CN118546994A - Transgenic breeding method for producing crocin in seed endosperm - Google Patents

Abstract

The invention discloses a transgenic breeding method for producing crocin in seed endosperm. According to the codon preference of rice, the invention optimizes zeaxanthin precursor synthesis-related genes CrtI, PSY, BHY, ^ORH , DXS, saffron-derived CsCCD2L, CsUGT74AD1, CsUGT91P3, CsALDH3I1 genes, and gardenia-derived GjCCD4a, GjUGT94E13, GjUGT74F8, and GjALDH2C3 genes, and the optimized zeaxanthin precursor synthesis-related genes are respectively combined with saffron-derived genes or gardenia-derived genes to construct plant transformation vectors and transform rice, thereby obtaining transgenic rice capable of producing crocin in rice seed endosperm, realizing the synthesis of crocin in rice seed endosperm, and the product thereof can be directly eaten, and can also be used as a raw material for crocin production, which is beneficial to the production of crocin.

Description

A transgenic breeding method for producing crocin in seed endosperm

Technical Field

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

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.

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.

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.

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

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.

The first objective of the present invention is to provide a combination of five genes, CrtI, PSY, BHY, ^ORH and DXS, with four genes, CsCCD2L, CsUGT74AD1, CsUGT91P3 and CsALDH3I1, derived from saffron, or with four genes, GjCCD4a, GjUGT94E13, GjUGT74F8 and GjALDH2C3, derived from gardenia, for use in transgenic breeding for the production of crocin in rice seed endosperm.

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

The third object of the present invention is to provide a plant transformation vector containing optimized five genes of CrtI, PSY, BHY, ^ORH , DXS and saffron derived genes of CsCCD2L, CsUGT74AD1, CsUGT91P3, and CsALDH3I1.

The fourth object of the present invention is to provide a plant transformation vector containing the optimized five genes of CrtI, PSY, BHY, ^ORH , DXS and the genes GjCCD4a, GjUGT94E13, GjUGT74F8, and GjALDH2C3 from Gardenia jasminoides.

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.

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

The invention uses zeaxanthin precursor synthesis-related genes CrtI, PSY, BHY, ^ORH , DXS, saffron-derived CsCCD2L, CsUGT74AD1, CsUGT91P3, CsALDH3I1 genes, and gardenia-derived GjCCD4a, GjUGT94E13, GjUGT74F8, GjALDH2C3 genes as templates, optimizes the above genes according to rice codon preferences, combines the optimized zeaxanthin precursor synthesis-related genes with saffron-derived genes or gardenia-derived genes to construct plant transformation vectors, and transforms rice, thereby obtaining transgenic rice capable of producing crocin in rice seed endosperm, and realizing the de novo synthesis of crocin in rice endosperm.

Therefore, the present invention claims protection for the use of five genes, CrtI, PSY, BHY, ^ORH and DXS, in combination with four genes, CsCCD2L, CsUGT74AD1, CsUGT91P3 and CsALDH3I1, derived from saffron, or in combination with four genes, GjCCD4a, GjUGT94E13, GjUGT74F8 and GjALDH2C3, derived 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 codon preference of rice, and gene expression cassettes of the five genes CrtI, PSY, BHY, OR ^H , and DXS after sequence optimization and the saffron-derived genes CsCCD2L, CsUGT74AD1, CsUGT91P3, and CsALDH3I1 were constructed respectively. The constructed gene expression cassettes were built into plant transformation vectors and transformed into rice to obtain transgenic rice that produces crocin in seed endosperm;

Alternatively, gene expression cassettes of the five genes CrtI, PSY, BHY, OR ^H , and DXS after sequence optimization and the genes GjCCD4a, GjUGT94E13, GjUGT74F8, and GjALDH2C3 from Gardenia are constructed respectively, and the constructed gene expression cassettes are built into plant transformation vectors and transformed into rice to obtain transgenic rice that produces crocin in the seed endosperm.

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

S1. According to the codon preference of rice, the sequences of five genes, namely CrtI, PSY, BHY, OR ^H and DXS, and the genes CsCCD2L, CsUGT74AD1, CsUGT91P3 and CsALDH3I1 from saffron 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. Optimize the sequences of five genes, namely CrtI, PSY, BHY, OR ^H and DXS, and the genes GjCCD4a, GjUGT94E13, GjUGT74F8 and GjALDH2C3 from Gardenia jasminoides according to the codon preference of rice, and construct the corresponding gene expression cassette using the optimized sequences;

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

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

Specifically, according to the codon preference of rice, the optimized zeaxanthin precursor synthesis-related genes OR ^H and DXS genes are recorded as sAtOR ^H and sAtDXS, respectively, and the optimized sequences are shown in SEQ ID NOs: 1 to 2; the optimized crocin synthesis-related genes CsCCD2L, CsUGT74AD1, CsUGT91P3, and CsALDH3I1 from saffron are recorded as sCsCCD2L, sCsUGT74AD1, sCsUGT91P3, and sCsALDH3I1, respectively, and the optimized sequences are shown in SEQ ID NOs: 1 to 2. IDNO:3~6; the optimized crocin synthesis-related genes GjCCD4a, GjUGT94E13, GjUGT74F8, and GjALDH2C3 genes from Gardenia are respectively recorded as sGjCCD4a, sGjUGT94E13, sGjUGT74F8, and sGjALDH2C3, and the optimized sequences are respectively shown in SEQ ID NO:7~10.

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.

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

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.

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

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

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.

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); the promoter used in the sAtOR ^H 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 was octopinesynthase terminator Tocs (SEQ ID NO: 16).

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).

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.

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:

CrtI-PSY-BHY-OR ^H -DXS-CsCCD2L-CsUGT74AD1-CsUGT91P3-CsALDH3I1.

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:

CrtI-PSY-BHY- ^ORH -DXS-GjCCD4a-GjUGT94E13-GjUGT74F8-GjALDH2C3.

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.

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

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

The present invention also claims protection for a plant transformation vector constructed in the above method containing the optimized five genes of CrtI, PSY, BHY, ^ORH , DXS and the genes CsCCD2L, CsUGT74AD1, CsUGT91P3, and CsALDH3I1 derived from saffron.

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

CrtI-PSY-BHY-OR ^H -DXS-CsCCD2L-CsUGT74AD1-CsUGT91P3-CsALDH3I1.

The present invention also claims protection for the plant transformation vector constructed in the above method containing the optimized five genes of CrtI, PSY, BHY, ^ORH , DXS and the genes of GjCCD4a, GjUGT94E13, GjUGT74F8, and GjALDH2C3 from Gardenia.

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

CrtI-PSY-BHY- ^ORH -DXS-GjCCD4a-GjUGT94E13-GjUGT74F8-GjALDH2C3.

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

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.

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

The present invention uses zeaxanthin precursor synthesis-related genes CrtI, PSY, BHY, ^ORH , DXS, saffron-derived CsCCD2L, CsUGT74AD1, CsUGT91P3, CsALDH3I1 genes, and gardenia-derived GjCCD4a, GjUGT94E13, GjUGT74F8, and GjALDH2C3 genes as templates, optimizes the above genes according to rice codon preference, and constructs plant transformation vectors by combining the optimized zeaxanthin precursor synthesis-related genes with saffron-derived genes or gardenia-derived genes, respectively, and transforms rice to obtain transgenic rice capable of producing crocin in rice seed endosperm. The present invention realizes the synthesis of crocin in rice seed endosperm, and the 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 shortcomings of the existing use of saffron stigma to extract crocin, such as complex procedures, low yield, and difficulty in chemical synthesis.

The present invention solves the problem of the shortage of traditional sources of crocin, provides a bioreactor for crocin, creates a new crop germplasm for producing high crocin content, and also explores the difference in the functions 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

FIG1 is a schematic diagram of the structure of a gene expression cassette constructed using the 11 optimized genes of the present invention on a supply vector.

Figure 2 is a schematic diagram of the structure of the plant transformation vector constructed during the multi-gene assembly process 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-CsCAU ₁ U ₂ 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-GjCAU ₁ U ₂ 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.

Figure 3 is a PCR detection diagram of exogenous genes in the _T0 generation transformed rice genomic DNA introduced with the plant transformation vectors pYLTAC380MF-DOBhPC-CsCAU ₁ U ₂ and pYLTAC380MF-DOBhPC-GjCAU ₁ U ₂ ; CK ⁺ is the plant transformation vector pYLTAC380MF-DOBhPC-CsCAU ₁ U ₂ or pYLTAC380MF-DOBhPC-GjCAU ₁ U ₂ ; WT is the wild-type control genomic DNA.

Figure 4 shows the appearance and color of the transgenic rice brown rice of pYLTAC380MF-DOBhPC for synthesizing zeaxanthin precursor, pYLTAC380MF-DOBhPC-CsCAU ₁ U ₂ , and pYLTAC380MF-DOBhPC-GjCAU ₁ U ₂ for synthesizing crocin; the wild type WT is the recipient rice variety Huaguang (HG); the yellow logo indicates the gene related to the maize yellow precursor; the red logo indicates the gene derived from saffron, and the purple logo indicates the gene derived from gardenia.

Figure 5 shows the UPLC-MS\MS detection results of crocin in the endosperm of rice seeds transformed with pYLTAC380MF-DOBhPC-CsCAU ₁ U ₂ .

Figure 6 shows the UPLC-MS\MS detection results of crocin in the endosperm of rice seeds transformed with pYLTAC380MF-DOBhPC-GjCAU ₁ U ₂ .

Figure 7 is a schematic diagram of the pathway for synthesizing crocin in the endosperm of transgenic rice seeds constructed by 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 red thick arrows represent reactions catalyzed by enzymes expressed by genes related to carotenoid synthesis; and the red dotted arrows represent reactions catalyzed by enzymes expressed by genes related to crocin synthesis.

DETAILED DESCRIPTION

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.

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

Example 1

The present invention uses zeaxanthin precursor synthesis-related genes CrtI, PSY, BHY, ^ORH , DXS and saffron-derived genes CsCCD2L, CsUGT74AD1, CsUGT91P3, and CsALDH3I1 as templates, optimizes the above genes according to rice codon preferences, combines the optimized zeaxanthin precursor synthesis-related genes with saffron-derived genes into a plant transformation vector, and transforms rice, thereby realizing the de novo synthesis of crocin in rice endosperm.

Among them, 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; the OR ^H gene and DXS gene are both derived from Arabidopsis thaliana, the GenBank No of the OR ^H gene is NM_125561.4, and the GenBank No of the DXS gene is U27099.1.

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

S1. According to the codon preference of rice, the sequences of five genes, namely CrtI, PSY, BHY, OR ^H and DXS, and the genes CsCCD2L, CsUGT74AD1, CsUGT91P3 and CsALDH3I1 from saffron were optimized, and the corresponding gene expression cassettes were constructed using the optimized sequences;

The details are as follows:

1. Synthesis of nine key gene coding regions:

The five genes CrtI, PSY, BHY, OR ^H and DXS were used as templates respectively, and codons were optimized using the Codon Optimization Tool program according to the codon preference of rice. 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), cloned into plasmid vectors, and their sequences were determined by sequencing.

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.

2. Synthesis of plastid transit peptide coding sequence TP:

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).

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

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 of the plastid transit peptide (SEQ ID NO: 11) 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 multiple fragments of plasmid vectors (Zhu et al., 2014), the supply vector (I) pYL322d1-sEuCrtI plasmid containing the sEuCrtI gene expression cassette was obtained (as shown in Figure 1).

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 and the one-step assembly method of plasmid vector multiple fragments, the supply vector (II) pYL322d2-sZmPSY plasmid containing the sZmPSY gene expression cassette was obtained (as shown in Figure 1).

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 and the one-step assembly method of multiple fragments of plasmid vectors, the supply vector (III) pYL322d1-sHpBHY plasmid containing the sHpBHY gene expression cassette was obtained (as shown in Figure 1).

Construction of the supply vector pYL322d2-sAtOR ^H containing the sAtOR ^H gene expression cassette: Reverse amplification of the multi-gene supply vector II (pYL322d2) to obtain the vector backbone fragment a; amplification of the manopinesynthase terminator Tmas (SEQ ID NO: 15) from the plasmid pSAT3 (GenBank No. DQ005465) as fragment b; amplification of the reverse sAtOR ^H gene as fragment c; using rice genomic DNA as a template, amplification of the rice endosperm-specific storage protein promoter Pens4 (GenBank No. MH748577.1) of about 2.4 kb as fragment d. Each fragment has 25 bp of homologous sequence on both sides, and using the principle of Gibson assembly, according to the method of one-step assembly of plasmid vector multiple fragments, the supply vector (IV) pYL322d2-sAtOR ^H plasmid containing the sAtOR ^H gene expression cassette was obtained (as shown in Figure 1).

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 method of one-step assembly of multiple fragments of the plasmid vector, the supply vector (V) pYL322d1-sAtDXS plasmid containing the sAtDXS gene expression cassette was obtained (as shown in Figure 1).

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, and using the principle of Gibson assembly, according to the method of one-step assembly of multiple fragments of the plasmid vector, the supply vector (VI) pYL322d2-sCsCCD2L plasmid containing the sCsCCD2L gene expression cassette is obtained (as shown in Figure 1).

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 sCsUGT91P3 gene expression cassette supply vector pYL322d1-sCsUGT91P3; 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 containing the sCsUGT74AD1 and sCsUGT91P3 gene expression cassettes (as shown in Figure 1).

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 homologous sequences on both sides, and using the principle of Gibson assembly, according to the method of one-step assembly of plasmid vector multiple fragments, the supply vector (VIII) pYL322d2-sCsALDH3I1 plasmid containing the CsALDH3I1 gene expression cassette is obtained (as shown in Figure 1).

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

The assembly of the plant transformation vector pYLTAC380MF-DOBhPC-CsCAU ₁ U ₂ for rice endosperm-specific crocin synthesis containing the optimized CrtI, PSY, BHY, OR ^H , DXS genes and the saffron-derived CsCCD2L, CsUGT74AD1, CsUGT91P3, and CsALDH3I1 genes is as follows:

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.

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

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 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 sEuCrtI gene expression cassette.

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 karatomycin and ampicillin double-resistance plate, 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 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.

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.

Assembly of sAtOR ^H gene expression cassette: Mix the supply vector (IV) pYL322d2-sAtOR ^H plasmid and the receiving vector pYLTAC380GW-BhPC plasmid, 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 the supply vector plasmid recombination and its backbone deletion. Wash the mixed colonies on the double-resistance plates and extract the plasmids, then use PI-SceI to eliminate the unrecombined plasmids, transform DH10B, and identify on karatomycin plates to obtain the receiving vector pYLTAC380GW-OBhPC plasmid containing sEuCrtI+sZmPSY+sHpBHY+sAtOR ^H gene expression cassette.

Assembly of sAtDXS gene expression cassette: The supply vector (V) pYL322d1-sAtDXS plasmid and the receiving vector pYLTAC380GW-OBhPC plasmid were mixed, and the competent cells of Escherichia coli strain NS3529 expressing Cre enzyme were transformed by electroporation together. 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 non-recombinant plasmids were eliminated by I-SceI enzyme digestion, DH10B was transformed and the receiving vector pYLTAC380GW-DOBhPC plasmid containing sEuCrtI+sZmPSY+sHpBHY+sAtOR ^H +sAtDXS gene expression cassette was obtained by identification on karatomycin plates.

(2) Construction of plant transformation vector pYLTAC380MF-DOBhPC-CsCAU ₁ U ₂ for rice endosperm-specific crocin synthesis:

The sCsCCD2L, sCsUGT74AD1, sCsUGT91P3 and sCsALDH3I1 gene expression cassettes from saffron were superimposed on the plant transformation vector pYLTAC380GW-DOBhPC for synthesizing zeaxanthin precursor to construct pYLTAC380GW-DOBhPC-CsCAU ₁ U ₂ . 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-CsCAU ₁ U ₂ which can realize the self-deletion of the hygromycin resistance selection marker.

Assembly of sCsCCD2L gene expression cassette: The supply vector (VI) pYL322d2-sCsCCD2L plasmid was mixed with the receiving vector pYLTAC380GW-DOBhPC plasmid containing 5 gene expression cassettes, and the NS3529 competent cells were transformed by electroporation. The transformants were screened on the karatomycin and ampicillin double-resistance plates, and the Cre enzyme endogenously expressed in NS3529 was used to achieve the supply vector plasmid recombination and the supply vector backbone deletion. The mixed colonies on the double-resistance plates were washed and the mixed plasmids were extracted. After the unrecombined plasmids were cut with PI-SceI, they were transformed into Escherichia coli DH10B, and the receiving vector pYLTAC380GW-DOBhPC-CsC plasmid containing sEuCrtI+sZmPSY+sHpBHY+sAtOR ^H +sAtDXS+sCsCCD2L gene expression cassettes was identified on the karatomycin plates.

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 unrecombined plasmids were eliminated by I-SceI enzyme digestion, DH10B was transformed, and the plasmids containing sEuCrtI+sZmPSY+sHpBHY+sAtOR ^H +sAtDXS+ were identified on karatomycin plates.

The recipient vector of the sCsCCD2L+sCsUGT74AD1+sCsUGT91P3 gene expression cassette is pYLTAC380GW-DOBhPC-CsCU ₁ U ₂ plasmid.

Assembly of sCsALDH3I1 gene expression cassette: The supply vector (VIII) pYL322d2-sCsALDH3I1 plasmid was mixed with the receiving vector pYLTAC380GW-DOBhPC-CsCU ₁ U ₂ plasmid containing 8 gene expression cassettes, and the NS3529 competent cells were transformed by electroporation. The transformants were selected on karatomycin and ampicillin double-resistant plates, and the Cre enzyme endogenously expressed in NS3529 was used to achieve supply vector plasmid recombination and supply vector backbone deletion. The mixed colonies on the double-resistance plate were washed and the mixed plasmids were extracted. The non-recombinant plasmids were cut with PI-SceI and transformed into Escherichia coli DH10B. The positive transformants were screened on karatomycin plates, and the plant transformation vector pYLTAC380GW-DOBhPC-Cs CAU ₁ U ₂ containing the sEuCrtI+sZmPSY+sHpBHY+sAtOR ^H +sAtDXS+sCsCCD2L+sCsUGT74AD1+sCsUGT91P3+sCsALDH3I1 gene expression cassette was identified. The schematic diagram of its structure is shown in Figure 2.

Assembly of marker-free elements: Incubate 5×BP Clonase II enzyme mix (ThermoFisher) on ice for about 2 minutes, vortex and centrifuge briefly. Mix the marker-free element donor vector pYLMF-H with the element vector to be inserted pYLTAC380GW-DOBhPC-CsCAU ₁ U ₂ , 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.

The result of enzyme digestion detection of the plant transformation vector pYLTAC380MF-DOBhPC-CsCAU ₁ U ₂ is shown in FIG2 . As can be seen from FIG2 , the present invention obtains the plant transformation vector pYLTAC380MF-DOBhPC-CsCAU ₁ U ₂ containing the HPT gene.

S3. Transformation of rice with the plant transformation vector to obtain transgenic rice plant transformation vector pYLTAC380MF-DOBhPC-CsCAU ₁ U ₂ producing crocin in seed endosperm and detection:

Genetic transformation of rice: The plant transformation vector pYLTAC380MF-DOBhPC-CsCAU ₁ U ₂ plasmid was transferred into Agrobacterium EHA105 and used to transform rice embryo callus tissue. Specifically, immature or mature rice seeds are induced to callus at 25° C. in the dark; an appropriate amount of Agrobacterium transformed with a multi-gene vector plasmid is suspended in an infection liquid medium added with 100 μmol/L acetosyringone, and shake-cultured at 28° C. (200 rpm, 0.5 h); an OD ₅₅₀ value is adjusted to 0.3-0.4 using a spectrophotometer, and then the callus can be used for infection; fresh, light yellow, and vigorously growing granular embryonic callus is selected, mixed with Agrobacterium bacterial solution, and soaked for 20 minutes, and after the bacterial solution is dried, it is transferred to a co-culture medium, and after dark culture for 3 days, it is transferred to a screening medium containing 50 mg/L hygromycin, and subcultured once every 2 weeks for 2 times; after resistance screening, the resistant callus with green dots is transferred to a differentiation medium, and transformed seedlings are differentiated to obtain transformed plants, i.e., transgenic rice.

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

Infection medium: 10×MS _macro stock solution 100mL, 1000×B5 _micro stock solution 1mL, 100×B5 _vit stock solution 10mL, 2,4-D 2mg, hydrolyzed casein 500mg, inositol 2g, sucrose 30g, acetosyringone 100μmol, pH adjusted to 5.5, and supplemented to 1L with _ddH2O .

Co-culture medium: 10×MS _macro stock solution 100mL, 1000×B5 _micro stock solution 1mL, 100×B5 _vit stock solution 10mL, 2,4-D 2mg, hydrolyzed casein 500mg, inositol 2g, sucrose 30g, acetosyringone 100μmol, agar 8g, pH adjusted to 5.5, and ddH ₂ O supplemented to 1L.

Screening medium: 10×N6 _macro stock solution 100mL, 1000×B5 _micro stock solution 1mL, 100×B5 _vit stock solution 10mL, 2,4-D 2mg, hydrolyzed casein 300mg, L-proline 500mg, L-glutamine 500mg, sucrose 30g, agar 8g, pH adjusted to 5.8, supplemented to 1L with ddH ₂ O;

After high temperature sterilization, cool and add 1 mL each of 1000× ceftriaxone sodium (cef), 1000× carbenicillin (carb), and 1000× hygromycin (Hm). Differentiation medium: 100 mL of 10×N6 _macro stock solution, 1 mL of 1000×MS _micro stock solution, 10 mL of 100×B5 _vit stock solution, 3 mg of BA, 1 mg of NAA, 18.2 g of sorbitol, 20 g of sucrose, 8 g of agar, pH adjusted to 5.8, and ddH ₂ O added to 1 L.

PCR detection of transgenic rice genome: The leaves of the _T0 transgenic plants were extracted by SDS method and used as templates. The 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.

Table 1 Primers used for PCR detection of transgenic rice genome

The amplification program used was: 94°C pre-denaturation for 4 min; 94°C denaturation for 30 sec, 58°C annealing for 30 sec, 72°C extension for 30 sec, for a total of 30 cycles; and finally 72°C extension for 5 min. The PCR detection diagram of the foreign gene introduced into the genomic DNA of the T ₀ rice transformant of pYLTAC380MF-DOBhPC-CsCAU ₁ U ₂ is shown in Figure 3. As can be seen from Figure 3, the wild type (WT) control could not amplify the foreign gene, and the transgenic positive plants could amplify the above gene.

Example 2

The present invention uses zeaxanthin precursor synthesis-related genes CrtI, PSY, BHY, ^ORH , DXS and gardenia-derived GjCCD4a, GjUGT94E13, GjUGT74F8, and GjALDH2C3 genes as templates, optimizes the above genes according to rice codon preference, combines the optimized zeaxanthin precursor synthesis-related genes with genes from gardenia to construct a plant transformation vector and transforms rice, thereby realizing the de novo synthesis of crocin in rice endosperm.

Specifically, the zeaxanthin precursor synthesis-related genes CrtI, PSY, BHY, OR ^H and DXS are those described in Example 1.

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

S1. The five genes CrtI, PSY, BHY, ^ORH , and DXS after sequence optimization were fused with the GjCCD4a, GjUGT94E13, GjUGT74F8, and GjALDH2C3 genes from Gardenia jasminoides respectively with the rice seed endosperm expression promoter, among which CrtI and BHY genes were also fused with the plastid transit peptide to construct the corresponding gene expression cassette.

The details are as follows:

1. Synthesis of nine key gene coding regions:

The five genes CrtI, PSY, BHY, OR ^H and DXS were used as templates respectively, and codons were optimized using the Codon Optimization Tool program according to the codon preference of rice. The optimized gene sequences were synthesized and named sEuCrtI (the optimized CrtI gene sequence cited from CN105907780B), sZmPSY (the optimized PSY gene sequence cited from CN105907780B), sHpBHY (the optimized BHY gene sequence cited from CN105907780B), sAtOR ^H (SEQ ID NO: 1), and sAtDXS (SEQ ID NO: 2), cloned into plasmid vectors, and their sequences were determined by sequencing.

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.

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

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.

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

The construction of the supply vector containing the sEuCrtI, sZmPSY, sHpBHY, sAtOR ^H and sAtDXS gene expression cassettes is the same as in Example 1.

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, and using the principle of Gibson assembly, according to the method of one-step assembly of plasmid vector multiple fragments, the supply vector (IX) pYL322d2-sGjCCD4a plasmid containing the GjCCD4a gene expression cassette was obtained (as shown in Figure 1).

Construction of the donor vector pYL322d1-GjUGT94E13-2A-GjUGT74F8 containing the sGjUGT94E13 and sGjUGT74F8 fusion 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, 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 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 (X) pYL322d1-GjUGT94E13-2A-GjUGT74F8 plasmid containing GjUGT94E13 and GjUGT74F8 gene expression cassettes was obtained (as shown in Figure 1).

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 homologous sequences on both sides. Using the principle of Gibson assembly and the method of one-step assembly of plasmid vector multiple fragments, the supply vector (XI) pYL322d2-GjALDH2C3 containing the GjALDH2C3 gene expression cassette was obtained (as shown in Figure 1).

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

The assembly of the plant transformation vector pYLTAC380MF-DOBhPC-CsCAU ₁ U ₂ for rice endosperm-specific crocin synthesis containing the optimized CrtI, PSY, BHY, OR ^H , DXS genes and the Gardenia-derived GjCCD4a, GjUGT94E13, GjUGT74F8, and GjALDH2C3 genes is as follows:

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

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

(2) Construction of plant transformation vector pYLTAC380MF-DOBhPC-CsCAU ₁ U ₂ : 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-GjCAU ₁ U ₂ 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-GjCAU ₁ U ₂ that can achieve self-deletion of the hygromycin resistance selection marker.

Assembly of sGjCCD4a gene expression cassette: The supply vector (IX) pYL322d2-sGjCCD4a plasmid was mixed with the receiving vector pYLTAC380GW-DOBhPC plasmid containing 5 gene expression cassettes, and transformed into NS3529 competent cells by electroporation. 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 supply vector backbone deletion. The mixed colonies on the double-resistance plates were washed and the mixed plasmids were extracted. After the unrecombined plasmids were cut with PI-SceI, they were transformed into Escherichia coli DH10B, and the receiving vector pYLTAC380GW-DOBhPC-GjC plasmid containing sEuCrtI+sZmPSY+sHpBHY+sAtOR ^H +sAtDXS+sGjCCD4a gene expression cassettes was identified on karatomycin plates.

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 unrecombined plasmids were eliminated by I-SceI digestion, DH10B was transformed, and the cells containing sEuCrtI+sZmPSY+sHpBHY+sAtOR ^H + were identified on karatomycin plates.

The recipient vector of the sAtDXS+sGjCCD4a+sGjUGT94E13+sGjUGT74F8 gene expression cassette is pYLTAC380GW-DOBhPC-GjCU ₁ U ₂ plasmid.

Assembly of sGjALDH2C3 gene expression cassette: Mix the supply vector (XI) pYL322d2-sGjALDH2C3 plasmid with the receiving vector pYLTAC380GW-DOBhPC-GjCU ₁ U ₂ 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

The plant transformation vector pYLTAC380GW-DOBhPC-GjCA U ₁ U ₂ containing +sGjUGT74F8+sGjALDH2C3 has a schematic diagram of its structure as shown in FIG2 .

The assembly of the marker-free element is specifically shown in Example 1. The result of enzyme digestion identification of the plant transformation vector pYLTAC380GW-DOBhPC-GjCAU ₁ U ₂ is shown in FIG2 . As can be seen from FIG2 , the present invention obtains the plant transformation vector pYLTAC380GW-DOBhPC-GjCAU ₁ U ₂ .

S3. Transformation of rice with the plant transformation vector to obtain the transgenic rice multi-gene vector pYLTAC380MF-DOBhPC-GjCAU ₁ U ₂ that produces crocin in seed endosperm and detection:

Genetic transformation of rice: The multi-gene vector pYLTAC380MF-DOBhPC-GjCAU ₁ U ₂ plasmids were respectively transferred into Agrobacterium EHA105 for transformation of rice embryo callus; see Example 1 for details.

PCR detection of transgenic rice genome: The leaves of the _T0 transgenic plants were extracted by SDS method and used as templates. The exogenous HPT, sEuCrtI, sZmPSY, sHpBHY, sAtOR ^H , sAtDXS, sGjCCD4a, sGjUGT94E13, sGjUGT74F8 and sGjALDH2C3 genes were detected by PCR amplification method. The primers used to detect the exogenous HPT, sEuCrtI, sZmPSY, sHpBHY, sAtOR ^H and sAtDXS genes were the same as those in Example 1, and the primers used to detect the exogenous sGjCCD4a, sGjUGT94E13, sGjUGT74F8 and sGjALDH2C3 genes were shown in Table 2.

Table 2 Primers used for PCR detection of exogenous genes

The amplification procedure used was the same as in Example 1. The PCR detection of the exogenous gene in the genomic DNA of the _T0 rice transformant of pYLTAC380MF-DOBhPC-GjCAU ₁ U ₂ is shown in FIG3 . As can be seen from FIG3 , the wild-type (WT) control was unable to amplify the exogenous gene, and the transgenic positive plants were able to amplify the above gene. Example 3 Observation of the appearance of transgenic rice seeds and UPLC-MS/MS detection of crocin in the endosperm

Observation of the appearance of transgenic rice seeds: The phenotype of pYL380MF-DOBhPC-CsCAU ₁ U ₂ and pYL380MF-DOBhPC-GjCAU ₁ U ₂ transgenic rice brown rice was observed, and the results are shown in Figure 4. As shown in Figure 4, the seeds of pYL380MF-DOBhPC introduced with the synthetic zeaxanthin precursor and pYL380MF-DOBhPC-CsCAU ₁ U ₂ introduced with the saffron pathway were yellow, while the seeds of pYL380MF-DOBhPC-GjCAU ₁ U ₂ introduced with the gardenia pathway were orange-yellow.

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, and kept on ice in a dark place for 20 min; 250 μL of water was added and vortexed for 1 min; the aqueous phase and organic phase were collected by centrifugation at 4°C and 4000 rpm for 5 min, and the color of the extract was observed. The results are shown in Figure 4. As can be seen from Figure 4, although the pYL380MF-DOBhPC seeds are yellow, their aqueous phase is colorless and transparent, while the aqueous phases of pYL380MF-DOBhPC-CsCAU ₁ U ₂ and pYL380MF-DOBhPC-GjCAU ₁ U ₂ are obviously yellow, indicating that polar substances are produced in pYL380MF-DOBhPC-CsCAU ₁ U ₂ and pYL380MF-DOBhPC-GjCAU ₁ U ₂ ; relative to the yellow organic phase of DOBhPC, the organic phases of pYL380MF-DOBhPC-CsCAU ₁ U ₂ and pYL380MF-DOBhPC-GjCAU ₁ U ₂ are orange-red, indicating that additional fat-soluble pigments are produced.

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.

The results of UPLC-MS\MS detection of crocin in the endosperm of rice seeds transformed with pYLTAC380MF-DOBhPC-CsCAU ₁ U ₂ and pYLTAC380MF-DOBhPC-GjCAU ₁ U ₂ are shown in Figures 5 and 6. The results show that the transgenic rice seeds of pYL380MF-DOBhPC-CsCAU ₁ U ₂ and pYL380MF-DOBhPC-GjCA U ₁ U ₂ have the same characteristic peaks as the four crocin standards, indicating that crocin has been successfully synthesized in the two transgenic rice. The crocin content in the endosperm of rice seeds transformed with pYLTAC380MF-DOBhPC-CsCAU ₁ U ₂ and pYLTAC380MF-DOBhPC-GjCAU ₁ U ₂ was also detected. Among them, the crocin content in the rice endosperm transformed by pYL380MF-DOBhPC-CsCAU ₁ U ₂ was 587.82 ng/g; the crocin content in the rice endosperm transformed by pYL380MF-DOBhPC-GjCAU ₁ U ₂ was 855.4 ng/g.

The present invention also provides a schematic diagram of the pathway for synthesizing crocin in the endosperm of transgenic rice seeds, as shown in Figure 7. 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, and 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, and then dehydrogenated by aldehyde dehydrogenase (ALDH) to crocetin, and finally catalyzed by UDP-glucosyltransferase (UGT) to obtain crocin.

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 (10)

1. Use of five genes crti, PSY, BHY, OR ^H and DXS in combination with four genes CsCCD2L, csUGT AD1, csUGT91P3 and CsALDH I1 from saffron or with four genes GjCCD a, gjUGT94E13, gjUGT74F8 and GjALDH C3 from gardenia in transgenic breeding for the endosperm production of crocin in rice seeds; the specific method for the application comprises the following steps:

   Optimizing the sequences of the genes according to the preference of rice codons, respectively constructing gene expression cassettes of CrtI, PSY, BHY, OR ^H and DXS five genes with CsCCD L, csUGT AD1, csUGT91P3 and CsALDH I1 genes which are derived from saffron, constructing the constructed gene expression cassettes into plant transformation vectors, and transforming the plant transformation vectors into rice to obtain transgenic rice for producing crocin in seed endosperm;

   Or respectively constructing gene expression cassettes of CrtI, PSY, BHY, OR ^H genes and DXS five genes with GjCCD a, gjUGT94E13, gjUGT74F8 and GjALDH C3 genes which are derived from gardenia, constructing the constructed gene expression cassettes into plant transformation vectors, and transforming the plant transformation vectors into rice to obtain the transgenic rice for producing crocin in seed endosperm.
2. 2. A transgenic breeding method for producing crocin in rice seed endosperm, which is characterized by comprising the following steps:

   s1, optimizing CrtI, PSY, BHY, OR ^H and DXS five genes and CsCCD2L, csUGT AD1, csUGT91P3 and CsALDH I1 gene sequences from saffron according to rice codon preference, and constructing corresponding gene expression cassettes by utilizing the optimized sequences;

   s2, constructing the gene expression cassette in the step S1 into a plant transformation vector;

   s3, transforming the plant transformation vector in the step S2 into rice to obtain transgenic rice for producing crocin in seed endosperm;

   Or alternatively, the first and second heat exchangers may be,

   S1, optimizing CrtI, PSY, BHY, OR ^H and DXS five genes and GjCCD a, gjUGT94E13, gjUGT F8 and GjALDH C3 gene sequences from gardenia sources according to rice codon preference, and constructing corresponding gene expression cassettes by utilizing the optimized sequences;

   s2, constructing the gene expression cassette in the step S1 into a plant transformation vector;

   s3, transforming the plant transformation vector in the step S2 into rice to obtain the transgenic rice for producing crocin in seed endosperm.
3. 3. The method of claim 2, wherein the sequence of the sequence-optimized OR ^H and DXS genes is shown in SEQ ID NO. 1-2;

   the sequence of CsCCD2L, csUGT74AD1, csUGT91P3 and CsALDH I1 genes after sequence optimization are sequentially shown as SEQ ID NO 3-6;

   The sequences of GjCCD a, gjUGT94E13, gjUGT74F8 and GjALDH C3 genes with optimized sequences are shown in SEQ ID NO 7-10 in sequence.
4. 4. The method according to claim 2, wherein when the plant transformation vector is constructed by using gene expression cassettes of five genes and four genes derived from saffron, the sequence of each gene is as follows: crtI-PSY-BHY-OR ^H -DXS-CsCCD2L-CsUGT74AD1-CsUGT91P3-CsALDH I1.
5. 5. The method according to claim 2, wherein when the gene expression cassettes of the five genes and the four genes derived from gardenia after the sequence optimization are constructed into a plant transformation vector, the sequence of each gene is as follows: crtI-PSY-BHY-OR ^H -DXS-GjCCD4a-GjUGT94E13-GjUGT74F8-GjALDH2C3.
6. 6. The method of claim 2, wherein the method of transforming rice with the plant transformation vector is agrobacterium-mediated.
7. 7. The method of claim 2, wherein the plant transformation vector is constructed using a TGSII multiple gene vector system.
8. 8. The plant transformation vector containing optimized CrtI, PSY, BHY, OR ^H genes and DXS five genes and saffron-derived CsCCD2L, csUGT AD1, csUGT91P3 and CsALDH I1 genes constructed in the method as described in claim 4.
9. 9. The plant transformation vector comprising the optimized CrtI, PSY, BHY, OR ^H and DXS five genes and the GjCCD a, gjUGT94E13, gjUGT F8 and GjALDH C3 genes derived from gardenia constructed in the method of claim 5.
10. 10. Use of the plant transformation vector of claim 8 or 9 in constructing transgenic rice for the production of crocin in seed endosperm.