Method for creating new germplasm of high-betacyanin sweet corn

Abstract

The invention discloses a method for creating a new germplasm of sweet corn with high betacyanin content. According to the method, a carrier containing a single expression cassette (CDGA) of CYP76AD1, DODA1, DOPA5GT and ADHalpha driven by a corn endosperm specific promoter PGt1 is converted into corn, the converted corn of endosperm specific accumulation betacyanin is obtained through screening, and the result shows that the efficiency of expressing betacyanin by the CDGA in the corn is obviously superior to that of a double expression cassette (CDG + A). Furthermore, a new transgenic sweet corn germplasm (ZCDGA) with endosperm specific efficient accumulation of betacyanin is obtained through a cross breeding technology, and the betacyanin content, the sugar content and the yield of the new transgenic sweet corn germplasm are remarkably improved compared with those of parents. According to the method, betacyanin is successfully and efficiently synthesized in the corn endosperm, a new sweet corn germplasm with high nutritional value and excellent eating quality is created, and a new thought and method are provided for biological enhancement of nutritional functional crops.

Description

Technical Field

[0001] This invention relates to the fields of crop genetic improvement and synthetic biology, and more specifically, to a method for creating new sweet corn germplasm with high betalain content. Background Technology

[0002] Betalains possess powerful coloring properties and have been used as a natural colorant in food, beverages, and fruit salads. Simultaneously, as an excellent antioxidant, betalains are increasingly being used in food preservation and are currently an FDA-approved food additive. Increasing research also indicates that betalains are widely used as a natural medicine in healthcare to improve human health. They can inhibit glycogen degradation and glycemic ischemia, thus lowering blood sugar. They also have a certain repair function for liver function and liver damage; they can affect the function, structure, and permeability of microbial cell membranes, leading to cell death; they can also inhibit the occurrence of tumors or cancer and promote human health. Furthermore, betalains are also widely used in the cosmetics industry.

[0003] Corn endosperm contains few impurities besides endogenous starch, allowing for the specific accumulation of betalains, and the extraction method for betalains is simple. Previous studies used constitutive promoters to drive the RUBY system in corn, resulting in the accumulation of betalains in leaves, flowers, and fruits, but this led to undesirable phenotypes such as stunted growth, reduced pollen count, and decreased grain setting rate. The paper "eRUBY rice: Co-expression of a feedback-insensitive TyrAarogenate dehydrogenase with RUBY enhances endosperm betalain levels" discloses the method of extracting betalains from sugar beets. BvCYP76AD1 and BvDODA1 And from American pokeweed MjDOPA5GT The gene underwent codon optimization and was linked into a single expression cassette using P2A peptide sequences to construct the RUBY gene expression cassette. To overcome the feedback inhibition of the upstream enzyme TyrA by tyrosine accumulation, a feedback-insensitive enzyme derived from sugar beets was introduced. ADHα Genes to enhance tyrosine biosynthesis. Transient expression of RUBY and [other genes] in tobacco leaves. ADHα (eRUBY system), the results showed that RUBY and ADHα The betaine content in the leaves of RUBY was significantly higher than that in the leaves expressing RUBY alone. RUBY and... ADHαCompared with rice lines that express the RUBY gene alone, the eRUBY rice has a significantly higher content of betaine in the endosperm, and the pigment is evenly distributed in the endosperm tissue, which makes the brown rice present a stable dark red appearance, solving the previous problem of "low content and uneven distribution" of betaine in rice endosperm.

[0004] Rice and maize are both monocotyledonous grasses, but they differ significantly in the molecular regulation, metabolic network, and genetic background of endosperm development. For example, rice endosperm primarily stores glutenin, while maize primarily stores prolamins; their gene expression regulatory elements (such as promoters) are not universal. Therefore, optimal techniques used in rice cannot be directly and predictably applied to maize, especially regarding the efficiency and stability of multi-gene expression systems, the results of which are unknown. For instance, whether the dual-expression cassette strategy, which performs better in rice, remains effective in the complex genomic context of maize, and the interaction and genetic laws between exogenous genes and desirable traits in sweet maize (such as high sugar content), are all pressing technical challenges that need to be addressed in this field. Furthermore, previous studies using constitutive promoters to express the RUBY system in maize, while successfully accumulating betaine, resulted in severe adverse phenotypes such as stunted plant growth and reduced seed setting rate, making it unsuitable for agricultural production. Therefore, developing a technical solution that can efficiently and specifically synthesize betalains in corn endosperm without affecting the normal growth and development of plants, and can be combined with excellent agronomic traits (such as sweetness and glutinousness), has significant theoretical and practical implications. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a method for creating new high-betalain maize germplasm. Specifically, by combining optimized gene expression strategies with hybridization breeding, betalains are efficiently accumulated in maize endosperm, and this method is used to create high-yield and high-quality high-betalain sweet maize germplasm.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a method for creating new corn germplasm with high betaine content, which includes a betaine synthesis gene: tyrosine hydroxylase gene. CYP76AD1 Dopa dioxygenase gene DODA1 Dopa glucosyltransferase gene DOPA5GT Tyrosine synthase gene: feedback-insensitive TyrA aromatic amino acid dehydrogenase gene ADHα The vector was transformed into maize, and after screening, transformed maize ... BvCYP76AD1-P2A-BvDODA1- P2A-MjDOPA5GT - F2A-BvADHα .

[0007] This invention constructs an eRUBY single open reading frame by linking P2A and F2A peptides, thus creating a single expression cassette. BvCYP76AD1- P2A-BvDODA1-P2A-MjDOPA5GT - F2A-BvADHα (abbreviated as: CDGA) and dual expression box BvCYP76AD1-P2A- BvDODA1-P2A-MjDOPA5GT, BvADHα (Abbreviation: CDG+A) (Utilizing a 2A linker peptide to tandemly link exogenous genes not only effectively avoids vector redundancy but also allows for higher betalain content in maize endosperm). The expression cassette is driven by the maize endosperm-specific promoter PGt1 (26-kDa γ-zein promoter PGt1). PGt1 can specifically express betalains in the maize endosperm, confining betalain synthesis to the maize endosperm tissue, effectively avoiding the impact of betalains on normal plant growth (avoiding undesirable phenotypes such as slow growth and decreased grain filling rate in maize caused by constitutive promoter-driven expression). Specifically, the single expression cassette design of this invention... CYP76AD1 , DODA1 , DOPA5GT as well as ADHα By linking peptide fusion, it is placed in an open reading frame and named eRUBY, forming a single-gene expression unit; the dual expression cassette design will... CYP76AD1 , DODA1 , DOPA5GT Place it in an open reading box, then ADHα Genes are expressed independently, driven by independent mechanisms ADHα Enhanced tyrosine precursor supply. The above expression cassette was loaded into a recipient vector and transformed into maize using Agrobacterium infection to obtain transgenic maize with endosperm-specific accumulation of betaine.

[0008] Among them, the single-cassette-driven expression form (CDGA) significantly increased the accumulation of betaine in maize seed endosperm compared to the dual-cassette-driven expression form (CDG+A). In CDGA transgenic plants, the betaine content ranged from 1.07 to 6.88 mg / g, and the isobetaine content ranged from 0.54 to 1.81 mg / g. CDGA-#1 showed the highest betaine content at 6.88 mg / g, while the isobetaine content was 1.81 mg / g. In CDG+A transgenic plants, the betaine content ranged from 0.88 to 0.92 mg / g, and the isobetaine content ranged from 0.41 to 0.42 mg / g. This invention is the first to demonstrate that the expression of related genes driven by a single promoter (CDGA) has a significantly higher betalain accumulation efficiency than the dual expression cassette (CDG+A) strategy. The study found that this high-efficiency expression is closely related to the higher T-DNA integration copy number (up to 5 copies) in CDGA-transformed lines, revealing the different response of different crop chassis to multi-gene expression vector configurations, and providing key technical insights and optimization schemes for complex metabolic pathway engineering in maize.

[0009] Furthermore, the corn is B104 background corn.

[0010] This invention further utilizes hybridization breeding, using the high-betaine-producing transgenic maize B104 (CDGA) as the male parent and crossing it with the superior sweet maize inbred line 1341, successfully creating a new sweet maize germplasm rich in betaine (ZCDGA). Its betaine content ranges from 2.34 to 11.40 mg / g, and the isobetaine content ranges from 1.48 to 1.92 mg / g. Among these, ZCDGA-#2 has the highest betaine content at 11.40 mg / g, and the isobetaine content is 1.92 mg / g. The betaine content in the endosperm of the hybrid offspring is higher than that of the male parent. Furthermore, the content of soluble sugars such as glucose, fructose, and sucrose in the offspring is also significantly higher than that of both parents, indicating that the biosynthesis of betaine does not consume fruit sugars but rather synergistically enhances its nutritional and edible quality. This discovery overcomes the negative effects of traditional metabolic engineering that may result in a trade-off, achieving the dual goals of nutritional fortification and superior taste, and creating a completely new functional sweet corn germplasm resource.

[0011] To further verify the advantages of the hybrid offspring, this invention precisely measured the 100-seed weight and betaine content per 100 seeds of the hybrid ZCDGA and its parents. The results strongly demonstrate that the hybrid offspring exhibit significant heterosis. Compared with the transgenic male parent (CDGA) and female parent (1341), the 100-seed weight of ZCDGA was significantly increased, indicating an improvement in its yield trait. More importantly, the increase in betaine content was not due to a "concentration illusion" caused by the high moisture content of sweet corn. By calculating the total betaine yield per 100 seeds, it was found that the total yield of ZCDGA was much higher than that of the male parent, and its betaine content per unit dry weight also surpassed that of the male parent. This data conclusively shows that the hybridization strategy of this invention not only did not dilute the functional components due to increased yield, but also achieved a dual synergistic improvement in yield and quality (content per unit weight), which is a significant advancement that the prior art could not foresee.

[0012] Furthermore, the method also includes crossing or backcrossing the obtained endosperm-specific betalain accumulation transformed maize with sweet maize, and screening to obtain sweet maize with further enhanced endosperm-specific betalain accumulation. Hybridization technology can overcome the barriers to transformation in certain species, accumulate betalain in sweet maize lines and create new germplasm, and further enhance the target trait by utilizing heterosis.

[0013] Preferably, the hybridization involves using transformed corn with endosperm-specific betaine accumulation as the male parent and sweet corn as the female parent to obtain hybrid offspring (F1) with betaine content higher than that of the male parent, and combining the excellent agronomic traits (sweetness and waxiness) of corn.

[0014] Preferably, the sweet corn is sweet corn 1341.

[0015] Furthermore, the gene combination is an optimized sugar beet-derived tyrosine hydroxylase gene. BvCYP76AD1 Dopa dioxygenase gene BvDODA1 Four o'clock flower source dopa glucosyltransferase gene MjDOPA5GT And the TyrA aromatic amino acid dehydrogenase gene that is insensitive to feedback from beet sources. BvADHα .

[0016] Furthermore, the optimized CYP76AD1, DODA1, DOPA5GT, ADHα The nucleotide sequences are shown in SEQ ID No. 1 to 4, respectively.

[0017] Furthermore, the nucleotide sequence of the P2A peptide is shown in SEQ ID No. 5.

[0018] Furthermore, the nucleotide sequence of the F2A peptide is shown in SEQ ID No. 6.

[0019] Furthermore, the nucleotide sequence of the PGt1 is shown in SEQ ID No. 7.

[0020] Specifically, the single expression box is: PGt1:: BvCYP76AD1-P2A-BvDODA1-P2A-MjDOPA5GT - F2A-BvADHα The carrier is made of CYP76AD1, DODA1, DOPA5GT and ADHα Genes are linked via linker peptides to obtain fusion genes, which are then constructed using the maize endosperm-specific promoter PGt1; the single... CYP76AD1, DODA1, DOPA5GT Genes through P2A Peptide linkage; ADHα Genes are linked by the F2A peptide.

[0021] Furthermore, the carrier is Cre / loxP Mediated marker-assisted self-deletion expression vector. This involves transferring the aforementioned expression cassette into a vector carrying Cre / loxP A marker-assisted self-deletion expression vector was used to construct a multi-gene recombinant vector. Using the marker-assisted self-deletion system, transgenic maize seeds without selection markers can be obtained, significantly improving biosafety.

[0022] Preferably, the Cre / loxP The mediated marker self-deletion expression vector also includes loxP Site (nucleotide sequence as shown in SEQ ID No. 8) screening markers, Arabidopsis thaliana 18.2 heat shock promoter (nucleotide sequence as shown in SEQ ID No. 9), Cre recombinase (nucleotide sequence as shown in SEQ ID No. 10).

[0023] As a preferred embodiment, the present invention provides a method for creating new high-yield betaine-producing sweet corn germplasm, comprising the following steps: S1. Optimize beet sources BvCYP76AD1 , BvDODA1 Origin of four o'clocks MjDOPA5GT And the source of beets BvADHα Genes were constructed and eRUBY single open reading frames were created by linking P2A and F2A peptides. BvCYP76AD1-P2A- BvDODA1-P2A-MjDOPA5GT - F2A-BvADHα ); S2. The single open reading frame in S1 is driven by the maize endosperm-specific promoter PGt1; the expression box is PGt1:: BvCYP76AD1-P2A-BvDODA1-F2A-MjDOPA5GT - F2A-BvADHα (CDGA); S3. Receptor vector selection with Cre / loxP The 1300MF-B-mediated marker-assisted automatic deletion system was used to assemble the expression cassette in S2 into the receptor vector; S4. Transform the plant transformation vector described in step S3 into maize B104 to obtain transgenic maize with endosperm-specific accumulation of betaine; S5. Using the transgenic corn B104 obtained in step S4, which has been screened and verified to have high betalain content and no screening marker, as the male parent and sweet corn 1341 as the female parent, hybridize them and harvest F1 generation seeds.

[0024] S6. After planting F1 generation seeds, backcrossing was performed with sweet corn 1341 for multiple generations. Combined with molecular marker-assisted selection and phenotypic identification, a new homozygous sweet corn germplasm with stable inheritance and rich in betalains was finally obtained through self-pollination.

[0025] The present invention also provides maize seeds with endosperm-specific accumulation of betaine prepared by any of the methods described above.

[0026] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for creating new sweet corn germplasm high in betaine. The method involves using a sweet corn germplasm containing the corn endosperm-specific promoter PGt1. CYP76AD1 , DODA1 , DOPA5GT , ADHα This invention transforms maize with a single expression cassette (CDGA) vector and screens for transgenic maize that specifically accumulates betaine in the endosperm. The optimized eRUBY system was used in this invention, and the results showed that the single expression cassette (CDGA) was significantly more efficient at expressing betaine in maize than the dual expression cassette (CDG+A). Further, through hybridization breeding technology, the above-mentioned exogenous gene was introduced into sweet maize and expressed preferentially, resulting in a new transgenic sweet maize germplasm (ZCDGA) that efficiently accumulates betaine in the endosperm. Its betaine content can reach up to 11.40 mg / g (dry weight), achieving ultra-high accumulation and exhibiting superior parental advantage. Simultaneously, its sugar content is significantly higher than that of the parent, and its yield is also significantly higher than that of the parent. This transgenic maize can be directly used as a functional food or a raw material for betaine biomanufacturing. This invention not only successfully synthesized betalains efficiently in corn endosperm, but also created a new sweet corn germplasm that combines high nutritional value (high betalains) and excellent eating quality (high sugar content, sweet and glutinous combination), providing new ideas and methods for the biofortification of nutritional functional crops. Attached Figure Description

[0027] Figure 1 To construct and validate the stability of a corn vector for enhancing beetroot red pigment production. Among other things, Figure 1 In diagram A, a gene expression cassette was constructed using the four optimized genes described in this invention. BvADHα B represents the function of CDG in different expression forms; C represents the amplification and detection of exogenous genes by PCR. Hind III Bgl II, Swa I vector digestion detection.

[0028] Figure 2 To identify marker-free selection markers in transgenic maize progeny. Figure 2 A in the diagram illustrates the structure and working principle of the self-deletion screening marker system; B shows the detection of marker gene deletion in T3 generation transgenic plants using the PF / P-R1 / P-R2 primer pair, indicating that the T3 generation transgenic maize no longer has screening markers.

[0029] Figure 3 The images show the whole ear, seeds, and their cross-sectional and longitudinal phenotypes of enhanced betaine red maize. From left to right, the images show kernels of wild-type maize B104, CDGA (#1, #6, #9), and CDG+A (#2, #7, #8). The scale bar is 1 cm.

[0030] Figure 4 Images show the seeds of hybrid maize and their cross-sectional and longitudinal phenotypes. From left to right, they are images of wild-type maize 1341 and hybrid sweet maize ZCDGA-#1, -#2, and -#3 kernels. The scale bar is 1 cm.

[0031] Figure 5 HPLC determination of betaine in corn to enhance betaine production. Among them, Figure 5 In the diagram, A represents the HPLC detection chromatogram of CDGA and CDG+A in transgenic maize; B represents the HPLC quantitative analysis of betaine in CDGA and CDG+A of transgenic maize.

[0032] Figure 6 This study describes the HPLC detection of betaine in hybrid maize. Among other things, Figure 6 In the diagram, A represents the HPLC detection chromatogram of hybrid maize ZCDGA; B represents the HPLC quantitative analysis of betaine in hybrid maize ZCDGA.

[0033] Figure 7 This study aimed to detect sugar content in transformed and hybrid maize. Using wild-type maize B104, sweet maize 1341, transgenic maize lines CDGA (#1, #6, #9), and hybrid maize lines ZCDGA (#1, #2, #3), glucose, fructose, and sucrose were measured sequentially from left to right.

[0034] Figure 8 The content of betalains per 100 kernels in transformed and hybrid maize was determined. Figure 8 In the chart, A represents the statistical graph of the 100-kernel weight (dry weight) of transformed and hybrid maize; B represents the statistical graph of the betaine content (dry weight) in 100 kernels of transformed and hybrid maize. From left to right, they are B104, 1341, CDGA, and ZCDGA. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0036] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0037] Table 1 Primer list used in this invention

[0038] Example 1: Obtaining Transgenic Maize with Enhanced Betaine I. Experimental Methods By co-expressing the tyrosine synthase gene and betaine synthesis pathway in two ways based on the RUBY system (single expression cassette driven expression (CDGA) and dual expression cassette driven expression (CDG+A)), the betaine content in maize endosperm was significantly increased. The specific steps are as follows: 1. Gene optimization and expression cassette construction S1. Key gene selection: Source of beets BvCYP76AD1 , BvDODA1 Origin of four o'clocks MjDOPA5GT —Using P2A peptide linkage, fused into a single expression cassette (CDG); a feedback-insensitive tyrosine dehydrogenase derived from beets. BvADHα It is connected in series with the aforementioned single expression box (CDG) in two ways.

[0039] S2. Codon optimization: The above genes were optimized. The optimized sequence is shown below. BvCYP76AD1 The nucleotide sequence of the gene is shown in SEQ ID No. 1. BvDODA1 The nucleotide sequence of the gene is shown in SEQ ID No. 2. MjDOPA5GT The nucleotide sequence of the gene is shown in SEQ ID No. 3. BvADHα The nucleotide sequence of the gene is shown in SEQ ID No. 4.

[0040] S3. Expression Box Design: (1) Single expression box driven expression form (CDGA) Will BvADHα The gene and CDG single open reading frame are tandemly linked using the F2A peptide to form a single gene expression unit, which is then driven by the maize endosperm-specific promoter Gt1 (nucleotide sequence shown in SEQ ID No. 7), i.e., PGt1:: BvCYP76AD1-P2A-BvDODA1-P2A-MjDOPA5GT - F2A-BvADHα (CDGA) Figure 1 A); The nucleotide sequences of P2A and F2A peptides are shown in SEQ ID No. 5 and SEQ ID No. 6.

[0041] (2) The form of independent expression driven by dual expression boxes (CDG+A) CDG single open reading frame expression is driven by the maize endosperm-specific promoter Gt1, i.e., PGt1:: BvCYP76AD1- P2A-BvDODA1-P2A-MjDOPA5GT ;Will BvADHα Genes are expressed independently and driven by the maize endosperm-specific promoter Gt1, i.e., PGt1:: BvADHα And place the expression box downstream (CDG+A) ( Figure 1 A).

[0042] 2. Construction of self-deleting vector S1. Carrier assembly: The expression boxes of the two designs mentioned above were reassembled into 1300-MF (Cre / loxP The self-deleting carrier (the structural diagram and working principle of the screening and marking self-deleting system) is as follows: Figure 2 As shown in A), the dual expression cassette vector is configured according to PGt1::RUBY expression cassette → PGt1:: BvADHα Expression cassettes (arranged from upstream to downstream); the above vectors contain hygromycin resistance markers. Bar and Cre Recombinase gene.

[0043] S2. Final carrier: The plant transformation vectors 1300MF-CDGA and 1300MF-CDG+A (structural diagram) were obtained. Figure 1 As shown in A, the nucleotide sequence of the loxP site is shown in SEQ ID No. 8, the nucleotide sequence of the Arabidopsis thaliana P18.2 heat shock promoter is shown in SEQ ID No. 9, and the nucleotide sequence of the Cre enzyme (including introns) is shown in SEQ ID No. 10.

[0044] 3. Maize genetic transformation and screening S1. Agrobacterium-mediated transformation: Host bacteria: Agrobacterium tumefaciens EHA105.

[0045] Corn variety: B104.

[0046] Callus induction: Immature seeds were cultured in N6 medium containing 2 mg / L 2,4-D in the dark.

[0047] Co-culture: Agrobacterium infection medium (OD) 600 =0.4) Soak for 20 min, and culture for a total of 3 days.

[0048] S2. Resistance screening: Screening medium: N6 medium containing 50 mg / L hygromycin.

[0049] Differentiation medium: MS medium containing 3 mg / L BA + 1 mg / L NAA.

[0050] S3. Mark Deletion Verification: The deletion of marker genes in T3 generation transgenic plants was detected using PF / P-R1 / P-R2 primers (see Table 1) combined with electrophoresis images and first-generation sequencing images of amplified products.

[0051] II. Experimental Results PCR amplification and detection of exogenous genes Hind III Bgl II, Swa The results of the vector enzyme digestion assay confirmed the successful construction of 1300MF-CDGA and 1300MF-CDG+A, and their successful introduction into Agrobacterium (I). Figure 1 (B and C).

[0052] The results of marker gene deletion detection in T3 generation transgenic plants are as follows: Figure 2 As shown in B, marker genes Bar The recombinase expression cassette has been deleted, resulting in a marker-free line.

[0053] Example 2: Hybridization of enhanced betaine transgenic maize and sweet maize This embodiment provides a method for hybridizing transformed maize B104 and sweet maize 1341, transferring exogenous genes from B104 into sweet maize 1341 to form a new hybrid maize ZCDGA rich in betaine. The specific steps are as follows: S1. Pretreatment of maize B104 before hybridization Transformed maize B104 was used as the male parent, primarily providing pollen. Before the tassels released pollen, they were covered with white paper bags to ensure pollen purity.

[0054] S2. Pretreatment of sweet corn 1341 hybridization Sweet corn 1341, used as the female parent, primarily receives pollen and therefore self-pollination must be prevented. Demasculinization should be performed on the female parent before the tassel emerges from the top leaves but before pollen is released; similarly, the uppermost female ear of the female parent should be bagged. Furthermore, the silks must be trimmed evenly before pollination.

[0055] S3. Cross treatment involving the transformation of maize B104 and sweet corn 1341 Gently shake the male tassel of maize B104 (male parent) to collect pollen; temporarily remove the isolation bag from the female ear of the female parent, gently shake the pollen of maize B104 into the silks, and then re-bag the ear.

[0056] Test Example 1: Quantitative Analysis and Functional Verification of Metabolites 1. Phenotypic Analysis like Figure 3 As shown, the T3 generation seeds of wild-type maize lines B104, CDGA, and CDG+A exhibit different colors of yellow, purple, and red. The endosperm color is positively correlated with the seed coat color; the darker the seed coat, the darker the endosperm color. In the CDGA and CDG+A lines, the embryo color is consistent with that of wild-type B104, indicating that betalains accumulate in the endosperm.

[0057] like Figure 4 As shown, the T3 generation seeds of sweet corn 1341 and ZCDGA strains exhibit yellow and dark purple colors. The endosperm color is positively correlated with the seed coat color; the darker the seed coat, the darker the endosperm color. In the ZCDGA strains, the embryo color is consistent with that of wild-type 1341, indicating that betalains accumulate in the endosperm.

[0058] 2. Pigment extract from genetically modified corn kernels The endosperm of dried corn kernels 30 days after grain filling was thoroughly ground under freezing conditions, and betalains were extracted from the endosperm of the transformed corn line using 2 mL of ultrasonic (30 Hz, 30 min in the dark) extraction solution (methanol:water:concentrated hydrochloric acid = 85:14:1 volume ratio).

[0059] The results are as follows Figure 5 and Figure 6 As shown, after betalains are extracted into the solvent, their color intensity changes significantly, indicating a positive correlation between the extract color and the endosperm color.

[0060] 3. Metabolite detection: HPLC-MS / MS: A C18 column was used at 40℃, with a flow rate of 1 mL / min and an injection volume of 10 µL. Detection was performed at OD 535 nm. Conditions A: 1.6% formic acid in methanol; B: 1.6% formic acid in aqueous solution. For the following conditions: 0-5 min, A=15%; 5-10 min, A=20%; 10-28 min, A=28%; 28-32 min, A=40%; 32-40 min, A=0%.

[0061] Content determination: betalain standard (Sigma, Shanghai, China) concentration gradients of 2.5 mg / mL, 3.125 mg / mL, 6.25 mg / mL, 12.5 mg / mL and 50 mg / mL methanol solution were set up. After detection, a standard curve was established with the total peak area (mAU*s) as y and the corresponding concentration (mg / mL) as x.

[0062] The results of betaine detection in transformed maize (background B104) are as follows: Figure 5 Wild-type maize B104 does not contain betalains. The highest betalain content in the CDGA transformant lines was 6.88 mg / g (dry weight), and the highest iso-betalain content was 1.81 mg / g. In the CDG+A transformant lines, the highest betalain content was 0.92 mg / g (dry weight), and the highest iso-betalain content was 0.42 mg / g. The betalain content in CDGA is seven times that of CDG+A.

[0063] The results of betaine detection in hybrid maize (background 1341) are as follows: Figure 6 Wild-type 1341 sweet corn does not contain betalains, while ZCDGA has the highest betalain content at 11.40 mg / g (dry weight) and the highest iso-betalain content at 1.92 mg / g. The betalain content in ZCDGA is 1.7 times that of the CDGA transformant line.

[0064] The above results indicate that... BvADHα By tandemly expressing the F2A peptide with the RUBY system, more betaine can be synthesized in the maize endosperm compared to its independent expression. In hybrid maize with CDGA as the male parent and 1341 as the female parent, the betaine content is higher than that in the male parent. The hybrid line ZCDGA has the highest betaine content, at 11.40 mg / g (dry weight).

[0065] 4. T-DNA integration copy number detection The results are shown in Table 2. The high efficiency of betalain expression in transgenic maize lines is closely related to the higher T-DNA integration copy number (up to 5 copies) during CDGA transformation, revealing the different response of different crop chassis to multi-gene expression vector configurations. This provides key technical insights and optimization schemes for complex metabolic pathway engineering in maize.

[0066] Table 2. Copy number statistics of T-DNA in transformed maize (B104) T3 generation.

[0067] 5. Detection of soluble sugars in transformed and hybrid maize lines S1. Construction of Standard Curve Take 2 mL of standard sugar solutions of different concentrations, add 6.00 mL of anthrone reagent to each solution, shake well, and immediately heat in a boiling water bath for 3.5 min. Remove the solutions and immediately cool them by shaking rapidly in cold water or ice water to stop the reaction. Measure the absorbance at a wavelength of 640 nm. Plot a standard curve with absorbance on the x-axis and sugar content on the y-axis, and derive the standard linear equation.

[0068] S2. Sample processing Weigh 0.5 g of corn seeds, grind them thoroughly, add 10 mL of 75% ethanol, incubate in a water bath at 80℃ for 15 min, remove and cool to room temperature, centrifuge, take the supernatant and make up to 25 mL. This filtrate is the soluble sugar extract.

[0069] The results are as follows Figure 7 As shown, the sugar content of ZCDGA is higher than that of wild-type B104 and 1341, as well as its parent CDGA, indicating that the hybrid corn ZCDGA has the best eating quality and taste. It also proves that the soluble sugar in corn is not reduced due to the synthesis of betaine, indicating that CDGA does not affect the taste and quality of corn.

[0070] 6. Detection of betalain content per 100 kernels and per 100 kernels in transformed and hybrid maize lines One hundred corn seeds were randomly selected and weighed; each sample was weighed three times, with random selections made each time. After weighing, the betalain content in the 100 corn seeds was determined.

[0071] Statistical analysis was performed on the obtained data. The results are as follows: Figure 8 As shown in Figure A, the weight of the hybrid sweet corn (ZCDGA) was significantly higher than that of its parent and wild-type corn, indicating that the hybrid corn yield was increased; Figure 8 As shown in B, this further illustrates that the betaine content per unit dry weight of ZCDGA has also surpassed that of its predecessors.

[0072] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for creating new corn germplasm with high betalain content, characterized in that, This will include the betaine synthesis gene: tyrosine hydroxylase gene. CYP76AD1 Dopa dioxygenase gene DODA1 Dopa glucosyltransferase gene DOPA5GT Tyrosine synthase gene: feedback-insensitive TyrA aromatic amino acid dehydrogenase gene ADHα The vector was transformed into maize, and after screening, transformed maize ... BvCYP76AD1-P2A-BvDODA1-P2A-MjDOPA5GT - F2A-BvADHα .

2. The method according to claim 1, characterized in that, The method further includes crossing or backcrossing the obtained endosperm-specific betaine accumulation transformed corn with sweet corn, and screening to obtain sweet corn with further enhanced endosperm-specific betaine accumulation.

3. The method according to claim 2, characterized in that, The hybridization involves using transformed corn with endosperm-specific betalain accumulation as the male parent and sweet corn as the female parent to obtain hybrid offspring with betalain content higher than that of the male parent.

4. The method according to claim 1, characterized in that, The CYP76AD1, DODA1, DOPA5GT, ADHα The nucleotide sequences are shown in SEQ ID No. 1 to 4, respectively.

5. The method according to claim 1, characterized in that, The nucleotide sequence of the P2A peptide is shown in SEQ ID No.

5.

6. The method according to claim 1, characterized in that, The nucleotide sequence of the F2A peptide is shown in SEQ ID No.

6.

7. The method according to claim 1, characterized in that, The nucleotide sequence of PGt1 is shown in SEQ ID No.

7.

8. The method according to claim 1, characterized in that, The carrier is Cre / loxP Mediated marker self-deletion expression vector.

9. The method according to claim 8, characterized in that, The carrier also includes loxP Site selection markers, Arabidopsis thaliana 18.2 heat shock promoter, Cre recombinase.

10. Maize seeds with endosperm-specific accumulation of betaine prepared by the method according to any one of claims 1 to 9.

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