CN118546994B - A transgenic breeding method for producing crocin in seed endosperm - Google Patents
A transgenic breeding method for producing crocin in seed endosperm Download PDFInfo
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Abstract
The invention discloses a transgenic breeding method for producing crocin in seed endosperm. According to the rice codon preference, the invention optimizes the related genes CrtI, PSY, BHY, OR H for synthesizing the zeaxanthin precursor, DXS, csCCD L, csUGT AD1 and CsUGT91P3 and CsALDH I1 genes from saffron and GjCCD a and GjUGT94E13, gjUGT F8 and GjALDH C3 genes from gardenia, combines the optimized related genes for synthesizing the zeaxanthin precursor with the saffron source genes or the gardenia source genes respectively to construct plant transformation vectors and transform rice, so as to obtain transgenic rice capable of producing the crocin in rice seed endosperm, realize the synthesis of the crocin rice seed endosperm, and can be directly eaten or used as a crocin production raw material, thereby being beneficial to the production of the crocin.
Description
Technical Field
The present invention relates to the field of genetic engineering. In particular to a transgenic breeding method for producing crocin in seed endosperm.
Background
Crocin (Crocins) is also called crocin, is a main medicinal active ingredient of rare traditional Chinese medicinal materials crocin and gardenia, belongs to apocarotenoid glycoside compounds, and comprises 5 different glycosylation forms (crocin-I, crocin-II, crocin-III, crocin-IV and crocin-V). Researches show that crocin has better curative effects on various central nervous system and cardiovascular system diseases, and also has the effects of resisting cancer, resisting inflammation, resisting oxidization, protecting liver, benefiting gallbladder, resisting diabetes and the like. Besides medicinal value, crocin has long been used as perfume, dye and food additive, and has wide application value.
In nature, crocin is mainly extracted from stigma croci, while triploid stigma croci has low reproduction rate, and is known as red gold because of complex stigma picking process, low yield (1 g of dry stigma croci is composed of about 150 stigma croci) and high labor cost. In addition, crocin has a complex structure and rich chiral centers, and is difficult to chemically synthesize. Therefore, there is a need to provide a relatively simple and efficient method for producing crocin.
The use of biosynthesis is a novel production route. 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, chinese patent publication No. CN105907780A discloses a transgenic breeding method for producing astaxanthin in rice seed endosperm, and the astaxanthin synthesis related gene is expressed in rice seed endosperm to construct transgenic rice for synthesizing astaxanthin, and the technology has the advantages of high yield, low cost, high safety and the like. However, the synthesis of crocin in rice endosperm has not been reported.
Since most enzymes are low or even non-expressed in rice endosperm, crocin synthesis involves multiple genes. Therefore, how to synthesize crocin from the head in endosperm by genetic engineering and synthetic biology methods is a very valuable scientific problem to be studied. The synthesis of crocin, however, involves multiple genes, which makes the objective of producing crocin in plants or microorganisms by genetic engineering methods difficult to achieve. In addition, there is a great uncertainty whether the genetic background of rice seeds is suitable for inserting and expressing crocin synthetic genes and whether crocin can be successfully expressed and produced.
Disclosure of Invention
The invention provides a transgenic breeding method for producing crocin in seed endosperm, which aims at the defects of the prior art.
The first object of the present invention is to provide the use of CrtI, PSY, BHY, OR H and DXS five genes in combination with four genes of CsCCD2L, csUGT AD1, csUGT91P3 and CsALDH I1 derived from saffron or with four genes of GjCCD a, gjUGT94E13, gjUGT74F8 and GjALDH C3 derived from gardenia in transgenic breeding for the endosperm production of crocin in rice seed.
A second object of the present invention is to provide a transgenic breeding method for producing crocin in rice seed endosperm.
A third object of the present invention is to provide plant transformation vectors containing optimized CrtI, PSY, BHY, OR H, DXS five genes and saffron-derived CsCCD2L, csUGT AD1, csUGT91P3, csALDH I1 genes.
A fourth object of the present invention is to provide plant transformation vectors containing optimized CrtI, PSY, BHY, OR H, DXS five genes and Gardenia-derived GjCCD a, gjUGT94E13, gjUGT74F8, gjALDH C3 genes.
A fifth object of the present invention is to provide the use of said plant transformation vector for constructing transgenic rice capable of producing crocin in seed endosperm.
The above object of the present invention is achieved by the following technical scheme:
The invention takes the corn gluten precursor synthesis related genes CrtI, PSY, BHY, OR H, DXS, saffron-derived CsCCD2L, csUGT AD1, csUGT91P3, csALDH I1 genes and gardenia-derived GjCCD a, gjUGT94E13, gjUGT F8 and GjALDH C3 genes as templates, optimizes the genes according to rice codon preference, respectively combines the optimized corn gluten precursor synthesis related genes with saffron-derived genes or gardenia-derived genes to construct plant transformation vectors and converts the plant transformation vectors into rice, so as to obtain transgenic rice capable of producing crocin rice seed endosperm and realize the head-from synthesis of crocin rice endosperm.
Therefore, the invention claims the application of CrtI, PSY, BHY, OR H and DXS five genes to transgenic breeding for producing crocin from rice seed endosperm by combining the five genes with CsCCD2L, csUGT AD1, csUGT91P3 and CsALDH I1 genes from saffron or combining the five genes with GjCCD a, gjUGT94E13, gjUGT74F8 and GjALDH C3 genes from gardenia, wherein the specific method of the application is as follows:
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.
The invention also provides a transgenic breeding method for producing crocin in rice seed endosperm, which comprises 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 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.
Specifically, according to rice codon preference, optimized zeaxanthin precursor synthesis related genes OR H and DXS genes are sequentially marked as sAtOR H and sAtDXS, optimized sequences are sequentially marked as SEQ ID NO. 1-2, optimized crocin synthesis related genes CsCCD2L, csUGT AD1, csUGT91P3 and CsALDH I1 are sequentially marked as sCsCCD2L, sCsUGT AD1, sCsUGT91P3 and sCsALDH I1, optimized sequences are sequentially marked as SEQ ID NO. 3-6, optimized crocin synthesis related genes GjCCD a, gjUGT94E13, gjUGT F8 and GjALDH C3 genes are sequentially marked as sGjCCD a, sGjUGT94E13, sGjUGT F8 and sGjALDH C3, and optimized sequences are sequentially marked as SEQ ID NO. 7-10.
Specifically, when the CrtI gene or BHY gene expression cassette is constructed, a plastid transit peptide is linked between the promoter and the optimized gene sequence.
Specifically, the plastid transit peptide is TP, and the sequence of the plastid transit peptide is shown as SEQ ID NO. 11.
In a specific embodiment of the invention, in order to reduce the assembly times or reduce the vector size when constructing the plant transformation vector, the invention constructs the optimized CsUGT74AD1 and CsUGT91P3 gene expression cassettes on the same donor vector. And connecting the optimized GjUGT E13 and GjUGT F8 genes in series by using connecting peptide, constructing the genes in the same gene expression cassette, and constructing a plant transformation vector by using the gene expression cassette.
Specifically, the connecting peptide is 2A peptide, and the coding sequence F2A of the 2A peptide is shown as SEQ ID NO. 12.
Specifically, the gene expression cassette contains a promoter and a terminator of rice endosperm specific storage protein genes.
Specifically, the promoter of the rice endosperm specific storage protein gene comprises Pens 1-Pens, and the GenBank No of the promoter is AY427571.1, AY427575.1, EU264107.1, MH748577.1, EU264106.1, CP132236.1, AY427572.1, AY427574.1 and CP141114.1 in sequence.
Specifically, pens (GenBank No. AY 427571.1) was used as the promoter in the sEuCrtI gene expression cassette, caMV 35S terminator T35S (SEQ ID NO: 13), pens2 (GenBank No. AY 427575.1) was used as the promoter in the sZmPSY gene expression cassette, agropine synthase terminator Tags (SEQ ID NO: 14), pens3 (GenBank No. EU264107.1) was used as the promoter in the sHpBHY gene expression cassette, manopine synthase terminator Tmas (SEQ ID NO: 15) was used as the promoter in the sAtOR H gene expression cassette, pens (GenBank No. MH748577.1) was used as the promoter in the manopine synthase terminator Tmas, pens (GenBank No. EU264106.1) was used as the promoter in the sAtDXS gene expression cassette, and octopine synthase terminator Tocs (SEQ ID NO: 16) was used as the promoter.
Specifically, the promoter used in the sCsCCD2L gene expression cassette was Pens6 (GenBank No. CP132262.1), the terminator used was nopaline synthase terminator Tnos (SEQ ID NO: 17), the promoter used in the sCsUGT74AD1 gene expression cassette was Pens7 (GenBank No. AY 427572.1), the terminator used was TGluA-2 (SEQ ID NO: 18), the promoter used in the sCsUGT91P3 gene expression cassette was Pens8 (GenBank No. AY 427574.1), the terminator used was TGluA-1 (SEQ ID NO: 19), the terminator used in the sCsALDH I1 gene expression cassette was Pens9 (GenBank No. CP141114.1), and the terminator used was TGluB4 (SEQ ID NO: 20).
Specifically, the promoter used in the sGjCCD a gene expression cassette is Pens6, the terminator used is nopaline synthase terminator Tnos, the promoter used in the sGjUGT E13 and sGjUGT F8 gene expression cassettes is Pens8, 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 with optimized sequences and four genes derived from saffron are built into plant transformation vectors, the sequence of each gene is as follows:
CrtI-PSY-BHY-ORH-DXS-CsCCD2L-CsUGT74AD1-CsUGT91P3-CsALDH3I1。
specifically, when the gene expression cassettes of the five genes with optimized sequences and the four genes from the gardenia are built into a plant transformation vector, the sequence of each gene is as follows:
CrtI-PSY-BHY-ORH-DXS-GjCCD4a-GjUGT94E13-GjUGT74F8-GjALDH2C3。
specifically, the method for transforming rice in step S3 is an Agrobacterium-mediated method. Specifically, a plant transformation vector is firstly transferred into agrobacterium, and then callus of rice is transformed to obtain transgenic rice.
Specifically, TGSII multiple gene vector systems were used in constructing the plant transformation vectors.
The invention also claims a vector containing any one of the genes shown in SEQ ID NO 1-10.
The invention also claims plant transformation vectors containing optimized CrtI, PSY, BHY, OR H and DXS five genes and saffron-derived CsCCD2L, csUGT AD1, csUGT91P3 and CsALDH I1 genes constructed in the method.
Specifically, in the plant transformation vector, the sequence of each gene is as follows:
CrtI-PSY-BHY-ORH-DXS-CsCCD2L-CsUGT74AD1-CsUGT91P3-CsALDH3I1。
The invention also claims plant transformation vectors containing optimized CrtI, PSY, BHY, OR H genes and DXS five genes and GjCCD a, gjUGT94E13, gjUGT74F8 and GjALDH2C3 genes which are derived from gardenia and constructed in the method.
Specifically, in the plant transformation vector, the sequence of each gene is as follows:
CrtI-PSY-BHY-ORH-DXS-GjCCD4a-GjUGT94E13-GjUGT74F8-GjALDH2C3。
Optionally, the vector backbone used to construct the plant transformation vector is pYLTAC to 380GW.
The invention also claims the application of the plant transformation vector in the transgenic rice breeding for producing crocin from rice seed endosperm.
Compared with the prior art, the invention has the following beneficial effects:
The invention uses the corn gluten precursor synthesis related genes CrtI, PSY, BHY, OR H, DXS, saffron-derived CsCCD2L, csUGT AD1, csUGT91P3, csALDH I1 genes and gardenia-derived GjCCD a, gjUGT94E13, gjUGT F8 and GjALDH C3 genes as templates, optimizes the genes according to rice codon preference, combines the optimized corn gluten precursor synthesis related genes with saffron-derived genes or gardenia-derived genes respectively to construct plant transformation vectors and transforms rice, and obtains transgenic rice capable of producing crocin rice seed endosperm. The invention realizes the synthesis of crocin in rice seed endosperm, and the product can be directly eaten or used as raw material for producing crocin, is beneficial to the production of crocin, and overcomes the defects of complex process, low yield, difficult chemical synthesis and the like existing in the prior art of extracting crocin by using crocin stigma.
The invention solves the problem of the lack of the traditional sources of crocin, provides a crocin bioreactor, creates and obtains a new crop germplasm for producing high crocin content, explores the difference of enzyme systems derived from crocin and gardenia in the function of heterologously synthesizing crocin, and provides a new thought and important basis for the development of new varieties of more functional crocin crops.
Drawings
FIG. 1 is a schematic diagram showing the structure of a gene expression cassette constructed using the optimized 11 genes according to the present invention on a supply vector.
FIG. 2 shows a schematic structure of a plant transformation vector constructed in a polygene assembly process and a cleavage detection result of a related vector, wherein A in the figure shows a schematic structure of a plant transformation vector pYLTAC MF-DOBhPC-CsCAU 1U2 and a Not I cleavage detection result of the related vector, B in the figure shows a schematic structure of a plant transformation vector pYLTAC MF-DOBhPC-GjCAU 1U2 and a Not I cleavage detection result of the related vector, an arrow represents a target gene expression cassette obtained after Not I cleavage, and HPT of a T-DNA region is a hygromycin resistant gene.
FIG. 3 shows PCR detection of foreign genes in T 0 generation transformed rice genomic DNA into which plant transformation vectors pYLTAC MF-DOBhPC-CsCAU 1U2 and pYLTAC MF-DOBhPC-GjCAU 1U2 are introduced, CK + is plant transformation vector pYLTAC MF-DOBhPC-CsCAU 1U2 or pYLTAC MF-DOBhPC-GjCAU 1U2, and WT is wild type control genomic DNA.
FIG. 4 is a color chart of the appearance and extract of pYLTAC MF-DOBhPC of the synthetic zeaxanthin precursor and pYLTAC MF-DOBhPC-CsCAU 1U2、pYLTAC380MF-DOBhPC-GjCAU1U2 of the synthetic crocin transgenic rice brown rice, wild WT being the acceptor rice variety Huaguang (HG), yellow markers representing maize yellow precursor related genes, red markers representing crocin-derived genes, purple markers representing gardenia-derived genes.
FIG. 5 shows the result of UPLC-MS/MS detection of crocin in pYLTAC MF-DOBhPC-CsCAU 1U2 transformed rice seed endosperm.
FIG. 6 shows the result of UPLC-MS/MS detection of crocin in pYLTAC MF-DOBhPC-GjCAU 1U2 transformed rice seed endosperm.
FIG. 7 is a schematic diagram showing the pathway of crocin synthesis in endosperm of transgenic rice seed constructed in the invention, wherein the small black arrow in the figure represents a possible reaction in the endosperm of rice, the arrow in dotted line represents a definite absence reaction in the endosperm of rice, the arrow in red bold line represents a reaction catalyzed by an enzyme introduced into the expression of a gene related to carotenoid synthesis, and the arrow in red dotted line represents a reaction catalyzed by an enzyme introduced into the expression of a gene related to crocin synthesis.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Example 1
The invention respectively takes corn zeaxanthin precursor synthesis related genes CrtI, PSY, BHY, OR H, DXS and saffron-derived CsCCD2L, csUGT AD1, csUGT91P3 and CsALDH I1 genes as templates, optimizes the genes according to rice codon preference, combines the optimized corn zeaxanthin precursor synthesis related genes with saffron-derived genes to construct plant transformation vectors and converts the plant transformation vectors into rice, and realizes de novo synthesis of crocin rice endosperm.
Wherein the CrtI gene is CrtI gene of Erwinia (Erwinia uredovora), the GenBank No of the gene is D90087, the PSY gene is PSY gene of corn, the GenBank No of the gene is U32636.1, the BHY gene is BHY gene of Haematococcus pluvialis (Haematococcus pluvialis), the GenBank No of the gene is BD250390.1, the OR H gene and the DXS gene are both derived from Arabidopsis thaliana (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 invention also establishes a transgenic breeding method for producing crocin in rice seed endosperm, which comprises 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;
The method comprises the following steps:
1. Synthesis of nine key gene coding regions:
The CrtI, PSY, BHY, OR H genes and the DXS genes are respectively used as templates, codons are optimized by utilizing a Codon Optimization Tool program according to rice codon preference, optimized gene sequences are synthesized, and are sequentially named as sEuCrtI (from an optimized CrtI gene sequence in CN 105907780B), sZmPSY (from an optimized PSY gene sequence in CN 105907780B), sHpBHY (from an optimized BHY gene sequence in CN 105907780B), sAtOR H (shown as SEQ ID NO: 1) and sAtDXS (shown as SEQ ID NO: 2), cloned into a plasmid vector, and the sequences are determined by sequencing.
The genes CsCCD L (GenBank No. KP887110), csUGT74AD1 (GenBank No. MF596166), csUGT91P3 (GenBank No. MZ 190170) and CsALDH3I1 (GenBank No. MF596165) which are derived from saffron (Crocus sativus L) are respectively used as templates, codons are optimized by using Codon Optimization Tool program according to rice codon preference, optimized gene sequences are synthesized and obtained, the sequences are sequentially named sCsCCD2L, sCsUGT AD1, sCsUGT91P3 and sCsALDH I1, the DNA sequences of the sequences are sequentially shown as SEQ ID NO: 3-6, the sequences of the sequences are cloned into plasmid vectors, and the sequences are determined by sequencing.
2. Synthesis of plastid transit peptide coding sequence TP:
The sequence encoding plastid transit peptide TP (Transit peptide) (shown as SEQ ID NO: 11) was synthesized with reference to the sequence of the pea RbcS small subunit gene (GenBank No. X00806).
3. Construction of the Gene expression cassette on the supply vector:
Construction of supply vector pYL d1-sEuCrtI containing sEuCrtI Gene expression cassette A multiple gene supply vector I (pYL d 1) was reverse amplified (Lin et al, 2003; ZL 02134869.3) to obtain vector backbone fragment a, a rice endosperm specific storage protein promoter Pens (GenBank No. AY 427571.1) of about 1.5kb was amplified using rice genomic DNA as a template as fragment b, a plastid transit peptide sequence TP (SEQ ID NO: 11) was amplified as fragment c, sEuCrtI gene as fragment d, and CaMV 35S terminator T35S (SEQ ID NO: 13) from plasmid pCAMBIA1300 (GenBank No. A234296.1) as fragment e. The supply vector (I) pYL d1-sEuCrtI plasmid containing the sEuCrtI gene expression cassette was obtained by a one-step assembly of plasmid vector fragments (Zhu et al, 2014) using the principle of Gibson assembly (Gibson, 2011), with 25bp homologous sequences on each side of each fragment.
Construction of sZmPSY Gene expression cassette-containing supply vector pYL d2-sZmPSY A multiple gene supply vector II (pYL d 2) was amplified in reverse (Lin et al, 2003; ZL 02134869.3) to obtain vector backbone fragment a, plasmid pSAT7 (GenBank No. DQ5453) was used as template, agropine synthase terminator Tags (SEQ ID NO: 14) was amplified as fragment b, reverse sZmPSY gene was amplified as fragment c, rice endosperm-specific storage protein-1 kb promoter Pens2 (GenBank No. AY 427575.1) was amplified as fragment d, using rice genomic DNA as template. The two sides of each fragment are respectively provided with a 25bp homologous sequence, and the plasmid of the supply vector (II) pYL322d2-sZmPSY containing sZmPSY gene expression cassette is obtained by a plasmid vector multi-fragment one-step assembly method according to the principle of Gibson assembly (shown in figure 1).
Construction of sHpBHY Gene expression cassette-containing supply vector pYL d1-sHpBHY A multiple gene supply vector I (pYL d 322d 1) was reverse amplified to obtain vector backbone fragment a, a rice endosperm-specific storage protein-amplified promoter Pens3 (GenBank No. EU264107.1) of about 2.4kb was used as fragment b, a plastid transit peptide sequence TP (SEQ ID NO: 11) was amplified as fragment c, sHpBHY gene was amplified as fragment d, and manopine synthase terminator Tmas (SEQ ID NO: 15) was amplified from plasmid pSAT3 (GenBank No. DQ005465) as fragment e. The two sides of each fragment are respectively provided with a 25bp homologous sequence, and the plasmid of the supply vector (III) pYL322d1-sHpBHY containing sHpBHY gene expression cassette is obtained by a plasmid vector multi-fragment one-step assembly method according to the principle of Gibson assembly (shown in figure 1).
Construction of sAtOR H Gene expression cassette-containing supply vector pYL d2-sAtOR H A multigenic supply vector II (pYL d 2) was reverse amplified to give vector backbone fragment a, a manopine synthase terminator Tmas (SEQ ID NO: 15) was amplified from plasmid pSAT3 (GenBank No. DQ005465) as fragment b, a reverse sAtOR H gene was amplified as fragment c, and a rice endosperm-specific storage protein-about 2.4kb promoter Pens (GenBank No. MH748777.1) was amplified as fragment d using rice genomic DNA as a template. The two sides of each fragment are respectively provided with a 25bp homologous sequence, and the plasmid of the supply vector (IV) pYL322d2-sAtOR H containing sAtOR H gene expression cassette is obtained by a plasmid vector multi-fragment one-step assembly method according to the principle of Gibson assembly (shown in figure 1).
Construction of sAtDXS Gene expression cassette-containing supply vector pYL d1-sAtDXS A multiple gene supply vector I (pYL d 322d 1) was reverse amplified to obtain vector backbone fragment a, a rice endosperm-specific storage protein-amplified promoter Pens5 (GenBank No. EU264106.1) of about 2.3kb was used as fragment b, sAtDXS gene was amplified as fragment c, and octopine synthase terminator Tocs (SEQ ID NO: 16) was amplified from plasmid pSAT1 (GenBank No. DQ005461) as fragment d. The two sides of each fragment are respectively provided with a 25bp homologous sequence, and the plasmid of a supply vector (V) pYL322d1-sAtDXS containing sAtDXS gene expression cassette is obtained by a plasmid vector multi-fragment one-step assembly method according to the principle of Gibson assembly (shown in figure 1).
Construction of sCsCCD L Gene expression cassette-containing supply vector pYL d2-sCsCCD L A multiple gene supply vector II (pYL d 2) was reverse amplified to obtain vector backbone fragment a, a rice endosperm-specific storage protein-amplified promoter Pens6 (GenBank No. CP132, 136.) of about 1.9kb was used as fragment b, sCsCCD L gene was amplified as fragment c, and a nopaline synthase terminator Tnos (SEQ ID NO: 17) was amplified from plasmid pSAT2 (GenBank No. DQ005463) as fragment d, using rice genomic DNA as a template. The two sides of each fragment are respectively provided with a 25bp homologous sequence, and a plasmid Vector (VI) pYL d2-sCsCCD2L containing sCsCCD L gene expression cassette is obtained by a plasmid vector multi-fragment one-step assembly method according to the principle of Gibson assembly (as shown in figure 1).
Construction of supply vector pYL d1-sCsUGT74AD1-sCsUGT P3 containing sCsUGT AD1 and sCsUGT91P3 Gene expression cassettes A vector backbone fragment a was obtained by reverse amplification of the polygene supply vector II (pYL 322d 2), a promoter Pens7 (GenBank No. AY 427572.1) of about 0.9kb of rice endosperm-specific storage protein was amplified using rice genomic DNA as a template as fragment b, sCsUGT AD1 gene was amplified as fragment c, and a terminator TGluA-2 (SEQ ID NO: 18) of about 0.8kb of rice endosperm-specific storage protein was amplified using rice genomic DNA as a template as fragment d. The two sides of each fragment are respectively provided with 25bp homologous sequences, the principle of Gibson assembly is utilized, a plasmid vector multi-fragment one-step assembly method is utilized to obtain a vector pYL d2-sCsUGT AD1 containing sCsUGT AD1 gene expression cassette, a multi-gene supply vector I is reversely amplified to obtain a vector skeleton fragment a, a rice genome DNA (deoxyribonucleic acid) is taken as a template, a promoter Pens8 (GenBank No. AY 427574.1) of about 1kb of rice endosperm specific storage protein is amplified to be taken as a fragment b, a sCsUGT P3 gene is amplified to be taken as a fragment c, and a rice endosperm specific storage protein terminator TGluA-1 (SEQ ID NO: 19) of about 0.5kb is amplified to be taken as a fragment d. The method comprises the steps of respectively carrying out two sides of each fragment with 25bp homologous sequences, obtaining a vector pYL d1-sCsUGT P3 containing sCsUGT P3 gene expression cassette by utilizing the principle of Gibson assembly and according to a plasmid vector multi-fragment one-step assembly method, carrying out restriction enzyme digestion on the vector pYL d1-sCsUGT P3 by utilizing restriction enzyme SacI to obtain a vector skeleton fragment a, and amplifying sCsUGT AD1 gene expression cassettes with 25bp homologous sequences on two sides by taking plasmid pYL d2-sCsUGT AD1 as templates to obtain a fragment b. The vector for supplying sCsUGT AD1 and sCsUGT P3 gene expression cassettes (VII) pYL d1-sCsUGT AD1-sCsUGT91P3 plasmids (shown in figure 1) are obtained by a plasmid vector multi-segment one-step assembly method according to the principle of Gibson assembly.
Construction of supply vector pYL d2-CsALDH I1 containing CsALDH I1 Gene expression cassette A multiple gene supply vector II (pYL d 2) was reverse amplified to obtain vector backbone fragment a, a rice endosperm specific storage protein promoter Pens of about 0.7kb (GenBank No. CP141114.1) was amplified using rice genomic DNA as a template as fragment b, csALDH I1 gene was amplified as fragment c, and a rice endosperm specific storage protein terminator TGluB4 of about 0.5kb (SEQ ID NO: 20) was amplified using rice genomic DNA as a template as fragment d. The two sides of each fragment are respectively provided with a 25bp homologous sequence, and the plasmid Vector (VIII) pYL d2-sCsALDH3I1 containing CsALDH I1 gene expression cassette is obtained by a plasmid vector multi-fragment one-step assembly method according to the principle of Gibson assembly (shown in figure 1).
S2, constructing the gene expression cassette in the step S1 into a plant transformation vector
Plant transformation vector pYLTAC MF-DOBhPC-CsCAU 1U2 containing optimized CrtI, PSY, BHY, OR H and DXS five genes and saffron-derived CsCCD2L, csUGT AD1, csUGT91P3 and CsALDH I1 genes for rice endosperm specific synthesis of crocin is assembled as follows:
The plant transformation vector is assembled by utilizing a polygene vector system TGSII. The multi-gene vector system TGSII is composed of 1 receiving vector based on convertible artificial chromosome (TAC) and 2 gene-loaded supply vectors, and the Cre/loxP site-specific recombination method is utilized to make different supply vectors and receiving vectors carry out gene assembly for more than 2 rounds alternately, so as to construct plant transformation vectors.
(1) Construction of vector pYLTAC GW-DOBhPC for specific synthesis of zeaxanthin precursor from rice endosperm:
And sEuCrtI, assembling a gene expression cassette, namely mixing a supply vector (I) pYL322d1-sEuCrtI plasmid with a receiving vector pYLTAC380GW plasmid, electrically exciting and transforming competent cells of an escherichia coli strain NS3529 for expressing Cre enzyme, screening transformants on a double-resistant plate of calicheamicin and chloramphenicol, and utilizing the Cre enzyme endogenously expressed by NS3529 to realize the recombination of the supply vector plasmid and the deletion of a supply vector skeleton. And washing out the mixed colony mixed-drawing plasmid on the double-antibody plate, then carrying out enzyme digestion by using I-SceI to eliminate non-recombinant plasmid, then converting escherichia coli DH10B without Cre gene, and identifying on the caliamycin plate to obtain a receiving carrier pYLTAC GW-C plasmid containing sEuCrtI gene expression cassette.
And sZmPSY, assembling a gene expression cassette, namely mixing a supply vector (II) pYL d2-sZmPSY plasmid with a receiving vector pYLTAC380GW-C plasmid containing the sEuCrtI gene expression cassette, carrying out electric excitation transformation on NS3529 competent cells, screening transformants on a double-antibody plate of calicheamicin and ampicillin, and utilizing Cre enzyme endogenously expressed by NS3529 to realize recombination of the supply vector plasmid and deletion of a supply vector skeleton. And washing out the mixed colony mixed-extraction plasmid on the double-antibody plate, then cutting off the non-recombinant plasmid by using PI-SceI enzyme, converting the E.coli DH10B, and identifying on a caliamycin plate to obtain a receiving vector pYLTAC GW-PC plasmid containing sEuCrtI + sZmPSY gene expression cassette.
And sHpBHY, assembling a gene expression cassette, namely mixing a supply vector (III) pYL d1-sHpBHY plasmid with a receiving vector pYLTAC380GW-PC plasmid, performing co-electric excitation transformation on competent cells of an escherichia coli strain NS3529 expressing Cre enzyme, screening transformants on a caliamycin and chloramphenicol double-antibody plate, utilizing Cre enzyme expressed endogenously by NS3529 to realize recombination of the supply vector plasmid and deletion of a skeleton thereof, washing a mixed colony mixed-extraction plasmid on the double-antibody plate, performing I-SceI digestion to eliminate non-recombinant plasmids, converting DH10B, and identifying on the caliamycin plate to obtain the receiving vector pYLTAC GW-BhPC plasmid containing the sEuCrtI + sZmPSY + sHpBHY gene expression cassette.
And sAtOR H, assembling a gene expression cassette, namely mixing a supply vector (IV) pYL322d2-sAtOR H plasmid with a receiving vector pYLTAC380GW-BhPC plasmid, carrying out electric excitation transformation on NS3529 competent cells, screening transformants on a double-resistant plate of calicheamicin and ampicillin, and utilizing Cre enzyme endogenously expressed by NS3529 to realize the recombination of the supply vector plasmid and the deletion of a framework thereof. The mixed colony on the double-antibody plate is washed out, plasmid is mixed and extracted, and after PI-SceI enzyme digestion is used for eliminating non-recombinant plasmid, DH10B is transformed, and the receiving vector pYLTAC380GW-OBhPC plasmid containing sEuCrtI + sZmPSY + sHpBHY + sAtOR H gene expression cassette is obtained by identification on a caliamycin plate.
Assembling sAtDXS gene expression cassette, mixing supplied carrier (V) pYL322d1-sAtDXS plasmid with receiving carrier pYLTAC380GW-OBhPC plasmid, co-electrically exciting and transforming colibacillus strain NS3529 competent cell expressing Cre enzyme, screening transformant on double-resistant plate of caliamycin and chloramphenicol, utilizing Cre enzyme endogenously expressed by NS3529 to realize supplied carrier plasmid recombination and framework deletion, washing mixed colony on double-resistant plate to mix-extract plasmid, cutting with I-SceI to eliminate non-recombined plasmid, transforming DH10B, identifying and obtaining receiving carrier pYLTAC GW-DOBhPC plasmid containing sEuCrtI + sZmPSY + sHpBHY + sAtOR H + sAtDXS gene expression cassette on caliamycin plate.
(2) Construction of plant transformation vector pYLTAC MF-DOBhPC-CsCAU 1U2 for specific Synthesis of crocin from Rice endosperm:
The plant transformation vector pYLTAC MF-DOBhPC-CsCAU 1U2 capable of realizing self-deleting hygromycin screening Marker is constructed by superposing sCsCCD L, sCsUGT AD1, sCsUGT91P3 and sCsALDH3I1 gene expression cassettes from saffron on the basis of a plant transformation vector pYLTAC GW-DOBhPC for synthesizing a zeaxanthin precursor, constructing pYLTAC380GW-DOBhPC-CsCAU 1U2, and assembling Marker-free elements containing an HPT expression cassette through Gateway-BP recombination reaction.
Assembly of sCsCCD L Gene expression cassettes the supply Vector (VI) pYL d2-sCsCCD2L plasmid was mixed with the receiving vector pYLTAC GW-DOBhPC plasmid containing 5 gene expression cassettes, the NS3529 competent cells were transformed by electric excitation, transformants were selected on a double antibody plate of calicheamicin and ampicillin, and recombination of the supply vector plasmid and deletion of the supply vector backbone were achieved by using Cre enzyme endogenously expressed by NS 3529. The mixed colony on the double-antibody plate is washed out, the plasmid is subjected to mixed extraction, PI-SceI is used for enzyme digestion and cutting, then the non-recombinant plasmid is transformed into escherichia coli DH10B, and a receiving vector pYLTAC380GW-DOBhPC-CsC plasmid containing sEuCrtI + sZmPSY + sHpBHY + sAtOR H + sAtDXS + sCsCCD2L gene expression cassette is obtained by identification on a caliamycin plate.
The assembly of sCsUGT AD1 and sCsUGT P3 gene expression cassettes comprises mixing a feed Vector (VII) pYL322d1-sCsUGT AD1-sCsUGT91P3 plasmid with a receiving vector pYLTAC GW-DOBhPC-CsC plasmid containing 6 gene expression cassettes, co-electrically transforming competent cells of an escherichia coli strain NS3529 expressing Cre enzyme, screening transformants on a dual-antibody plate of calicheamicin and chloramphenicol, utilizing Cre enzyme endogenously expressed by NS3529 to realize the recombination of the feed vector plasmid and the deletion of the skeleton thereof, washing mixed colony on the dual-antibody plate to mix-extract plasmids, and then performing I-SceI digestion to eliminate non-recombined plasmids, transforming DH10B, and identifying on the calicheamicin plate to obtain the recombinant vector containing sEuCrtI + sZmPSY + sHpBHY + sAtOR H + sAtDXS +
The receiving vector pYLTAC GW-DOBhPC-CsCU 1U2 plasmid of the sCsCCD L+ sCsUGT74AD1+ sCsUGT P3 gene expression cassette.
Assembly of sCsALDH I1 Gene expression cassettes the supply Vector (VIII) pYL322d2-sCsALDH I1 plasmid was mixed with the receiving vector pYLTAC380GW-DOBhPC-CsCU 1U2 plasmid containing 8 gene expression cassettes, the competent cells of NS3529 were transformed by electric excitation, transformants were selected on a double antibody plate of calicheamicin and ampicillin, and recombination of the supply vector plasmid and deletion of the supply vector backbone were achieved by means of Cre enzyme endogenously expressed in NS 3529. Washing out mixed colony mixed-drawing plasmid on the double-antibody plate, cutting off non-recombinant plasmid by PI-SceI enzyme, transforming escherichia coli DH10B, screening positive transformant on the caliamycin plate, and identifying to obtain plant transformation vector pYLTAC380GW-DOBhPC-Cs CAU 1U2 containing sEuCrtI + sZmPSY + sHpBHY + sAtOR H + sAtDXS + sCsCCD L+ sCsUGT74 7AD 1+ sCsU GT91P3+ sCsALDH I1 gene expression cassette, the structure of which is shown in figure 2.
Assembly of Marker-free elements after incubation on ice for 5X BP Clonase IIenzyme mix (ThermoFisher) for about 2min, vortexing and brief centrifugation. The Marker-free element donor vector pYLMF-H is mixed with the element vector pYLTAC380GW-DOBhPC-CsCAU 1U2 to be inserted, proteinase K is added after the reaction for 6 hours at 25 ℃ and the reaction is stopped at 37 ℃ for 10 minutes, the reaction product is subjected to electric excitation to transform escherichia coli DH10B, positive transformants are screened on a kanamycin flat plate containing 5% sucrose (sacB is a sucrose lethal gene), and plasmids contained in the positive transformants are extracted for enzyme digestion identification.
As shown in FIG. 2, the result of the digestion of plant transformation vector pYLTAC MF-DOBhPC-CsCAU 1U2 shows that the plant transformation vector pYLTAC MF-DOBhPC-CsCAU 1U2 containing the HPT gene is obtained according to the present invention as shown in FIG. 2.
S3, transforming the plant transformation vector into rice to obtain transformation and detection of a transgenic rice plant transformation vector pYLTAC MF-DOBhPC-CsCAU 1U2 for producing crocin in seed endosperm:
Genetic transformation of Rice plant transformation vector pYLTAC MF-DOBhPC-CsCAU 1U2 plasmid is transferred into Agrobacterium EHA105 for transformation of rice embryo callus. The method comprises the steps of inducing callus of immature seeds or mature seeds of rice under a dark condition at 25 ℃, suspending agrobacterium transferred with polygenic vector plasmids in a proper amount of infection liquid culture medium added with 100 mu mol/L acetosyringone, performing shake culture at 28 ℃ (200 rpm,0.5 h), adjusting an OD 550 value to 0.3-0.4 by using a spectrophotometer, namely, dip-dying the callus, selecting granular embryogenic callus which is light yellow and has vigorous growth, mixing the granular embryogenic callus with agrobacterium liquid, soaking for 20min, transferring the granular embryogenic callus into a co-culture medium after drying the bacterial liquid, transferring the granular embryogenic callus into a screening culture medium containing 50mg/L hygromycin after dark culture for 3 days, transferring the granular embryogenic callus to a differentiation culture medium after 2 weeks of secondary culture for 2 times, and transferring the resistant callus with green spots to the differentiation culture medium after resistant screening, differentiating transformed seedlings, namely, obtaining transformed plants.
The components and the amounts of the culture medium are as follows:
10 XMS macro mother liquor 100mL,1000 XB 5 micro mother liquor 1mL,100 XB 5 vit mother liquor 10mL,2, 4-D2 mg, hydrolyzed casein 500mg, inositol 2g, sucrose 30g, acetosyringone 100. Mu. Mol, pH adjusted to 5.5, ddH 2 O make up to 1L.
Co-culture medium 10 XMS macro mother liquor 100mL,1000 XB 5 micro mother liquor 1mL,100 XB 5 vit mother liquor 10mL,2, 4-D2 mg, hydrolyzed casein 500mg, inositol 2g, sucrose 30g, acetosyringone 100. Mu. Mol, agar 8g, pH adjusted to 5.5, ddH 2 O make up to 1L.
Screening media 10 XN 6 macro mother liquor 100mL,1000 XB 5 micro mother liquor 1mL,100 XB 5 vit mother liquor 10mL,2, 4-D2 mg, hydrolyzed casein 300mg, L-proline 500mg, L-glutamine 500mg, sucrose 30g, agar 8g, pH adjusted to 5.8, ddH 2 O make up to 1L;
after high temperature sterilization, the mixture was cooled and 1000 Xceftriaxone sodium (cef), 1000 Xcarbenicillin (carb) and 1000 Xhygromycin (Hm) antibiotics were added 1mL each. Differentiation medium 10 XN 6 macro mother liquor 100mL,1000 XMS micro mother liquor 1mL,100 XB 5 vit mother liquor 10mL, BA3mg, NAA1mg, sorbitol 18.2g, sucrose 20g, agar 8g, pH adjusted to 5.8, ddH 2 O make up to 1L.
The PCR detection of transgenic rice genome includes extracting genome DNA from T 0 -generation transgenic plant leaf with SDS method as template, and PCR amplification to detect whether exogenous gene contains HPT, sEuCrtI, sZmPSY, sHpBHY, sAtOR H, sAtDXS, sCsCCD2L, sCsUGT AD1, sCsUGT91P3 and sCsALDH3I1 genes. The primers used for PCR detection of the genome of transgenic rice are shown in Table 1.
Table 1 primers for PCR detection of transgenic rice genome
The amplification procedure used was 94℃pre-denaturation for 4min, 94℃denaturation for 30sec,58℃fire off for 30sec,72℃extension for 30sec for 30 cycles, and finally 72℃extension for a further 5min. A PCR detection diagram of the foreign gene introduced into the genomic DNA of the T 0 generation rice transformant of pYLTAC MF-DOBhPC-CsCAU 1U2 is shown in FIG. 3. As can be seen from FIG. 3, none of the Wild Type (WT) controls can amplify the foreign gene and none of the transgenic positive plants can amplify the gene.
Example 2
The invention uses corn gluten precursor synthesis related genes CrtI, PSY, BHY, OR H and DXS and gardenia derived GjCCD a, gjUGT94E13, gjUGT F8 and GjALDH C3 genes as templates, optimizes the genes according to rice codon preference, combines the optimized corn gluten precursor synthesis related genes with gardenia derived genes to construct plant transformation vectors and transforms rice, and realizes de novo synthesis of crocin in rice endosperm.
Specifically, the zeaxanthin precursor synthesis-related genes CrtI, PSY, BHY, OR H and DXS are described in example 1.
The invention also establishes a transgenic breeding method for producing crocin in rice seed endosperm, which comprises the following steps:
S1, respectively fusing the CrtI, PSY, BHY, OR H genes and DXS five genes with GjCCD a, gjUGT94E13, gjUGT F8 and GjALDH C3 genes which are derived from gardenia and are subjected to sequence optimization with rice seed endosperm expression promoters, wherein CrtI and BHY genes are fused with plastid transit peptides, and constructing corresponding gene expression cassettes.
The method comprises the following steps:
1. Synthesis of nine key gene coding regions:
The five genes CrtI, PSY, BHY, OR H and DXS are respectively used as templates, codons are optimized by utilizing a Codon Optimization Tool program according to rice codon preference, optimized gene sequences are synthesized, and are named sEuCrtI (from the optimized CrtI gene sequence in CN 105907780B), sZmPSY (from the optimized PSY gene sequence in CN 105907780B), sHpBHY (from the optimized BHY gene sequence in CN 105907780B), sAtOR H (SEQ ID NO: 1) and sAtDXS (SEQ ID NO: 2) in sequence, cloned into a plasmid vector and the sequences are determined by sequencing.
The gene GjCCD a (GenBank No. KY 631925), gjUGT E13 (GenBank No. MN 944055), gjUGT F8 (GenBank No. MN 944054) and GjALDH C3 (GenBank No. KY 631926) derived from Gardenia jasminoides ellis (Gardenia jasminoides Ellis) are respectively used as templates, codons are optimized according to rice codon preference, optimized gene sequences are synthesized by using a Codon Optimization Tool program, the sequences are sequentially named as sGjCCD a, sGjUGT94E13, sGjUGT74F8 and sGjALDH C3, the sequences are sequentially shown as SEQ ID NO: 7-10, and the sequences are determined by cloning into plasmid vectors and sequencing.
2. Synthesis of plastid Transporter peptide coding sequence TP and 2A peptide coding sequence F2A:
The sequence encoding plastid transit peptide TP (Transit peptide) (SEQ ID NO: 11) 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 codon is optimized according to rice codon preference by utilizing Codon Optimization Tool program, and the F2A sequence shown in SEQ ID NO.12 is obtained through synthesis.
3. Construction of the Gene expression cassette on the supply vector:
Construction of supply vector containing sEuCrtI, sZmPSY, sHpBHY, sAtOR H, sAtDXS gene expression cassette was the same as in example 1.
Construction of donor vector pYL d2-sGjCCD a containing sGjCCD a Gene expression cassette A multiple gene supply vector II (pYL d 2) was reverse amplified to obtain vector backbone fragment a, a rice endosperm-specific storage protein-amplified promoter Pens6 (GenBank No. CP132, 136.1) of about 1.9kb was used as fragment b, gjCCD a gene was amplified as fragment c, and nopaline synthase terminator Tnos (SEQ ID NO: 17) was amplified from plasmid pSAT2 (GenBank No. DQ005463) as fragment d using rice genomic DNA as a template. The two sides of each fragment are respectively provided with 25bp homologous sequences, and the plasmid of the supply vector (IX) pYL d 322d2-sGjCCD4a containing GjCCD a gene expression cassette is obtained by a plasmid vector multi-fragment one-step assembly method according to the principle of Gibson assembly (shown in figure 1).
Construction of donor vector pYL d1-GjUGT E13-2A-GjUGT F8 containing sGjUGT E13 and sGjUGT F8 fusion gene expression cassette A vector backbone fragment a was obtained by reverse amplification of the polygene supply vector I (pYL d 1), a promoter Pens (GenBank No. AY 427574.1) of about 1kb of rice endosperm specific storage protein was amplified as fragment b using rice genomic DNA as a template, a GjUGT E13-2A-GjUGT F8 fusion gene was amplified as fragment c, and a terminator TGluA-1 (SEQ ID NO: 19) of about 0.5kb of rice endosperm specific storage protein was amplified as fragment d using rice genomic DNA as a template. The homologous sequences of 25bp are respectively arranged at two sides of each fragment, and the plasmid vector (X) pYL322d1-GjUGT E13-2A-GjUGT F8 containing GjUGT E94 and GjUGT F8 gene expression cassettes is obtained by a plasmid vector multi-fragment one-step assembly method according to the principle of Gibson assembly (as shown in figure 1).
Construction of supply vector pYL d2-GjALDH C3 containing sGjALDH C3 Gene expression cassette A method of reverse amplifying Multi-Gene supply vector II (pYL d 2) to obtain vector backbone fragment a, amplifying Rice endosperm-specific storage protein approximately 0.7kb promoter Pens9 (GenBank No. CP141114.1) as fragment b, amplifying GjALDH C3 gene as fragment C, amplifying Rice endosperm-specific storage protein approximately 0.5kb terminator TGluB4 (SEQ ID NO: 20) with Rice genomic DNA as template as fragment d. The two sides of each fragment are respectively provided with a 25bp homologous sequence, and the plasmid containing GjALDH C3 gene expression cassette supply vector (XI) pYL d2-GjALDH2C3 is obtained by a plasmid vector multi-fragment one-step assembly method according to the principle of Gibson assembly (as shown in figure 1).
S2, constructing the gene expression cassette in the step S1 into a plant transformation vector
Plant transformation vector pYLTAC MF-DOBhPC-CsCAU 1U2 containing optimized CrtI, PSY, BHY, OR H and DXS five genes and GjCCD a, gjUGT94E13, gjUGT F8 and GjALDH C3 genes derived from gardenia for rice endosperm specific synthesis of crocin is assembled as follows:
The polygene vector is assembled by utilizing a polygene vector system TGSII.
(1) Construction of the polygene vector pYLTAC380,380 GW-DOBh PC for specific synthesis of zeaxanthin precursor from rice endosperm is described in example 1.
(2) Construction of plant transformation vector pYLTAC MF-DOBhPC-CsCAU 1U2, namely, superposing four genes sGjCCD4a, sGjUGT94E13, sGjUGT F8 and sGjALDH C3 from capejasmine sources on the basis of vector pYLTAC GW-DOBhPC for synthesizing zeaxanthin precursor, constructing pYLTAC GW-DOBhPC-GjCAU 1U2 for specifically synthesizing crocin in rice endosperm, and constructing a polygene expression vector pYLTAC MF-DOBhPC-GjCAU 1U2 capable of realizing self-deleting hygromycin screening mark by assembling Marker-free element containing HPT expression cassette through Gateway-BP recombination reaction.
Assembly of sGjCCD a Gene expression cassettes the supply vector (IX) pYL d2-sGjCCD a plasmid was mixed with the receiving vector pYLTAC GW-DOBhPC plasmid containing 5 gene expression cassettes, the NS3529 competent cells were transformed by electric excitation, transformants were selected on a double antibody plate of calicheamicin and ampicillin, and recombination of the supply vector plasmid and deletion of the supply vector backbone were achieved by Cre enzyme endogenously expressed by NS 3529. The mixed colony on the double-antibody plate is washed out, the plasmid is subjected to mixed extraction, PI-SceI is used for enzyme digestion and cutting, then the non-recombinant plasmid is transformed into escherichia coli DH10B, and a receiving vector pYLTAC380GW-DOBhPC-GjC plasmid containing sEuCrtI + sZmPSY + sHpBHY + sAtOR H + sAtDXS + sGjCCD4a gene expression cassette is obtained by identification on a caliamycin plate.
The assembly of sGjUGT E13 and sGjUGT F8 gene expression cassettes comprises mixing a supply vector (X) pYL322d1-GjUGT E13-2A-GjUGT F8 plasmid with a receiving vector pYLTAC GW-DOBhPC-GjC plasmid containing 6 target gene expression cassettes, co-electrically transforming competent cells of an escherichia coli strain NS3529 expressing Cre enzyme, screening transformants on a dual-antibody plate of calicheamicin and chloramphenicol, utilizing Cre enzyme endogenously expressed by NS3529 to realize recombination of the supply vector plasmid and deletion of a skeleton thereof, washing a mixed colony mixed drawing plasmid on the dual-antibody plate, performing I-SceI digestion to eliminate non-recombined plasmids, transforming DH10B, and identifying on the calicheamicin plate to obtain the recombinant vector containing sEuCrtI + sZmPSY + sHpBHY + sAtOR H +
The receiving vector pYLTAC380GW-DOBhPC-GjCU 1U2 plasmid of sAtDXS + sGjCCD a+ sGjUGT94E13+ sGjUGT74F8 gene expression cassette.
Assembly of sGjALDH C3 Gene expression cassettes the supply vector (XI) pYL322d2-sGjALDH C3 plasmid was mixed with the receiving vector pYLTAC380GW-DOBhPC-GjCU 1U2 plasmid containing 8 gene expression cassettes, the competent cells of NS3529 were transformed by electric excitation, transformants were selected on a double antibody plate of calicheamicin and ampicillin, and recombination of the supply vector plasmid and deletion of the supply vector backbone were achieved by Cre enzyme endogenously expressed in NS 3529. Washing mixed colony mixed-extraction plasmid on a double-antibody plate, cutting the non-recombinant plasmid by using PI-SceI enzyme, then converting the E.coli DH10B, screening positive transformants on a caliamycin plate, and identifying to obtain the recombinant plasmid containing sEuCrtI + sZmPSY + sHpBHY + sAtOR H + sAtDXS + sGjCCD4a+ sGjUGT94E13
The structural schematic diagram of the plant transformation vector pYLTAC380GW-DOBhPC-GjCA U 1U2 of + sGjUGT74 7F8+ sGjALDH C3 is shown in figure 2.
The assembly of Marker-free elements is described in particular in example 1. The result of the enzyme digestion identification of the plant transformation vector pYLTAC GW-DOBhPC-GjCAU 1U2 is shown in FIG. 2, and as can be seen from FIG. 2, the plant transformation vector pYLTAC GW-DOBhPC-GjCAU 1U2 is obtained.
S3, transforming the plant transformation vector into rice to obtain the transformation and detection of the transgenic rice polygene vector pYLTAC MF-DOBhPC-GjCAU 1U2 for producing crocin in seed endosperm:
Genetic transformation of Rice the polygenic vector pYLTAC MF-DOBhPC-GjCAU 1U2 plasmid was transformed into Agrobacterium EHA105 for transformation of rice embryo callus, see in particular example 1.
PCR detection of transgenic rice genome, namely, extracting genomic DNA from leaves of obtained T 0 -generation transgenic plants by using an SDS method as a template, and respectively detecting exogenous HPT, sEuCrtI, sZmPSY, sHpBHY, sAtOR H, sAtDXS, sGjCCD a, sGjUGT94E13, sGjUGT74F8 and sGjALDH2C3 genes by using a PCR amplification method. The primers used for detecting exogenous HPT, sEuCrtI, sZmPSY, sHpBHY, sAtOR H and sAtDXS genes are the same as those in example 1, and the primers used for detecting exogenous sGjCCD a, sGjUGT94E13, sGjUGT F8 and sGjALDH2C3 genes are shown in Table 2.
TABLE 2 primers for PCR detection of exogenous genes
The amplification procedure used was the same as in example 1. The PCR detection of exogenous gene of the genomic DNA of the T 0 generation rice transformant of pYLTAC MF-DOBhPC-GjCAU 1U2 is shown in FIG. 3. As can be seen from FIG. 3, none of the Wild Type (WT) controls can amplify the foreign gene and none of the transgenic positive plants can amplify the gene. Example 3 appearance observation of transgenic Rice seed and UPLC-MS/MS detection of crocin in endosperm
Appearance observation of transgenic Rice seeds phenotypic observations were performed on pYL MF-DOBhPC-CsCAU 1U2 and pYL MF-DOBhPC-GjCAU 1U2 transgenic Rice brown rice, and the results are shown in FIG. 4. As can be seen from FIG. 4, pYL MF-DOBhPC, which had been introduced into the synthetic zeaxanthin precursor, and pYL MF-DOBhPC-CsCAU 1U2, which had been introduced into the saffron pathway, exhibited yellow, while pYL MF-DOBhPC-GjCAU 1U2, which had been introduced into the gardenia pathway, exhibited orange-yellow.
The extraction and UPLC-MS/MS identification of the crocin of the transgenic rice seeds comprise the steps of freeze-drying rice seeds for 2days, weighing 0.3g, grinding into powder by a freeze grinder, adding 250 mu L of methanol, swirling for 1min, adding 500 mu L of chloroform, swirling for 1min on light-shielding ice for 20min, adding 250 mu L of water, swirling for 1min, centrifuging at 4 ℃ for 5min, respectively collecting aqueous phase and organic phase, and observing the color of an extract, wherein the result is shown in figure 4. As can be seen from FIG. 4, although pYL MF-DOBhPC seeds exhibited yellow color, the aqueous phase was colorless and transparent, while the aqueous phases of pYL MF-DOBhPC-CsCAU 1U2 and pYL MF-DOBhPC-GjCAU 1U2 were visibly yellow, indicating that pYL MF-DOBhPC-CsCAU 1U2 and pYL MF-DOBhPC-GjCAU 1U2 had polar material formation, and the organic phases of pYL MF-DOBhPC-CsCAU 1U2 and pYL MF-DOBhPC-GjCAU 1U2 were orange-red relative to the organic phase of DOBhPC yellow color, indicating additional liposoluble pigment formation.
The aqueous phase of the sample to be tested was filtered through a 0.22 μm aqueous filter, and then poured into a 2mL brown sample bottle with a sample size of 1. Mu.L, and analyzed by a AGILENT EC-C18 (2.1X 100mm,2.7 μm) column. Mobile phase A is primary water and B is 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 the identification of crocin in the endosperm of transformed rice seeds of UPLC-MS\MS detection pYLTAC380MF-DOBhPC-CsCAU 1U2 and pYLTAC380MF-DOBhPC-GjCAU 1U2 are shown in figures 5 and 6, and the results show that the transgenic rice seeds of pYL MF-DOBhPC-CsCAU 1U2 and pYL380MF-DOBhPC-GjCA U 1U2 all have the same characteristic peaks as four crocin standard samples, indicating that crocin was successfully synthesized in both transgenic rice plants. The crocin content of pYLTAC MF-DOBhPC-CsCAU 1U2 and pYLTAC MF-DOBhPC-GjCAU 1U2 transformed rice seed endosperm was also examined. Wherein the crocin content in the pYL MF-DOBhPC-CsCAU 1U2 transformed rice endosperm is 587.82ng/g, and the crocin content in the pYL MF-DOBhPC-GjCAU 1U2 transformed rice endosperm is 855.4ng/g.
The invention also provides a schematic diagram of the way of synthesizing crocin in endosperm of transgenic rice seeds, as shown in figure 7. The biosynthesis of crocin requires a high degree of coordination of a variety of pathways, including the upstream methylerythritol phosphate (MEP) pathway, the midstream carotenoid biosynthesis pathway, and the downstream crocin biosynthesis pathway. Wherein the MEP pathway provides a precursor geranylgeranyl pyrophosphate (GGPP) of the core carotenoid biosynthesis pathway, and the carotenoid biosynthesis pathway provides an important precursor zeaxanthin (Zeaxanthin) for the crocin biosynthesis pathway, which is oxidized and cleaved into crocin dialdehyde (crocetin dialdehyde) by a Carotenoid Cleavage Dioxygenase (CCD), dehydrogenated into crocin (Crocetin) by an aldehyde dehydrogenase (ALDH), and finally catalyzed by UDP-glucosyltransferase (UGT) to obtain crocin.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
- The application of five genes of CrtI, PSY, BHY, OR H and DXS in transgenic breeding for producing crocin from rice seed endosperm by combining the five genes with four genes of CsCCD2L, csUGT AD1, csUGT91P3 and CsALDH I1 from saffron or combining the four genes of GjCCD a, gjUGT94E13, gjUGT74F8 and GjALDH C3 from gardenia comprises the following specific methods: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 after sequence optimization, constructing the constructed gene expression cassettes into plant transformation vectors and transforming rice to obtain transgenic rice for producing crocin in seed endosperm;The CrtI gene is CrtI gene of Erwinia (Erwinia uredovora), the GenBank No is D90087, the PSY gene is PSY gene of corn, the GenBank No is U32636.1, the BHY gene is BHY gene of Haematococcus pluvialis (Haematococcus pluvialis), the GenBankNo is BD250390.1, the OR H gene and the DXS gene are both derived from Arabidopsis thaliana (Arabidopsisthaliana), the GenBank No of OR H gene is NM_125561.4, and the GenBank No of the DXS gene is U27099.1;The GenBank No of CsCCD2L, csUGT AD1, csUGT91P3 and CsALDH I1 genes of saffron sources are KP887110, MF596166, MZ190170 and MF596165 in sequence;The GenBank No of GjCCD a, gjUGT94E13, gjUGT74F8, gjALDH C3 genes from gardenia are KY631925, MN944055, MN944054 and KY631926 in sequence.
- 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 transgenic rice for producing crocin in seed endosperm;The CrtI gene is CrtI gene of Erwinia (Erwinia uredovora), the GenBank No is D90087, the PSY gene is PSY gene of corn, the GenBank No is U32636.1, the BHY gene is BHY gene of Haematococcus pluvialis (Haematococcus pluvialis), the GenBankNo is BD250390.1, the OR H gene and the DXS gene are both derived from Arabidopsis thaliana (Arabidopsisthaliana), the GenBank No of OR H gene is NM_125561.4, and the GenBank No of the DXS gene is U27099.1;The GenBank No of CsCCD2L, csUGT AD1, csUGT91P3 and CsALDH I1 genes of saffron sources are KP887110, MF596166, MZ190170 and MF596165 in sequence;The GenBank No of GjCCD a, gjUGT94E13, gjUGT74F8, gjALDH C3 genes from gardenia are KY631925, MN944055, MN944054 and KY631926 in sequence.
- 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 sequentially shown as SEQ ID NO 7-10.
- 4. The method of claim 2, wherein when the plant transformation vector is constructed by using gene expression cassettes of the five genes with optimized sequences and four genes derived from saffron, the sequence of each gene is CrtI-PSY-BHY-OR H -DXS-CsCCD2L-CsUGT74AD1-CsUGT91P3-CsALDH I1.
- 5. The method of claim 2, wherein the sequence-optimized gene expression cassettes of the five genes and the four genes derived from gardenia are constructed into plant transformation vectors, and the sequence of the genes is CrtI-PSY-BHY-OR H -DXS-GjCCD4a-GjUGT94E13-GjUGT74F8-GjALDH2C3.
- 6. The method of claim 2, wherein the method of transforming rice with the plant transformation vector is agrobacterium-mediated.
- 7. The method of claim 2, wherein the plant transformation vector is constructed using a TGSII multiple gene vector system.
- 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. 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. Use of the plant transformation vector of claim 8 or 9 in constructing transgenic rice for the production of crocin in seed endosperm.
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| CN107142276B (en) * | 2017-05-23 | 2020-11-13 | 华南农业大学 | Transgenic breeding method for synthesizing anthocyanin in crop seed endosperm |
| CN110791512B (en) * | 2018-08-02 | 2022-05-17 | 中国医学科学院药用植物研究所 | Screening, identification and application of glycosyltransferases GjUGT94E13 and GjUGT74F8 involved in crocin synthesis |
| WO2020093285A1 (en) * | 2018-11-07 | 2020-05-14 | 中国医学科学院药用植物研究所 | Enzyme gene for biosynthesis of crocin and use thereof |
| EP4151732A1 (en) * | 2021-09-15 | 2023-03-22 | Universidad de Castilla-La Mancha | Transgenic plants producing high levels of apocarotenoids compounds and uses thereof |
| CN114686501A (en) * | 2022-05-05 | 2022-07-01 | 长沙学院 | Prokaryotic expression and application of gardenia phytoene synthase gene (GjPSY) |
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| CN105907780A (en) * | 2016-04-29 | 2016-08-31 | 华南农业大学 | Transgenic breeding method producing astaxanthin in crop seed endosperm |
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