A transgenic breeding method for producing crocin in seed endosperm

Abstract

The present invention discloses a transgenic breeding method for producing crocin in seed endosperm. The present invention modifies the genes CrtI, PSY, BHY, OR H , DXS, CsCCD2L, CsUGT74AD1, CsUGT91P3, CsALDH3I1 genes derived from saffron and GjCCD4a, GjUGT94E13, GjUGT74F8, GjALDH2C3 genes derived from gardenia were optimized, and the optimized zeaxanthin precursor synthesis-related genes were combined with saffron-derived genes or gardenia-derived genes to construct plant transformation vectors and transformed into rice, to obtain transgenic rice that can produce crocin in the endosperm of rice seeds, thereby realizing the synthesis of crocin in the endosperm of rice seeds. The product can be eaten directly or used as a raw material for crocin production, which is beneficial to the production of crocin.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to a transgenic breeding method for producing crocin in seed endosperm. Background Art

[0002] Crocin, also known as crocin, is the main active ingredient of the precious Chinese medicinal materials saffron and gardenia. It belongs to the apocarotenoid glycoside compound, including 5 different glycosylation forms (crocin-Ⅰ, crocin-Ⅱ, crocin-Ⅲ, crocin-Ⅳ and crocin-Ⅴ). Studies have shown that crocin has a good therapeutic effect on a variety of central nervous system and cardiovascular system diseases, and also has anti-cancer, anti-inflammatory, antioxidant, liver-protecting and choleretic and anti-diabetic effects. In addition to its medicinal value, crocin has long been used as a spice, dye and food additive, and has broad application value.

[0003] In nature, crocin is mainly extracted from the stigma of saffron, and the triploid saffron itself has a low reproduction rate, and due to the complex process of picking stigma, low yield (1g of dry saffron is composed of the stigma of about 150 saffron flowers) and high labor costs, saffron is expensive and is therefore known as "red gold". In addition, crocin has a complex structure and abundant chiral centers, making it difficult to chemically synthesize. Therefore, it is necessary to provide a relatively simple and efficient method for producing crocin.

[0004] The use of biosynthesis is a new production approach. The applicant team has previously developed a technology for synthesizing specific functional substances based on rice (Oryza sativa L.) seed endosperm as a bioreactor. For example, the Chinese patent with publication number CN105907780A discloses a transgenic breeding method for producing astaxanthin in rice seed endosperm, in which astaxanthin synthesis-related genes are expressed in rice seed endosperm to construct transgenic rice that synthesizes astaxanthin. This technology has the advantages of high yield, low cost and high safety. However, there is currently no report on the synthesis of crocin in rice endosperm.

[0005] Since most enzymes in rice endosperm are lowly expressed or even not expressed, and the synthesis of crocin involves multiple genes. Therefore, how to synthesize crocin from scratch in the endosperm through genetic engineering and synthetic biology methods is a scientific issue that is very worthy of research and exploration. The synthesis of crocin involves multiple genes, which makes it difficult to achieve the goal of producing crocin in plants or microorganisms using genetic engineering methods. In addition, there is great uncertainty as to whether the genetic background of rice seeds is suitable for inserting and expressing crocin synthesis genes and whether crocin can be successfully expressed and produced. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a transgenic breeding method for producing crocin in seed endosperm.

[0007] The first object of the present invention is to provide CrtI, PSY, BHY, OR H The five genes, CsCCD2L, CsUGT74AD1, CsUGT91P3 and CsALDH3I1 from saffron, or the four genes, GjCCD4a, GjUGT94E13, GjUGT74F8 and GjALDH2C3 from Gardenia, were combined for application in transgenic breeding of rice seed endosperm for crocin production.

[0008] The second object of the present invention is to provide a transgenic breeding method for producing crocin in the endosperm of rice seeds.

[0009] The third object of the present invention is to provide a method comprising optimizing CrtI, PSY, BHY, OR H , DXS and the plant transformation vector of the saffron-derived genes CsCCD2L, CsUGT74AD1, CsUGT91P3, and CsALDH3I1.

[0010] The fourth object of the present invention is to provide a method comprising optimizing CrtI, PSY, BHY, OR H , DXS and the plant transformation vector containing the five genes GjCCD4a, GjUGT94E13, GjUGT74F8, and GjALDH2C3 from Gardenia.

[0011] The fifth object of the present invention is to provide the use of the plant transformation vector in constructing transgenic rice capable of producing crocin in seed endosperm.

[0012] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0013] The invention uses zeaxanthin precursor synthesis-related genes CrtI, PSY, BHY, OR H , DXS, CsCCD2L, CsUGT74AD1, CsUGT91P3, CsALDH3I1 genes from saffron and GjCCD4a, GjUGT94E13, GjUGT74F8, GjALDH2C3 genes from gardenia were used as templates, and the above genes were optimized according to the codon preference of rice. The optimized genes related to the synthesis of zeaxanthin precursors were combined with genes from saffron or gardenia to construct plant transformation vectors and transformed into rice. Transgenic rice that can produce crocin in the endosperm of rice seeds was obtained, realizing the de novo synthesis of crocin in rice endosperm.

[0014] Therefore, the present invention claims protection of CrtI, PSY, BHY, OR H The application of five genes, CsCCD2L, CsUGT74AD1, CsUGT91P3 and CsALDH3I1 from saffron, or GjCCD4a, GjUGT94E13, GjUGT74F8 and GjALDH2C3 from gardenia, in transgenic breeding for the production of crocin in rice seed endosperm; the specific method of the application is:

[0015] The sequences of the above genes were optimized according to the rice codon preference, and the optimized sequences of CrtI, PSY, BHY, and OR were constructed respectively. H The gene expression cassettes of five genes, DXS and CsCCD2L, CsUGT74AD1, CsUGT91P3 and CsALDH3I1 from saffron were constructed into plant transformation vectors and transformed into rice to obtain transgenic rice that produces crocin in seed endosperm;

[0016] Or construct the sequence-optimized CrtI, PSY, BHY, OR H The gene expression cassettes of five genes, namely, GjCCD4a, GjUGT94E13, GjUGT74F8, and GjALDH2C3, derived from Gardenia were constructed into plant transformation vectors and transformed into rice to obtain transgenic rice that produces crocin in the seed endosperm.

[0017] The present invention also provides a transgenic breeding method for producing crocin in rice seed endosperm, comprising the following steps:

[0018] S1. Based on the codon preference of rice, CrtI, PSY, BHY, OR H The five genes of , DXS and saffron-derived CsCCD2L, CsUGT74AD1, CsUGT91P3, and CsALDH3I1 were optimized, and the corresponding gene expression cassettes were constructed using the optimized sequences;

[0019] S2. constructing the gene expression cassette described in step S1 into a plant transformation vector;

[0020] S3. Transforming rice with the plant transformation vector described in step S2 to obtain transgenic rice that produces crocin in seed endosperm;

[0021] Or, S1. According to the rice codon preference, CrtI, PSY, BHY, OR HThe five genes of , DXS and GjCCD4a, GjUGT94E13, GjUGT74F8, and GjALDH2C3 from Gardenia were optimized, and the corresponding gene expression cassettes were constructed using the optimized sequences;

[0022] S2. constructing the gene expression cassette described in step S1 into a plant transformation vector;

[0023] S3. Transform rice with the plant transformation vector described in step S2 to obtain transgenic rice that produces crocin in the seed endosperm.

[0024] Specifically, the present invention optimizes the zeaxanthin precursor synthesis-related gene OR according to the rice codon preference. H , DXS genes are denoted as sAtOR H and sAtDXS, and the optimized sequences are shown in SEQ ID NOs: 1-2; the optimized crocin synthesis-related genes CsCCD2L, CsUGT74AD1, CsUGT91P3, and CsALDH3I1 from saffron are respectively recorded as sCsCCD2L, sCsUGT74AD1, sCsUGT91P3, and sCsALDH3I1, and the optimized sequences are shown in SEQ ID NOs: 3-6; the optimized crocin synthesis-related genes GjCCD4a, GjUGT94E13, GjUGT74F8, and GjALDH2C3 from Gardenia are respectively recorded as sGjCCD4a, sGjUGT94E13, sGjUGT74F8, and sGjALDH2C3, and the optimized sequences are shown in SEQ ID NOs: 7-10.

[0025] Specifically, when constructing the CrtI gene or BHY gene expression cassette, a plastid transit peptide is connected between the promoter and the optimized gene sequence.

[0026] Specifically, the plastid transit peptide is TP, and its sequence is shown in SEQ ID NO:11.

[0027] In a specific embodiment of the present invention, in order to reduce the number of assemblies or reduce the size of the vector when constructing a plant transformation vector, the present invention constructs the optimized CsUGT74AD1 and CsUGT91P3 gene expression cassettes on the same donor vector. In addition, the optimized GjUGT94E13 and GjUGT74F8 genes are connected in series with a connecting peptide and constructed in the same gene expression cassette, and then the gene expression cassette is used to construct a plant transformation vector.

[0028] Specifically, the connecting peptide is a 2A peptide, and the 2A peptide encoding sequence F2A is shown in SEQ ID NO:12.

[0029] Specifically, the gene expression box contains the promoter and terminator of the rice endosperm-specific storage protein gene.

[0030] Specifically, the promoters of the rice endosperm-specific storage protein genes include Pens1 to Pens9, and their GenBank Nos are AY427571.1, AY427575.1, EU264107.1, MH748577.1, EU264106.1, CP132236.1, AY427572.1, AY427574.1, and CP141114.1, respectively.

[0031] Specifically, the promoter used in the sEuCrtI gene expression cassette is Pens1 (GenBank No.AY427571.1), and the terminator used is CaMV 35S terminator T35S (SEQ ID NO: 13); the promoter used in the sZmPSY gene expression cassette is Pens2 (GenBank No.AY427575.1), and the terminator used is agropine synthase terminator Tags (SEQ ID NO: 14); the promoter used in the sHpBHY gene expression cassette is Pens3 (GenBank No.EU264107.1), and the terminator used is manopine synthase terminator Tmas (SEQ ID NO: 15); H The promoter used in the gene expression cassette is Pens4 (GenBank No. MH748577.1), and the terminator used is manopine synthase terminator Tmas; the promoter used in the sAtDXS gene expression cassette is Pens5 (GenBank No. EU264106.1), and the terminator used is octopine synthase terminator Tocs (SEQ ID NO: 16).

[0032] Specifically, the promoter used in the sCsCCD2L gene expression cassette is Pens6 (GenBank No. CP132236.1), and the terminator used is nopaline synthase terminator Tnos (SEQ ID NO: 17); the promoter used in the sCsUGT74AD1 gene expression cassette is Pens7 (GenBank No. AY427572.1), and the terminator used is TGluA-2 (SEQ ID NO: 18); the promoter used in the sCsUGT91P3 gene expression cassette is Pens8 (GenBank No. AY427574.1), and the terminator used is TGluA-1 (SEQ ID NO: 19); the promoter used in the sCsALDH3I1 gene expression cassette is Pens9 (GenBank No. CP141114.1), and the terminator used is TGluB4 (SEQ ID NO: 20).

[0033] Specifically, the promoter used in the sGjCCD4a gene expression cassette is Pens6, and the terminator used is nopalinesynthase terminator Tnos; the promoter used in the sGjUGT94E13 and sGjUGT74F8 gene expression cassettes is Pens8, and the terminator used is TGluA-1; the promoter used in the sGjALDH2C3 gene expression cassette is Pens9, and the terminator used is TGluB4.

[0034] Specifically, when the gene expression cassettes of the five genes after sequence optimization and the four genes from saffron were constructed into a plant transformation vector, the order of arrangement of the genes was as follows:

[0035] CrtI-PSY-BHY-OR H -DXS-CsCCD2L-CsUGT74AD1-CsUGT91P3-CsALDH3I1.

[0036] Specifically, when the gene expression cassettes of the five genes after sequence optimization and the four genes from Gardenia jasminoides were used to construct a plant transformation vector, the order of arrangement of the genes was as follows:

[0037] CrtI-PSY-BHY-OR H -DXS-GjCCD4a-GjUGT94E13-GjUGT74F8-GjALDH2C3.

[0038] Specifically, the method for transforming rice in step S3 is the Agrobacterium-mediated method, which includes firstly transferring the plant transformation vector into Agrobacterium, and then transforming the rice callus to obtain transgenic rice.

[0039] Specifically, when constructing the plant transformation vector, the TGSII multi-gene vector system was used.

[0040] The present invention also claims protection for a vector containing a gene shown in any one of SEQ ID NOs: 1 to 10.

[0041] The present invention also claims the optimized CrtI, PSY, BHY, OR H , DXS and the plant transformation vector of the saffron-derived genes CsCCD2L, CsUGT74AD1, CsUGT91P3, and CsALDH3I1.

[0042] Specifically, in the plant transformation vector, the order of arrangement of the genes is as follows:

[0043] CrtI-PSY-BHY-OR H -DXS-CsCCD2L-CsUGT74AD1-CsUGT91P3-CsALDH3I1.

[0044] The present invention also claims the optimized CrtI, PSY, BHY, OR H , DXS and the plant transformation vector containing the five genes GjCCD4a, GjUGT94E13, GjUGT74F8, and GjALDH2C3 from Gardenia.

[0045] Specifically, in the plant transformation vector, the order of arrangement of the genes is as follows:

[0046] CrtI-PSY-BHY-OR H -DXS-GjCCD4a-GjUGT94E13-GjUGT74F8-GjALDH2C3.

[0047] Optionally, the vector backbone used to construct the above-mentioned plant transformation vector is pYLTAC380GW.

[0048] The present invention also claims to protect the use of the plant transformation vector in the breeding of transgenic rice that produces crocin in rice seed endosperm.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] The invention uses zeaxanthin precursor synthesis-related genes CrtI, PSY, BHY, OR H, DXS, CsCCD2L, CsUGT74AD1, CsUGT91P3, CsALDH3I1 genes from saffron, and GjCCD4a, GjUGT94E13, GjUGT74F8, GjALDH2C3 genes from gardenia are used as templates, and the above genes are optimized according to the rice codon preference, and the optimized zeaxanthin precursor synthesis-related genes are respectively combined with saffron-derived genes or gardenia-derived genes to form a plant transformation vector and transform rice, and obtain transgenic rice that can produce crocin in rice seed endosperm. The present invention realizes the synthesis of crocin in rice seed endosperm, and its product can be directly eaten, and can also be used as a raw material for producing crocin, which is beneficial to the production of crocin, and overcomes the existing shortcomings of complex procedures, low yield, and difficulty in chemical synthesis, etc., in extracting crocin using the stigma of saffron.

[0051] The present invention solves the problem of the shortage of traditional sources of crocin, provides a crocin bioreactor, creates a new crop germplasm for producing high crocin content, and also explores the difference in the function of the enzyme systems derived from saffron and gardenia in the heterologous synthesis of crocin, providing new ideas and important basis for the development of more new varieties of functional crocin crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The schematic diagram is a structural diagram of a gene expression cassette constructed using the 11 optimized genes of the present invention on a supply vector.

[0053] Figure 2 It is a schematic diagram of the structure of the plant transformation vector constructed in the process of multi-gene assembly and the enzyme digestion detection results of the related vectors; Figure A is a schematic diagram of the structure of the plant transformation vector pYLTAC380MF-DOBhPC-CsCAU1U2 and the Not I enzyme digestion detection results of the related vectors; Figure B is a schematic diagram of the structure of the plant transformation vector pYLTAC380MF-DOBhPC-GjCAU1U2 and the Not I enzyme digestion detection results of the related vectors; the arrow represents the target gene expression cassette obtained after Not I digestion; HPT in the T-DNA region is the hygromycin resistance gene.

[0054] Figure 3 Figure 2 is a PCR detection diagram of foreign genes in the genomic DNA of T0 transformed rice introduced with plant transformation vectors pYLTAC380MF-DOBhPC-CsCAU1U2 and pYLTAC380MF-DOBhPC-GjCAU1U2; CK +is the plant transformation vector pYLTAC380MF-DOBhPC-CsCAU1U2 or pYLTAC380MF-DOBhPC-GjCAU1U2; WT is the wild-type control genomic DNA.

[0055] Figure 4 The appearance and color of the extract of transgenic rice brown rice containing pYLTAC380MF-DOBhPC for synthesizing zeaxanthin precursor, pYLTAC380MF-DOBhPC-CsCAU1U2 and pYLTAC380MF-DOBhPC-GjCAU1U2 for synthesizing crocin; the wild type WT is the recipient rice variety Huaguang (HG); the yellow logo indicates genes related to maize yellow precursor; the red logo indicates genes derived from saffron, and the purple logo indicates genes derived from gardenia.

[0056] Figure 5 UPLC-MS\MS detection results of crocin in the endosperm of rice seeds transformed with pYLTAC380MF-DOBhPC-CsCAU1U2.

[0057] Figure 6 UPLC-MS\MS detection results of crocin in the endosperm of rice seeds transformed with pYLTAC380MF-DOBhPC-GjCAU1U2.

[0058] Figure 7 Schematic diagram of the pathway for synthesizing crocin in the endosperm of transgenic rice seeds constructed in the present invention; the small black arrows in the figure represent reactions that may exist in the rice endosperm; the dotted arrows represent reactions that are definitely not present in the rice endosperm; the thick red arrows represent reactions catalyzed by enzymes expressed by genes related to carotenoid synthesis; and the dotted red arrows represent reactions catalyzed by enzymes expressed by genes related to crocin synthesis. DETAILED DESCRIPTION

[0059] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0060] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0061] Example 1

[0062] The invention uses zeaxanthin precursor synthesis-related genes CrtI, PSY, BHY, OR H, DXS and saffron-derived CsCCD2L, CsUGT74AD1, CsUGT91P3, and CsALDH3I1 genes were used as templates, and the above genes were optimized according to the codon preference of rice. The optimized lutein precursor synthesis-related genes were combined with saffron-derived genes to construct a plant transformation vector and transformed into rice, realizing the de novo synthesis of crocin in rice endosperm.

[0063] The CrtI gene is the CrtI gene of Erwinia uredovora, and its GenBank No is D90087; the PSY gene is the PSY gene of corn, and its GenBank No is U32636.1; the BHY gene is the BHY gene of Haematococcus pluvialis, and its GenBank No is BD250390.1; H Both the gene and the DXS gene are from Arabidopsis thaliana. H The GenBank No of the gene is NM_125561.4, and the GenBank No of the DXS gene is U27099.1.

[0064] The present invention also establishes a transgenic breeding method for producing crocin in rice seed endosperm, comprising the following steps:

[0065] S1. Based on the codon preference of rice, CrtI, PSY, BHY, OR H The five genes of , DXS and saffron-derived CsCCD2L, CsUGT74AD1, CsUGT91P3, and CsALDH3I1 were optimized, and the corresponding gene expression cassettes were constructed using the optimized sequences;

[0066] The details are as follows:

[0067] 1. Synthesis of nine key gene coding regions:

[0068] Respectively with the CrtI, PSY, BHY, OR H , DXS five genes as templates, according to the rice codon preference, the codon was optimized using the Codon Optimization Tool program, and the optimized gene sequences were synthesized and named sEuCrtI (from the optimized CrtI gene sequence in CN105907780B), sZmPSY (from the optimized PSY gene sequence in CN105907780B), sHpBHY (from the optimized BHY gene sequence in CN105907780B), sAtOR H(as shown in SEQ ID NO: 1) and sAtDXS (as shown in SEQ ID NO: 2) were cloned into a plasmid vector and their sequences were determined by sequencing.

[0069] The CsCCD2L (GenBank No. KP887110), CsUGT74AD1 (GenBank No. MF596166), CsUGT91P3 (GenBank No. MZ190170) and CsALDH3I1 (GenBank No. MF596165) genes from saffron (Crocus sativus L.) were used as templates, and the codons were optimized using the Codon Optimization Tool program according to the codon preference of rice. The optimized gene sequences were synthesized and named sCsCCD2L, sCsUGT74AD1, sCsUGT91P3 and sCsALDH3I1, respectively. Their DNA sequences are shown in SEQ ID NOs: 3 to 6, respectively. They were cloned into plasmid vectors and their sequences were confirmed by sequencing.

[0070] 2. Synthesis of plastid transit peptide coding sequence TP:

[0071] With reference to the sequence of the pea RbcS small subunit gene (GenBank No. X00806), a sequence encoding plastid transit peptide TP (Transit peptide) was synthesized (as shown in SEQ ID NO: 11).

[0072] 3. Construction of gene expression cassette on the supply vector:

[0073] Construction of the supply vector pYL322d1-sEuCrtI containing the sEuCrtI gene expression cassette: The multigene supply vector I (pYL322d1) (Lin et al., 2003; ZL02134869.3) was reversely amplified to obtain the vector backbone fragment a; using rice genomic DNA as a template, the promoter Pens1 (GenBank No.AY427571.1) of about 1.5 kb of rice endosperm-specific storage protein was amplified as fragment b; the sequence TP (SEQ ID NO: 11) of the plastid transit peptide was amplified as fragment c; the sEuCrtI gene was amplified as fragment d; the CaMV 35S terminator T35S (SEQ ID NO: 13) was amplified from the plasmid pCAMBIA1300 (GenBank No. AF234296.1) as fragment e. Each fragment has 25 bp of homologous sequence on both sides. Using the principle of Gibson assembly (Gibson, 2011) and the method of one-step assembly of plasmid vector multiple fragments (Zhu et al., 2014), the supply vector (I) pYL322d1-sEuCrtI plasmid containing sEuCrtI gene expression cassette (such as Figure 1 shown).

[0074] Construction of the supply vector pYL322d2-sZmPSY containing the sZmPSY gene expression cassette: The multigene supply vector II (pYL322d2) (Lin et al., 2003; ZL02134869.3) was reversely amplified to obtain the vector backbone fragment a; using plasmid pSAT7 (GenBank No.DQ5453) as a template, the agropine synthase terminator Tags (SEQ ID NO: 14) was amplified as fragment b; the reverse sZmPSY gene was amplified as fragment c; using rice genomic DNA as a template, the promoter Pens2 of rice endosperm-specific storage protein of about 1 kb (GenBank No.AY427575.1) was amplified as fragment d. Each fragment has 25 bp homologous sequences on both sides. Using the principle of Gibson assembly, the plasmid vector multi-fragment one-step assembly method was used to obtain the supply vector (II) pYL322d2-sZmPSY plasmid containing the sZmPSY gene expression cassette (such as Figure 1 shown).

[0075] Construction of the supply vector pYL322d1-sHpBHY containing the sHpBHY gene expression cassette: reverse amplification of the multigene supply vector I (pYL322d1) to obtain the vector backbone fragment a; using rice genomic DNA as a template, amplify the approximately 2.4 kb promoter Pens3 (GenBank No.EU264107.1) of the rice endosperm-specific storage protein as fragment b; amplify the sequence TP (SEQ ID NO:11) of the plastid transit peptide as fragment c; amplify the sHpBHY gene as fragment d; amplify the manopine synthase terminator Tmas (SEQ ID NO:15) from the plasmid pSAT3 (GenBank No.DQ005465) as fragment e. Each fragment has 25 bp homologous sequences on both sides. Using the principle of Gibson assembly, the plasmid vector multi-fragment one-step assembly method was used to obtain the supply vector (III) pYL322d1-sHpBHY plasmid containing the sHpBHY gene expression cassette (such as Figure 1 shown).

[0076] sAtOR H Gene expression cassette delivery vector pYL322d2-sAtOR H Construction of the multigene supply vector II (pYL322d2) was reversely amplified to obtain the vector backbone fragment a; the manopinesynthase terminator Tmas (SEQ ID NO: 15) was amplified from the plasmid pSAT3 (GenBank No. DQ005465) as fragment b; the reverse sAtOR H The gene was used as fragment c; the promoter Pens4 (GenBank No. MH748577.1) of rice endosperm-specific storage protein of about 2.4 kb was amplified using rice genomic DNA as a template as fragment d. Each fragment was flanked by 25 bp of homologous sequences. Using the principle of Gibson assembly and the method of assembling multiple fragments of plasmid vector in one step, the sAtOR was obtained. H Gene expression cassette delivery vector (IV) pYL322d2-sAtOR H Plasmids (such as Figure 1 shown).

[0077] Construction of the supply vector pYL322d1-sAtDXS containing the sAtDXS gene expression cassette: Reverse amplification of the multi-gene supply vector I (pYL322d1) to obtain the vector backbone fragment a; using rice genomic DNA as a template, amplify the promoter Pens5 (GenBank No.EU264106.1) of about 2.3 kb of rice endosperm-specific storage protein as fragment b; amplify the sAtDXS gene as fragment c; amplify the octopine synthase terminator Tocs (SEQ IDNO: 16) from the plasmid pSAT1 (GenBank No.DQ005461) as fragment d. Each fragment has 25 bp of homologous sequences on both sides. Using the principle of Gibson assembly and the one-step assembly method of the plasmid vector multi-fragment, the supply vector (V) pYL322d1-sAtDXS plasmid containing the sAtDXS gene expression cassette (such as Figure 1 shown).

[0078] Construction of the supply vector pYL322d2-sCsCCD2L containing the sCsCCD2L gene expression cassette: Reverse amplification of the multi-gene supply vector II (pYL322d2) to obtain the vector backbone fragment a; using rice genomic DNA as a template, amplify the promoter Pens6 (GenBank No. CP132236.) of about 1.9 kb of rice endosperm-specific storage protein as fragment b; amplify the sCsCCD2L gene as fragment c; amplify the nopaline synthase terminator Tnos (SEQ ID NO: 17) from the plasmid pSAT2 (GenBank No. DQ005463) as fragment d. Each fragment has 25 bp of homologous sequence on both sides. Using the principle of Gibson assembly and the one-step assembly method of the plasmid vector multi-fragment, the supply vector (VI) pYL322d2-sCsCCD2L plasmid (such as Figure 1 shown).

[0079] Construction of the supply vector pYL322d1-sCsUGT74AD1-sCsUGT91P3 containing the sCsUGT74AD1 and sCsUGT91P3 gene expression cassettes: reverse amplification of the multi-gene supply vector II (pYL322d2) to obtain the vector backbone fragment a; using rice genomic DNA as a template, amplifying the promoter Pens7 (GenBank No.AY427572.1) of rice endosperm-specific storage protein of about 0.9 kb as fragment b; amplifying the sCsUGT74AD1 gene as fragment c; using rice genomic DNA as a template, amplifying the terminator TGluA-2 (SEQ ID NO: 18) of rice endosperm-specific storage protein of about 0.8 kb as fragment d. Each fragment has 25bp homologous sequences on both sides. Using the principle of Gibson assembly and the one-step assembly method of multiple fragments of plasmid vectors, the supply vector pYL322d2-sCsUGT74AD1 containing the sCsUGT74AD1 gene expression cassette was obtained; the multi-gene supply vector I was reversely amplified to obtain the vector backbone fragment a; using rice genomic DNA as a template, the promoter Pens8 (GenBank No.AY427574.1) of rice endosperm-specific storage protein with a length of about 1kb was amplified as fragment b; the sCsUGT91P3 gene was amplified as fragment c; using rice genomic DNA as a template, the terminator TGluA-1 (SEQ IDNO: 19) of rice endosperm-specific storage protein with a length of about 0.5kb was amplified as fragment d. Each fragment has 25bp homologous sequences on both sides. Using the principle of Gibson assembly, the plasmid vector multi-fragment one-step assembly method was used to obtain the supply vector pYL322d1-sCsUGT91P3 containing the sCsUGT91P3 gene expression cassette; the vector pYL322d1-sCsUGT91P3 was digested with restriction endonuclease SacI to obtain the vector backbone fragment a; the plasmid pYL322d2-sCsUGT74AD1 was used as a template to amplify the sCsUGT74AD1 gene expression cassette with 25bp homologous sequences on both sides as fragment b. Using the principle of Gibson assembly, the plasmid vector multi-fragment one-step assembly method was used to obtain the supply vector (VII) pYL322d1-sCsUGT74AD1-sCsUGT91P3 plasmid (such as Figure 1 shown).

[0080] Construction of the supply vector pYL322d2-CsALDH3I1 containing the CsALDH3I1 gene expression cassette: Reverse amplification of the multi-gene supply vector II (pYL322d2) to obtain the vector backbone fragment a; using rice genomic DNA as a template, amplify the promoter Pens9 (GenBank No. CP141114.1) of about 0.7 kb of rice endosperm-specific storage protein as fragment b; amplify the CsALDH3I1 gene as fragment c; using rice genomic DNA as a template, amplify the terminator TGluB4 (SEQ ID NO: 20) of about 0.5 kb of rice endosperm-specific storage protein as fragment d. Each fragment has 25 bp of homologous sequence on both sides. Using the principle of Gibson assembly and the one-step assembly method of plasmid vector multi-fragment, the supply vector (VIII) pYL322d2-sCsALDH3I1 plasmid containing the CsALDH3I1 gene expression cassette (such as Figure 1 shown).

[0081] S2. Construct the gene expression cassette described in step S1 into a plant transformation vector

[0082] Contains optimized CrtI, PSY, BHY, OR H The assembly of the plant transformation vector pYLTAC380MF-DOBhPC-CsCAU1U2 for rice endosperm-specific crocin synthesis containing five genes, CsCCD2L, CsUGT74AD1, CsUGT91P3, and CsALDH3I1 from saffron is as follows:

[0083] The plant transformation vector of the present invention is assembled using the multi-gene vector system TGSII. The multi-gene vector system TGSII is composed of a receiving vector based on a transformable artificial chromosome (TAC) and two supply vectors loaded with genes. The Cre / loxP site-specific recombination method is used to alternately assemble different supply vectors with the receiving vector for more than two rounds of gene assembly to construct a plant transformation vector.

[0084] (1) Construction of the pYLTAC380GW-DOBhPC plasmid for rice endosperm-specific synthesis of zeaxanthin precursor:

[0085] Assembly of sEuCrtI gene expression cassette: Mix the supply vector (I) pYL322d1-sEuCrtI plasmid with the receiving vector pYLTAC380GW plasmid, electroporate and transform competent cells of E. coli strain NS3529 expressing Cre enzyme, screen transformants on karatomycin and chloramphenicol double-resistant plates, and use the Cre enzyme endogenously expressed in NS3529 to achieve the supply vector plasmid recombination and supply vector backbone deletion. Wash the mixed colonies on the double-resistant plates and extract the plasmids, then use I-SceI to eliminate the unrecombined plasmids, transform E. coli DH10B without Cre gene, identify on karatomycin plates, and obtain the receiving vector pYLTAC380GW-C plasmid containing the sEuCrtI gene expression cassette.

[0086] Assembly of sZmPSY gene expression cassette: Mix the supply vector (II) pYL322d2-sZmPSY plasmid with the receiving vector pYLTAC380GW-C plasmid containing the sEuCrtI gene expression cassette, electroporate and transform NS3529 competent cells, screen transformants on a double-resistant plate of karatomycin and ampicillin, and use the Cre enzyme endogenously expressed in NS3529 to achieve the supply vector plasmid recombination and supply vector backbone deletion. Wash the mixed colonies on the double-resistant plate and extract the plasmid, then cut the unrecombined plasmid with PI-SceI enzyme, transform Escherichia coli DH10B, identify on a karatomycin plate, and obtain the receiving vector pYLTAC380GW-PC plasmid containing the sEuCrtI+sZmPSY gene expression cassette.

[0087] Assembly of sHpBHY gene expression cassette: The supply vector (III) pYL322d1-sHpBHY plasmid was mixed with the receiving vector pYLTAC380GW-PC plasmid, and the competent cells of the Escherichia coli strain NS3529 expressing the Cre enzyme were transformed by electroporation together. The transformants were screened on karatomycin and chloramphenicol double-resistant plates, and the Cre enzyme endogenously expressed in NS3529 was used to achieve the recombination of the supply vector plasmid and the deletion of its backbone. The mixed colonies on the double-resistant plates were washed and the mixed plasmids were extracted. After the non-recombinant plasmids were eliminated by I-SceI enzyme digestion, DH10B was transformed and identified on karatomycin plates to obtain the receiving vector pYLTAC380GW-BhPC plasmid containing the sEuCrtI+sZmPSY+sHpBHY gene expression cassette.

[0088] sA H Assembly of gene expression cassette: Supply vector (IV) pYL322d2-sAtOR HThe plasmid was mixed with the receiving vector pYLTAC380GW-BhPC plasmid, and electroporated to transform NS3529 competent cells. Transformants were screened on karatomycin and ampicillin double-resistance plates, and the Cre enzyme endogenously expressed in NS3529 was used to achieve the supply vector plasmid recombination and its backbone deletion. The mixed colonies on the double-resistance plates were washed off and the mixed plasmids were extracted. After the unrecombined plasmids were eliminated by PI-SceI enzyme digestion, DH10B was transformed, and the plasmids containing sEuCrtI+sZmPSY+sHpBHY+sAtOR were identified on karatomycin plates. H The recipient vector of the gene expression cassette is the pYLTAC380GW-OBhPC plasmid.

[0089] Assembly of sAtDXS gene expression cassette: The supply vector (V) pYL322d1-sAtDXS plasmid was mixed with the receiving vector pYLTAC380GW-OBhPC plasmid, and the competent cells of the Escherichia coli strain NS3529 expressing the Cre enzyme were transformed by electroporation together. The transformants were screened on karatomycin and chloramphenicol double-resistant plates. The Cre enzyme endogenously expressed in NS3529 was used to achieve the recombination of the supply vector plasmid and the deletion of its backbone. The mixed colonies on the double-resistant plates were washed and the mixed plasmids were extracted. After the unrecombined plasmids were eliminated by I-SceI enzyme digestion, DH10B was transformed, and the plasmids containing sEuCrtI+sZmPSY+sHpBHY+sAtOR were obtained by identification on karatomycin plates. H +pYLTAC380GW-DOBhPC plasmid, the recipient vector of the sAtDXS gene expression cassette.

[0090] (2) Construction of plant transformation vector pYLTAC380MF-DOBhPC-CsCAU1U2 for rice endosperm-specific crocin synthesis:

[0091] The sCsCCD2L, sCsUGT74AD1, sCsUGT91P3 and sCsALDH3I1 gene expression cassettes derived from saffron were superimposed on the plant transformation vector pYLTAC380GW-DOBhPC for synthesizing zeaxanthin precursor to construct pYLTAC380GW-DOBhPC-CsCAU1U2. Then, through the Gateway-BP recombination reaction, the Marker-free element containing the HPT expression cassette was assembled to construct the plant transformation vector pYLTAC380MF-DOBhPC-CsCAU1U2 which can realize the self-deletion of the hygromycin resistance selection marker.

[0092] Assembly of sCsCCD2L gene expression cassette: Mix the supply vector (VI) pYL322d2-sCsCCD2L plasmid with the receiving vector pYLTAC380GW-DOBhPC plasmid containing 5 gene expression cassettes, electroporate and transform NS3529 competent cells, screen transformants on karatomycin and ampicillin double-resistance plates, and use the Cre enzyme endogenously expressed in NS3529 to achieve supply vector plasmid recombination and supply vector backbone deletion. Wash the mixed colonies on the double-resistance plates and extract the plasmid, then cut the unrecombined plasmid with PI-SceI, transform E. coli DH10B, and identify the plasmid containing sEuCrtI+sZmPSY+sHpBHY+sAtOR on the karatomycin plate. H The recipient vector pYLTAC380GW-DOBhPC-CsC plasmid for the +sAtDXS+sCsCCD2L gene expression cassette.

[0093] Assembly of sCsUGT74AD1 and sCsUGT91P3 gene expression cassettes: The supply vector (VII) pYL322d1-sCsUGT74AD1-sCsUGT91P3 plasmid was mixed with the receiving vector pYLTAC380GW-DOBhPC-CsC plasmid containing 6 gene expression cassettes, and the competent cells of the Escherichia coli strain NS3529 expressing the Cre enzyme were transformed by electroporation together. The transformants were screened on karatomycin and chloramphenicol double-resistant plates, and the Cre enzyme endogenously expressed in NS3529 was used to achieve the recombination of the supply vector plasmid and the deletion of its backbone. The mixed colonies on the double-resistant plates were washed and the mixed plasmids were extracted. After the non-recombinant plasmids were eliminated by I-SceI enzyme digestion, DH10B was transformed, and the cells containing sEuCrtI+sZmPSY+sHpBHY+sAtOR were identified on karatomycin plates. H +sAtDXS+

[0094] The receiving vector pYLTAC380GW-DOBhPC-CsCU1U2 plasmid for the sCsCCD2L+sCsUGT74AD1+sCsUGT91P3 gene expression cassette.

[0095] Assembly of sCsALDH3I1 gene expression cassette: Mix the supply vector (VIII) pYL322d2-sCsALDH3I1 plasmid with the receiving vector pYLTAC380GW-DOBhPC-CsCU1U2 plasmid containing 8 gene expression cassettes, electroporate and transform NS3529 competent cells, screen transformants on karatomycin and ampicillin double-resistance plates, and use the Cre enzyme endogenously expressed in NS3529 to achieve supply vector plasmid recombination and supply vector backbone deletion. Wash the mixed colonies on the double-resistance plates and extract the plasmid, then cut the unrecombined plasmid with PI-SceI enzyme, transform Escherichia coli DH10B, screen positive transformants on karatomycin plates, and identify the plasmid containing sEuCrtI+sZmPSY+sHpBHY+sAtOR H The plant transformation vector pYLTAC380GW-DOBhPC-Cs CAU1U2 containing the gene expression cassette of +sAtDXS+sCsCCD2L+sCsUGT74AD1+sCsUGT91P3+sCsALDH3I1 is shown in the schematic diagram of its structure. Figure 2 shown.

[0096] Assembly of marker-free elements: Incubate 5×BP Clonase II enzyme mix (ThermoFisher) on ice for about 2 minutes, vortex and briefly centrifuge. Mix the marker-free element donor vector pYLMF-H with the element vector to be inserted pYLTAC380GW-DOBhPC-CsCAU1U2, react at 25°C for 6 hours, add proteinase K and react at 37°C for 10 minutes to terminate the reaction, and transform the reaction product into Escherichia coli DH10B by electroporation, screen positive transformants on kanamycin plates containing 5% sucrose (sacB is a sucrose lethal gene), and extract the plasmid contained therein for enzyme digestion identification.

[0097] The results of enzyme digestion of plant transformation vector pYLTAC380MF-DOBhPC-CsCAU1U2 are as follows Figure 2 As shown by Figure 2 It can be seen that the present invention has obtained the plant transformation vector pYLTAC380MF-DOBhPC-CsCAU1U2 containing the HPT gene.

[0098] S3. Transformation of rice with the plant transformation vector to obtain the transformation and detection of the transgenic rice plant transformation vector pYLTAC380MF-DOBhPC-CsCAU1U2 producing crocin in the seed endosperm:

[0099] Genetic transformation of rice: The plant transformation vector pYLTAC380MF-DOBhPC-CsCAU1U2 plasmid was transferred into Agrobacterium EHA105 for transformation of rice embryo callus. Specifically, immature or mature rice seeds were induced to callus at 25°C in the dark; an appropriate amount of Agrobacterium transformed with a multi-gene vector plasmid was suspended in an infection liquid medium supplemented with 100 μmol / L acetosyringone, and cultured at 28°C with shaking (200 rpm, 0.5 h), and the OD was adjusted using a spectrophotometer. 550 When the value is 0.3-0.4, it can be used for callus infection; select fresh, light yellow, vigorously growing granular embryonic callus, mix it with Agrobacterium solution, soak it for 20 minutes, absorb the solution and transfer it to co-cultivation medium, culture it in the dark for 3 days, transfer it to the screening medium containing 50 mg / L hygromycin, subculture it every 2 weeks, and subculture it twice; after resistance screening, transfer the resistant callus with green dots to differentiation medium, differentiate the transformed seedlings, and obtain the transformed plants, i.e., transgenic rice.

[0100] The above-mentioned culture medium components and dosages are as follows:

[0101] Infection medium: 10×MS macro Mother solution 100mL, 1000×B5 micro 1 mL of mother solution, 100×B5 vit Mother solution 10mL, 2,4-D 2mg, hydrolyzed casein 500mg, inositol 2g, sucrose 30g, acetosyringone 100μmol, pH adjusted to 5.5, ddH2O added to 1L.

[0102] Co-culture medium: 10×MS macro Mother solution 100mL, 1000×B5 micro 1 mL of mother solution, 100×B5 vit 10 mL of mother solution, 2 mg of 2,4-D, 500 mg of hydrolyzed casein, 2 g of inositol, 30 g of sucrose, 100 μmol of acetosyringone, 8 g of agar, pH adjusted to 5.5, and made up to 1 L with ddH2O.

[0103] Screening medium: 10×N6 macro Mother solution 100mL, 1000×B5 micro 1 mL of mother solution, 100×B5 vit 10 mL of mother solution, 2 mg of 2,4-D, 300 mg of hydrolyzed casein, 500 mg of L-proline, 500 mg of L-glutamine, 30 g of sucrose, 8 g of agar, pH adjusted to 5.8, and supplemented to 1 L with ddH2O;

[0104] After high temperature sterilization, cool and add 1 mL each of 1000× ceftriaxone sodium (cef), 1000× carbenicillin (carb), and 1000× hygromycin (Hm) antibiotics. Differentiation medium: 10×N6 macro Mother solution 100mL, 1000×MS micro 1 mL of mother solution, 100×B5 vit Mother solution 10mL, BA 3mg, NAA 1mg, sorbitol 18.2g, sucrose 20g, agar 8g, pH adjusted to 5.8, ddH2O added to 1L.

[0105] PCR detection of transgenic rice genome: The leaves of T0 transgenic plants were extracted by SDS method and used as templates. PCR amplification method was used to detect whether the exogenous genes contained HPT, sEuCrtI, sZmPSY, sHpBHY, sAtOR H , sAtDXS, sCsCCD2L, sCsUGT74AD1, sCsUGT91P3 and sCsALDH3I1 genes. The primers used for PCR detection of transgenic rice genome are shown in Table 1.

[0106] Table 1 Primers used for PCR detection of transgenic rice genome

[0107]

[0108]

[0109] The amplification program used was: 94℃ pre-denaturation for 4min; 94℃ denaturation for 30sec, 58℃ annealing for 30sec, 72℃ extension for 30sec, for a total of 30 cycles; and finally 72℃ extension for 5min. The PCR detection of the exogenous gene introduced into the genomic DNA of the T0 rice transformant of pYLTAC380MF-DOBhPC-CsCAU1U2 is shown in the figure. Figure 3 As shown. Figure 3 It can be seen that the wild type (WT) control cannot amplify the exogenous gene, while the transgenic positive plants can amplify the above genes.

[0110] Example 2

[0111] The invention uses zeaxanthin precursor synthesis-related genes CrtI, PSY, BHY, OR H , DXS and GjCCD4a, GjUGT94E13, GjUGT74F8, and GjALDH2C3 genes from gardenia were used as templates, and the above genes were optimized according to the codon preference of rice. The optimized genes related to the synthesis of lutein precursor were combined with genes from gardenia to construct a plant transformation vector and transformed into rice, thus realizing the de novo synthesis of crocin in rice endosperm.

[0112] Specifically, the zeaxanthin precursor synthesis-related genes CrtI, PSY, BHY, OR H , DXS is as described in Example 1.

[0113] The present invention also establishes a transgenic breeding method for producing crocin in rice seed endosperm, comprising the following steps:

[0114] S1. After sequence optimization, CrtI, PSY, BHY, OR H The five genes, DXS, GjCCD4a, GjUGT94E13, GjUGT74F8, and GjALDH2C3 from Gardenia were fused with the rice seed endosperm expression promoter, respectively. Among them, CrtI and BHY genes were also fused with the plastid transit peptide to construct the corresponding gene expression cassette.

[0115] The details are as follows:

[0116] 1. Synthesis of nine key gene coding regions:

[0117] Respectively with the CrtI, PSY, BHY, OR H , DXS five genes as templates, according to the rice codon preference, the codon was optimized using the Codon Optimization Tool program, and the optimized gene sequences were synthesized and named sEuCrtI (from the optimized CrtI gene sequence in CN105907780B), sZmPSY (from the optimized PSY gene sequence in CN105907780B), sHpBHY (from the optimized BHY gene sequence in CN105907780B), sAtOR H (SEQ ID NO: 1) and sAtDXS (SEQ ID NO: 2) were cloned into a plasmid vector and their sequences were confirmed by sequencing.

[0118] The GjCCD4a (GenBank No.KY631925), GjUGT94E13 (GenBank No.MN944055), GjUGT74F8 (GenBank No.MN944054), and GjALDH2C3 (GenBank No.KY631926) genes from Gardenia jasminoides Ellis were used as templates, and codons were optimized using the CodonOptimization Tool program according to the codon preference of rice. The optimized gene sequences were synthesized and named sGjCCD4a, sGjUGT94E13, sGjUGT74F8 and sGjALDH2C3, respectively. Their sequences are shown in SEQ ID NOs: 7 to 10, respectively. They were cloned into plasmid vectors and their sequences were confirmed by sequencing.

[0119] 2. Synthesis of plastid transit peptide coding sequence TP and 2A peptide coding sequence F2A:

[0120] The sequence encoding the plastid transit peptide TP (Transit peptide) was synthesized with reference to the sequence of the pea RbcS small subunit gene (GenBank No. X00806). According to the F2A protein sequence (GSVKQTLNFDLLKLAGDVESNPGPGS), the codons were optimized using the Codon Optimization Tool program according to the rice codon preference, and the F2A sequence shown in SEQ ID NO: 12 was synthesized.

[0121] 3. Construction of gene expression cassette on the supply vector:

[0122] Contains sEuCrtI, sZmPSY, sHpBHY, sAtOR H , The construction of the supply vector of the sAtDXS gene expression cassette is the same as in Example 1.

[0123] Construction of the donor vector pYL322d2-sGjCCD4a containing the sGjCCD4a gene expression cassette: Reverse amplification of the multi-gene supply vector II (pYL322d2) to obtain the vector backbone fragment a; using rice genomic DNA as a template, amplify the promoter Pens6 (GenBank No. CP132236.1) of about 1.9 kb of rice endosperm-specific storage protein as fragment b; amplify the GjCCD4a gene as fragment c; amplify the nopaline synthase terminator Tnos (SEQ ID NO: 17) from the plasmid pSAT2 (GenBank No. DQ005463) as fragment d. Each fragment has 25 bp of homologous sequence on both sides. Using the principle of Gibson assembly and the one-step assembly method of the plasmid vector multi-fragment, the supply vector (IX) pYL322d2-sGjCCD4a plasmid containing the GjCCD4a gene expression cassette (such as Figure 1 shown).

[0124] Construction of the donor vector pYL322d1-GjUGT94E13-2A-GjUGT74F8 containing the sGjUGT94E13 and sGjUGT74F8 fusion gene expression cassette: reverse amplification of the multi-gene supply vector I (pYL322d1) to obtain the vector backbone fragment a; using rice genomic DNA as a template, amplifying the promoter Pens8 (GenBank No.AY427574.1) of rice endosperm-specific storage protein of about 1 kb as fragment b; amplifying the GjUGT94E13-2A-GjUGT74F8 fusion gene as fragment c; using rice genomic DNA as a template, amplifying the terminator TGluA-1 (SEQ ID NO: 19) of rice endosperm-specific storage protein of about 0.5 kb as fragment d. Each fragment has 25 bp homologous sequences on both sides. Using the principle of Gibson assembly, the supply vector (X) pYL322d1-GjUGT94E13-2A-GjUGT74F8 plasmid containing GjUGT94E13 and GjUGT74F8 gene expression cassettes was obtained according to the one-step assembly method of plasmid vector multi-fragment. Figure 1 shown).

[0125] Construction of the supply vector pYL322d2-GjALDH2C3 containing the sGjALDH2C3 gene expression cassette: Reverse amplification of the multi-gene supply vector II (pYL322d2) to obtain the vector backbone fragment a; using rice genomic DNA as a template, amplify the promoter Pens9 (GenBank No. CP141114.1) of about 0.7 kb of rice endosperm-specific storage protein as fragment b; amplify the GjALDH2C3 gene as fragment c; using rice genomic DNA as a template, amplify the terminator TGluB4 (SEQ ID NO: 20) of about 0.5 kb of rice endosperm-specific storage protein as fragment d. Each fragment has 25 bp of homologous sequence on both sides. Using the principle of Gibson assembly and the method of one-step assembly of multiple fragments of plasmid vectors, the supply vector (XI) pYL322d2-GjALDH2C3 containing the GjALDH2C3 gene expression cassette (such as Figure 1 shown).

[0126] S2. Construct the gene expression cassette described in step S1 into a plant transformation vector

[0127] Contains optimized CrtI, PSY, BHY, OR H The assembly of the plant transformation vector pYLTAC380MF-DOBhPC-CsCAU1U2 for rice endosperm-specific crocin synthesis containing five genes, DXS, and GjCCD4a, GjUGT94E13, GjUGT74F8, and GjALDH2C3 from Gardenia is as follows:

[0128] The multi-gene vector of the present invention is assembled using the multi-gene vector system TGSII.

[0129] (1) Construction of the multi-gene vector pYLTAC380GW-DOBh PC for rice endosperm-specific synthesis of zeaxanthin precursor: see Example 1.

[0130] (2) Construction of plant transformation vector pYLTAC380MF-DOBhPC-CsCAU1U2: Four genes, sGjCCD4a, sGjUGT94E13, sGjUGT74F8 and sGjALDH2C3, from Gardenia jasminoides were superimposed on the vector pYLTAC380GW-DOBhPC for synthesizing zeaxanthin precursor to construct pYLTAC380GW-DOBhPC-GjCAU1U2 for specifically synthesizing crocin in rice endosperm. Then, through the Gateway-BP recombination reaction, marker-free elements containing the HPT expression cassette were assembled to construct the multi-gene expression vector pYLTAC380MF-DOBhPC-GjCAU1U2 that can achieve self-deletion of the hygromycin resistance selection marker.

[0131] Assembly of sGjCCD4a gene expression cassette: Mix the supply vector (IX) pYL322d2-sGjCCD4a plasmid with the receiving vector pYLTAC380GW-DOBhPC plasmid containing 5 gene expression cassettes, electroporate and transform NS3529 competent cells, screen transformants on karatomycin and ampicillin double-resistance plates, and use the Cre enzyme endogenously expressed in NS3529 to achieve supply vector plasmid recombination and supply vector backbone deletion. Wash the mixed colonies on the double-resistance plates and extract the plasmid, then cut the unrecombined plasmid with PI-SceI enzyme, transform E. coli DH10B, and identify the plasmid containing sEuCrtI+sZmPSY+sHpBHY+sAtOR on the karatomycin plate. H The receiving vector pYLTAC380GW-DOBhPC-GjC plasmid for the +sAtDXS+sGjCCD4a gene expression cassette.

[0132] Assembly of sGjUGT94E13 and sGjUGT74F8 gene expression cassettes: The supply vector (X) pYL322d1-GjUGT94E13-2A-GjUGT74F8 plasmid was mixed with the receiving vector pYLTAC380GW-DOBhPC-GjC plasmid containing 6 target gene expression cassettes, and the competent cells of the Escherichia coli strain NS3529 expressing the Cre enzyme were transformed by electroporation together. The transformants were screened on karatomycin and chloramphenicol double-resistant plates, and the Cre enzyme endogenously expressed in NS3529 was used to achieve the recombination of the supply vector plasmid and the deletion of its backbone. The mixed colonies on the double-resistant plates were washed and the mixed plasmids were extracted. After the non-recombinant plasmids were eliminated by I-SceI enzyme digestion, DH10B was transformed, and the cells containing sEuCrtI+sZmPSY+sHpBHY+sAtOR were identified on karatomycin plates. H +

[0133] The receiving vector pYLTAC380GW-DOBhPC-GjCU1U2 plasmid for the sAtDXS+sGjCCD4a+sGjUGT94E13+sGjUGT74F8 gene expression cassette.

[0134] Assembly of sGjALDH2C3 gene expression cassette: Mix the supply vector (XI) pYL322d2-sGjALDH2C3 plasmid with the receiving vector pYLTAC380GW-DOBhPC-GjCU1U2 plasmid containing 8 gene expression cassettes, electroporate and transform NS3529 competent cells, screen transformants on karatomycin and ampicillin double-resistance plates, and use the Cre enzyme endogenously expressed in NS3529 to achieve supply vector plasmid recombination and supply vector backbone deletion. Wash the mixed colonies on the double-resistance plates and extract the plasmid, then cut the unrecombined plasmid with PI-SceI enzyme, transform Escherichia coli DH10B, screen positive transformants on karatomycin plates, and identify the plasmid containing sEuCrtI+sZmPSY+sHpBHY+sAtOR H +sAtDXS+sGjCCD4a+sGjUGT94E13

[0135] +sGjUGT74F8+sGjALDH2C3 plant transformation vector pYLTAC380GW-DOBhPC-GjCA U1U2, its structure diagram is as follows Figure 2 shown.

[0136] The assembly of the marker-free element is specifically described in Example 1. The results of enzyme digestion identification of the plant transformation vector pYLTAC380GW-DOBhPC-GjCAU1U2 are as follows: Figure 2 As shown by Figure 2 It can be seen that the present invention obtains the plant transformation vector pYLTAC380GW-DOBhPC-GjCAU1U2.

[0137] S3. Transformation of the plant transformation vector into rice to obtain the transformation and detection of the transgenic rice multi-gene vector pYLTAC380MF-DOBhPC-GjCAU1U2 that produces crocin in the seed endosperm:

[0138] Genetic transformation of rice: The multi-gene vector pYLTAC380MF-DOBhPC-GjCAU1U2 plasmid was respectively transferred into Agrobacterium EHA105 for transformation of rice embryo callus; see Example 1 for details.

[0139] PCR detection of transgenic rice genome: The leaves of T0 transgenic plants were extracted by SDS method and used as templates. The exogenous HPT, sEuCrtI, sZmPSY, sHpBHY, sAtOR were detected by PCR amplification. H, sAtDXS, sGjCCD4a, sGjUGT94E13, sGjUGT74F8 and sGjALDH2C3 genes. Among them, exogenous HPT, sEuCrtI, sZmPSY, sHpBHY, sAtOR H The primers used for detecting exogenous sGjCCD4a, sGjUGT94E13, sGjUGT74F8 and sGjALDH2C3 genes are shown in Table 2.

[0140] Table 2 Primers used for PCR detection of exogenous genes

[0141]

[0142] The amplification procedure used was the same as in Example 1. The PCR detection of exogenous genes in the genomic DNA of the T0 rice transformants of pYLTAC380MF-DOBhPC-GjCAU1U2 is shown in FIG. Figure 3 As shown. Figure 3 It can be seen that the wild type (WT) control cannot expand the exogenous gene, and the transgenic positive plants can expand the above gene. Example 3 Observation of the appearance of transgenic rice seeds and UPLC-MS / MS detection of crocin in endosperm

[0143] Observation of the appearance of transgenic rice seeds: The phenotype of pYL380MF-DOBhPC-CsCAU1U2 and pYL380MF-DOBhPC-GjCAU1U2 transgenic brown rice was observed. Figure 4 As shown. Figure 4 It can be seen that the seeds of pYL380MF-DOBhPC introduced with the synthetic zeaxanthin precursor and pYL380MF-DOBhPC-CsCAU1U2 introduced with the saffron pathway are yellow, while the seeds of pYL380MF-DOBhPC-GjCAU1U2 introduced with the gardenia pathway are orange-yellow.

[0144] Extraction and UPLC-MS / MS identification of crocin from transgenic rice seeds: After freeze-drying the rice seeds for 2 days, 0.3 g was weighed and ground into powder using a cryo-grinder. 250 μL of methanol was added and vortexed for 1 min. 500 μL of chloroform was added and vortexed for 1 min. The mixture was kept on ice and protected from light for 20 min. 250 μL of water was added and vortexed for 1 min. The aqueous and organic phases were collected by centrifugation at 4000 rpm at 4°C for 5 min. The color of the extract was observed. The results are as follows: Figure 4 As shown. Figure 4It can be seen that although the pYL380MF-DOBhPC seeds are yellow, their aqueous phase is colorless and transparent, while the aqueous phases of pYL380MF-DOBhPC-CsCAU1U2 and pYL380MF-DOBhPC-GjCAU1U2 are obviously yellow, indicating that polar substances are produced in pYL380MF-DOBhPC-CsCAU1U2 and pYL380MF-DOBhPC-GjCAU1U2; relative to the yellow organic phase of DOBhPC, the organic phases of pYL380MF-DOBhPC-CsCAU1U2 and pYL380MF-DOBhPC-GjCAU1U2 are orange-red, indicating that additional fat-soluble pigments are produced.

[0145] The aqueous phase of the above-mentioned sample to be tested was filtered through a 0.22μm water filter membrane, and then injected into a 2mL brown sampling bottle with an injection volume of 1μL. The sample was analyzed using an Agilent EC-C18 (2.1*100mm, 2.7μm) column. Mobile phase: A: primary water; B: acetonitrile. Gradient elution conditions: 0-1min, 10% B; 1-8min, 10%-50% B; 8-8.1min, 50%-90% B; 8.1-10min, 90% B; 10-10.1min, 90%-10% B; 10.1-12min, 10% B.

[0146] UPLC-MS\MS detection of crocin in the endosperm of rice seeds transformed with pYLTAC380MF-DOBhPC-CsCAU1U2 and pYLTAC380MF-DOBhPC-GjCAU1U2 Figure 5 and Figure 6 As shown, the results showed that the transgenic rice seeds of pYL380MF-DOBhPC-CsCAU1U2 and pYL380MF-DOBhPC-GjCA U1U2 had the same characteristic peaks as the four crocin standards, indicating that crocin was successfully synthesized in the two transgenic rice. The crocin content in the endosperm of rice seeds transformed with pYLTAC380MF-DOBhPC-CsCAU1U2 and pYLTAC380MF-DOBhPC-GjCAU1U2 was also detected. Among them, the crocin content in the endosperm of rice transformed with pYL380MF-DOBhPC-CsCAU1U2 was 587.82ng / g; the crocin content in the endosperm of rice transformed with pYL380MF-DOBhPC-GjCAU1U2 was 855.4ng / g.

[0147] The present invention also provides a schematic diagram of the pathway for the synthesis of crocin in the endosperm of transgenic rice seeds, such as Figure 7As shown. The biosynthesis of crocin requires a high degree of coordination of multiple pathways, including the upstream methylerythritol phosphate (MEP) pathway, the midstream carotenoid biosynthesis pathway, and the downstream crocin biosynthesis pathway. Among them, the MEP pathway provides the precursor geranylgeranyl pyrophosphate (GGPP) of the core carotenoid biosynthesis pathway, while the carotenoid biosynthesis pathway provides an important precursor zeaxanthin for the crocin biosynthesis pathway, which is oxidatively cleaved by carotenoid cleavage dioxygenase (CCD) to crocetin dialdehyde, then dehydrogenated to crocetin by aldehyde dehydrogenase (ALDH), and finally catalyzed by UDP-glucosyltransferase (UGT) to obtain crocetin.

[0148] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. CrtI,PSY,BHY,OR H The application of five genes, CsCCD2L, CsUGT74AD1, CsUGT91P3 and CsALDH3I1 from saffron, or GjCCD4a, GjUGT94E13, GjUGT74F8 and GjALDH2C3 from gardenia, in transgenic breeding for the production of crocin in rice seed endosperm; the specific method of the application is: The sequences of the above genes were optimized according to the rice codon preference, and the optimized sequences of CrtI, PSY, BHY, and OR were constructed respectively. H The gene expression cassettes of five genes, DXS and CsCCD2L, CsUGT74AD1, CsUGT91P3 and CsALDH3I1 from saffron were constructed into plant transformation vectors and transformed into rice to obtain transgenic rice that produces crocin in seed endosperm; Or construct the sequence-optimized CrtI, PSY, BHY, OR H The gene expression cassettes of five genes, DXS, and the genes GjCCD4a, GjUGT94E13, GjUGT74F8, and GjALDH2C3 from Gardenia were constructed into plant transformation vectors and transformed into rice to obtain transgenic rice that produces crocin in seed endosperm; The CrtI gene is the CrtI gene of Erwinia uredovora, and its GenBank No is D90087; the PSY gene is the PSY gene of corn, and its GenBank No is U32636.1; the BHY gene is the BHY gene of Haematococcus pluvialis, and its GenBank No is BD250390.1; H Both the gene and the DXS gene are from Arabidopsis thaliana. H The GenBank No of the gene is NM_125561.4, and the GenBank No of the DXS gene is U27099.1; The GenBank Nos of the saffron-derived CsCCD2L, CsUGT74AD1, CsUGT91P3, and CsALDH3I1 genes are KP887110, MF596166, MZ190170, and MF596165, respectively; The GenBank Nos of the GjCCD4a, GjUGT94E13, GjUGT74F8 and GjALDH2C3 genes derived from Gardenia jasminoides are KY631925, MN944055, MN944054 and KY631926, respectively.

2. A transgenic breeding method for producing crocin in rice seed endosperm, characterized in that: The following steps are involved: S1. Based on the codon preference of rice, CrtI, PSY, BHY, OR H The five genes of , DXS and saffron-derived CsCCD2L, CsUGT74AD1, CsUGT91P3, and CsALDH3I1 were optimized, and the corresponding gene expression cassettes were constructed using the optimized sequences; S2. constructing the gene expression cassette described in step S1 into a plant transformation vector; S3. Transforming rice with the plant transformation vector described in step S2 to obtain transgenic rice that produces crocin in seed endosperm; or, S1. Based on the codon preference of rice, CrtI, PSY, BHY, OR H The five genes of , DXS and GjCCD4a, GjUGT94E13, GjUGT74F8, and GjALDH2C3 from Gardenia were optimized, and the corresponding gene expression cassettes were constructed using the optimized sequences; S2. constructing the gene expression cassette described in step S1 into a plant transformation vector; S3. Transforming rice with the plant transformation vector described in step S2 to obtain transgenic rice that produces crocin in seed endosperm; The CrtI gene is the CrtI gene of Erwinia uredovora, and its GenBank No is D90087; the PSY gene is the PSY gene of corn, and its GenBank No is U32636.1; the BHY gene is the BHY gene of Haematococcus pluvialis, and its GenBank No is BD250390.1; H Both the gene and the DXS gene are from Arabidopsis thaliana. H The GenBank No of the gene is NM_125561.4, and the GenBank No of the DXS gene is U27099.1; The GenBank Nos of the saffron-derived CsCCD2L, CsUGT74AD1, CsUGT91P3, and CsALDH3I1 genes are KP887110, MF596166, MZ190170, and MF596165, respectively; The GenBank Nos of the GjCCD4a, GjUGT94E13, GjUGT74F8 and GjALDH2C3 genes derived from Gardenia jasminoides are KY631925, MN944055, MN944054 and KY631926, respectively.

3. The method according to claim 2, characterized in that: OR after sequence optimization H , the sequences of DXS genes are shown in SEQ ID NOs: 1 to 2; The sequences of CsCCD2L, CsUGT74AD1, CsUGT91P3, and CsALDH3I1 genes after sequence optimization are shown in SEQ ID NOs: 3 to 6, respectively; The sequences of GjCCD4a, GjUGT94E13, GjUGT74F8 and GjALDH2C3 genes after sequence optimization are shown in SEQ ID NOs: 7 to 10 respectively.

4. The method according to claim 2, characterized in that: When the gene expression cassettes of the five genes after sequence optimization and the four genes from saffron were constructed into a plant transformation vector, the order of arrangement of the genes was: CrtI-PSY-BHY-OR H -DXS-CsCCD2L-CsUGT74AD1-CsUGT91P3-CsALDH3I1.

5. The method according to claim 2, characterized in that: When the gene expression cassettes of the five genes after sequence optimization and the four genes from Gardenia were used to construct a plant transformation vector, the order of arrangement of the genes was: CrtI-PSY-BHY-OR H -DXS-GjCCD4a-GjUGT94E13-GjUGT74F8-GjALDH2C3.

6. The method according to claim 2, characterized in that: The method of transforming rice with the plant transformation vector is Agrobacterium-mediated method.

7. The method according to claim 2, characterized in that: The TGSII multi-gene vector system is used when constructing the plant transformation vector.

8. The method of claim 4 comprising optimized CrtI, PSY, BHY, OR H , DXS and the plant transformation vector of the saffron-derived genes CsCCD2L, CsUGT74AD1, CsUGT91P3, and CsALDH3I1.

9. The method of claim 5 comprising optimized CrtI, PSY, BHY, OR H , DXS and the plant transformation vector containing the five genes GjCCD4a, GjUGT94E13, GjUGT74F8, and GjALDH2C3 from Gardenia.

10. Use of the plant transformation vector according to claim 8 or 9 in constructing transgenic rice that produces crocin in seed endosperm.

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