CN110592124A - A gene combination expressing betalain in rice and its application - Google Patents

Abstract

A gene combination for expressing and producing betalain in rice and application thereof, including melo gene, BvDODA1 gene and BvCYP76AD1 gene, according to the codon preference of rice, the melo gene is optimized to the meloS gene, and its nucleotide sequence is as shown in SEQ ID NO.1; BvDODA1 gene is optimized to BvDODA1S gene, and its nucleotide sequence is shown in SEQ ID NO.2; BvCYP76AD1 gene is optimized to BvCYP76AD1S gene, and its nucleotide sequence is shown in SEQ ID NO.3 , respectively fused with the 35S promoter of the cauliflower mosaic virus and the NOS terminator of Agrobacterium rhizogenes, respectively constructing the corresponding gene expression cassette, and then connecting into the plant expression vector to obtain the multigene plant transformation vector containing the above-mentioned three gene expression cassettes, Transform into rice to obtain transgenic rice rich in betalain, and the content of betalain is about 2.0-2.6 mg/g FW.

Description

A gene combination expressing betalain in rice and its application

technical field

The invention belongs to the field of genetic engineering, in particular to a gene combination for expressing betalain in rice and its application.

Background technique

The main component of betalain is betanin, which is a natural pigment extracted from sugar beet. It has no toxic side effects, bright color and contains nutrients necessary for human metabolism and growth and development. The coloring of powder, juice, soft drink, candy, cake, ice cream, canned food, concentrated juice, ice cream, jelly, sausage food not only increases the beautiful appearance of the food, but also improves the nutritional value of the food.

(Am.J.Clin.Nutr.(2004) 80, 391-395, 2005) found that consumption of thorn pear fruits containing betalain can significantly reduce lipid damage caused by peroxidative stress and improve the body's resistance to Oxidation levels; in addition, Kapadia et al. (Cancer Lett (1996) 100, 211-214) found that beetroot had a significant inhibitory effect on skin and lung cancer in mice.

Because of the above effects, betalain is approved for use as a food additive (Moreno et al., Phytochemistry Reviews, (2008) 7(2):261-280), and it also has antioxidant and health-promoting properties. , resulting in a significant increase in its demand.

The main source of betacyanin is sugar beet (Beta vulgaris), but many factors can significantly affect the yield of betacyanin in sugar beet. According to literature reports, the current betacyanin from sugar beet can only meet 10% of the global demand for food additives ( Manchali et al., Stability of betalain pigments of red beet. In RedBeet Biotechnology; Neelwarne, B., Ed.; Springer US: Boston, MA, 55-74.2013). Therefore, there is a need to find another way to produce betalain to meet the global demand for food additives.

Rice is an important food crop and model plant, and at the same time, it is also an excellent bioreactor, such as the production of β-carotene and human serum protein in rice. However, there is no beta-carotene synthesis pathway in rice. Therefore, it is necessary to construct a betalain synthesis pathway by genetic engineering and transfer it into rice, so as to obtain a new rice material rich in betalain.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a combination of genes expressing and producing betalain in rice and application thereof, the gene is fused with the 35S promoter of cauliflower mosaic virus and the NOS terminator of Agrobacterium rhizogenes to construct a gene expression cassette, and then Connected into a plant expression vector to obtain a multi-gene plant transformation vector, transformed into rice, to obtain transgenic rice rich in betalain, the content of betalain is about 2.0-2.6mg/g FW, and the rice can not only be used for the production of beet red It can also be used as a functional rice to promote health.

In order to achieve the above object, the present invention provides the following technical solutions:

A combination of genes expressed in rice to produce betalain, including melo gene, BvDODA1 gene and BvCYP76AD1 gene.

Further, according to the codon preference of rice, the melo gene is optimized as a meloS gene, and its nucleotide sequence is shown in SEQ ID NO. 1; the BvDODA1 gene is optimized as a BvDODA1S gene, and its nucleotide sequence is as shown in SEQ ID NO. .2; the BvCYP76AD1 gene is optimized to the BvCYP76AD1S gene, and its nucleotide sequence is shown in SEQ ID NO.3.

A multi-gene plant transformation vector includes a plant expression vector and gene expression cassettes of meloS gene, BvDODA1S gene and BvCYP76AD1S gene fused with 35S promoter of cauliflower mosaic virus and NOS terminator of Agrobacterium rhizogenes respectively.

Further, the plant expression vector is pCAMBIA1301.

The application of the combination of genes expressed in rice to produce betalain in rice.

A method for obtaining a betalain-containing transgenic rice, comprising the following steps:

1) According to the codon preference of rice, the sequences of the three genes of melo, BvDODA1 and BvCYP76AD1 were optimized, and the three genes after sequence optimization were respectively fused with the 35S promoter of cauliflower mosaic virus and the NOS terminator of Agrobacterium tumefaciens, Construct gene expression cassettes respectively;

2) connecting the three gene expression cassettes constructed in step 1) into a plant expression vector to obtain a multigene plant transformation vector containing the three gene expression cassettes of melo, BvDODA1 and BvCYP76AD1;

3) The multigene plant transformation vector described in step 2) is transformed into Agrobacterium, and transformed into rice to obtain betalain-rich transgenic rice.

Further, in step 1), the nucleotide sequences of the 35S promoter of the cauliflower mosaic virus are respectively shown in SEQ ID NO.4; the NOS terminator sequence of the Agrobacterium rhizogenes is shown in SEQ ID NO.5.

Preferably, in step 2), the plant expression vector is pCAMBIA1301.

Also, in step 3), the variety of the rice is Zhonghua 11.

In the present invention, the melo gene of Aspergillus oryzae, the BvDODA1 gene of sugar beet (Beta vulgaris) and the BvCYP76AD1 gene of sugar beet (Beta vulgaris) are optimized according to the preference of rice codons, and the optimization is carried out according to the following principles: ( 1) Optimize gene codons to improve gene translation efficiency according to rice codon preference; (2) Eliminate the recognition sites of commonly used restriction endonucleases in genes to facilitate the construction of expression cassettes; (3) Eliminate inverted repeats, stems The loop structure and transcription termination signal balance the GC/AT within the gene and improve the stability of RNA; (4) Make the protein encoded by the gene conform to the N-terminal principle to improve the stability of the translated protein; (5) Optimize the secondary structure of mRNA free energy to improve gene expression efficiency.

In the present invention, the meloS gene, BvDODA1S gene and BvCYP76AD1S gene obtained by optimizing melo, BvDODA1 and BvCYP76AD1 are respectively fused with the 35S promoter of cauliflower mosaic virus and the NOS terminator of Agrobacterium rhizogenes, respectively constructing corresponding gene expression cassettes, and then It is connected into a plant expression vector to obtain a multi-gene plant transformation vector containing the above-mentioned three-gene expression cassette, and transformed into rice to obtain betalain-rich transgenic rice.

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

In the present invention, the melo gene, the BvDODA1 gene and the BvCYP76AD1 gene are combined and expressed in rice after optimization to produce betalain, and the content of betalain in the obtained transgenic rice plant is about 2.0-2.6 mg/g FW, which can be either As a raw material for producing betalain, it can also be eaten as a functional rice promoting health, and has important production and application value.

Description of drawings

Fig. 1 is a schematic diagram of the structure of the three-gene (meloS, BvDODA1S and BvCYP76AD1S) plant transformation vector in Example 1 of the present invention.

Figure 2 is the rice callus in Example 2 of the present invention, left: wild-type rice; right: transgenic rice.

Figure 3 is the PCR detection result of the exogenous gene of the T ₀ generation rice genomic DNA in Example 2 of the present invention, wherein, 1, 2 and 3 are transgenic rice; WT is wild-type rice.

Figure 4 shows the results of RT-PCR detection of foreign genes in T ₁ generation rice plants, wherein 1, 2 and 3 are transgenic rice; WT is wild-type rice.

Figure 5 is the appearance phenotype of the transgenic rice plant in Example 3 of the present invention; wherein, BR1, BR2 and BR3 are transgenic rice; WT is wild-type rice.

Figure 6 is a mass spectrometry identification diagram of betalain in plants in Example 3 of the present invention, wherein a is a standard mass spectrogram; b is a transgenic plant mass spectrogram; c is a wild-type plant mass spectrogram.

Detailed ways

The present invention will be further elaborated below with reference to the accompanying drawings and specific embodiments of the description, and the embodiments are only used to explain the present invention, but not 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, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

The mother liquor in the embodiment of the present invention and each culture medium formula:

1. The formula of stock solution

MSmax mother liquor (10X): NH ₄ NO ₃ 16.5 g, KNO ₃ 19.0 g, MgSO 4 ·7H ₂ O, 3.7 g, CaCl ₂ ·2H ₂ O 4.4 g, add water to make up to 1000 ml.

_MSmin mother liquor (100X): KI _0.083g , _{H3BO4 0.62g} , MnSO4 _· 2H2O _2.23g , ZnSO4 _{· 7H2O} 0.86g, Na2MoO4 _· 2H2O 0.025g, _CuSO4 ·5H ₂ O 0.0025g, CoCl ₂ ·2H ₂ O 0.0025g, add water to make up to 1000ml.

N6max mother liquor (10X): KNO ₃ 28.3g, KH ₂ PO ₄ 4.0g, (NH ₄ ) ₂ ·SO ₄ 4.63g, MgSO ₄ ·7H ₂ O 1.85g, CaCl ₂ ·2H ₂ O 1.66g, add water to volume to 1000ml.

N6min mother liquor (100X): KI 0.08g, H ₃ BO ₄ 0.16g, MnSO ₄ ·2H ₂ O 0.44g, ZnSO ₄ ·7H ₂ O 0.15g, add water to make up to 1000ml.

Fe ²⁺ -EDTA mother solution (100X): FeSO ₄ ·7H ₂ O 2.78g, Na ₂ EDTA·2H ₂ O 3.73g, dissolved separately, then mixed, and water was added to make up to 1000ml.

Vitamin mother solution (100X): niacin (Nicotinic acid) 0.1g, vitamin B6 (Pyridoxine HCl, VB6) 0.1g, vitamin B1 (Thiaminc HCl, vb1) 0.1g, glycine (Glycine) 0.2g, Inositol (Inositol) 10g , add water to 1000ml.

2. Culture medium formula

Co-cultivation medium: N6max stock solution (10X): 12.5ml, N6min stock solution (100X) 1.25ml, Fe ²⁺ -EDTA stock solution (100X) 2.5ml, vitamin stock solution (100X): 2.5ml, dichlorophenoxyacetic acid 2g/ L(2,4-D) 0.75ml, Casein Enzymatic Hydrolysate 0.2g, Sucrose 5g, Agarose 1.75g, add water to 250ml, adjust pH=5.6, melt in microwave before use, add 5ml 50% glucose and 250 μl 20 g/L acetosyringone.

Selection medium: N6max stock solution (10X) 25ml, N6min stock solution (100X) 2.5ml, Fe ²⁺ -EDTA stock solution (100X) 2.5ml, vitamin stock solution (100X) 2.5ml, dichlorophenoxyacetic acid 2g/L (2, 4-D) 0.625ml, CaseinEnzymatic Hydrolysate 0.15g, sucrose 7.5g, agar powder 1.75g, add water to 250ml to adjust pH=6.0, melt and add hygromycin and carbenzyl before use.

Pre-differentiation medium: MSmax stock solution (10X) 25ml: MSmin stock solution (100X) 2.5ml, Fe ²⁺ -EDTA stock solution (100X) 2.5ml, vitamin stock solution (100X) 2.5ml, 6-benzylaminopurine 2g/L ( 6-BA) 0.5ml, kinetin 2g/L (KT) 0.5ml, indole acetic acid 1mg/ml (IAA) 50μl, Casein Enzymatic Hydrolysate 0.15g, sucrose 7.5g, agar powder 1.75g, Add water to 250m to adjust pH=5.9, and add hygromycin and carbenzyl before use.

Differentiation medium: MSmax stock solution (10X) 100ml, MSmin stock solution (100X) 10ml, Fe ²⁺ -EDTA stock solution (100X) 10ml, vitamin stock solution (100X) 10ml, 6-benzylaminopurine 2g/L (6-BA) 2.0ml, kinetin 2g/L (KT) 2.0ml, indole acetic acid 1mg/ml (IAA) 0.2ml, naphthalene acetic acid 1g/L (NAA) 0.2ml, Casein EnzymaticHydrolysate 1g, sucrose 30g, Phytagel (Phytagel) 3g, add water to 1000ml to adjust pH=6.0, and divide into vials.

Rooting medium: MSmax stock solution (10X) 50ml, MSmin stock solution (100X) 5ml, Fe2+-EDTA (100X) 10ml, vitamin stock solution (100X) 10ml, agar powder (Sucrose) 20g, Phytagel (Phytagel) 3g, add water to 1000ml was adjusted to pH=5.8 and divided into vials.

Example 1 Construction of multigene plant expression vector

1. Optimized synthesis of three genes

The optimization is carried out according to the following principles: (1) Optimize the gene codons according to the rice codon preference to improve the gene translation efficiency; (2) Eliminate the recognition sites of the commonly used restriction endonucleases inside the gene to facilitate the construction of the expression cassette; (3) Eliminate inverted repeat sequences, stem-loop structures and transcription termination signals to balance GC/AT within the gene and improve the stability of RNA; (4) Make the protein encoded by the gene conform to the N-terminal principle to improve the stability of the translation protein; (5) ) to optimize the free energy of mRNA secondary structure to improve gene expression efficiency.

Using the melo gene (GenBank No. D37929.1) of Aspergillus oryzae as a template, according to the rice codon preference, the DNA sequence meloS shown in SEQ ID NO.1 was synthesized, cloned into a plasmid vector, and sequenced to determine its Sequence; using the BvDODA1 gene (GenBank No.HQ656027.1) of sugar beet (Beta vulgaris) as a template, according to the rice codon preference, the DNA sequence BvDODA1S shown in SEQ ID NO.2 was synthesized and cloned into a plasmid vector, and sequenced to determine its Sequence; using BvCYP76AD1 (GenBank No. HQ656023.1) of sugar beet (Beta vulgaris) as a template, according to the codon preference of rice, the DNA sequence BvCYP76AD1S shown in SEQ ID NO.3 was synthesized, cloned into a plasmid vector, and its sequence was determined by sequencing ; Take the 35S promoter of cauliflower mosaic virus as a template, synthesize the DNA sequence 35S promoter shown in SEQ ID NO.4, clone it into a plasmid vector, and determine its sequence by sequencing; take the NOS terminator of Agrobacterium rhizogenes as a template, synthesize The NOS terminator of the DNA sequence shown in SEQ ID NO. 5 was obtained, cloned into a plasmid vector, and its sequence was determined by sequencing.

2. Construction of three gene expression cassette elements: splicing of elements was performed according to the modified "overlap extension PCR" technique (Appl Microbiol Biotechnol. 2006, 73(1): 234-40).

2.1 Construction of the meloS gene expression cassette: three pairs of primers P1 and P2, P3 and P4, P5 and P6 were designed according to the above-mentioned chemically synthesized 35S promoter, meloS gene and NOS terminator sequences, and the primers between adjacent elements had 20bp of primers. In the overlapping region, primer P1 has an EcoRI restriction site, and primer P6 has a BamHI restriction site. The specific sequences are as follows:

P1: GAATTCATGGAGTCAAAGATTCAAATAGAGGACCTAACAGAACTCGCCGTAAAGACTGGC;

P2:ATTGGTTCAACAGATGCCATAGTCCCCCGTGTTCTCTCCAAATGAAATGAACTTCCTTAT;

P3:TGGAGAGAACACGGGGGGACTATGGCATCTGTTGAACCAATCAAAACTTTCGAGATTCGTC;

P4:GAGGGGTAGTTCGATGTTGGTTAACGCATAGTACCAACAGTGACGTTACGAACACGTTGA;

P5: CTGTTGGTACTATGCGTTAACCAACATCGAACTACCCCTCACGCATGCATTCATCAATAT;

P6: GGATCCCATCAACGCAAGACATGCGCACCACGACCGTCTGACAGGAGAGGAATTTCCGAC.

Take the plasmid of the above-mentioned chemically synthesized 35S promoter, meloS gene and NOS terminator as template, use P1 and P2, P3 and P4, P5 and P6 respectively to carry out three elements (35S promoter, meloS gene and NOS terminator) PCR amplification.

Reaction system: 35S promoter (meloS gene or NOS terminator) plasmid 1μl, 2.5mmol/L dNTPs 4μl, Buffer 25μl, KOD Plus (Toyobo Japan) polymerase 1U, primer P1 (corresponding to meloS gene is P3, corresponding to NOS terminator) P5) 1 μl, primer P2 (corresponding to meloS gene is P4, corresponding to NOS terminator is P6) 1 μl, ddH ₂ O is supplemented to 50 μl.

The reaction program was: 94 °C for 30 s, 54 °C for 30 s, 72 °C for 90 s, a total of 45 cycles, and 72 °C for another 10 min of extension, PAGE electrophoresis was used to separate and recover 3 PCR amplified fragments, and then the 3 amplified fragments were equimolar. Mix and use the mixed fragment as a template, and then perform PCR amplification with primers P1 and P6.

Reaction system: mixed fragment 1μl, 2.5mmol/L dNTPs 4μl, Buffer 25μl, KOD Plus (Toyobo Japan) polymerase 1U, primer P1 1μl, primer P6 1μl, ddH ₂ O to 50μl; reaction program: 94℃ for 30s, 54 30 s at ℃, 240 s at 72 ℃, a total of 45 cycles, and then extended at 72 ℃ for 10 min to obtain the expression cassette of the meloS gene, and the expression cassette was cloned by T-vector and the complete nucleotide sequence of the recombinant plasmid was determined.

2.2 Construction of BvDODA1S gene expression cassette

Three pairs of primers P7 and P8, P9 and P10, P11 and P12 were designed according to the above chemically synthesized 35S promoter, BvDODA1S gene and NOS terminator sequences. The primers between adjacent elements have an overlapping region of 20 bp, and primer P7 has BamHI restriction site, primer P12 has KpnI restriction site, the specific sequence is as follows:

P7: GGATCCATGGAGTCAAAGATTCAAATAGAGGACCTAACAGAACTCGCCGTAAAGACTGGC;

P8:TCACCATTCATCATCTTCATAGTCCCCCGTGTTCTCTCCAAATGAAATGAACTTCCTTAT;

P9:TTGGAGAGAACACGGGGGACTATGAAGATGATGAATGGTGAAGATGCAACTGATCAGATG;

P10:GAGGGGTAGTTCGATGTTGGTTAAGCAGAGGTGAACTTGTAGGAACCATGACACAGAGTA;

P11: ACAAGTTCACCTCTGCTTAACCAACATCGAACTACCCCTCACGCATGCATTCATCAATAT;

P12: GGTACCTTGCTTCATCAACGCAAGACATGCGCACCACGACCGTCTGACAGGAGAGGAAT.

With the above-mentioned chemically synthesized 35S promoter, BvDODA1S gene and the plasmid of NOS terminator as template, use the above-mentioned three pairs of primers P7 and P8, P9 and P10, P11 and P12 to carry out three originals (35S promoter, BvDODA1S gene and NOS respectively). terminator) PCR amplification.

Reaction system: chemically synthesized 35S promoter (BvDODA1S gene or NOS terminator) plasmid 1μl, 2.5mmol/L dNTPs 4μl, Buffer 25μl, KOD Plus (Toyobo Japan) polymerase 1U, primer P7 (corresponding to BvDODA1S gene is P9, corresponding to NOS terminator is P11) 1 μl, primer P8 (corresponding to BvDODA1S gene is P10, corresponding NOS terminator is P12) 1 μl, ddH ₂ O is supplemented to 50 μl.

The reaction program was: 94°C for 30s, 54°C for 30s, 72°C for 90s, a total of 45 cycles, and then extended at 72°C for 10 min, PAGE electrophoresis was used to separate and recover 3 PCR amplified fragments, and then the 3 amplified fragments were equimolar. The mixed fragments were used as templates, and PCR amplification was carried out using primers P7 and P12.

Reaction system: mixed fragment 1μl, 2.5mmol/L dNTPs 4μl, Buffer 25μl, KOD Plus (Toyobo Japan) polymerase 1U, primer P7 1μl, primer P12 1μl, ddH ₂ O to 50μl.

The reaction program was: 94°C for 30s, 54°C for 30s, 72°C for 180s, a total of 45 cycles, and then extended at 72°C for 10 min to obtain the expression cassette of the BvDODA1S gene, and the expression cassette was cloned by T-vector and the nucleosides of the recombinant plasmid. Acid full sequence determination.

2.3 Construction of BvCYP76AD1S gene expression cassette

Three pairs of primers P13 and P14, P15 and P16, P17 and P18 were designed according to the chemically synthesized 35S promoter, BvCYP76AD1S gene and NOS terminator sequences. The primers between adjacent elements have an overlapping region of 20 bp. KpnI restriction site, primer P18 has XhoI restriction site, the specific sequence is as follows:

P13: GGTACCATGGAGTCAAAGATTCAAATAGAGGACCTAACAGAACTCGCCGTAAAGACTGGC;

P14: GCCAAAGTTGCATGATCCATAGTCCCCCGTGTTCTCTCCAAATGAAATGAACTTCCTTAT;

P15:TGGAGAGAACACGGGGGGACTATGGATCATGCAACTTTGGCAATGATCCTTGCAATCTGGT;

P16:GAGGGGTAGTTCGATGTTGGTTAGTAACGTGGGATTGGAATCAGTTTCAATGGCTTGGTC;

P17:CCAACATCGAACTACCCCTCCCAACATCGAACTACCCCTCACGCATGCATTCATCAATAT;

P18: CTCGAGTTGCTTCATCAACGCAAGACATGCGCACCACGACCGTCTGACAGGAGAGGAATT.

With the above-mentioned chemically synthesized 35S promoter, BvCYP76AD1S gene and the plasmid of NOS terminator as template, use three pairs of primers P13 and P14, P15 and P16, P17 and P18 to carry out three elements (35S promoter, BvCYP76AD1S gene and NOS termination respectively). sub) PCR amplification.

Reaction system: chemically synthesized 35S promoter (BvCYP76AD1S gene or NOS terminator) plasmid 1μl, 2.5mmol/L dNTPs 4μl, Buffer 25μl, KOD Plus (Toyobo Japan) polymerase 1U, P13 (corresponding to BvCYP76AD1S gene is P15, corresponding to NOS Terminator is P17, the same below) 1 μl, primer P14 (corresponding to BvCYP76AD1S gene is P16, corresponding to NOS terminator is P18) 1 μl, ddH ₂ O is supplemented to 50 μl.

The reaction program was: 94°C for 30s, 54°C for 30s, 72°C for 120s, a total of 45 cycles, and then extended at 72°C for 10 min. The 3 PCR amplified fragments were separated and recovered by PAGE electrophoresis, and then each amplified fragment was mixed in equimolar numbers. , using the mixed fragment as a template, using primers P13 and P18 for PCR amplification.

Reaction system: mixed fragment 1μl, 2.5mmol/L dNTPs 4μl, Buffer 25μl, KOD Plus (Toyobo Japan) polymerase 1U, P13 1μl, P18 1μ, ddH ₂ O to 50μl; reaction program: 94°C for 30s, 54°C for 30s , 72 ℃ for 240 s, a total of 45 cycles, and then extended at 72 ℃ for 10 min to obtain the expression cassette of BvCYP76AD1S gene, and the expression cassette was cloned by T-vector and the complete nucleotide sequence of the recombinant plasmid was determined.

3. Construction of multigene plant transformation vector

The meloS gene expression cassette recombinant plasmid obtained above was digested with EcoRI and BamHI and ligated into the pCAMBIA1301 vector digested with the same enzyme to obtain the pCAMBIA1301-meloS recombinant plasmid.

The BvDODA1S gene expression cassette recombinant plasmid obtained above was digested with BamHI and KpnI and connected to the pCAMBIA1301-meloS recombinant plasmid digested by the same enzyme to obtain the pCAMBIA1301-meloS-BvDODA1S recombinant plasmid.

Finally, the recombinant plasmid of the BvCYP76AD1S gene expression cassette obtained above was digested with KpnI and XhoI and connected to the pCAMBIA1301-meloS-BvDODA1S recombinant plasmid that had been digested with the same enzymes to obtain a multigene plant transformation vector pCAMBIA1301-meloS containing three genes. -BvDODA1S-BvCYP76AD1S (Figure 1).

Example 2 Transformation of Rice

1. Acquisition and identification of transgenic rice

1.1 Agrobacterium preparation and electric shock

1) A single Agrobacterium was picked and inoculated into 5 mL of LB liquid medium (rifampicin 50 μg/mL, chloramphenicol 100 μg/mL), and cultured at 28°C at 250 rpm for 20 h.

2) Transfer 1 mL of bacterial liquid into 20-30 mL of LB liquid medium (rifampicin 50 μg/mL, chloramphenicol 100 μg/mL), and culture at 28°C for about 12 hours at 250 rpm, and measure OD600≈1.5.

3) 8000 rpm, 4°C, 10 min centrifugation to collect bacterial cells, resuspend in Agrobacterium transformation permeate (5wt% sucrose, 0.05wt% Silwet L-77) and dilute to OD600≈0.8.

4) Resuspend the bacterial cells with 0.5 times the volume of sterile water pre-cooled at 4°C, centrifuge at 3500g for 10 minutes, and carefully pour off the supernatant. Sufficiently resuspend the bacterial cells in 1-2 mL of sterile 10% glycerol pre-chilled at 4°C. The bacterial solution can be used immediately or stored at -70°C.

5) In a 1.5 mL eppendorf, add 40 μL bacterial solution and 1-2 μL plasmid DNA (0.4 pg-0.3 μg), mix well in a 0.2 cm diameter electric shock cup, and place on ice for about 1 minute. Adjust the shock parameters to 25μF, 2.5kV/cm, 400Ω. Electric shock, the discharge time is 4-5msec.

6) After electroshock transformation, add 1.0 mL of expression culture medium to the bacterial cells, culture at 29°C for 1 hour, and spread on YEB plate (rifampicin 50 μg/mL + kanamycin 50 μg/mL) added with antibiotics. Using the above transformation procedure, the plant expression vector pCAMBIA1301-meloS-BvDODA1S-BvCYP76AD1S was electroporated into Agrobacterium tumefaciens EHA105 to obtain Agrobacterium tumefaciens strain EHA105 (pCAMBIA1301-meloS-BvDODA1S-BvCYP76AD1S).

1.2 Agrobacterium infection and co-culture with rice callus

1) Rice callus induction:

Select Zhonghua 11 rice mature seeds (shelled) first soaked in 70% ethanol for 1 min, rinsed with sterilized water, then soaked in 2% sodium hypochlorite for 20 min to disinfect, rinsed repeatedly with sterilized water), placed in a superheated In a sterile petri dish on a clean bench, use sterilized filter paper to absorb water, and then use a dismantling knife to peel off the mature embryos, inoculate them on the medium with the scutellum facing up, and place the embryos on the callus induction medium. (including MS 4.4g/L, 2,4-D 2.5mg/L, casein 600mg/L, sucrose 30g/L and phytogel 5g/L, adjust pH to 5.8 with KOH, autoclave) Wound culture, dark culture at 25-28°C for 8-10 days to induce callus, when the embryo grows to 1cm, the scutellum callus with good quality is strictly screened in NBD2 (including NB and 2,4-D 2mg/L) Subculture was carried out on the culture medium and cultured in the dark for 4-7 days at 25-28°C.

2) Infection with Agrobacterium tumefaciens culture:

Agrobacterium EHAl05 with plant expression vector (pCAMBIA1301-meloS-BvDODA1S-BvCYP76AD1S) was picked from the YEB plate, inoculated into 5 mL of YEB liquid medium containing 50 mg/L kanamycin, at 28°C on a shaker with Shaking at a speed of 200 rpm to OD600=0.5, and then re-inoculated into fresh YEB medium at a ratio of 1:100 for shaking culture. The antibiotics and culture conditions contained in YEB medium are the same as before. When OD600=0.5, the bacterial liquid was centrifuged at 6000×g with relative centrifugal force for 10 minutes at 4°C to collect Agrobacterium cells, and resuspended Agrobacterium with 2/3MS+1/3YEB to OD600=0.5 for use.

3) Co-culture of Agrobacterium tumefaciens:

Take the vigorously growing rice embryogenic callus and divide it into small pieces of 2 mm. As for the sterilized petri dish, add the Agrobacterium tumefaciens solution containing 100 μmol/L acetosyringone with OD600=0.5, soak for 25 minutes, and then use After blotting the surface bacterial liquid with sterile filter paper, the rice callus was inoculated on the co-cultivation medium, and cultured in the dark at 28°C for 2-3 days.

4) Rice callus differentiation and plant regeneration

Collect the rice callus after co-cultivation, rinse three times with sterile water containing 500 mg/L cephalosporin, and absorb excess water.

The callus (Figure 2) was transferred to the selection medium (including NB medium, 2,4-D 0.5mg/L, cefotaxime 600mg/L and hygromycin 25mg/L) for 12-15 days, The first 6-7 days were cultured in dark, and the latter 8-9 days were cultured in light, and the light intensity was 45-55 mmoL·m ^-2 ·S ^-1 . Subsequently, the callus that had differentiated adventitious buds was transferred to the differentiation medium RE2-H for 15 days, and the adventitious buds could grow into 2-4 cm seedlings, wherein the differentiation medium RE2-H included MS, sucrose 30 g/ L, sorbose 10-20g/L, hydrolyzed casein 500mg/L, 6-benzylaminopurine 1mg/L, naphthalene acetic acid 0.5mg/L, kinetin 0.5mg/L, zeatin 0.2mg/L, hygromycin 50mg/L of agar and 8mg/L of agar powder, pH 5.8.

Finally, these seedlings were transferred to rooting medium, rooted after 2 weeks, transplanted into long test tubes for 20-30 days, and the larger rice plants were transplanted into the field of Baihe Transgenic Base of Shanghai Academy of Agricultural Sciences.

2. Identification of transgenic rice

2.1 PCR detection of transformed plant genomic DNA

For the leaves of the obtained T ₀ generation plants, the genomic DNA was extracted by SDS method as a template, and the exogenous genes meloS, BvDODA1S and BvCYP76AD1S were detected by PCR amplification method.

The primers used are as follows:

meloZ: atggcatctgttgaaccaatc and meloF: ttaacgcatagtaccaacagt;

BvDODAZ: atgaagatgattgaatggtgaa and BvDODAF: ttaagcagaggtgaacttgta;

BvCYP76ADZ: atggatcatgcaactttggca and BvCYP76ADF: ttagtaacgtgggattggaat.

The amplification program used: 94°C for 30s, 54°C for 30s, 72°C for 120s, a total of 45 cycles, and a final extension at 72°C for 10 min. The results are shown in Figure 3.

It can be seen from Figure 3 that none of the wild-type (WT) controls can amplify the exogenous genes, and the three transgenic lines can amplify the above three genes, indicating that the exogenous genes are completely integrated into the rice genome.

2.2 RT-PCR detection of T1 seeds of transgenic plants:

The T ₁ generation transgenic plants were ground into powder under liquid nitrogen conditions, and then total RNA was extracted by Trizol method, reverse transcribed into cDNA with MMV reverse transcription kit, oligDT primer, and exogenous meloS was carried out with the following primers and amplification conditions , BvDODA1S and BvCYP76AD1S gene RT-PCR detection, rice endogenous Actin 1 gene as an internal reference.

The primers used are as follows:

meloZ1:ctctgataccatctccgt;

meloF1:gttcgtgagttggttgca;

BvDODAZ1:tcaggacctgggtgggct;

BvDODAF1:ggttcagaccgaagtgat;

BvCYP76ADZ1:tgaacactctgctgagct;

BvCYP76ADF1: catctccatacccagaag.

The amplification program used was: 94°C for 30s, 54°C for 30s, 72°C for 30s, a total of 45 cycles, and a final extension at 72°C for 10 min. The results are shown in Figure 4.

The results showed that the control wild-type seeds could not amplify the exogenous genes, and the three transgenic lines could amplify the above three genes (see Figure 4), indicating that the exogenous genes were correctly transcribed and expressed in the transgenic seeds.

Example 3 Appearance observation of transgenic rice plants and detection of betalain content

The rice seeds harvested from T1 generation in Example 2 were normally germinated, and when the plant grew to about 20 cm, it was obvious that the transgenic rice plant had a dark red color (Fig. 5), and it was initially shown that the above-mentioned 3 genes were introduced to make the transgenic rice plant. Synthesis of betalain.

Mass spectrometry analysis and content determination of betalains in transgenic rice plants: Take 0.1 g of rice plants and grind them into powder on ice, add 4.5 mL of 32% ethanol with a pH of 5.3, and extract for 30 min in the dark with low temperature shaking (100 rpm); 4 ℃ The supernatant was collected by centrifugation at 8000 rpm for 5 min; the supernatant was centrifuged at 4° C. 8000 rpm for 5 min, and the collected supernatant was concentrated and dried at low temperature in the dark; finally, the dried substance was added with 200 μL of ultrapure water for determination.

The above-mentioned sample to be tested was passed through a 0.22 μm organic filter membrane, and then injected into a 2 mL brown sampling bottle with a sample injection volume of 20 μL, and high-performance liquid chromatography was used for mass spectrometry analysis. Mass spectrometry conditions: Capillary, 3000V; sample cone, 30V; source temp, 100 ℃; desolvation temp, 350℃; IE, 1.0V; Lock mass, LE (1μg/mL); molecular weight scan range (m/z), 50～1200.

The content of betalains was expressed in betanin equivalents, and the standard betalains were purchased from Sigma.

Betaine content (mg/g FW)=A538(MW)V(DF)/εLW.

In the formula: A538 represents the absorbance value at 538nm (λmax); L=1.0cm; DF is the dilution factor; V is the volume (mL); W is the dry weight of the pigment (g); =65000, MW=551.

It can be seen from the mass spectrometry that the presence of betalains was clearly detected in the transgenic plants (Fig. 6), but not in CK. At the same time, it was calculated that the transgenic plants contained about 2.0-2.6 mg/g FW of betaine. betaine.

sequence listing

<110> Shanghai Academy of Agricultural Sciences

<120> A kind of gene combination expressing and producing betalain in rice and its application

<130> 1911280

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1620

<212> DNA

<213> Aspergillus oryzae

<400> 1

atggcatctg ttgaaccaat caaaactttc gagattcgtc agaagggtcc tgttgagact 60

aaagctgaac gtaagtccat tcgtgatctg aacgaagaag agttggacaa actgattgag 120

gcatggcgtt ggattcaaga tcctgctcgt actggtgaag attccttctt ctaccttgct 180

ggtctgcatg gtgaaccatt ccgtggtgct ggttaccagt gtctgttgac cgaagagtac 240

actgtcttct ccaacaccac ctctgctcaa cgttggaatg atgaacagct gatggctgtt 300

gagaaggcat tgcgtaaggc atgtcctgat gtttctctgc catactggga tgagtctgac 360

gatgagactg caaagaaggg tattccattg atcttcactc agaaagagta caagggtaag 420

ccaaacccac tgtactccta caccttctct gaacgtattg ttgatcgttt ggctaagttc 480

cctgatgctg actactctaa gccacaaggt tacaagacct gtcgttaccc atactctggt 540

ctgtgtggtc aggatgacat cgctatcgct caacagcaca acaacttcct ggatgctaac 600

ttcaaccagg aacagatcac tggtctgctg aactccaatg tcacctcttg gctgaatctg 660

ggtcagttca ctgacatcga gggtaagcaa gtcaaggctg atactcgttg gaagatccgt 720

aacaactctc attggtgggg tggttactgt catcatggta acatcttgtt cccaacctgg 780

catcgtgcat acttccatcc actggagtct ggtggtaaag agactgaggc taaagccacc 840

tctcttgctg ttccactgga gtctccacac aatgacatgc atcttgctat tggtggtgtc 900

cagattcctg gtttcaacgt tgatcagtat gctggtgcta acggtgatat gggtgagaac 960

gatactgctt ccttcgatcc aatcttctac ttccatcact gcttcatcga ctacctgttc 1020

tggacttggc agaccatgca caagaagact gatgcatccc agatcaccat cttgcctgaa 1080

ctgccataca cctacaaggc accaacctct ggtactggtt ccgtcttcaa cgatgttcca 1140

cgtctgaact acgatactcc acttgatcca tttcgtgaga atggtgacaa ggtcacttcc 1200

aacaaactgc tgaccttgaa ggacctgcca tacacctaca aggcaccaac ctctggtact 1260

ggttccgtct tcaacgatgt tccacgtctg aactacccac tgtctccacc aatcctgcgt 1320

gtctctggta tcaatcgtgc atctatcgct ggttcttttg cactggcaat ctctcagact 1380

gatcacactg gtaaggctca agtcaaaggt atcgagtctg tcctgtctcg ttggcatgtc 1440

caaggttgtg caaactgtca gactcacctg tccaccactg cattcgtccc actgttcgag 1500

ctgaacgagg atgatgccaa acgtaaacat gctaacaacg aactggctgt ccatctgcac 1560

actcgtggta atcctggtgg tcaacgtgtt cgtaacgtca ctgttggtac tatgcgttaa 1620

<210> 2

<211> 828

<212> DNA

<213> Beta vulgaris

<400> 2

atgaagatga tgaatggtga agatgcaact gatcagatga tcaaagaatc cttcttcatc 60

actcatggta atccaatctt gactgttgaa gacacccatc cattgcgtcc attcttcgag 120

acttggcgtg agaaaatctt ctctaagaag cctaaggcaa tcctgatcat ctctggtcat 180

tgggagactg tcaaaccaac tgtcaatgct gtccatatca acgacactat ccatgacttc 240

gatgactacc ctgctgctat gtacctgttc aagtaccctg cacctggtgc accagaactg 300

gcacgtaaag tcgaggagat tctgaagaag tctggtttcg agactgctga gactgatgaa 360

aagcgtggtc tggatcatgg tgcatgggtt ccactgatgc tgatgtatcc tgaggctgac 420

atccctgtct gccagctgtc tgttcaacca catctggatg gtacttacca ctacaacttg 480

ggtcgtgcac tggcaccatt gaagaatgat ggtgtcttga tcatcggttc tggttctgca 540

actcatccac tggatgagac tccacactac ttcgacggtg ttgcaccttg ggctgctgca 600

ttcgactctt ggcttcgtaa agcactgatc aacggtcgtt tcgaagaagt caacatctac 660

gagaccaaag caccaaactg gaaactggca catccattcc ctgaacactt ctatccactg 720

catgttgttc ttggtgctgc tggtgagaag tggaaggctg agctgattca ttcttcttgg 780

gatcatggta ctctgtgtca tggttcctac aagttcacct ctgcttaa 828

<210> 3

<211> 1494

<212> DNA

<213> Beta vulgaris

<400> 3

atggatcatg caactttggc aatgatcctt gcaatctggt tcatctcttt tcacttcatc 60

aaactgctgt tctcccaaca gactaccaaa ctgcttccac ctggtccaaa gccattgcca 120

atcatcggta acatcttgga agtcaccacc accaccactg atgatgtcct tgatgttctt 180

cttcagctgt tcaagcagaa cgaactgact atgggttctg tcaccaccat tgttgtctcc 240

tctgctgatg tcgctaaaga gatgttcttg aagaaggatc atccactgtc caaccgtacc 300

attccaaact ctgttactgc tggtgatcat cacaaactga ctatgtcttg gttgcctgtc 360

tctcctaagt ggcgtaactt ccgtaagatc actgctgtcc acttgctgtc tcctcaacgt 420

cttgatgctt gccaaacctt ccgtcatgct aaggtccaac aactgtacga gtacgtccaa 480

gagtgtgcac agaaaggtca agctgttgac atcggtaaag ctgcattcac tacttctctg 540

aatctgttgt ccaaactgtt cttctccgtc gaactggcac accacaagtc tcatacttct 600

caagagttca aggaactgat ctggaacatc atggaagaca ttggtaagcc taactacgct 660

gactatcttc caatcttggg ttgtgttgat ccatctggta ttcgtcgtcg tttggcatgt 720

tccttcgata agctgatcgc tgtcttccag ggtatcatct gtgaacgtct tgcacctgac 780

tcttccacca ccggtaagaa gcctcatcgt tccttcgcta acctggctaa gattcatggt 840

ccattgatct ccttgcgtct tggtgagatc aaccatctgc ttgttgacat cttcgatgct 900

ggtactgaca ccacttcttc caccttcgaa tgggtcatga ccgagttgat ccgtaaccct 960

gagatgatgg agaaggcaca agaagagatc aagcaagtct tgggtaagga caaacagatc 1020

caggagtctg acatcatcaa cctgccatac ttgcaagcta tcatcaaaga aaccctgcgt 1080

cttcatccac caactgtctt cctgttgcca cgtaaagctg acactgatgt tgaactgtac 1140

ggttacattg tccctaaaga tgcacagatc ctggtcaact tgtgggcaat cggtcgtgat 1200

cctaacgcat ggcagaacgc tgacatcttc tctcctgaac gtttcatcgg ttgtgagatc 1260

gatgtcaaag gtcgtgactt cggtctgttg ccattcggtg ctggtcgtcg tatctgtcct 1320

ggtatgaatc tggctattcg tatgttgacc ttgatgctgg ctaccttgct tcagttcttc 1380

aactggaagc tggaaggtga catctctcca aaggacttgg acatggatga gaagttcggt 1440

atcgcattgc agaagaccaa gccattgaaa ctgattccaa tcccacgtta ctaa 1494

<210> 4

<211> 537

<212> DNA

<213> Cauliflower virus

<400> 4

atggagtcaa agattcaaat agaggaccta acagaactcg ccgtaaagac tggcgaacag 60

ttcatacaga gtctcttacg actcaatgac aagaagaaaa tcttcgtcaa catggtggag 120

cacgacacac ttgtctactc caaaaatatc aaagatacag tctcagaaga ccaaagggca 180

attgagactt ttcaacaaag ggtaatatcc ggaaacctcc tcggattcca ttgcccagct 240

atctgtcact ttattgtgaa gatagtggaa aaggaaggtg gctcctacaa atgccatcat 300

tgcgataaag gaaaggccat cgttgaagat gcctctgccg acagtggtcc caaagatgga 360

cccccaccca cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa 420

gtggattgat gtgatatctc cactgacgta agggatgacg cacaatccca ctatccttcg 480

caagaccctt cctctatata aggaagttca tttcatttgg agagaacacg ggggact 537

<210> 5

<211> 865

<212> DNA

<213> Agrobacterium

<400> 5

ccaacatcga actacccctc acgcatgcat tcatcaatat tattcatgcg gggaaaggca 60

agattaatcc aactggcaaa tcatccagcg tgattggtaa cttcagttcc agcgacttga 120

ttcgttttgg tgctacccac gttttcaata aggacgagat ggtggagtaa agaaggagtg 180

cgtcgaagca gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc 240

cggtcttgcg atgattatca tataatttct gttgaattac gttaagcatg taataattaa 300

catgtaatgc atgacgttat ttatgagatg ggttttttatg attagagtcc cgcaattata 360

catttaatac gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc 420

ggtgtcatct atgttactag atcgatcaaa cttcggtact gtgtaatgac gatgagcaat 480

cgagaggctg actaacaaaa ggtatgccca aaaacaacct ctccaaactg tttcgaattg 540

gaagttttctg ctcatgccga caggcataac ttagatattc gcgggctatt cccactaatt 600

cgtcctgctg gtttgcgcca agataaatca gtgcatctcc ttacaagttc ctctgtcttg 660

tgaaatgaac tgctgactgc cccccaagaa agcctcctca tctcccagtt ggcggcggct 720

gatacaccat cgaaaaccca cgtccgaaca cttgatacat gtgcctgaga aataggccta 780

cgtccaagag caagtccttt ctgtgctcgt cggaaattcc tctcctgtca gacggtcgtg 840

cgcatgtctt gcgttgatga agctt 865

Claims (10)

What is claimed is: 1. A gene combination expressing betalain in rice, comprising melo gene, BvDODA1 gene and BvCYP76AD1 gene. 2. according to the described gene combination that expresses and produces betalain in rice according to claim 1, it is characterized in that, according to the codon preference of rice, described melo gene is optimized as meloS gene, and its nucleotide sequence is such as SEQ ID NO .1; the BvDODA1 gene is optimized to be the BvDODA1S gene, and its nucleotide sequence is shown in SEQ ID NO.2; the BvCYP76AD1 gene is optimized to be the BvCYP76AD1S gene, and its nucleotide sequence is shown in SEQ ID NO.3. 3. A multigene plant transformation vector, comprising a plant expression vector, and a gene expression cassette of meloS gene, BvDODA1S gene and BvCYP76AD1S gene fused with the 35S promoter of cauliflower mosaic virus and the NOS terminator of Agrobacterium rhizogenes respectively. 4. The multigene plant transformation vector according to claim 3, wherein the plant expression vector is pCAMBIA1301. 5. The application in rice of expressing the combination of genes for producing betalain in rice according to claim 1 or 2. 6. A method for obtaining a transgenic rice containing betalain, comprising the following steps: 1) According to the codon preference of rice, the sequences of the three genes melo, BvDODA1 and BvCYP76AD1 were optimized, and the three genes after sequence optimization were respectively fused with the 35S promoter of cauliflower mosaic virus and the NOS terminator of Agrobacterium rhizogenes, Construct gene expression cassettes respectively; 2) connecting the three gene expression cassettes in step 1) into a plant expression vector to obtain a multigene plant transformation vector containing the three gene expression cassettes of melo, BvDODA1 and BvCYP76AD1; 3) The multi-gene plant transformation vector described in step 2) is transformed into Agrobacterium, and then transformed into rice to obtain transgenic rice rich in betalain. 7. the method for obtaining the transgenic rice containing betalain according to claim 6, is characterized in that, in step 1), described melo gene is optimized as meloS gene, and its nucleotide sequence is as shown in SEQ ID NO.1 The BvDODA1 gene is optimized as a BvDODA1S gene, and its nucleotide sequence is shown in SEQ ID NO.2; the BvCYP76AD1 gene is optimized as a BvCYP76AD1S gene, and its nucleotide sequence is shown in SEQ ID NO.3. 8. the method for obtaining the transgenic rice containing betalain according to claim 6 or 7, it is characterized in that, in step 1), the nucleotide sequence of the 35S promoter of described cauliflower mosaic virus is respectively as SEQ ID NO .4; the NOS terminator sequence of the Agrobacterium rhizogenes is shown in SEQ ID NO.5. 9. The method for obtaining betalain-containing transgenic rice according to claim 6, wherein in step 2), the plant expression vector is pCAMBIA1301. 10 . The method for obtaining betalain-containing transgenic rice according to claim 6 , wherein in step 3), the variety of the rice is Zhonghua 11. 11 .