A transgenic breeding method for synthesizing catechins in rice seed endosperm

Abstract

The invention discloses a transgenic breeding method for synthesizing catechins in rice seed endosperm, and relates to the field of biotechnology. The transgenic breeding method comprises the steps of transforming a recombinant vector into rice callus tissue, and then cultivating a transgenic rice plant; the recombinant vector comprises a gene expression cassette for expressing CsCHI protein, a gene expression cassette for expressing CsCHS protein, a gene expression cassette for expressing CsF3H protein, a gene expression cassette for expressing CsDFR protein, a gene expression cassette for expressing CsLAR protein, a gene expression cassette for expressing CcF3'5'H protein, a gene expression cassette for expressing transcription factor OsMYBP1 protein, and a gene expression cassette for expressing transcription factor OsRc protein. The invention realizes the synthesis of catechins in crop (rice) endosperm for the first time, and its product can be used as a raw material for producing catechins, which has important guiding significance for new functional breeding.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a transgenic breeding method for synthesizing catechins in rice seed endosperm. Background Art

[0002] Rice is one of the main food crops, and about half of the world's population relies on rice as their staple food. In recent years, with the reduction of agricultural diversity, the nutritional elements in crops have become unbalanced. At the same time, rice has further lost nutrients during deep processing, which ultimately increases the risk of insufficient intake of trace nutrients for humans. Catechins are a class of highly active flavonols that have multiple functions such as preventing and treating cardiovascular diseases, lowering blood sugar and anti-oxidation. They are abundant in tea. Rice lacks flavonoids in its endosperm due to the lack of OsDFR, a key gene for flavonoid metabolism, and the low expression level of key genes in the flavonoid metabolic pathway in the endosperm. Therefore, it is impossible to achieve the synthesis of catechins in the endosperm using traditional breeding methods. If catechins can be produced in the rice endosperm, the above problems can be solved and the nutritional value of rice (polished rice) can be increased. This is also one of the main goals of functional rice genetic engineering breeding. Summary of the invention

[0003] The purpose of the present invention is to provide a transgenic breeding method for synthesizing catechins in rice seed endosperm to solve the problems existing in the above-mentioned prior art. The present invention realizes the synthesis of catechins in crop (rice) endosperm for the first time, and its product can be used as a raw material for producing catechins, which has important guiding significance for new functional breeding.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The present invention provides a recombinant vector, comprising a gene expression cassette for expressing CsCHI protein, a gene expression cassette for expressing CsCHS protein, a gene expression cassette for expressing CsF3H protein, a gene expression cassette for expressing CsDFR protein, a gene expression cassette for expressing CsLAR protein, a gene expression cassette for expressing CcF3'5'H protein, a gene expression cassette for expressing transcription factor OsMYBP1 protein and a gene expression cassette for expressing transcription factor OsRc protein;

[0006] The amino acid sequence of the CsCHI protein is shown in SEQ ID NO.2;

[0007] The amino acid sequence of the CsCHS protein is shown in SEQ ID NO.4;

[0008] The amino acid sequence of the CsF3H protein is shown in SEQ ID NO.6;

[0009] The amino acid sequence of the CsDFR protein is shown in SEQ ID NO.8;

[0010] The amino acid sequence of the CsLAR protein is shown in SEQ ID NO.10;

[0011] The amino acid sequence of the CcF3'5'H protein is shown in SEQ ID NO.12;

[0012] The amino acid sequence of the transcription factor OsRc protein is shown in SEQ ID NO.14;

[0013] The amino acid sequence of the transcription factor OsMYBP1 protein is shown in SEQ ID NO.16.

[0014] Furthermore, the gene expression cassette for expressing the CsCHI protein includes a promoter PgluB4, a gene encoding the CsCHI protein, and a TAtubq3 terminator;

[0015] The gene expression cassette for expressing the CsCHS protein comprises a promoter P16, a gene encoding the CsCHS protein and a Tact2 terminator;

[0016] The gene expression cassette for expressing the CsF3H protein comprises a promoter P10, a gene encoding the CsF3H protein and a Tnos terminator;

[0017] The gene expression cassette for expressing the CsDFR protein includes a promoter P10, a gene encoding the CsDFR protein and a Thsp18.2 terminator;

[0018] The gene expression cassette for expressing the CsLAR protein comprises a promoter PA2, a gene encoding the CsLAR protein and a T35S terminator;

[0019] The gene expression cassette for expressing the CcF3'5'H protein comprises a promoter PgluC, a gene encoding the CcF3'5'H protein and a Tnos terminator;

[0020] The gene expression cassette for expressing the transcription factor OsMYBP1 protein comprises a promoter P16, a gene encoding the transcription factor OsMYBP1 protein and a Tnos terminator;

[0021] The gene expression box for expressing the transcription factor OsRc protein comprises a promoter PgluB4, a gene encoding the transcription factor OsRc protein and a T35S terminator.

[0022] Furthermore, the CDS sequence of the gene encoding the CsCHI protein is shown in SEQ ID NO.1;

[0023] The CDS sequence of the gene encoding the CsCHS protein is shown in SEQ ID NO.3;

[0024] The CDS sequence of the gene encoding the CsF3H protein is shown in SEQ ID NO.5;

[0025] The CDS sequence of the gene encoding the CsDFR protein is shown in SEQ ID NO.7;

[0026] The CDS sequence of the gene encoding the CsLAR protein is shown in SEQ ID NO.9;

[0027] The CDS sequence of the gene encoding the CcF3'5'H protein is shown in SEQ ID NO.11;

[0028] The CDS sequence of the gene encoding the transcription factor OsRc protein is shown in SEQ ID NO.13;

[0029] The CDS sequence of the gene encoding the transcription factor OsMYBP1 protein is shown in SEQ ID NO.15.

[0030] The present invention also provides a recombinant host cell, comprising the above-mentioned recombinant vector.

[0031] Furthermore, the host cell is Agrobacterium.

[0032] The present invention also provides the use of the above-mentioned recombinant vector or recombinant host cell in preparing transgenic rice capable of synthesizing catechins in seed endosperm.

[0033] The present invention also provides a transgenic breeding method for synthesizing catechins in rice seed endosperm, comprising the steps of transforming the above-mentioned recombinant vector into rice callus tissue and then cultivating transgenic rice plants.

[0034] Furthermore, the recombinant vector is transformed into rice callus tissue using Agrobacterium transformation method.

[0035] The present invention also provides a method for preparing rice with a high catechin content, comprising the steps of obtaining rice seeds by planting transgenic rice, and then performing hulling treatment to obtain the rice;

[0036] The transgenic rice is cultivated by adopting the above transgenic breeding method.

[0037] The present invention also provides rice with high catechin content prepared according to the above preparation method.

[0038] The present invention discloses the following technical effects:

[0039] The present invention provides a transgenic breeding method for producing catechins in the endosperm of crop seeds, which realizes the synthesis of catechins in the endosperm of crops (rice) for the first time. Its products can be used as raw materials for producing catechins, which has important guiding significance for new functional breeding. The present invention also realizes the accumulation of active substances catechins in the endosperm of crop seeds, and provides a new idea of ​​technical methods for the synthesis of key substances with complex metabolic pathways. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0041] Figure 1 To reconstruct the catechin synthesis pathway in the endosperm of transgenic crop seeds;

[0042] Figure 2 Schematic diagram of the structure of the multi-gene assembly process; A is a schematic diagram of the structure of the TR1 vector; B is a schematic diagram of the structure of the TR2 vector;

[0043] Figure 3 The signal peak values ​​and change multiples of the top 20 metabolites transiently expressed in tobacco and the catechin ion flow diagram of transiently transformed tobacco leaves detected by HPLC; wherein, A is a bar graph of the signal peak values ​​and change multiples of the top 20 metabolites with higher metabolite changes in transiently expressed transcription factors OsRc and OsMYBP1, and flavonoid biosynthesis structural genes CsCHS, CsCHI, CsF3H, CcF3'5'H, CsDFR and CsLAR in tobacco; B is a catechin ion flow diagram of transiently transformed tobacco leaves detected by high performance liquid chromatography;

[0044] Figure 4 Figure 1 is a graph showing the results of PCR detection of rice; A is a graph showing the results of PCR identification of a TR1 transgenic rice material into which two genes were introduced, and 1-24 are 24 transgenic rice plants of the TR1 material; B is a graph showing the results of PCR identification of a TR2 transgenic rice material into which eight exogenous genes were introduced, and 1-24 are 24 transgenic rice plants of the TR2 material; in A and B, the sizes of the Marker bands are 5KB, 3KB, 2KB, 1KB, 0.75KB, 0.5KB, 0.3KB, 0.2KB and 0.1KB from top to bottom;

[0045] Figure 5The results of using RT-PCR to detect the expression of exogenous genes in seeds of rice transformants 15 days after flowering; A is a result of detecting the expression of exogenous genes in TR1 transgenic rice materials with 2 genes introduced; B is a result of detecting the expression of exogenous genes in TR2 transgenic rice materials with 8 genes introduced;

[0046] Figure 6 The results of the expression detection of endogenous genes in transgenic rice are shown in Figure 1. Among them, AL represents the expression level of rice phenylalanine ammonia lyase gene (OsPAL), rice cinnamate 4-hydroxylase gene (OsC4H), rice chalcone synthase gene (OsCHS), rice chalcone isomerase gene (OsCHI), rice flavonoid-3-hydroxylase gene (OsF3H), rice flavonoid-3'-hydroxylase gene (OsF3'H), rice flavonoid-3',5'-hydroxylase gene (OsF3'5'H), rice colorless anthocyanidin reductase gene (OsLAR), rice flavonoid synthase gene (OsFNS2), rice anthocyanidin reductase gene (OsANR), rice anthocyanidin transporter gene (OsANP) and rice WD40 repeat protein gene (OsW40), respectively.

[0047] Figure 7 The figure is the result of the identification of gallocatechin in the seeds of TR2 transgenic rice material by HPLC; A is the chromatogram; B is the ion flow diagram; C is the quantitative analysis result diagram;

[0048] Figure 8 The figure is the result of the identification of catechins in the seeds of TR2 transgenic rice material by HPLC; A is the chromatogram; B is the ion flow diagram; C is the quantitative analysis result diagram;

[0049] Fig. 9 This is the result of HPLC detection of Afzelechin in TR2 transgenic rice seeds; A is the chromatogram; B is the ion flow diagram; C is the quantitative analysis result diagram;

[0050] Fig.10 This is the result of detecting catechins in rice seeds by staining method;

[0051] Fig.11 This is the result of the rice seed antioxidant test; A is ABTS + A is the result diagram of free radical scavenging experiment; B is the result diagram of DPPH free radical scavenging experiment. DETAILED DESCRIPTION

[0052] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0053] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0054] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0055] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0056] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0057] The present invention obtains transgenic crops that synthesize catechins in seed endosperm by transferring a gene combination containing 6 genes expressing structural proteins and 2 genes expressing transcription factors into crops: the 6 structural proteins are from tea trees, namely CsCHI, CsCHS, CsF3H, CsDFR, CsLAR, and the structural protein CcF3'5'H of Crocosmia x crocosmiiflora; the 2 transcription factors are OsMYBP1 and OsRc. ​​The specific steps are to fuse the above genes with crop endosperm expression promoters to construct a multi-gene expression cassette, and finally obtain a plant transformation vector containing genes CsCHI, CsCHS, CsF3H, CsDFR, CsLAR, CcF3'5'H, OsMYBP1 and OsRc; transform the vector into breeding crops to obtain transgenic crops with rich catechins in seed endosperm (the synthesis pathway of catechins in seed endosperm is shown in Figure 1 ). Metabolite detection of the transgenic crop seeds obtained revealed that more than 20 flavonoids with increased content were found in TR1 polished rice, and catechins were detected in TR2 polished rice, mainly catechin, afzelechin and gallocatechin, with contents of 107.5 μg / g, 339.5 μg / g and 285.6 μg / g, respectively.

[0058] The information of the protein, its encoding gene and promoter involved in the present invention is as follows:

[0059] The CDS sequence of the chalcone isomerase gene (CsCHI) is shown in SEQ ID NO.1, and the amino acid sequence of the CsCHI protein is shown in SEQ ID NO.2;

[0060] The CDS sequence of the chalcone synthase gene (CsCHS) is shown in SEQ ID NO.3, and the amino acid sequence of the CsCHS protein is shown in SEQ ID NO.4;

[0061] The CDS sequence of the flavanone-3-hydroxylase gene (CsF3H) is shown in SEQ ID NO.5, and the amino acid sequence of the CsF3H protein is shown in SEQ ID NO.6;

[0062] The CDS sequence of the dihydroxyflavonol 4-reductase gene (CsDFR) is shown in SEQ ID NO.7, and the amino acid sequence of the CsDFR protein is shown in SEQ ID NO.8;

[0063] The CDS sequence of the leucocyanidin reductase gene (CsLAR) is shown in SEQ ID NO.9, and the amino acid sequence of the CsLAR protein is shown in SEQ ID NO.10;

[0064] The CDS sequence of the flavonoid-3',5'-hydroxylase gene (CcF3'5'H) is shown in SEQ ID NO.11, and the amino acid sequence of the CcF3'5'H protein is shown in SEQ ID NO.12;

[0065] The CDS sequence of the rice transcription factor OsRc gene is shown in SEQ ID NO.13, and the amino acid sequence of the OsRc protein is shown in SEQ ID NO.14;

[0066] The CDS sequence of the rice transcription factor OsMYBP1 gene is shown in SEQ ID NO.15, and the amino acid sequence of the OsMYBP1 protein is shown in SEQ ID NO.16;

[0067] The nucleotide sequence of promoter P16 is shown in SEQ ID NO.17; the nucleotide sequence of promoter P10 is shown in SEQ ID NO.18; the nucleotide sequence of promoter PA2 is shown in SEQ ID NO.19; the nucleotide sequence of promoter PgluC is shown in SEQ ID NO.20; and the nucleotide sequence of promoter PgluB4 is shown in SEQ ID NO.21.

[0068] The terminators used in the present invention were purchased from the Addgene website (https: / / www.addgene.org / ).

[0069] The primer sequences involved in the following examples are shown in Table 1.

[0070] Table 1 Primer sequences

[0071]

[0072] Example 1

[0073] The present invention firstly transiently overexpresses the key genes for catechin synthesis CsCHI, CsCHS, CsF3H, CsDFR, CsLAR, CcF3'5'H, OsMYBP1 and OsRc in tobacco, and detects that gallocatechin, catechin and afoetidin are produced, which are increased by nearly 9000 times, 700 times and 40 times respectively compared with the control group. Based on this study, the present embodiment constructs the genes CsCHI, CsCHS, CsF3H, CsDFR, CsLAR, CcF3'5'H, OsMYBP1 and OsRc into a multi-gene expression cassette vector driven by an endosperm-specific promoter, and the specific steps are as follows:

[0074] (1) The eight essential genes included the cloning of six structural genes, CsCHI, CsCHS, CsF3H, CsDFR, CsLAR, CcF3'5'H, and two transcription factors, OsMYBP1 and OsRc; in addition, the cloning of the rice endosperm expression promoter was also included.

[0075] The endosperm expression promoter pGluB4 was amplified using the primer pair pGluB4-F / R and the gDNA of rice ZH11 as a template; the endosperm expression promoter P16 was amplified using the primer pair P16-F / R and the gDNA of rice ZH11 as a template; the endosperm expression promoter P10 was amplified using the primer pair P10-F / R and the gDNA of rice ZH11 as a template; the endosperm expression promoter PA2 was amplified using the primer pair PA2-F / R and the endosperm expression promoter PgluC was amplified using the gDNA of rice ZH11 as a template.

[0076] The primer pair CsCHI-F / R was used to amplify the gene CsCHI using the cDNA of tea plant as a template; the primer pair CsCHS-F / R was used to amplify the gene CsCHS using the cDNA of tea plant as a template; the primer pair CsF3H-F / R was used to amplify the gene CsF3H using the cDNA of tea plant as a template; the primer pair CsDFR-F / R was used to amplify the gene CsDFR using the cDNA of tea plant as a template; the primer pair CsLAR-F / R was used to amplify the gene CsLAR using the cDNA of tea plant as a template; the primer pair CcF3'5'HF / R was used to amplify the gene CcF3'5'H using the cDNA of Kite odorifera as a template; the primer pair OsRc-F / R was used to amplify the gene OsRc using the cDNA of red rice as a template; the primer pair OsMYBP1-F / R was used to amplify the gene OsMYBP1 using the cDNA of ZH11 as a template.

[0077] (2) Construction of gene expression cassette

[0078] The present invention first constructs the endosperm promoters PgluB4, P16, P10, PA2, PgluC, and Pnos into the pUPD2 vector to obtain the primary expression element pUPD2-promotors; then constructs the genes CsCHI, CsCHS, CsF3H, CsDFR, CsLAR, CcF3'5'H, OsMYBP1, OsRc, and Hyg into the primary expression element pUPD2-genes, and ensures the sequence is correct by second-generation sequencing analysis. Subsequently, the primary elements of pUPD2-promotors, pUPD2-genes, and pUPD2-ternimators are further combined using the GoldenBraid vector assembly principle (Sarrion et al., 2011). Finally, the following gene expression cassette is constructed:

[0079] CsCHI gene expression cassette PgluB4::CsCHI::TAtubq3: contains the rice endosperm storage protein gene promoter PgluB4, the CsCHI gene encoding chalcone isomerase, and the TAtubq3 terminator;

[0080] CsCHS gene expression cassette P16::CsCHS::Tact2: contains the rice endosperm storage protein gene promoter P16, the CsCHS gene encoding chalcone synthase, and the Tact2 terminator;

[0081] CsF3H gene expression cassette P10::CsF3H::Tnos: contains rice endosperm storage protein gene promoter P10, CsF3H gene encoding flavanone-3-hydroxylase and Tnos terminator;

[0082] CsDFR gene expression cassette P10::CsDFR::Thsp18.2: contains rice endosperm storage protein gene promoter P10, CsDFR gene encoding dihydroxyflavonol 4-reductase and Thsp18.2 terminator;

[0083] CsLAR gene expression cassette PA2::CsLAR::T35S: contains rice endosperm storage protein gene promoter PA2, CsLAR gene encoding colorless anthocyanin reductase and T35S terminator;

[0084] CcF3'5'H gene expression cassette PgluC::CcF3'5'H::Tnos: contains the rice endosperm storage protein gene promoter PgluC, the CcF3'5'H gene encoding flavonoid-3',5'-hydroxylase, and the Tnos terminator;

[0085] The gene expression cassette of transcription factor OsMYBP1, P16::OsMYBP1::Tnos, contains the rice endosperm storage protein gene promoter P16, the OsMYBP1 gene encoding the rice transcription factor, and the Tnos terminator;

[0086] The gene expression cassette of transcription factor OsRc PgluB4::OsRC::T35S: contains the rice endosperm storage protein gene promoter PgluB4, the OsRc gene encoding the rice transcription factor and the T35S terminator.

[0087] In addition, an expression cassette Pnos::Hyg::Tnos with a promoter of Pnos, linked to a screening marker gene Hyg (NCBI accession number AUO29335.1), and a terminator of Tnos was constructed for screening transgenic positive materials. The nucleotide sequence of Pnos is shown in SEQ ID NO.66, and the nucleotide sequence of Tnos is shown in SEQ ID NO.67.

[0088] (3) Assembly of the multi-gene vector J24 for rice endosperm-specific catechin synthesis:

[0089] TeaRice-1 (TR1) vector construction: P16::OsMYBP1::Tnos and PgluB4::OsRC::T35S were combined as vector a, and then Pnos::Hyg::Tnos was combined with vector a to form OsRc::OsMYBP1::HygR (see the schematic diagram for the structure) Figure 2 Middle A).

[0090] TeaRice-2 (TR2) vector construction: P16::CsCHS::Tact2 and PgluB4::CsCHI::TAtubq3 were combined into the same vector and sequenced as vector b; P16::OsMYBP1::Tnos and PgluC::CcF3'5'H::Tnos were combined into the same vector and sequenced as vector c; P10::CsDFR::Thsp18.2 and PA2::CsLAR::T35S were combined into the same vector and sequenced as vector d; P10::CsF3H::Tnos and PgluB4::OsRC::T35S were combined into the same vector and sequenced as vector e. The next round of reaction was to combine vector b and vector c into the same vector and sequenced as vector f; vector d and vector e were combined into the same vector and sequenced as vector g. Vector f and vector g were combined into the same vector and sequenced as vector h. Vector h was combined with Pnos::Hyg::Tnos to obtain TR2 vector (see the schematic diagram for the structure). Figure 2 (B) The vector contains six structural genes and two transcription factors.

[0091] Example 2 Rice transformation and detection of multi-gene vectors TR1 and TR2

[0092] Tobacco transient expression: transcription factors OsRc and OsMYBP1, as well as flavonoid biosynthesis structural genes CsCHS, CsCHI, CsF3H, CcF3'5'H, CsDFR and CsLAR were subcloned into the PJF754 vector driven by the P35s promoter through the Gateway method and transformed into GV3101 Agrobacterium. Agrobacterium liquids from colonies identified as positive were mixed in a 1:1 ratio and transiently co-expressed in tobacco leaves. After 3 days, the changes in metabolites in tobacco leaves were detected. The results are shown in Figure 2. Figure 3 .

[0093] Genetic transformation of rice: callus was induced from mature seeds of rice variety Zhonghua 11 at 28°C in the dark (reference: Nishimura et al. 2006, A protocol for Agrobacterium-mediated transformation in rice. Nat Protoc., 1: 2796-2802) and the multi-gene expression vectors TR1 and TR2 were respectively transferred into EHA105 Agrobacterium. Agrobacterium was streaked and added to a suspension medium containing 100 μmol / L acetosyringone and 50% glucose and cultured to an OD value of 0.4. Fresh, light yellow, vigorously growing granular embryonic callus was selected and the bacterial solution was poured into the callus to fully infect it. After the bacterial liquid on the callus surface was dried with sterile filter paper, it was transferred to the co-culture medium and cultured for 2 days. It was then transferred to the screening medium containing 50 mg / L hygromycin. After resistant callus grew, it was transferred to the differentiation medium for differentiation and seedling emergence, thereby obtaining TR1 transgenic plants and TR2 transgenic plants.

[0094] The formulas of the co-culture medium, screening medium and differentiation medium are shown in Tables 2 to 4, respectively.

[0095] Table 2 Co-culture medium formula

[0096]

[0097] Table 3 Screening medium formula

[0098]

[0099] Table 4 Differentiation medium formula

[0100]

[0101] The transgenic seedlings were obtained, and their leaves were taken and DNA was extracted using the CTAB method, as follows: 2-3 cm long fresh rice leaf tissue was taken and placed in a 2 mL centrifuge tube containing 3 small steel balls. The sample grinder was set to 30 HZ frequency for 30 seconds to grind the rice leaves into powder. 800 μL of CTAB extraction solution was added to the 2 mL centrifuge tube containing the rice leaf powder. After vigorous shaking for 15 seconds, it was placed in a 65°C environment in order to allow CTAB to fully lyse the leaf tissue. After that, the sample was vigorously mixed every 5 minutes and repeated 3 times. An equal volume of chloroform / isoamyl alcohol (24:1) was added and vigorously shaken and allowed to stand until the DNA and organic phase separated, and then high-speed centrifugation was performed. The upper aqueous phase was aspirated in 500 μL of isopropanol and slowly flipped up and down. After about 10 minutes, the DNA precipitated to the bottom of the tube. 600 μL of 75% ethanol was added twice to wash the DNA precipitate, and the supernatant was discarded and aired until the DNA at the bottom of the tube became colorless and transparent. Add an appropriate amount of sterile water to dissolve the DNA and store it in a -20℃ refrigerator for later use. Use the DNA extracted by CTAB method as a template to amplify and detect the exogenous gene by PCR.

[0102] Identification of TR1 transgenic plants: Primers F1 and R1 were used to detect the OsRC gene, and a fragment of about 2004 bp was amplified; primers F2 and R2 were used to detect the OsMYBP1 gene, and a fragment of about 931 bp was amplified.

[0103] TR2 transgenic plant identification: Primers F1 and R1 were used to detect the OsRC gene, and a fragment of about 2004 bp was amplified; primers F3 and R3 were used to detect the CcF3'5'H gene, and a fragment of about 1584 bp was amplified

[0104] The positive rate of material identification was statistically analyzed, and the results showed that the positive rate of TR1 material was 50%, and the positive rate of TR2 material was 46% ( Figure 4 ).

[0105] Example 3 RT-PCR detection of transgenic plant seeds

[0106] RT-PCR tests were performed on TR1 and TR2 materials that were positive in DNA identification, respectively, using the following method:

[0107] Seeds taken 15 days after flowering were used for gene expression level identification, so as to analyze the expression level of related genes introduced into rice endosperm. The seeds need to be ground into powder under liquid nitrogen conditions, extracted with Trizol kit to extract seed RNA, and then reverse transcribed into cDNA with reverse transcription kit. RT-PCR detection of endogenous and exogenous genes was performed, using rice endogenous Actin as an internal reference and Zhonghua 11 seeds as a control. The results of exogenous gene detection are shown in Figure 5 The detection results of endogenous genes are shown in Figure 6The endogenous genes included: rice phenylalanine ammonia lyase gene (OsPAL), rice cinnamate 4-hydroxylase gene (OsC4H), rice chalcone synthase gene (OsCHS), rice chalcone isomerase gene (OsCHI), rice flavonoid-3-hydroxylase gene (OsF3H), rice flavonoid-3'-hydroxylase gene (OsF3'H), rice flavonoid-3',5'-hydroxylase gene (OsF3'5'H), rice colorless anthocyanidin reductase gene (OsLAR), rice flavonoid synthase gene (OsFNS2), rice anthocyanidin reductase gene (OsANR), rice anthocyanidin transporter gene (OsANP) and rice WD40 repeat protein gene (OsW40).

[0108] The detection primers are as follows:

[0109] The detection primers for CsCHS gene are F4 and R4; the detection primers for CsCHI gene are F5 and R5; the detection primers for CsDFR gene are F6 and R6; the detection primers for CcF3'5'H gene are F7 and R7; the detection primers for CsF3H gene are F8 and R8; the detection primers for CsLAR gene are F9 and R9; the detection primers for rice endogenous Actin gene are Actin-F and Actin-R.

[0110] The results show:

[0111] like Figure 5 As shown in B, compared with the wild-type control Zhonghua 11 (ZH11), the introduced exogenous genes were expressed in TR2. The expression level of CsCHI in TR2 / 3 and TR2 / 4 increased by about 4.5×10 4 times and 5.5×10 4 The expression level of CsCHS in TR2 / 3 and TR2 / 4 increased by about 4.9×10 6 times and 4.9×10 5 The expression level of CsDFR in TR2 / 3 and TR2 / 4 increased by about 5.2×10 4 times and 3.7×10 4 The expression levels of CcF3'5'H in TR2 / 3 and TR2 / 4 increased by about 66 times and 1.8×10 4 The expression level of CsLAR in TR2 / 3 and TR2 / 4 increased by about 3.6×10 4 times and 4.5×10 3 The expression level of CsF3H in TR2 / 3 and TR2 / 4 increased by about 7.4×10 4 times and 5.5×10 4The expression levels of OsMYBP1 in TR2 / 3 and TR2 / 4 increased by about 277 and 107 times compared with the wild-type control, and the expression levels of OsRC in TR2 / 3 and TR2 / 4 increased by about 1.9×10 5 times and 2.3×10 4 times.

[0112] Compared with the control Zhonghua 11, the transferred foreign genes were also expressed in TR1. Figure 5 As shown in A, the expression levels of OsMYBP1 in TR1 / 4 and TR1 / 6 increased by about 300 and 157 times compared with the wild-type control, and the expression levels of OsRC in TR2 / 3 and TR2 / 4 increased by about 1.9×10 5 times and 6.5×10 4 times.

[0113] Example 4 Detection of catechins in transgenic rice endosperm by high performance liquid chromatography

[0114] In order to detect the content of metabolites in transgenic rice endosperm, the transgenic rice seeds with 8 genes were ground into powder, 0.1 g of powder was taken, and 70% methanol was added at a ratio of 1 g: 8000 mL. After the sample was mixed, it was fully vortexed for 1 min with a vortexer and placed on ice. After extraction, it was centrifuged in a low-temperature high-speed centrifuge; then the sample supernatant was filtered by microporous membrane, and the filtered sample was stored in a metabolic bottle with an inner liner tube for subsequent HPLC-MS loading analysis. Since transcriptional regulation plays a very important role in metabolic regulation, the transcription factors OsRc and OsMYBP1 that regulate the flavonoid synthesis pathway in rice were transferred into the TR1 material, which can activate the expression of multiple genes for flavonoid synthesis. Metabolic level detection showed that compared with ZH11, a total of more than 20 flavonoid substances with increased content were detected in the transgenic rice endosperm TR1 material, indicating that the transcription factor successfully activated the flavonoid synthesis pathway, and the results were consistent with expectations. In the strategy of metabolic engineering, overexpression of key enzymes that produce catechins can improve the acquisition of target metabolites. TR2 material is a material that has been transformed with transcription factors OsRc and OsMYBP1 and added with five more active exogenous genes. Eleven flavonoids with increased content were detected in the transgenic rice endosperm TR2 material, mainly catechins such as catechin, afzelechin and gallocatechin ( Figure 7-Figure 9). This indicates that the five foreign genes introduced by the present invention play an important role in the production of catechins. Then, a quantitative analysis was performed on the catechins contained in the transgenic rice endosperm TR2 material. The total catechin content in TR2-4 was 401.8 μg / g, and the total catechin content in TR2-3 was 532.2 μg / g, indicating that the rice with rich endosperm catechins, namely "tea rice", was successfully created.

[0115] Example 5: Verification of the Antioxidant Effect of “Tea Rice” Material

[0116] 1. Staining Analysis

[0117] In order to more intuitively illustrate that transgenic rice seeds are rich in catechins, the present invention uses two specific catechin dyes, vanillin and dimethylaminophenol aldehyde (DMACA), to perform staining analysis on TR1 material and TR2 material. The catechin standard with a concentration of 100 ppm reacts with vanillin to present bright red, while Taxifolin as a negative control is colorless, indicating that vanillin does not react with flavonoids to form a color reaction with certain specificity.

[0118] Test results are shown in Fig.10 The results showed that the polished rice of TR2 material seeds was observed to be red when dyed with vanillin, the polished rice of TR1 material seeds was observed to be yellow, and ZH11 was colorless. The catechins at a concentration of 100 ppm reacted with DMACA to produce bright green, proanthocyanidin B2 (PRrocyanidin B2) as a positive control was black, and Taxifolin as a negative control was colorless, indicating that DMACA would not react with flavonoids and had a certain specificity. The results showed that the polished rice of TR2 material seeds reacted with DMACA to produce bright green. The above results show that there is a certain content of catechins in the polished rice of transgenic rice seeds.

[0119] 2. ABTS + Free radical scavenging experiment

[0120] Experimental steps: Take 0.1 g of rice seed polished rice sample and extract it with 70% methanol at a solid-liquid ratio of 1:10000. Vortex thoroughly and ultrasonicate in an ice water bath for 10 min, repeat the operation 3 times, and extract overnight in a dark environment at 4°C. Take 88 μL of 140 mM potassium persulfate solution and 5 mL of 7 mM ABTS solution and mix thoroughly. After reacting in the dark for 12 hours, ABTS is obtained. + Free radical solution. Dilute ABTS with anhydrous ethanol before use +The free radical solution was adjusted to an absorbance of 0.7 ± 0.002 at 400 nm. Trolox was dissolved in ethanol in a gradient of 400 ppm, 300 ppm, 200 ppm, 150 ppm, 100 ppm, 80 ppm, 50 ppm, and 20 ppm as a standard. Take 25 μL of the sample and add 5 mL of the pre-mixed ABTS. + The reaction solution was mixed thoroughly and reacted at room temperature in the dark for 10 min. The corresponding absorbance value was measured as At. Take 25 μL of the extract and add 5 mL of the pre-mixed ABTS+ reaction solution to get A0. Take 25 μL of the standard sample with different concentrations and add 5 mL of the pre-mixed ABTS+ reaction solution to get A0. + The absorbance of the reaction solution was measured at a wavelength of 734 nm using an enzyme reader.

[0121] ABTS + Free radical scavenging = (A0-At) / A0

[0122] Results: The present invention uses Trolox (a water-soluble vitamin E antioxidant) as a standard to detect the antioxidant capacity of tea rice. The results show that as a control, the antioxidant capacity of ZH11 is equivalent to 50ppm / g of Trolox; the antioxidant capacity of TR1 is equivalent to 180ppm / g of Trolox, and the antioxidant capacity of TR2 is equivalent to 220ppm / g of Trolox ( Fig.11 Middle A).

[0123] 3. DPPH free radical scavenging experiment

[0124] Experimental steps: Take 0.1 g of rice seed polished rice sample and extract it with 70% methanol at a solid-liquid ratio of 1:10000. Vortex thoroughly and ultrasonicate in an ice-water bath for 10 min, repeat the operation 3 times, and extract overnight in a dark environment at 4 °C. Weigh 1 mg DPPH and dissolve it in 100% methanol and dilute it to 20 mL to make its concentration 0.05%. The reagent is prepared immediately before use. Take 100 μL of sample, add 100 μL DPPH solution, and mix thoroughly to obtain Ai. Take 100 μL of sample and add 100 μL methanol to obtain Aj. Take 100 μL DPPH solution and add 100 μL methanol to obtain AO. Set up 2 biological replicates and 2 technical replicates for each sample. Incubate for 1 h in a dark room temperature environment, and measure the absorbance at a wavelength of 517 nm using an enzyme reader.

[0125] DPPH free radical scavenging rate / % =[AO-(Ai-Aj)] / A0×100%

[0126] Results: Fig.11As shown in B, in the DPPH free radical scavenging experiment, the free radical scavenging rate of ZH11 as a control was about 30%, the free radical scavenging rate of TR1 was about 50%, and the free radical scavenging rate of TR2 was about 70%.

[0127] The above two antioxidant experiments consistently show that the tea rice material of the present invention has a strong antioxidant capacity.

[0128] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A recombinant vector, characterized in that: Including a gene expression cassette for expressing CsCHI protein, a gene expression cassette for expressing CsCHS protein, a gene expression cassette for expressing CsF3H protein, a gene expression cassette for expressing CsDFR protein, a gene expression cassette for expressing CsLAR protein, a gene expression cassette for expressing CcF3'5'H protein, a gene expression cassette for expressing transcription factor OsMYBP1 protein and a gene expression cassette for expressing transcription factor OsRc protein; The amino acid sequence of the CsCHI protein is shown in SEQ ID NO.2; The amino acid sequence of the CsCHS protein is shown in SEQ ID NO.4; The amino acid sequence of the CsF3H protein is shown in SEQ ID NO.6; The amino acid sequence of the CsDFR protein is shown in SEQ ID NO.8; The amino acid sequence of the CsLAR protein is shown in SEQ ID NO.10; The amino acid sequence of the CcF3'5'H protein is shown in SEQ ID NO.12; The amino acid sequence of the transcription factor OsRc protein is shown in SEQ ID NO.14; The amino acid sequence of the transcription factor OsMYBP1 protein is shown in SEQ ID NO.16; The gene expression cassette for expressing the CsCHI protein comprises a promoter PgluB4, a gene encoding the CsCHI protein and a TAtubq3 terminator; The gene expression cassette for expressing the CsCHS protein comprises a promoter P16, a gene encoding the CsCHS protein and a Tact2 terminator; The gene expression cassette for expressing the CsF3H protein comprises a promoter P10, a gene encoding the CsF3H protein and a Tnos terminator; The gene expression cassette for expressing the CsDFR protein includes a promoter P10, a gene encoding the CsDFR protein and a Thsp18.2 terminator; The gene expression cassette for expressing the CsLAR protein comprises a promoter PA2, a gene encoding the CsLAR protein and a T35S terminator; The gene expression cassette for expressing the CcF3'5'H protein comprises a promoter PgluC, a gene encoding the CcF3'5'H protein and a Tnos terminator; The gene expression cassette for expressing the transcription factor OsMYBP1 protein comprises a promoter P16, a gene encoding the transcription factor OsMYBP1 protein and a Tnos terminator; The gene expression cassette for expressing the transcription factor OsRc protein comprises a promoter PgluB4, a gene encoding the transcription factor OsRc protein and a T35S terminator; The coding gene sequence of the CsCHI protein is shown in SEQ ID NO.1; The coding gene sequence of the CsCHS protein is shown in SEQ ID NO.3; The coding gene sequence of the CsF3H protein is shown in SEQ ID NO.5; The coding gene sequence of the CsDFR protein is shown in SEQ ID NO.7; The coding gene sequence of the CsLAR protein is shown in SEQ ID NO.9; The coding gene sequence of the CcF3'5'H protein is shown in SEQ ID NO.11; The coding gene sequence of the transcription factor OsRc protein is shown in SEQ ID NO.13; The coding gene sequence of the transcription factor OsMYBP1 protein is shown in SEQ ID NO.

15.

2. A recombinant host cell, characterized in that Comprising the recombinant vector according to claim 1; The recombinant host cell is Agrobacterium.

3. Use of the recombinant vector according to claim 1 or the recombinant host cell according to claim 2 in preparing transgenic rice having the ability to synthesize catechins in seed endosperm.

4. A transgenic breeding method for synthesizing catechins in rice seed endosperm, characterized in that: The method comprises the steps of transforming the recombinant vector according to claim 1 into rice callus tissue and then cultivating transgenic rice plants.

5. The transgenic breeding method according to claim 4, characterized in that: The recombinant vector is transformed into rice callus tissue by Agrobacterium transformation method.

6. A method for preparing rice with high catechin content, characterized in that: The method comprises the steps of obtaining rice seeds by planting transgenic rice and then obtaining the rice by hulling. The transgenic rice is cultivated by the transgenic breeding method according to claim 4 or 5.

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