg gaccgcgcca tcgccgaaag acgcgcccgg 240

aggaaaagag aacagctctc ataccaggct gctgcgattg ctgcgtcgat cggggtttcc 300

ggcatcgcaa tctttgcgac ttatctccgt ttcgcaatgc acatgaccgt tggaggcgcg 360

gtgccgtggg gcgaagtggc tggtacgttg ttgctcgtcg taggaggggc tcttggcatg 420

gagatgtatg cccggtacgc acacaaggcc atttggcacg agtcaccatt gggctggttg 480

ctgcataagt cccatcacac gccaaggacg gggccgttcg aggccaacga tctctttgcc 540

atttattaacg gtctgccggc tatgctgctc tgcacgttcg gattctggct tccgaacgtg 600

cttggagccg cttgctttgg agccggcctt ggcattacgc tgtatgggat ggcgtacatg 660

ttcgtgcacg atggcctggt gcatcgtcgg ttccccaccg gaccgattgc gggtctgcct 720

tacatgaagc gtctgacggt ggcccatcaa ttgcaccaca gtggcaaata cggcggagcc 780

ccttggggca tgtttctcgg accgcaagag cttcagcata taccaggtgc cgcagaggag 840

gtggagcgct tggttcttga gttggactgg agtaagaggt aa 882

<210> 4

<211> 987

<212>DNA

<213> Chlamydomonas reinhardtii

<400> 4

atgggtccag gcatccagcc tacatctgcc cggccgtgct ctcgtaccaa gcactcccgt60

tttgccctct tggccgctgc gctgactgca aggcgggtca aacagtttac taagcagttc 120

cgctcacgcc gtatggccga ggacattctt aaactttggc agagacaata tcatctgcca 180

cgggaggaca gcgacaagag aaccctccgg gaaagggtgc atctttacag gcctcctaga 240

tcggatctgg gcggtatcgc agtggccgtc acggtgatcg cgttgtgggc cacactcttt 300

gtgtatggtc tctggtttgt caagcttcct tgggcattga aggtgggcga gacggccacc 360

tcatgggcta cgattgctgc tgtgtttttc tccttggagt tcttgtacac tggactcttc 420

atcactaccc atgacgcaat gcatggcact attgccctca ggaacaggag gctcaacgac 480

ttcctgggcc agttggcgat ttcgttgtac gcttggttcg actactcagt ccttcaccgg 540

aagcattggg agcatcacaa tcataccgggg gaacccagggg ttgatcccga ttttcaccgc 600

ggcaatccaa atctcgcggt ctggttcgcg cagttcatgg tgagctatat gacgctctcg 660

caatttctta aaatagcagt atggagtaat ctgctccttt tggctggcgc tccactcgct 720

aaccagctgc tcttcatgac tgccgcgccg atactctcag cttttcggtt gttttactac 780

ggcacctacg tccctcacca cccagagaaa ggacacacag gggcgatgcc atggcaagtg 840

agccgcactt cgtcagcgag tcggctgcag agctttctta cgtgttatca cttcgatctt 900

cattgggaac atcaccggtg gccttacgcc ccgtggtggg aactcccaaa gtgcagacag 960

atagctcgcg gggctgcgct cgcctag 987

<210> 5

<211> 1526

<212>DNA

<213> rice

<400> 5

agtagctctt tgcatgtaac attaacagag gagggtcagc tgtggcaagc aagcaagcag 60

acgcgggagg ggaggcgcgc agcaggcatg gtgacggggt agggcgcagc ggatcgagat 120

gcgaggattt ttttttgggg gggtggggtggggggagaga aacgagtcat ccaacccaac 180

gcggatttag ctagtttcca gtgccttgga acagccgccg aaacggtgtc gcgcatgcga 240

cctggtttct atgtttttgc ttctttctct tcttttattc cgttgccaca tcaacttttt 300

gtctagttgg cagcctaatt aattctatgg aaaccaggtg acatggaggg ttgggggacat 360

ggtggaaaaa accggaacgg gccgacagtt caaccggaaa aaaccagaac ccgtt cagtt 420

caaaagaaag accggacatg catatgaccc gctttgaacc ggcagaaccg gtcggttttt 480

ctatgaaccg gtcattaaac cgtccccggt tagaccgaac aagccacaat aatcttgaaa 540

tgggccttga tgtggcccaa ttggtctgcc tagagcgttt tggttggcaa aaatcaatct 600

cctattctcg gcacgtgtga tatacaatgg taagtgagat atacaattct cggcacggct 660

acattacaag gtgtcgcatt gtgtcaatgt ttggttaatt tgctagattc acatataca 720

tgccaggaag ttcagaacaa tgtgttgcct ttcaccggaa aactttgttg gagcaaatgc 780

cttcttcttt tttgcttctg cttcttgagt ccatgtggag gaagcagtag atagctgatg 840

atatcaggat tccttctgtg tctgtgtagg tgtagcaaca ccactataat ttttatttag 900

caacacaata tcaatttggt ctataaaagt atgaattaaa tcaatcccca accacaatta 960

gagtaagttg gtgagttatt gtaaagctct gcaaagttaa tttaaaagtt attgcattaa 1020

cttatttcgt atcacaaaca agttttcaca agagttattaa tggaacaatg aaaaccattg 1080

aacatactat aatttttttt cttactgaaa ttatataatt caaagagcat aaacccacac 1140

agtcgtaaag ttccacgtgt agtgcattat caaaataata gcttacaaaa cataacaaac 1200

ttagtttcaa aagttgcaat ccttatcaca ttgacacata aagtgagcga tgagtcatgt 1260

cattattttt ttgctcacca tcatgtatat atgatgggca taaaagttac tttgatgatg 1320

atatcaaaga aatttttag gtgcacctaa cagaatatcc aaataatatg actcacttag 1380

atcctaatat agcatcaagc aaaactaaca ctctaaagca accgataggg aaacatctat 1440

aaatagacaa gcataatgaa aaccctcctc atccttcaca caattcaaac attagttg 1500

aagcatagta gtagaatcct acaaaa 1526

<210> 6

<211> 1059

<212>DNA

<213> rice

<400> 6

tttgtcacct ctgacggacc taacacccgtc aaaggtgagt taagtcatca gtgaccattg 60

atctgtgacg agggagtcta tccgtcacag aagagggttt gagcccgtca ctgctagctt 120

gttctgctgt agtgtgtgcg ttatgtcctt tctgaggaac cgatttagtt tatgactcat 180

caattccact ttacatatca aaaggtttat tatgggggca atgcttttgt gaaattgaat 240

ttgtattttg tgtcacgttg tggacataca tatcttcaat ttataaataa Tattacaaat 300

aggtggaact gaagtaactt cgtttgtaag gatcaaaatc tgatagtggc atgagcttgg 360

agtttcaaaa actttttggt caaatccgtt tttataaggc tggtgtattg ctacagaagt 420

aaggaagaat gtcatctcgt gttccaaagc ccaaaatgac aaactaaagc ttggaggttga 480

tccatactta agtgtcagaa aatcttttga gtgtacaaca gatatgtgta ttctgaagtt 540

aaacactacc tacctaacgt tacaaaacgt catttcacaa aacaatgtat ctagataaaa 600

aaatatgaca tgtaaagtga gtaatgactc atatttataa tcaaaaactg ataacaataa 660

gatgagatag ttactaaagt acctttgacg atggcatgtc caagtatgtg tacctccacc 720

tagcacaaca tcccaaatga tcatattaaa aggcatatga atacaagcaa ccccaagatg 780

cacacaagaa acaacacaaa ttgcacaaaa ccaaaagcaa ccgatgtctt gagcgtagag 840

atcatgctat ccccactata aatacaaatg aactatatca aagatgctcc ttatccttat 900

ggaaaaatca caaacatcaa aacggtataa gagttctcta acatcaatct Catagctata 960

aagatctttg tcatcctctc tctcctcgcc ctcgcagcga gcagcgcctc agcacagttt 1020

gatgcttgca cctatggggca aagccaacag cagccgttc 1059

<210> 7

<211> 2433

<212>DNA

<213> rice

<400> 7

tgttatacat ttgattcacc tatgcatgtt catgggtatt ataagataga ataatatata 60

ttgtcaaaat tatttgtgtt aattattaac attatataga tgagaatgac ataattaaaa 120

aagatataat atggttccat aagacacttt caattgaatg cgtatatgtt ttttctggca 180

aatactaagc acgtactttt tcctcttctt ttggagaaat atatgttttt ctcttagggt 240

aggtgtgcgt atgtgtattt atatgatgga tgtgcatacg tttatatatg cgctcgtgaa 300

tgccttttat atcattagta aaaaaatact ttcaagaaaa ttggtgagat agagtgaagt 360

ggtgctgcgg caacgtggat tctttgacaa cctcgtcagg agtggtcgct actcatatgc 420

acaatttgag gaaaatcgat ctcatagctg caaagaaaga aagaaagaga aaactgctcg 480

atgcagctgt tcgtatacac actcttctta ttatttttcc tttatgacat ctctttatac 540

gtatatagtg agggattact tgaaatagata tatatatata tatataatgg ttgaaataat 600

atatccacgg gttatgtata aataaataac tagattttac ctttttatat atttttctt 660

agataaacac gggttatgta atatataata cttaattaat cgatgatgat atgctttgtt 720

tcgcgtgtcc tagatatgct cttccaatga ggtagaccaa atgattctta aaagataaaa 780

ggacatccac atgctgtaaa aaaatgattg ttaatttttc aaaaaaaata tatgaataaa 840

gaaacatata atatatgaaa atacattcat ggtgattgca tatatctgta tgtattacac 900

aatatggagt gagtaaattt ttagagtgct actcacaatt taaattgttt ttatttttga 960

aaatataaat ccaatttaac tatgctattc agtgatatac aatcatataa tccacaattt 1020

taattttatta ttatttttta tggatgattt gatgagatgc gttatttaga ataaaacttg 1080

aaataacata tacttttaca acttaaaaca tatatggtta acatggaatt tttttagttt 1140

ttcatctaat ctaagtatac aagaaagaaa aagtgaaaag acatataggg gatatgtacg 1200

tacacggacg tacaggtcca tggggacacg tacggccgga taacgtggga tttttttaga 1260

tagattgatt tggtattttc ttcaaataaa gatctgatgg ttaaaaataa tgagtccacc 1320

agattaaatg aaaaccgatg gctagatgtt tttctttttt attagaattt ctatgatta 1380

ttaaaacgcc acgtggtgat ctaggagcat ttgtaggaat gccacgtagc ggtttaggag 1440

cgtttgtaga aagtttaatg ggtttttagt atataataga aagttgtat aagacagcgt 1500

actagctata catgtacaag ggagaagtga ggataacaaa gataagtatg gcgcaattct 1560

gattttccat aattccatta acttttggaa ttgcgcgaag ttagttcccc ccccccccct 1620

ttttttcacg ttttcattta taaagtaaca gtgttcgatc cttccatttc ctgttaagaa 1680

ctcaatgtac atgccaaact acaaatatgt atgtattcta ttaaagcact tttactggaa 1740

aaaaaaactc taacgtaaca atcacctagg ctaaatgtta cttcctccgt ttcacaatgt 1800

aagtcattct agcatttccc acatttatat tgatgcatct agattcatta acatcaatat 1860

gaatgtggga aatgctagaa tgtctagatt cattaacatc aatatgaatg tgggaaatgc 1920

tagaatgact tacattgtga aacggaggga gtatatatgt aagtcgtgac ttatgtaaca 1980

cgctcttaaa attggacaaa ttatatatat gcttataatc aaaatactac ttcacccatc 2040

taaaagaata ttgttagaat acaaatttag tttaacatag tgccgagcca atatggtata 2100

tctttatcat cagaatttgt ttccacgcaa ttctcaataa agcaccaatg caccattgat 2160

caaacacctt tatatctaca gcatcatctc gccacgttct tccaaaatat tggttgaaca 2220

aatgtccaaa tacacctcat gactcattgt ttctagctta cttgacctcc accaggtagt 2280

tttgcaaaaa tcaaatcctt tttggcgtgt aatattgcta ttacctataa ataatcccct 2340

agagcaattg ttatctcatc accctcaaca tataatcttc tacatttaca ccatctagta 2400

atcttgttaa gcatctccca tacgctgtca aca 2433

<210> 8

<211> 2462

<212>DNA

<213> rice

<400> 8

ggatacatct cgatgctttg ctaggtcgct ctagctagct agccagccaa ccgctccatt 60

ccaatggcta tgcatccacg cctcttgccg ccagtcacca tgcacggctg caattaaatc 120

tgcaagtgaa acattgattg acgctgacga tcatctggtt gcatctacgt gtaaaagaga 180

atgtgagaag cagcaccagc acatcacgtt cgtcgtccga tctttcttca tccttgccac 240

agctcaatcc aatttgccat atgttgttag tgagtagtga caagcgagga ccttttagct 300

tactttgtta ggtttctttc tccgatcctc ttctcagagc ttagagattg agcttggatc 360

attggacgaa gctgcatcaa gcccattttg ccgactcact tgccatatgc atccatgatc 420

aactaattgt tgaatggtaa gatttacacc aatatttgta gttttgcagc ctttgtaatt 480

tatatgagtt caaccactaa ttaaaagcat tactcttttc attagtaatt taacctgccc 540

ttaatatcat gtcattgaga aagtttgtat cggttggcaa aaaaaggaaa aggagaggga 600

aaacagtgga tgatttgata gaatcataga agagggcaat ggataaattt taaggtaata 660

ttataaagcc tattacgtaa atattgtttt tgacgtaatt caagtgctaa gcagtaacac 720

cttatatatc tatataaatt atttattaga tgttatatta aggggctcct attttgatag 780

gaacattaat agctcacttc gacggtagaa aatactcacc atactttgtg gagaaaatat 840

gaaggaggag aagaaccgtt catcctcact tcattatctt cttaattaaa agtatatagt 900

gtttgtgtat atgcgctgaa ttcggaagta gattcagaaa tgttttccat tggttacacc 960

tttagacatt gtttacaaat tgcttgttca agaaacaacc gaattttatt gtagcacaca 1020

aggactagtc tgctccctga acagataatt ggacttggat aggggcttct ttcctgatgc 1080

atcctaaccc taccgcacac tgattacta gctagaggtt atctttctgc atgaacagaa 1140

gtcatcactg cagaaactga attagttcaa tgtgagatga actggtgagg atcagtcttt 1200

gtgtatggat gtacataaat ggatgtggtt caccgcattt catgggcttc aacagccttc 1260

aattagttga tttgatatat gatagctttt catttggaga gtactattgg tgaaatggaa 1320

gagagacagt ggagaaaaga taattgttgc tacatatgac aacagtgaga gtcaactttt 1380

agcatttatc aaaggaaatt ttgatgtatg gagtgaagaa aagaaaacaa gagtgataaa 1440

aaaaattatt tcggtgcagt aataattaag aggctttgtt gtgtcaacta ttatagaaag 1500

atagttttta agtgatattt attagatata taagagcttt cctacatgcc ttacattaat 1560

tctgcaagct tggttagttt ctaccctgct caaataatta aatataatta aattata act 1620

aaattaatac ttcgttgtaa aaaggatgat gtactacagt acagtgtgta ggcaaacact 1680

caaactataa atcataaata aacacaaaat attcaggatt taaatcacca aatatcaaat 1740

ccaatataaa ttaaatgtct cattttgttc atcacaaaat atataaatca aaaccattga 1800

tttcacagga ttgagctcaa tttcaagcaa gagaaaggac atgaagacaa actcactcga 1860

tcacttgtat taatcgatta ccttttcttt tgttgtaata gttttttttt ttttgctttc 1920

gtataattat gttcgattcg cttggctttg atacggaaaa gaacccatac actaaaatca 1980

cacgtttctt aattttgtgc aatctagtgt gttaaagctt cattgctgaa cactttttaa 2040

ctgccagagc atattttcta acaatatttg ttctgagctc ttctttttct ttgtcttgca 2100

aattggatca ctatacaagc catggaaata aagatcaaca gattttgtta ccttgcgtaa 2160

ctcgacgtct gttgacagta tacgcattaa ttttcttgca cggaagaatc aaaacaagtt 2220

gaaaaaatgt ggtgcataca gtccacttta acaaactttt atccatatca tgtccaaatc 2280

gattaggtgt gagtcacaat aaatgttcta tgcaaacaag ctaatcacaa ttatgaacag 2340

caaaaaaaat tgtgtccgtt cttgagatca ctttagtctt tatagctata tatagaaacc 2400

acccatagat agctatccct ctcataaaca aattgatagt taagattttc tgcaacaaaa 2460

at 2462

<210> 9

<211> 171

<212>DNA

<213> pea

<400> 9

atggcttcta tgatatcctc ttccgctgtg acaacagtca gccgtgcctc tagggggcaa 60

tccgccgcag tggctccatt cggcggcctc aaatccatga ctggattccc agtgaagaag 120

gtcaacactg aattacttc cattacaagc aatggtggaa gagtaaagtg c 171

Claims (9)

1. The application of four genes PSY , CrtI , BHY and BKT in the transgenic breeding of crop seed endosperm producing astaxanthin. 2. A transgenic breeding method for producing astaxanthin in crop seed endosperm, characterized in that, comprising the following steps: S1. According to the codon preference of the breeding crop, optimize the sequences of the 4 genes of PSY , CrtI , BHY and BKT , and fuse the sequence-optimized 4 genes with the endosperm expression promoter and plastid transit peptide of the breeding crop respectively, Construction of gene expression cassettes; S2. Constructing the four gene expression cassettes in S1 into plant transformation vectors to obtain plant transformation vectors containing PSY , CrtI , BHY and BKT ; S3. Transforming the plant transformation vector described in S2 into a breeding crop to obtain a transgenic crop producing astaxanthin in the seed endosperm. 3. The breeding method according to claim 2, characterized in that, the breeding crops are rice, corn, wheat. 4. according to the described breeding method of claim 2 or 3, it is characterized in that, described PSY gene is the PSY gene of corn, and CrtI gene is the CrtI gene of Erwinia , and BHY gene is the BHY gene of Haematococcus pluvialis, The BKT gene is the BKT gene of Chlamydomonas reinhardtii. 5. The breeding method according to claim 4, wherein, when the breeding crop is rice, according to the codon preference of monocotyledonous plants and rice, optimize the PSY gene of corn, the CrtI gene of Erwinia, Pluvialis The optimized sequences of the BHY gene of Haematococcus and the BKT gene of Chlamydomonas reinhardtii are shown in SEQ ID NO: 1-4. 6. The method according to claim 1, wherein when the breeding crop is rice, the promoter used is the rice endosperm-specific storage protein gene promoter Pens1～Pens4, and the sequence is as shown in SEQ ID NO:5～8. shown; the plastid transit peptide coding sequence used is TP, as shown in SEQ ID NO:9. 7. method according to claim 2, is characterized in that, when breeding crop is paddy rice, breeding method comprises the steps: S1. According to the codon preference of monocots and rice, optimize the PSY gene of maize, the CrtI gene of Erwinia, the BHY gene of Haematococcus pluvialis, and the BKT gene sequence of Chlamydomonas reinhardtii. The optimized gene is called They are PSY-r , CrtI-p , BHY-p and BKT-p , the sequences of which are shown in SEQ ID NO: 1-4; the four genes after sequence optimization are respectively linked to the rice endosperm-specific storage protein gene promoter Pens1- The coding sequences of Pens4 and the plastid transit peptide TP respectively construct gene expression cassettes; the rice endosperm-specific storage protein gene promoters Pens1-Pens4 and the plastid transit peptide TP coding sequences are respectively shown in SEQ ID NO: 5-9; S2. constructing a plant transformation vector with 4 gene expression cassettes in S1 to obtain a multigene vector containing PSY-r , CrtI-p , BHY-p and BKT-p gene expression cassettes; S3. Transforming the multigene vector into Agrobacterium, transforming the callus of the breeding crop rice, and obtaining the transgenic rice. 8. The method according to claim 7, wherein, when 4 gene expression cassettes in S2 are used to construct plant transformation vectors, a multigene carrier system is used to construct a T-DNA region containing PSY, Crt I, BHY, BKT A polygenic vector of four genes. 9. A vector containing four genes PSY-r , CrtI-p , BHY-p and BKT-p shown in SEQ ID NO: 1-4.