Application of Kale BoaBEE2 Gene in Regulating Carotenoid Content in Plants

Abstract

The present invention relates to the field of gene editing and breeding, and in particular to the application of the kale BoaBEE2 gene in regulating the carotenoid content in plants. In a specific embodiment of the present invention, the transgenic plant containing the gene obtained by genetic engineering has a significantly increased carotenoid content. It can be seen that the kale BoaBEE2 gene can effectively increase the carotenoid content in plants, and has application prospects in cultivating high-quality cruciferous plant varieties, especially cultivating kale varieties with high carotenoid content.

Description

Technical Field

[0001] The present invention relates to the field of gene editing and breeding, and in particular to the application of the kale BoaBEE2 gene in regulating the carotenoid content in plants. Background Art

[0002] In addition to their color and health effects on the human body, carotenoids are also widely involved in the physiological processes in plants and play the role of signal molecules in the interaction between plants and the external environment, including plant growth and development and plant responses to environmental stimuli. The biosynthesis pathway of carotenoids is relatively conservative and has been revealed in many plants. Carotenoids are a class of fat-soluble terpenoid pigments composed of isoprenoid units, which are widely present in various animals, plants and microorganisms. As secondary metabolites, carotenoids not only participate in the regulation of many important physiological and biochemical processes during the growth and development of plants, but are also important nutrients for humans and animals because of their strong antioxidant and anti-tumor activities. In addition, carotenoids, as an essential component of human dietary nutrition, play an important role in protecting vision, anti-oxidation, anti-aging, anti-cancer and defense against cardiovascular and cerebrovascular diseases.

[0003] Cruciferous plants (Brassicaceae Burnett) are annual, biennial or perennial plants of the order Brassicaceae, rarely subshrubs. Cruciferous plants often have cells containing black mustard glucosinolate that produce a special spicy smell. Most are herbaceous, rarely subshrubs; roots sometimes swell into thick tubers; stems are upright or spread; basal leaves are spiral or rosette-shaped; flowers are neat, bisexual, and rarely degenerate into unisexuality; fruits are long or short siliques; seeds are generally small, with smooth or textured surfaces. Cruciferous plants have economic value, among which Brassica and Radish are the main vegetables and oil crops in China. Among them, kale is a Brassica vegetable rich in carotenoids, native to China. Due to its unique flavor, rich nutrients, and effects such as tumor prevention and blood lipid reduction, the market demand continues to increase. Therefore, increasing the content of natural carotenoids in kale and cultivating high-quality kale varieties have broad development prospects. Summary of the invention

[0004] The purpose of the present invention is to provide an application of the kale BoaBEE2 gene in regulating the carotenoid content in plants to solve the problems existing in the above-mentioned prior art. Overexpression of the kale BoaBEE2 gene of the present invention can increase the carotenoid content in plants, which has guiding significance for cultivating plants with high carotenoid content.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides the use of a kale BoaBEE2 gene, a biological material containing the BoaBEE2 gene, or a protein encoded by the BoaBEE2 gene in regulating the carotenoid content in a plant. The nucleotide sequence of the kale BoaBEE2 gene is shown in SEQ ID NO.1; the amino acid sequence of the protein of the kale BoaBEE2 gene is shown in SEQ ID NO.2.

[0007] Preferably, the plant comprises a plant of the family Cruciferae.

[0008] Preferably, the cruciferous plants include mustard, cabbage and kale.

[0009] Further preferably, the cruciferous plants include mustard, cabbage and kale, but are not limited to mustard, cabbage and kale.

[0010] Preferably, the biological material includes a recombinant vector or a recombinant bacterium.

[0011] The present invention provides a method for increasing the carotenoid content in a plant, comprising: overexpressing a BoaBEE2 gene in a plant to increase the carotenoid content in the plant; the nucleotide sequence of the BoaBEE2 gene is shown in SEQ ID NO.1.

[0012] Preferably, the plant comprises a plant of the family Cruciferae.

[0013] Preferably, the cruciferous plants include mustard, cabbage and kale.

[0014] Further preferably, the cruciferous plants include mustard, cabbage and kale, but are not limited to mustard, cabbage and kale.

[0015] The present invention provides the use of the kale BoaBEE2 gene, biological materials containing the BoaBEE2 gene or proteins encoded by the BoaBEE2 gene in cultivating high carotenoid content and / or improving transgenic kale. The nucleotide sequence of the kale BoaBEE2 gene is shown as SEQ ID NO.1; the amino acid sequence of the protein of the kale BoaBEE2 gene is shown as SEQ ID NO.2.

[0016] Preferably, the plant comprises a cruciferous plant;

[0017] The cruciferous plants include mustard, cabbage and kale.

[0018] Further preferably, the cruciferous plants include mustard, cabbage and kale, but are not limited to mustard, cabbage and kale.

[0019] Preferably, the biological material includes a recombinant vector or a recombinant bacterium.

[0020] The present invention discloses the following technical effects:

[0021] The present invention provides the use of the kale BoaBEE2 gene in regulating the carotenoid content in plants, wherein the carotenoid biosynthesis regulatory gene BoaBEE2 gene is cloned from the wild type kale 'Four Seasons Thick Strips' using PCR amplification technology, and the BoaBEE2 gene can increase the carotenoid content in kale. The kale BoaBEE2 gene provided by the present invention is conducive to a deeper understanding of the regulatory mechanism of kale carotenoid biosynthesis. In a specific embodiment of the present invention, the transgenic plant containing the gene obtained by genetic engineering has significantly increased carotenoid content. It can be seen that the kale BoaBEE2 gene can effectively increase the carotenoid content in plants, and has application prospects in cultivating and improving high-quality kale varieties, especially cultivating kale varieties with high carotenoid content. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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.

[0023] Figure 1 Comparison of leaf colors between WT plants and BoaBEE2-OE (BEE2-OE) plants;

[0024] Figure 2 The results of pigment content analysis of WT plants and BoaBEE2-OE plants are shown in Figure 2.

[0025] Figure 3 The fluorescence quantitative PCR results of WT plants and BoaBEE2-OE plants are shown;

[0026] Figure 4 A comparison of the colors of WT callus and BoaBEE2-OE callus;

[0027] Figure 5 The results of the analysis of the pigment content in WT callus and BoaBEE2-OE callus are shown;

[0028] In the figure, WT is the wild-type kale material, and OE is the kale material overexpressing BoaBEE2. DETAILED DESCRIPTION

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] Example 1: Kale BoaBEE2 gene and its application in Kale plants

[0035] 1. Test methods

[0036] 1. Extraction of total RNA from Chinese kale and synthesis of cDNA

[0037] Total RNA was extracted from mature leaves of wild-type kale 'Si Ji Cu Tiao' using an RNA extraction kit, and the extracted total RNA was reverse transcribed into cDNA using a reverse transcription kit;

[0038] 2. Cloning of BoaBEE2 gene from Kale:

[0039] The nucleotide sequence of the BoaBEE2 gene is shown in SEQ ID NO.1, specifically: ATGGACTTGTCTGTACT TGAAAGAGTTAAATGGCTGCAGCAGCAACAAATGGTCTCACCTGATTTTTTTGAGATACTTGGCTCAGATGGGAGAGAAGAGCTCAAAAGGGTTGAGAGTTATTTGGGAAACAATAATGATGAGTTGCAGAGTTTCAGACATTATGAAACTATTGATGGCTGCATCTCCAGGACAAGTAGCTTCCAAATTGAGCAAGTGAAGAACAATGAAGAAAACAGAGTCATTGCCTTGCAGCACAAGAGAAAAACAGAGGGTAAGACAGAAAAGATAGAGAACAAGAAGAAGAAGAAGATCAAAACAGAGGATGAAACAGAGTCAAGCATGAAAGGGAAATCAAACATGAGCAACTCAGAGACATCCTCAGAGATTCAGAAGCGAGACTACATCCATGTGAGAGCTAGAAGAGGTGAAGCCACAGACAGACATAGCTTAGCAGAAAGGGCAAGGAGAGAAAAGTTAAGCAAGAAGATGAAATGTCTACAAGACATTGTTCCTGGATGCAACAAAGTTACCGGTAAAGCCGGTATGCTTGATGAGATCATCAACTATGTCCAATCTCTGCAACAACAAGTCGAGTTCTTGTCTATGAAACTCTCTGTCTTAAATCCAGAGCTCGAGTATCACATCAATGAATTATCCACCAAACAGTTTCAGGCTTGCTTCACTGATCTACCAGAAGCCGTCTCGAAGCAGTTAATGATGGTGGATGCAAGTTCTTTTCCATTACAACATCAAGAATCTCTAGATTACTCTGTCATAAACTCAATCCAAACCACAACTCTCGGCTCTAAAGATCAAACATCTTCAGGCTGGGAAATTCACTCACAATCTCTTTACAACAACTTGAGAACCGATTCAGTTTCAAGTTTCTTCAGCCTCGAGTAA;

[0040] The amino acid sequence of the protein encoded by the BoaBEE2 gene is shown in SEQ ID NO.2, specifically: MDLSVLERVKWLQQQQMVSPDFFEILGSDGREELKRVESYLGNNNDELQSFRHYETIDGCISRTSSFQIEQVKNNEENRVIALQHKRKTEGKTEKIENKKKKKIKTEDETESSMKGKSNMSNSETSSEIQKRDYIHVRARRGEATDRHSLAERARREKLSKKMKCLQDIVPGCNKVTGKAGMLDEIINYVQSLQQQVEFLSMKLSVLNPELEYHINELSTKQFQACFTDLPEAVSKQLMMVDASSFPLQHQESLDYSVINSIQTTTLGSKDQTSSGWEIHSQSLYNNLRTDSVSSFFSLE;

[0041] Design specific primers for BoaBEE2 gene cloning based on the nucleotide sequence of BoaBEE2 gene:

[0042] Forward primer: 5'-ATGGACTTGTCTGTACTTGAAAGAG-3' (SEQ ID NO. 3), reverse primer: 5'-TTACTCGAGGCTGAAGAAACTTG-3' (SEQ ID NO. 4).

[0043] The cDNA of 'Four Seasons Thick Strip' was used as template for amplification. The PCR reaction conditions were: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 57℃ annealing for 5s, 72℃ extension for 5s, 34 cycles in total; 72℃ extension for 1min. The PCR product was subjected to 1.0% mass volume fraction agarose gel electrophoresis to recover the target band, ligated to pClone007 vector (Beijing Qingke Biotechnology Co., Ltd.) and transformed into Escherichia coli DH5α, the plasmid was extracted, and after PCR identification, it was entrusted to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0044] 3. Construction of recombinant expression vector of BoaBEE2 gene

[0045] (1) First, the pMDC83 linearized vector was obtained by double restriction digestion (Spe I and Kpn I (Thermo Scientific)), and then purified and recovered;

[0046] (2) CE Design V1.04 was used to design the homologous recombination primers of the BoaBEE2 gene (SEQ ID NO.29 and SEQ ID NO.30, SEQ ID NO.29 is specifically: 5'-gacctcgactctagaactagtATGGACTTGTCTGTACTTGAAAGAGTTAA-3' (upstream primer), SEQ ID NO.30 is specifically: 5'-ctcgagggggggcccggtaccTTACTCGAGGCTGAAGAAACTTGA-3' (downstream primer), the target fragment of the BoaBEE2 gene was amplified and recovered (the method is the same as above), the target fragment DNA and the linearized vector pMDC83 were recombined by homologous recombination, 5 μL of the reaction solution was added to 50 μL of DH5a competent cells, and the cells were gently mixed with a pipette, incubated on ice for 30 min, heat-shocked in a 42°C water bath for 45 s, and then quickly cooled on ice for 2 min;

[0047] (3) Add 700 μL LB liquid medium and incubate at 37°C for 1 h. Collect the cells by centrifugation at 5,000 rpm for 1 min, discard part of the supernatant, resuspend the cells with the remaining medium, and gently spread them on the LB solid medium containing Kan resistance (recombination reaction transformation plate) with a sterile spreader stick, and incubate inverted in a 37°C incubator for 16-24 h;

[0048] (4) Pick several clones on the recombination reaction transformation plate for colony PCR identification. Pick the positive colonies and culture them in liquid LB medium containing Kan antibiotics in an incubator at 37°C and 200 rpm overnight. The bacterial solution is entrusted to Beijing Qingke Biotechnology Co., Ltd. for sequencing to identify the accuracy of the vector. The vector with accurate sequencing is the recombinant vector pMDC83-BoaBEE2;

[0049] (5) After successful identification, save the recombinant vector pMDC83-BoaBEE2.

[0050] 4. Transform the recombinant vector into Agrobacterium GV3101:

[0051] (1) Add 0.5 μg of the recombinant vector pMDC83-BoaBEE2 to every 50 μL of GV3101 Agrobacterium competent cells, stir the bottom of the tube by hand to mix, and place on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min. Add 700 μL of LB liquid medium without antibiotics and culture at 28°C with shaking for 2 h;

[0052] (2) Collect the cells by centrifugation at 6000 rpm for 1 min, and keep about 100 μL of the supernatant. Gently blow and resuspend the cells, and evenly spread them on LB solid medium containing kan and rif (the concentration of kan in the medium is 50 μg / mL, and the concentration of rif is 20 μg / mL). Invert and place in a 28°C incubator for 2 days, and select several positive clones to simply verify the results by colony PCR;

[0053] 5. Explant culture of Chinese kale

[0054] Sterile kale seeds were sown on 1 / 2MS solid culture medium and cultured in a tissue culture room. The conditions in the tissue culture room were: temperature 25 / 20°C (day / night), photoperiod 16 / 8h (day / night), and light intensity 36μmol·m -2 ·S -1 After 7 days of culture, kale cotyledons with 1-2 mm petioles were cut and used as explants for Agrobacterium-mediated transformation.

[0055] 6. Genetic transformation of Chinese kale

[0056] (1) Explants were pre-cultured in MS medium for 3 days with a photoperiod of 16 h light / 8 h dark and a light intensity of 80 μmol·m -2 ·S -1 ;

[0057] (2) Infect with Agrobacterium strain GV3101 into which the recombinant vector has been transferred for 1-2 minutes;

[0058] (3) The explants were co-cultured with Agrobacterium in MS medium for 3 days under dark conditions;

[0059] (4) The explants were transferred to delayed selection medium (0.03 mg / L NAA + 0.75 mg / L 6-BA + 0.8% agar + 325 mg / L Carb + 325 mg / L Timentin) and grown for 7 days;

[0060] (5) The explants were transferred to a resistance screening medium (0.03 mg / L NAA + 0.75 mg / L 6-BA + 0.8% agar + 325 mg / L Carb + 325 mg / L Timentin + 12 mg / L Hyg) containing selective antibiotics for resistance screening. The explants were subcultured every 20 days or so, and the subculture medium used was the same as the resistance screening medium.

[0061] (6) After the explants grew for 3 months, hygromycin-resistant plants were obtained and then transplanted into a matrix composed of nutrient soil, perlite, and vermiculite in a volume ratio of 3:1:1. The explants were placed under a light intensity of 80 μmol·m -2 ·S-1 , cultured in an artificial climate box with a temperature of 25 / 20°C (day / night), a photoperiod of 12 / 12h (day / night), and a relative humidity of 75%.

[0062] 7. Positive plant screening

[0063] Specific primers Hyg-F and Hyg-R (Table 1) were designed according to the hygromycin sequence contained in the vector, and PCR amplification was performed using the genomic DNA of each transgenic plant as a template. After detection by agarose gel electrophoresis, transgenic positive plants were screened and kept as seeds to obtain the T1 generation of kale overexpressing the BoaBEE2 gene;

[0064] Table 1 Detailed information of primers

[0065] name sequence SEQ ID NO. Hyg-F CGATTGCGTCGCATCGACC 5 Hyg-R TTCTACAACCGGTCGCGGAG 6

[0066] 8. Phenotypic observation

[0067] (1) Wild-type kale (WT) and BoaBEE2-overexpressing kale T1 (BoaBEE2-OE) seeds were sown simultaneously in a matrix composed of nutrient soil, perlite, and vermiculite in a volume ratio of 3:1:1. The seeds were grown under a light intensity of 80 μmol·m -2 ·S -1 , cultured in an artificial climate chamber with a temperature of 25 / 20°C (day / night), a photoperiod of 12 / 12h (day / night), and a relative humidity of 75%;

[0068] (2) When the leaves of the overexpression plants and the control plants were grown for 30 days, the color difference was analyzed using a colorimeter. Three positions were randomly selected for color difference measurement on the leaves of each plant, and the three color difference values ​​of L*, a* and b* were statistically analyzed; the results are shown in Tables 2 and Figure 1 shown.

[0069] Table 2 Statistical results of the three color difference values ​​of L*, a* and b*

[0070] Plant type L* a* b* WT <![CDATA[39.85 a ]]> <![CDATA[-7.51 a ]]> <![CDATA[20.96 a ]]> BoaBEE2-OE <![CDATA[37.54 b ]]> <![CDATA[-8.65 b ]]> <![CDATA[16.52 b ]]>

[0071] Note: Different shoulder letters indicate significant differences.

[0072] From Table 2 and Figure 1 It can be seen that compared with WT plants, the leaf color of T1 generation kale plants overexpressing the BoaBEE2 gene is darker.

[0073] 9. Pigment content analysis

[0074] (1) Leaves of WT and BoaBEE2-overexpressing kale T1 plants grown for 30 days were collected, 200 mg per plant, and carotenoids were extracted with 25 mL of acetone. After ultrasonic treatment for 20 min, the leaves were centrifuged at 4000 × g for 5 min at room temperature, and the supernatant was filtered with a 0.22 μm cellulose acetate filter and then analyzed by high performance liquid chromatography (HPLC). (2) Chlorophyll and carotenoids were analyzed by HPLC using isopropanol and 80% acetonitrile-water at 30°C at 0.5 mL min -1 The flow rate is in Waters C 18 The sample (10 μL) was separated on a chromatographic column (150×3.9 mm inner diameter; particle size 3 μm), and the absorbance was detected at 448 and 428 nm. The HPLC analysis results showed that the carotenoid content of the T1 generation of kale overexpressing the BoaBEE2 gene was significantly higher than that of the WT plants ( Figure 2 ).

[0075] 10. Gene expression analysis

[0076] (1) The third true leaf of WT and BoaBEE2-OE plants (BoaBEE2-OE is the T1 generation of BoaBEE2 gene-overexpressing kale) grown for 30 days was used as the material, immediately frozen with liquid nitrogen, and stored in a -80°C refrigerator;

[0077] (2) RNA and cDNA were obtained according to the method in “1. Extraction of total RNA from kale and synthesis of cDNA”;

[0078] (3) 10 genes of Chinese kale and the internal reference gene β-actin were selected to design expression detection primers, as shown in Table 3;

[0079] Table 3 Primers

[0080]

[0081] (3) Real-time quantitative PCR using TB Green Premix Ex Taq TM The expression of carotenoid biosynthesis genes in kale was detected according to the instructions of the II qPCR Kit (Tli RNaseHPlus, Takara);

[0082] (4) The formula for calculating the relative transcriptional expression level of the target gene is 2 -ΔΔCt , ΔΔCt=(Ct 目的基因 -Ct Actin ) treatment group-(Ct 目的基因 -Ct Actin )Control group.

[0083] Fluorescence quantitative PCR results showed that the expression of 10 carotenoid biosynthesis genes in BoaBEE2-OE plants was higher than that in WT ( Figure 3 ).

[0084] The above-mentioned fluorescence quantitative expression and physiological index analysis results prove that overexpression of kale BoaBEE2 gene can significantly increase the carotenoid content of kale.

[0085] Example 2: Kale BoaBEE2 gene and its application in Kale callus

[0086] 1. Test methods

[0087] 1. Extraction of total RNA from Chinese kale and synthesis of cDNA

[0088] The total RNA was extracted from mature leaves of wild-type kale 'Si Ji Cu Tiao' using the CTAB method.

[0089] (1) Add 15 mL of CTAB extraction buffer (300 μL of β-mercaptoethanol is required) to a 50 mL centrifuge tube and preheat to 65°C.

[0090] (2) Weigh 2-3 g of fresh plant material and grind it into powder in liquid nitrogen;

[0091] (3) The sample was transferred to a 50 mL centrifuge tube and rapidly shaken to mix until no lumps formed. The sample was incubated at 65°C for 10 min, and then 15 mL of chloroform was added and mixed.

[0092] (4) Centrifuge at 10,000 rpm for 15 min at room temperature, transfer the upper aqueous phase into a new centrifuge tube, add an equal volume of chloroform, mix well, and extract again;

[0093] (5) Transfer the upper aqueous phase to another centrifuge tube, add 8 M LiCl to a final concentration of 2 M (approximately 1 / 3 of the aqueous phase), and incubate at 4 °C overnight;

[0094] (6) Centrifuge at 12,000 rpm for 20 min at 4°C to precipitate RNA, discard the supernatant, wash the precipitate with 70% (volume percentage) ethanol, and air dry;

[0095] (7) Add 500 μL SSTE buffer to dissolve the precipitate, transfer to a 1.5 mL centrifuge tube, add an equal volume of acid phenol / chloroform (the volume ratio of acid phenol to chloroform is 5:1) and mix well;

[0096] (8) Centrifuge at 13,000 rpm for 10 min at room temperature, transfer the upper aqueous phase to another 1.5 mL centrifuge tube, add an equal volume of chloroform and mix;

[0097] (9) Centrifuge at 13,000 rpm for 10 min at room temperature, transfer the upper aqueous phase to a new tube, add 1 / 10 volume of 3 M NaAC and 2 volumes of anhydrous ethanol, and incubate at -80 °C for 30 min;

[0098] (10) Centrifuge at 13,000 rpm for 20 min at 4°C, wash the precipitate with 70% (volume percentage) ethanol, dry it, add 50 μL of DEPC-treated H2O to dissolve the RNA, take 1 μL for electrophoresis, and store it at -80°C for later use.

[0099] The extracted total RNA was reverse transcribed into cDNA using the PrimeScript 1st Strand cDNA Synthesis Kit (Baoriyi Biotechnology (Beijing) Co., Ltd.);

[0100] 2. Cloning of BoaBEE2 gene from Kale:

[0101] According to the nucleotide sequence of BoaBEE2 gene (SEQ ID NO.1), specific primers for BoaBEE2 gene cloning were designed:

[0102] The forward primer is shown in SEQ ID NO.3, and the reverse primer is shown in SEQ ID NO.4.

[0103] The cDNA of 'Four Seasons Thick Strip' was used as template for amplification. The PCR reaction conditions were: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 57℃ annealing for 5s, 72℃ extension for 5s, 34 cycles in total; 72℃ extension for 1min. The PCR product was subjected to 1.0% mass volume fraction agarose gel electrophoresis to recover the target band, ligated to pTPOP-Blunt Simple vector (Beijing Ailaide Biotechnology Co., Ltd.) and transformed into Escherichia coli DH5α. The extracted plasmid was identified by PCR and then commissioned to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0104] 3. Construction of recombinant expression vector of BoaBEE2 gene

[0105] (1) First, the linearized vector pCAMBIA1301-35S-Nos (purchased from Beijing Solebow Technology Co., Ltd.) was obtained by double enzyme digestion (BamH I and Sal I (Thermo Scientific)), and then purified and recovered using a magnetic bead micro-gel recovery kit;

[0106] (2) CE Design V1.04 was used to design the homologous recombination primers of BoaBEE2 gene (SEQ ID NO.31 and SEQ ID NO.32, SEQ ID NO.31 is specifically: 5'-gatctgcagcccgggggatccATGGACTTGTCTGTACTTGAAAGAGTTAA-3' (upstream primer) SEQ ID NO.32 is specifically: 5'-aagcttatcgataccgtcgacTTACTCGAGGCTGAAGAAACTTGA-3') (downstream primer), the target fragment of BoaBEE2 gene was amplified and recovered (the method is the same as above), and the target fragment DNA and linearized vector pCAMBIA1301-35S-Nos were recombined by homologous recombination method, 5 μL of the reaction solution was added to 50 μL of DH5a competent cells, and gently mixed with a pipette, incubated on ice for 30 min, heat shocked in a 42°C water bath for 45 s, and then quickly cooled on ice for 2 min;

[0107] (3) Add 700 μL LB liquid medium and incubate at 37°C for 1 h. Collect the cells by centrifugation at 5,000 rpm for 1 min, discard part of the supernatant, resuspend the cells with the remaining medium, and gently spread them on the LB solid medium containing Kan resistance with a sterile spreader stick. Incubate inverted in a 37°C incubator for 16-24 h;

[0108] (4) Pick several clones on the recombination reaction transformation plate for colony PCR identification. Pick the positive colonies and culture them in liquid LB medium containing Kan antibiotics at 37°C and 200 rpm in an incubator overnight. The bacterial solution is entrusted to Beijing Qingke Biotechnology Co., Ltd. for sequencing to identify the accuracy of the vector. The vector with accurate sequencing is the recombinant vector pCAMBIA1301-BoaBEE2;

[0109] (5) After successful identification, save the recombinant vector pCAMBIA1301-BoaBEE2.

[0110] 4. Transform the recombinant vector into Agrobacterium EHA105:

[0111] (1) Add 0.5 μg of the recombinant vector pCAMBIA1301-BoaBEE2 to every 50 μL of EHA105 Agrobacterium competent cells, stir the bottom of the tube by hand to mix, place on ice for 5 min, place in liquid nitrogen for 5 min, place in a 37°C water bath for 5 min, and place in an ice bath for 5 min, then add 700 μL of LB liquid medium without antibiotics and culture at 28°C with shaking for 2 h;

[0112] (2) Collect the cells by centrifugation at 6000 rpm for 1 min, and keep about 100 μL of the supernatant. Gently blow and resuspend the cells, and evenly spread them on LB solid medium containing kan and rif (the concentration of kan in the medium is 50 μg / mL, and the concentration of rif is 20 μg / mL). Invert and place in a 28°C incubator for 2 days, and select several positive clones to simply verify the results by colony PCR;

[0113] 5. Callus culture of Chinese kale

[0114] Sterile seeds were sown on 1 / 2MS solid medium and cultured in a tissue culture room. The conditions in the tissue culture room were: temperature 25 / 20°C (day / night), photoperiod 16 / 8h (day / night), and light intensity 36μmol·m -2 ·S -1 After 7 days of culture, 0.5-1.0 cm of kale hypocotyls were cut and placed as explants for Agrobacterium-mediated transformation.

[0115] 6. Genetic transformation of Chinese kale callus

[0116] (1) The explants were precultured in MS medium containing 2,4-D and 6-BA for 7 days, wherein the concentration of 2,4-D was 0.5 mg / L and the concentration of 6-BA was 2 mg / L;

[0117] (2) Infect with Agrobacterium strain EHA105 into which the recombinant vector has been transferred for 1-2 minutes;

[0118] (3) The explants were co-cultured with Agrobacterium for 3 days in MS medium containing 2,4-D, 6-BA, and AS (acetosyringone), wherein the concentration of 2,4-D was 0.5 mg / L, the concentration of 6-BA was 2 mg / L, and the concentration of AS was 0.2 mM;

[0119] (4) The explants were transferred to screening medium (0.5 mg / L 2,4-D + 2 mg / L 6-BA + 0.8% agar + 200 mg / L Carb + 300 mg / L Timentin + 12 mg / L Hyg) and grown for 7 days.

[0120] 7. Phenotypic observation

[0121] Wild-type kale callus (WT) and BoaBEE2-overexpressing kale callus (BoaBEE2-OE) were placed under a NSZ-608T stereo microscope and photographed. Figure 4 When the BoaBEE2 gene cloned by the present invention was transferred into wild-type kale callus, the BoaBEE2-OE kale callus was yellow, while the uninfected wild-type kale callus was white ( Figure 4 ).

[0122] 8. Pigment content analysis

[0123] (1) 200 mg of WT and BoaBEE2-OE kale callus were taken, carotenoids were extracted with 25 mL of acetone, and then ultrasonicated for 20 min. The carotenoids were centrifuged at 4000 × g for 5 min at room temperature. The supernatant was filtered with a 0.22 μm cellulose acetate filter and then analyzed by high performance liquid chromatography (HPLC);

[0124] (2) Chlorophyll and carotenoids were analyzed by HPLC using an Agilent 1260 instrument with a VWD detector at 30°C using isopropanol and 80% acetonitrile-water at a rate of 0.5 mL min -1 The flow rate is in Waters C 18 The sample (10 μL) was separated on a chromatographic column (150×3.9 mm inner diameter; particle size 3 μm), and the absorbance was detected at 448 and 428 nm.

[0125] HPLC analysis results showed that the carotenoid content in BoaBEE2-OE kale callus was higher than that in wild type ( Figure 5 ).

[0126] The above phenotypic and pigment analysis results prove that overexpression of the kale BoaBEE2 gene can significantly increase the carotenoid content of kale.

[0127] In summary, it can be seen that the carotenoid content of the transgenic plant containing the gene obtained by genetic engineering in the specific embodiment of the present invention is significantly increased. It can be seen that the kale BoaBEE2 gene can effectively increase the carotenoid content in the plant, and has application prospects in cultivating and improving high-quality cruciferous plant varieties, especially cultivating kale varieties with high carotenoid content.

[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. Use of the BoaBEE2 gene of kale, a biological material containing the BoaBEE2 gene, or a protein encoded by the BoaBEE2 gene in regulating the carotenoid content in kale, characterized in that: The nucleotide sequence of the kale BoaBEE2 gene is shown as SEQ ID NO.1; the amino acid sequence of the protein of the kale BoaBEE2 gene is shown as SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The biological material includes a recombinant vector or a recombinant bacterium.

3. A method for increasing the carotenoid content in kale, characterized in that: include: The BoaBEE2 gene is overexpressed in kale to increase the content of carotenoids in the kale; the nucleotide sequence of the BoaBEE2 gene is shown in SEQ ID NO:

1.

4. Use of the BoaBEE2 gene of kale, a biological material containing the BoaBEE2 gene, or a protein encoded by the BoaBEE2 gene in cultivating transgenic kale with high carotenoid content and / or improving transgenic kale, characterized in that: The nucleotide sequence of the kale BoaBEE2 gene is shown in SEQ ID NO.1; the amino acid sequence of the protein of the kale BoaBEE2 gene is shown in SEQ ID NO.

2.

5. The use according to claim 4, characterized in that: The biological material includes a recombinant vector or a recombinant bacterium.

Images