Gene combination and method for biosynthesizing crocin

Abstract

The invention belongs to the technical field of crop genetic engineering, and provides a gene combination for biosynthesis of crocin, which is characterized in that a tHMG1S gene, an HpPSY1S gene, a PscrtZS gene, a PacrtIS gene, a CsCCD2S gene, an NcALD8S gene, a GjUGT74F8S gene and a UGT94E13S gene are optimized according to rice codon preference, and the eight genes are fused with a rice endosperm specific phenotypic promoter NRP 33, an RbcS plastid transport peptide sequence and a Nos terminator, so as to obtain the crocin biosynthesis gene combination. The method comprises the following steps: constructing gene expression cassettes, connecting all the expression cassettes in series to construct a polygene plant vector, transforming the polygene plant vector to rice to obtain transgenic rice rich in crocin, and determining that the content of crocin I in the dry weight of rice seeds is 2.24 mu g / g, the content of crocin II in the dry weight of rice seeds is 1.65 mu g / g, and the content of crocin III in the dry weight of rice seeds is 5.36 mu g / g. The rice seeds can be used as a molecular farm for producing crocin.

Description

Technical Field

[0001] The present invention belongs to the technical field of crop genetic engineering, and particularly relates to a gene combination for biosynthesis of crocins and a method for producing crocins in a molecular farm. Background Art

[0002] Crocins are the main active ingredients of the precious traditional Chinese medicine saffron. They belong to water-soluble glycosylated carotenoid compounds. Modern pharmacological studies have shown that crocins have properties such as antioxidant, anti-inflammatory, lipid-lowering, blood sugar-lowering, neuroprotective, and anti-tumor effects. Clinical trials have shown that crocins have a positive effect in the treatment of depression and Alzheimer's disease. Due to these excellent pharmacological functions, the market demand for crocins is increasing continuously. However, due to resource limitations, it is far from meeting the market demand.

[0003] Currently, crocins are mainly obtained by extraction from expensive and rare saffron, but its manual harvesting cost greatly limits its large-scale production and wide application. Moreover, crocins are difficult to obtain by chemical synthesis due to their complex structure and abundant chiral centers. Therefore, it is necessary to seek sustainable alternative methods for obtaining crocins. Summary of the Invention

[0004] The purpose of the present invention is to provide a gene combination and method for biosynthesis of crocins. This gene combination can be used to construct a multi-gene plant vector and transform it into plants to obtain transgenic plants rich in crocins.

[0005] To achieve the above purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a gene combination for biosynthesis of crocins, which includes tHMG1S gene, HpPSY1S gene, PacrtIS gene, PscrtZS gene, CsCCD2S gene, NcALD8S gene, GjUGT74F8S gene and UGT94E13S gene, and their nucleotide sequences are respectively shown as SEQ ID No.1 - 8.

[0007] Among them, the tHMG1S gene, HpPSY1S gene, PacrtIS gene, PscrtZS gene, CsCCD2S gene, NcALD8S gene, GjUGT74F8S gene and UGT94E13S gene are respectively optimized from the following genes according to the codon preference of rice: tHMG1 gene, HpPSY1 gene, PacrtI gene, PscrtZ gene, CsCCD2 gene, NcALD8 gene, GjUGT74F8 gene and UGT94E13 gene.

[0008] The present invention also provides a multi-gene plant transformation vector, which includes a plant expression vector and gene expression cassettes of the aforementioned gene combinations. The gene expression cassettes are respectively fused with an NRP 33 promoter and a Nos terminator based on the tHMG1S gene, HpPSY1S gene, PscrtZS gene, PacrtIS gene, CsCCD2S gene, NcALD8S gene, GjUGT74F8S gene, and UGT94E13S gene.

[0009] As an embodiment, the nucleotide sequence of the NRP 33 promoter in the multi-gene plant transformation vector is as shown in SEQ ID No.9, and the nucleotide sequence of the Nos terminator is as shown in SEQ ID No.10.

[0010] As an embodiment, the multi-gene plant transformation vector further includes a gene expression cassette of a 35S promoter of cauliflower mosaic virus and a Nos terminator fused with a marker gene SbG2S, and the nucleotide sequence of the marker gene SbG2S is as shown in SEQ ID No.11.

[0011] Preferably, the PacrtIS gene, PscrtZS gene, CsCCD2S gene, NcALD8S gene, GjUGT74F8S gene, and UGT94E13S gene are also respectively connected to the Arabidopsis thaliana RbcS plastid transit peptide coding sequence. The Arabidopsis thaliana RbcS plastid transit peptide coding sequence is as shown in SEQ ID No.12.

[0012] As an embodiment, the plant expression vector is a plant binary vector pYP694 obtained by introducing a multiple cloning site with pCamBIA-1301 as the backbone.

[0013] The present invention provides a method for biosynthesizing crocin, that is, the aforementioned multi-gene plant transformation vector is transformed into a plant, and crocin is expressed in the plant.

[0014] The present invention also provides a method for biosynthesizing crocin, and the specific operation steps are as follows:

[0015] 1) The expression cassettes of eight genes, namely tHMG1S, HpPSY1S, PscrtZS, PacrtIS, CsCCD2S, NcALD8S, GjUGT74F8S, and UGT94E13S, and the SbG2S gene expression cassette are successively cloned into the plant expression vector pYP694 to obtain a recombinant plasmid vector pYR48;

[0016] 2) The recombinant plasmid vector pYR48 is transferred into a host bacterium;

[0017] 3) The host bacterium infects the callus, and the resistant callus is screened out;

[0018] 4) The resistant callus is subjected to differentiation culture to obtain a transgenic plant expressing crocin.

[0019] As an embodiment, the plant is a gramineous plant or a leguminous plant.

[0020] Preferably, the host bacterium in step 2) is Agrobacterium tumefaciens EHA105.

[0021] Preferably, the callus in step 3) is selected from gramineous plants or leguminous plants, including rice, wheat, corn, sorghum, millet and soybean.

[0022] Beneficial effects: In the present invention, the tHMG1 gene, HpPSY1 gene, HpcrtZ gene, PacrtI gene, CsCCD2 gene, NcALD8 gene, GjUGT74F8 gene and UGT94E13 gene are combined and expressed in plants after optimization to biosynthesize crocin. The obtained transgenic rice has a crocin I content of 2.24 μg / g, a crocin II content of 1.65 μg / g, and a crocin III content of 5.36 μg / g in the endosperm. This transgenic rice germplasm can be used as a molecular farm for producing crocin raw materials, and can also be used as a functional rice for promoting health. Brief Description of the Drawings

[0023] Figure 1 Schematic diagram of the vector construction for the biosynthesis of crocin in rice endosperm.

[0024] Figure 2 Phenotype diagram of rice green callus.

[0025] Figure 3 PCR detection of the expression of crocin biosynthesis genes in rice. M: marker; CK: wild-type rice; EH887-2, 3, 4: namely 887-2, 887-3, 887-4, different lines of rice created in the present invention.

[0026] Figure 4 Schematic diagram of rice seed color. The first three rows: the color of the rice seeds created in the present invention; the last row: the color of wild-type rice seeds.

[0027] Figure 5 LC / MS detection of the crocin content in rice seeds. A is the mass spectrum of the crocin standard product. From left to right, they are crocin I, crocin II and crocin III respectively; B is the mass spectrum of the crocin in the rice seeds created in the present invention. From left to right, they are crocin I, crocin II, crocin III respectively. Detailed Embodiments

[0028] The present invention provides a gene combination for biosynthesizing crocin, and the gene combination includes the tHMG1S gene, the HpPSY1S gene, the PacrtIS gene, the PscrtZS gene, the CsCCD2S gene, the NcALD8S gene, the GjUGT74F8S gene and the UGT94E13S gene.

[0029] The above genes are respectively optimized from the tHMG1 gene, the HpPSY1 gene, the PscrtZ gene, the PacrtI gene, the CsCCD2 gene, the NcALD8 gene, the GjUGT74F8 gene and the UGT94E13 gene according to the codon preference of rice. The optimization principles are as follows: First, optimize the gene codons, improve the gene translation efficiency according to the rice codon preference. Second, eliminate the recognition sites of common restriction endonucleases inside the gene to facilitate the construction of expression cassettes. Third, eliminate reverse repeat sequences, stem-loop structures and transcription termination signals to make the GC / AT inside the gene balanced and improve the stability of RNA, and the ratio of GC / AT inside the gene should reach 1:1 as much as possible. Fourth, make the gene-encoded protein conform to the N-terminal principle to improve the stability of the translated protein. Fifth, optimize the free energy of the mRNA secondary structure to improve the gene expression efficiency. When optimizing the secondary structure, avoid 6 or more consecutive A+T sequences and avoid 5 or more G+C sequences; avoid CG and TA double oligonucleotides at positions 2 and 3; at the same time, make the free energy ΔG at the 5' end of the gene increase to more than 5.4 Kcal and make the free energy at the 3' end decrease to less than 10.8 Kcal. Through the above optimization principles, the optimized genes can be highly expressed in the rice seed endosperm, and finally eight optimized genes are obtained, namely tHMG1S, HpPSY1S, PacrtIS, PscrtZS, CsCCD2S, NcALD8S, GjUGT74F8S, UGT94E13S, and their nucleotide sequences are shown in SEQ ID No.1-8.

[0030] The present invention also provides a multi-gene plant transformation vector, and the multi-gene plant transformation vector includes a plant expression vector and gene expression cassettes containing the tHMG1S gene, HpPSY1S gene, PscrtZS gene, PacrtIS gene, CsCCD2S gene, NcALD8S gene, GjUGT74F8S gene, and UGT94E13S gene. The gene expression cassettes are formed by respectively connecting the rice endosperm-specific phenotype promoter NRP 33 promoter and the Nos terminator at both ends of the above eight genes, and fusing them to form an expression unit. Among them, the nucleotide sequence of the NRP 33 promoter is as shown in SEQ ID No.9, and the nucleotide sequence of the Nos terminator is as shown in SEQ ID No.10. Among them, the PacrtIS gene, PscrtZS gene, CsCCD2S gene, NcALD8S gene, GjUGT74F8S gene, and UGT94E13S gene are also respectively connected to the Arabidopsis thaliana RbcS plastid transit peptide coding sequence. The Arabidopsis thaliana RbcS plastid transit peptide coding sequence is as shown in SEQ ID No.12. The multi-gene plant transformation vector of the present invention also contains a gene expression cassette of a marker gene SbG2S that can form green callus and is fused with the 35S promoter and Nos terminator of cauliflower mosaic virus, and the nucleotide sequence of the gene of the marker gene SbG2S is as shown in SEQ ID No.11. The plant expression vector of the present invention can be selected as pCamBIA-1301 or a plant binary vector pYP694 obtained by modifying the vector by introducing a new multiple cloning site with pCamBIA-1301 as the backbone. Preferably, the plant expression vector can be selected as the plant binary vector pYP694. The above eight optimized gene expression units together with the marker gene SbG2S are cloned into the plant expression vector in sequence to obtain a recombinant plasmid vector, and the cloning method refers to Patent CN114592000B.

[0031] The present invention also provides a method for biosynthesizing crocin. The multi-gene plant transformation vector described in the present invention is transformed into a plant, and crocin is expressed in the plant. As an implementation manner, the specific operation steps are as follows: 1) The expression cassettes of the above eight genes, namely tHMG1S, HpPSY1S, PscrtZS, PacrtIS, CsCCD2S, NcALD8S, GjUGT74F8S, and UGT94E13S, and the SbG2S gene expression cassette are successively cloned into the plant expression vector pYP694 to obtain the recombinant plasmid vector pYR48; 2) The recombinant plasmid vector pYR48 is transferred into a host bacterium; 3) The host bacterium infects callus, and after screening out the resistant callus; 4) The resistant callus is subjected to differentiation culture to obtain a transgenic plant expressing crocin. As an implementation manner, the host bacterium in step 2) is Agrobacterium tumefaciens EHA105. As an implementation manner, the callus in step 3) is selected from gramineous plants including rice, wheat, corn, sorghum, millet, etc.; it can also be selected from leguminous plants including soybeans, etc. Preferably, the callus is rice embryogenic callus. The method for detecting the crocin content in rice in the present invention refers to the reference Mi, Jianing et al. "A rapid LC-MS method for qualitative and quantitative profiling of plant apocarotenoids." Analytica chimica acta vol. 1035 (2018): 87-95. doi: 10.1016 / j.aca.2018.07.002.

[0032] In the present invention, unless otherwise specified, all raw material components are commercially available products well-known to those skilled in the art. The present invention relates to molecular biology experiments. If not otherwise specified, they are all referenced from the book "Molecular Cloning" (written by J. Sambrook, E.F. Fritsch, and T. Maniatis, 1994, Science Press). The rice seeds (Nipponbare) used are preserved by the Agricultural Synthetic Biology Research Center of the Biotechnology Research Institute of the Shanghai Academy of Agricultural Sciences. The reagents used in the present invention, if not otherwise stated, are all purchased from Sangon Biotech (Shanghai) Co., Ltd. or Shanghai National Pharmaceutical Group Co., Ltd. The culture media used in the present invention, if not otherwise specified, can refer to Patent CN 114592000A.

[0033] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

[0034] Example 1

[0035] Structural optimization and chemical synthesis of 8 genes in the crocin synthesis pathway

[0036] The 8 genes before optimized synthesis were: tHMG1 gene (GenBank No: M22002.1, 1771..3285), HpPSY1 gene (GenBank No: DQ057355.1), PacrtI gene (GenBank No: D90087.2), PscrtZ gene (GenBank No: KP866868.1), CsCCD2 gene (GenBank No: KJ541749.1), NcALD8 gene (GenBank No: XM_011396597.1), GjUGT74F8 gene (GenBank No: MN944054.1), UGT94E13 gene (GenBank No: KY631935.1).

[0037] The 8 genes after optimized synthesis were named as: tHMG1S gene, HpPSY1S gene, PacrtIS gene, PscrtZS gene, CsCCD2S gene, NcALD8S gene, GjUGT74F8S gene and UGT94E13S gene; their sequence numbers corresponded to SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 respectively.

[0038] Example 2

[0039] Construct gene expression cassette

[0040] Using the sequence of Gene Bank No: D63901.1 as a template, the rice (Oryza sativa) endosperm-specific expression promoter NRP33 promoter with the DNA sequence shown in SEQ ID No.9 was synthesized.

[0041] Using the sequence of GenBank No: AB830573.1 as a template, the Agrobacterium tumefaciens Nos terminator with the DNA sequence shown in SEQ ID No.10 was synthesized.

[0042] Referring to the sequence of the Arabidopsis thaliana RbcS small subunit gene (Gene Bank No: NM_105379.4), the sequence encoding the plastid transit peptide RbcS TP (SEQ ID No.11) was optimized and synthesized.

[0043] Using the sequence of GenBank No: XM_021456623.1 as a template, the SbG2S gene that can promote chloroplast development was optimized and synthesized to obtain the DNA sequence shown in SEQ ID No. 12.

[0044] The fusion of the gene with plastid transit peptide, promoter, and terminator was carried out using the improved overlap extension PCR technique. The specific references for this improved overlap extension PCR technique are: (Rihe Peng et al., Applied Microbiology Biotechnology. 2006, 73: 234 - 240 and ZL 202011411342.4). The sequences of the NRP33 promoter, tHMG1S gene, and Nos terminator were fused in this order to construct the NRP33tHMG1S gene expression cassette; the sequences of the NRP33 promoter, HpPSY1S gene, and Nos terminator were fused in this order to construct the NRP33HpPSY1S gene expression cassette; the sequences of the NRP33 promoter, RbcS, PacrtIS gene, and Nos terminator were fused in this order to construct the NRP33RPacrtIS gene expression cassette; the sequences of the NRP33 promoter, RbcS, PscrtZS gene, and Nos terminator were fused in this order to construct the NRP33RPscrtZS gene expression cassette; the sequences of the NRP33 promoter, RbcS, CsCCD2S gene, and Nos terminator were fused in this order to construct the NRP33R3CsCCD2S gene expression cassette; the sequences of the NRP33 promoter, RbcS, NcALD8S gene, and Nos terminator were fused in this order to construct the NRP33RNcALD8S gene expression cassette; the sequences of the NRP33 promoter, RbcS, GjUGT74F8S gene, and Nos terminator were fused in this order to construct the NRP33RGjUGT74F8S gene expression cassette; the sequences of the NRP33 promoter, RbcS, UGT94E13S gene, and Nos terminator were fused in this order to construct the NRP33RUGT94E13S gene expression cassette; the sequences of the 35S promoter, SbG2S gene, and Nos terminator were fused in this order to construct the 35S SbG2S gene expression cassette.

[0045] PCR amplification was performed using Phanta Max Super-Fidelity DNA Polymerase from Vazyme Biotech Co., Ltd, which is suitable for high-fidelity amplification of long genes. The PCR amplification program was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 45 s, annealing at 56 - 72°C for 45 s, extension at 72°C for 5 - 20 min (depending on the fragment length), with 25 - 35 cycles of amplification; final extension at 72°C for 10 min. The complete sequence was determined by nucleotide full-sequence analysis by Sangon Biotech (Shanghai) Co., Ltd.

[0046] Example 3

[0047] Construction of plant expression vector

[0048] The 9 correctly sequenced gene expression cassettes constructed in Example 2 were ligated into the pYP694 vector according to the method of reference patent CN114592000B to obtain the recombinant plant expression vector pYR48 containing a total of 9 genes for the crocin biosynthesis pathway and marker genes, as Figure 1 shown, where the pYP694 vector refers to Tian YS et al. Enhancing carotenoid biosynthesis in rice endosperm by metabolic engineering. Plant Biotechnol J. 2019 May;17(5):849 - 851.

[0049] Example 4

[0050] Rice transformation

[0051] 1) Preparation of Agrobacterium

[0052] Single colonies of Agrobacterium tumefaciens EHA105 were picked and inoculated into 5 mL of LB liquid medium (rifampicin 50 μg / mL, chloramphenicol 100 μg / mL), and cultured at 28°C and 250 rpm for 20 h. 1 mL of the bacterial solution was transferred into 20 mL of LB liquid medium (rifampicin 50 μg / mL, chloramphenicol 100 μg / mL), and cultured at 28°C and 250 rpm for about 12 h, and the OD 600 was measured ≈ 1.5. The cells were collected by centrifugation at 8000 rpm, 4°C for 10 min, and resuspended in Agrobacterium transformation infiltration solution (5 wt% sucrose, 0.05 wt% Silwet L - 77) and diluted to OD 600≈0.8. Mix 40 μl of the bacterial solution and 2 μl of the recombinant plant expression vector pYR48 in an electroporation cuvette with a diameter of 0.2 cm, and place it on ice for 1 min. Adjust the electroporation parameters to 25 μF, 2.5 kV / cm, 400 Ω, and the discharge time is 4 - 5 ms. After electroporation, immediately add 1 ml of LB culture medium to the cells, culture at 28 °C for 1 h, and spread on a YEB plate (rifampicin 50 μg / mL, kanamycin 100 μg / mL). Using the above transformation procedure, transfer the expression vector pYR48 into Agrobacterium tumefaciens EHA105 to obtain Agrobacterium tumefaciens EHA105(pYR48).

[0053] 2) Agrobacterium infection and co - culture with rice callus

[0054] Immerse the rice embryogenic callus in the prepared Agrobacterium suspension. After 30 min of infection, then blot the excess bacterial solution from the callus on sterile filter paper, and transfer it to the co - culture medium N6 (containing 2,4 - D 1.5 mg / L), and culture it at 28 °C in the dark for 3 - 4 d.

[0055] 3) Screening of resistant callus

[0056] Transfer the co - cultured callus out, rinse it 3 - 4 times with sterile water, then blot the excess water with sterile filter paper, and transfer the callus to the selection medium N6 (containing 2,4 - D 1.5 mg / L, hygromycin 25 mg / L), and culture it in the dark at 28 °C, sub - culture once every two weeks.

[0057] 4) Plant regeneration

[0058] After 2 - 3 generations of screening, select the vigorously growing green callus, as Figure 2 shown, transfer it to the pre - differentiation medium MS (containing 6 - BA 1 mg / L, KT 1 mg / L, IAA 0.05 mg / L, hygromycin 25 mg / L) for pre - differentiation treatment: after 5 - 7 days of dark culture, then transfer the resistant callus to the differentiation medium MS (containing 6 - BA 4 mg / L, KT 4 mg / L, IAA 0.2 g / L, hygromycin 25 mg / L), and carry out differentiation under the conditions of 16 h light, 8 h dark, and 28 °C every day. Cut off the original roots of the regenerated seedlings, root and strengthen the seedlings on the rooting medium MS, and then transfer them to a pot in the artificial climate chamber to obtain transgenic rice. Keep the humidity in the first few days, and carry out subsequent cultivation management according to the conventional method.

[0059] Example 5

[0060] Verification of transgenic rice

[0061] For the T obtained in Example 4 0Seeds harvested from the first-generation rice plants were used to extract genomic DNA by the SDS method as a template, and the exogenous genes were detected by PCR. The primer sequences designed are as follows:

[0062] tHMG1S-F (SEQ ID No.15): 5’-GGTACTAACGCACGTCAACTG;

[0063] tHMG1S-R (SEQ ID No.16): 5’-GGATTTGATGCAGGTGACGGA.

[0064] HpPSY1S-F (SEQ ID No.17): 5’-CAGATCGCTAGAGCTAGACAGTG;

[0065] HpPSY1S-R (SEQ ID No.18): 5’-ACCTCTGTGCTGTGGAAGGAGTG.

[0066] PscrtZS-F (SEQ ID No.19): 5’-TGGTGCATGATGGTCTGGTTCATC;

[0067] PscrtZS-R (SEQ ID No.20): 5’-CTTGCCAGATGAGGAGGTATCCAC.

[0068] PacrtIS-F (SEQ ID No.21): 5’-TTCGACTTCCGTGATCAGCTG;

[0069] PacrtIS-R (SEQ ID No.22): 5’-GATCAGGTCCTCCAGCATCAG.

[0070] CsCCD2S-F (SEQ ID No.23): 5’-AGGTACGGCTCCGAGGCCATCTTC;

[0071] CsCCD2S-R (SEQ ID No.24): 5’-CTCAGCCTGGTGCTTCTGCAACTC.

[0072] NcALD8S-F (SEQ ID No.25): 5’-GTTCGTTACATGCCATACGACTGG;

[0073] NcALD8S-R (SEQ ID No.26): 5’-ACGACGTGCCTTCATGTAGGCAG.

[0074] GjUGT74F8S-F (SEQ ID No.27): 5’-TGGCTGCTCCTCAATGGACTGATC;

[0075] GjUGT74F8S-R (SEQ ID No.28): 5’-AGCCAACTCAGCCACGAACTCATC. UGT94E13S-F (SEQ ID No.29): 5’-TGAACGCTAGATTGATCGAGGCTG;

[0076] UGT94E13S-R (SEQ ID No.30): 5’-CAGCATCCACCGTTGTTCTTCCTG.

[0077] Amplification procedure: 94°C for 30 s, 54°C for 30 s, 72°C for 30 s, for a total of 45 cycles, and finally an extension at 72°C for 10 min. The results are as Figure 3 shown. The wild type (CK) could not amplify the exogenous gene, while the transgenic lines could amplify the above 8 genes, indicating that the exogenous genes were completely integrated into the rice genome.

[0078] Example 6

[0079] Determination of crocin content

[0080] Plant the rice plants that were detected as positive in Example 5 in the farmland according to the following steps:

[0081] 1) Select suitable land, plow the land, apply fertilizers, irrigate, etc. to ensure that the soil fertility and moisture meet the growth needs of rice.

[0082] 2) Sow the selected rice seeds in the field. There are two sowing methods: direct seeding and seedling raising. Direct seeding is to directly scatter the seeds in the field, and seedling raising is to first plant in the seedling nursery and then transplant the seedlings to the field when they grow to a certain height.

[0083] 3) After sowing, the field needs to be regularly managed, including weeding, irrigation, fertilization, pest and disease control, etc.

[0084] 4) When the rice is mature, harvest it. The harvest can be done manually or by machine.

[0085] 5) Thresh the harvested paddy. It can be clearly seen that the color of the seeds is yellow (see Figure 4 ), indicating that crocin has been biosynthesized in the rice seeds.

[0086] 6) Repeat steps 2)-5) for the seeds with yellow color until homozygous seeds are obtained

[0087] 7) Shell the homozygous seeds of different strains, weigh 1 - 1.5 g, crush them, take about 0.5 g of the powder, add 1 mL of 50% methanol aqueous solution as the extract, add steel beads for homogenization, extract ultrasonically for 60 min, centrifuge at 8000 g for 10 min, take the supernatant, filter through a syringe filter and then perform LC / MS qualitative and quantitative analysis. The analysis method refers to the reference (J. Mi et al. (2018) Analytica Chimica Acta 1035, 87 - 95.). The results of the seeds of the rice strain with the highest crocin content are as follows: the content of crocin I is 2.24 μg / g, the content of crocin II is 1.65 μg / g, and the content of crocin III is 5.36 μg / g (dry weight), as Figure 5 shown.

[0088] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A gene combination for biosynthesis of crocin, characterized in that: The gene combination includes tHMG1S gene, HpPSY1S gene, PacrtIS gene, PscrtZS gene, CsCCD2S gene, NcALD8S gene, GjUGT74F8S gene and UGT94E13S gene, and the nucleotide sequences thereof are shown in SEQ ID No. 1 to 8 respectively.

2. The gene combination according to claim 1, characterized in that: The tHMG1S gene, HpPSY1S gene, PacrtIS gene, PscrtZS gene, CsCCD2S gene, NcALD8S gene, GjUGT74F8S gene and UGT94E13S gene are obtained by optimizing the following genes according to the codon preference of rice: tHMG1 gene, HpPSY1 gene, PacrtI gene, PscrtZ gene, CsCCD2 gene, NcALD8 gene, GjUGT74F8 gene and UGT94E13 gene.

3. A multi-gene plant transformation vector, characterized in that: The multi-gene plant transformation vector comprises a plant expression vector and a gene expression cassette of the gene combination of claim 1 or 2, wherein the gene expression cassette is fused with an NRP 33 promoter and a Nos terminator on the basis of the tHMG1S gene, HpPSY1S gene, PscrtZS gene, PacrtIS gene, CsCCD2S gene, NcALD8S gene, GjUGT74F8S gene and UGT94E13S gene, respectively.

4. The multi-gene plant transformation vector according to claim 3, characterized in that: The nucleotide sequence of the NRP 33 promoter is shown in SEQ ID No.9, and the nucleotide sequence of the Nos terminator is shown in SEQ ID No.

10.

5. The multi-gene plant transformation vector according to claim 1, characterized in that: The multi-gene plant transformation vector also includes a gene expression box of a 35S promoter and a Nos terminator fused with a marker gene SbG2S of cauliflower mosaic virus; the nucleotide sequence of the marker gene SbG2S is shown in SEQ ID No.

11.

6. The multi-gene plant transformation vector according to claim 3, characterized in that: The PacrtIS gene, PscrtZS gene, CsCCD2S gene, NcALD8S gene, GjUGT74F8S gene and UGT94E13S gene are also connected to the Arabidopsis thaliana RbcS plastid transit peptide coding sequence respectively; the Arabidopsis thaliana RbcS plastid transit peptide coding sequence is shown in SEQ ID No.

12.

7. The multi-gene plant transformation vector according to claim 3, characterized in that: The plant expression vector is a plant binary vector pYP694 obtained by introducing a multiple cloning site using pCamBIA-1301 as a skeleton.

8. A method for biosynthesizing crocin, characterized in that: The multi-gene plant transformation vector according to any one of claims 3 to 7 is transformed into a plant to express crocin in the plant.

9. The method for biosynthesizing crocin according to claim 8, characterized in that: The specific steps are as follows: 1) The expression cassettes of eight genes, tHMG1S, HpPSY1S, PscrtZS, PacrtIS, CsCCD2S, NcALD8S, GjUGT74F8S and UGT94E13S, and the SbG2S gene expression cassette were cloned into the plant expression vector pYP694 in sequence to obtain the recombinant plasmid vector pYR48; 2) Transform the recombinant plasmid vector pYR48 into the host bacteria; 3) The host bacteria infects the callus tissue and selects the resistant callus tissue; 4) Differentiating and culturing the resistant callus to obtain transgenic plants expressing crocin.

10. The method for biosynthesizing crocin according to claim 8, characterized in that: The plant is a grass plant or a leguminous plant.

11. The method for biosynthesizing crocin according to claim 9, characterized in that: In step 2), the host bacteria is Agrobacterium tumefaciens EHA105.

12. The method for biosynthesizing crocin according to claim 9, characterized in that: The callus tissue in step 3) is selected from grass plants or leguminous plants, including rice, wheat, corn, sorghum, millet and soybean.

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