Eggplant anthocyanin regulation gene SmCytb5 and application thereof

Abstract

The invention discloses a regulatory gene SmCytb5 of anthocyanin in eggplants and application of the regulatory gene SmCytb5, and relates to the technical field of biotechnology and plant genetic breeding. The SmCytb5 overexpression transgenic plant is constructed through experiments, compared with a wild type, the leaf color of the transgenic plant is changed from green to purple, the anthocyanin content is remarkably increased, and therefore it is proved that the SmCytb5 gene regulates expression of the anthocyanin in the eggplant, and a foundation is laid for a graduate to produce the eggplant rich in anthocyanin.

Description

Technical Field

[0001] The present invention relates to the technical fields of biotechnology and plant genetic breeding, and more specifically to an anthocyanin regulatory gene SmCytb5 in eggplant and its applications. Background Art

[0002] Eggplant (Solanum melongena L.) belongs to the genus Solanum of the Solanaceae family, is native to tropical Asia, and is a common vegetable rich in anthocyanins. Currently, it is one of the vegetables widely cultivated globally, with the cultivation output in Asia accounting for about 74% of the global total output. China has a long history of eggplant cultivation, and in recent years, the eggplant output in China has been continuously increasing. Eggplant is rich in nutrients, containing not only basic nutrients, but also many kinds of trace elements and various alkaloids. The eggplant fruit can be used as a vegetable, with various effects such as protecting the cardiovascular system, clearing heat and promoting blood circulation, anti-ascorbic acid, detumescence and analgesia, and anti-aging; the roots, stems, leaves, and seeds can all be used as medicine. Most importantly, the purple eggplant peel is rich in a large amount of anthocyanins, so it is known as one of the ten healthiest vegetables.

[0003] Anthocyanins are a class of water-soluble pigments widely present in plant stems, leaves, petals, and fruit peels. They not only endow plants with colorful colors but also protect them from various stress damages such as drought, extreme temperatures, and high salt. In addition to being very important for plants, anthocyanins also have important nutritional and pharmacological effects on humans and are increasingly favored by people as natural pigments and anti-aging foods.

[0004] Therefore, mining anthocyanin regulatory genes in eggplant is of great significance for eggplant breeding. Summary of the Invention

[0005] In view of this, the present invention provides an anthocyanin regulatory gene SmCytb5 in eggplant and its applications.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The SmCytb5 gene, the amino acid sequence encoded by it is as shown in SEQ ID NO.2.

[0008] Preferably, the nucleotide sequence of the SmCytb5 gene is as shown in SEQ ID NO.1.

[0009] Another object of the present invention is to provide the SmCytb5 protein, the amino acid sequence of which is as shown in SEQ ID NO.2.

[0010] Another object of the present invention is to provide a biological material, and the biological material is any one of the following:

[0011] A: An expression cassette capable of overexpressing the SmCytb5 gene with a nucleotide sequence as shown in SEQ ID NO.1;

[0012] B: A recombinant vector containing the expression cassette described in A;

[0013] C: A recombinant microorganism containing the expression cassette described in A or the recombinant vector described in B;

[0014] D: A non-renewable plant part containing the expression cassette described in A or the recombinant vector described in B or the recombinant microorganism described in C.

[0015] Another object of the present invention is to provide the application of the above gene, or the above protein, or the above biological material, and the application is to regulate the anthocyanin content in eggplants or cultivate eggplant varieties with high anthocyanin content.

[0016] Another object of the present invention is to provide a method for cultivating eggplant germplasm with high anthocyanin content, which improves the expression level of the SmCytb5 gene; wherein, the nucleotide sequence of the SmCytb5 gene is as shown in SEQ ID NO.1.

[0017] Another object of the present invention is to provide a method for increasing the anthocyanin content in eggplants, which improves the expression level of the SmCytb5 gene; wherein, the nucleotide sequence of the SmCytb5 gene is as shown in SEQ ID NO.1.

[0018] Another object of the present invention is to provide a method for identifying plants, whether the plant contains eggplants of the above biological material, or eggplants obtained by the above method, and it includes the following steps: determining whether the plant contains the above gene SmCytb5.

[0019] Preferably, the method is PCR verification, and the required primers include SmCytb5-F and vector primer pHB-R, and their nucleotide sequences are:

[0020] SmCytb5-F: 5'-ATGGACACAAAAAATATGTTC-3', as shown in SEQ ID NO.5;

[0021] pHB-R: 5'-GCATTGAACTTGACGAACGTTGTCGA-3', as shown in SEQ ID NO.6.

[0022] Preferably, the PCR reaction program:

[0023] Pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 s, annealing at 55°C for 5 s, extension at 72°C for 10 s, 35 cycles; final extension at 72°C for 5 min.

[0024] Beneficial effects: The present invention discloses and provides the SmCytb5 gene, the SmCytb5 protein and their applications. By experiments, the present invention constructs SmCytb5 overexpression transgenic plants. Compared with the wild type, the leaf color of the transgenic plants changes from green to purple, and the anthocyanin content increases significantly, thereby proving that the SmCytb5 gene regulates the expression of anthocyanin in eggplants and laying a foundation for the research and production of eggplants rich in anthocyanin. Brief description of the drawings

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0026] Figure 1 Schematic diagram of 'Jinyangwang No. 8' provided by the present invention.

[0027] Figure 2 Schematic diagram of PCR amplification of the full length of SmCytb5 provided by the present invention. Among them, Marker is 2000bp, and lanes 1 and 2 are PCR amplification of the full length of SmCytb5 of 441bp.

[0028] Figure 3 Gel electrophoresis diagram for verification of transgenic plants provided by the present invention. Among them, the corresponding Marker is 2000bp, lane 1 is the verification result of the wild type WT; lane 2 is the verification result of the transgenic plant overexpressing SmCytb5; lane 4 is the verification result of the SmCytb5 positive plasmid.

[0029] Figure 4 Results of investigation on the relative expression level of SmCytb5 overexpression transgenic plants provided by the present invention.

[0030] Figure 5 Schematic diagram showing a significant increase in anthocyanin content in SmCytb5 overexpression plants provided by the present invention. Among them, WT is the 'Jinyangwang No. 8' plant; OE-SmCytb5 is the transgenic purple plant of 'Jinyangwang No. 8'.

[0031] Figure 6 Schematic diagram showing a significant increase in the expression levels of the structural genes SmDFR, SmF3’5’H, SmMYB113 and Sm5GT related to anthocyanin synthesis, while the expression levels of the early anthocyanin synthesis genes SmCHI, SmCHS, SmF3H and SmF3’H are significantly down-regulated. Among them, WT is the 'Jinyangwang No. 8' plant; OE-SmCytb5 is the transgenic purple plant of 'Jinyangwang No. 8'. Detailed implementation manners

[0032] For the convenience of understanding the present invention, the present invention will be described more comprehensively below with reference to the embodiments. The following are the preferred embodiments of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. It should be understood that the experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. All common reagents used in the embodiments are commercially available products.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0034] Example 1

[0035] Cloning method of SmCytb5 gene

[0036] 1. Use the FastPure Universal Plant Total RNA Isolation Kit (Vazyme) kit to extract the total RNA of the leaves of 'Jinyangwang No. 8' (see the appendix Figure 1 ). The specific operation steps are as follows:

[0037] (1) Take 100 mg of plant tissue and grind it thoroughly into powder with liquid nitrogen. Immediately add 600 μl of Buffer EL and vortex vigorously for 30 s to fully mix the sample with the lysis solution. Centrifuge at 12,000 rpm for 5 min;

[0038] (2) Take the supernatant into FastPure gDNA-Filter Columns lll (about 500 μl) and centrifuge at 12,000 rpm for 30 s. Discard FastPure gDNA-Filter Columns lll and collect the filtrate;

[0039] (3) Add anhydrous ethanol with a volume 0.5 times that of the filtrate (about 250 μl, adjusted according to the actual situation of the supernatant) to the collection tube and mix well by shaking for 15 s;

[0040] (4) Transfer the above-mentioned mixed solution into FastPure RNA Columns V (FastPure RNA Columns V has been placed in the collection tube), centrifuge at 12,000 rpm for 30 s, and discard the filtrate;

[0041] (5) Add 700 μl of Buffer RWA to FastPure RNA Columns V, centrifuge at 12,000 rpm for 30 s, and discard the filtrate;

[0042] (6) Add 500 μl of Buffer RWB to FastPure RNA Columns V, centrifuge at 12,000 rpm for 30 s, and discard the filtrate;

[0043] (7) Repeat the previous step;

[0044] (8) Place FastPure RNA Columns V back into the collection tube and centrifuge at 12,000 rpm for 2 min;

[0045] (9) Transfer FastPure RNA Columns V to a new RNase-free Collection Tubes 1.5 ml centrifuge tube, and suspend and drip 60 μl of RNase-free ddH2O onto the center of the membrane of the adsorption column;

[0046] (10) Centrifuge at 12,000 rpm for 1 min, take an appropriate amount of RNA solution, measure the RNA concentration, and detect the RNA integrity using 1% agarose.

[0047] 2. PCR amplification of the full length of the SmCytb5 gene

[0048] Take 1 μg of total RNA, perform reverse transcription according to the instructions of the HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme) reagent, and dilute the reaction dilution 4 times as the template for PCR amplification. Primer sequences: SmCytb5-F: 5'-tctctctctcaagcttATGGACACAAAAAATATGTTC-3', as shown in SEQ ID NO.3; SmCytb5-R: 5'-tgcagctcgaggatccTCAAATCTGGAGAGCTCCAGTGG-3', as shown in SEQ ID NO.4. The PCR amplification system is shown in Table 1.

[0049] Table 1

[0050]

[0051] PCR reaction procedure: Pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 s, annealing at 55°C for 5 s, extension at 72°C for 10 s for a total of 35 cycles; final extension at 72°C for 5 min.

[0052] The PCR product was purified and recovered, and the fragment size was approximately 441 bp (electrophoresis results are shown in the appendix Figure 2 . The nucleotide sequence is:

[0053] ATGGACACAAAAAATATGTTCACCCTTTCTCAAGTTGCACAACACAAGTCAAAGCAAGATTGTTGGATCATCATCCATGGCAGAGTAATAGACGTAACGAAGTTTCTGGAAGAACATCCTGGAGGAGAGGAAGTGTTGATTGAATCAGCTGGAAAGGATGCAACTAAAGAATTTGAAGATATTGGGCACAGTAAAGCTGCCAAGAACTTTCTCTTGAAATACCAGATTGGATATCTTCAAGGCTATAAAATCCAAGATGATGATGATAATTTGTTCTCTGATTCCTACAAAGAACCAATAAAGGCAAAAGAAATGGAAGCTTTTGTGATCAAAGAAGATTCCAAGCCCAAGTATCTGGTTTTTGTTGAGTATTTTGTTCCTTTCTTGGCTGCTGCATTCTTTCTGTATTATCAATATCCCACTGGAGCTCTCCAGATTTGA, as shown in SEQ ID NO.1.

[0054] The amino acid sequence of SmCytb5: MDTKNMFTLSQVAQHKSKQDCWIIIHGRVIDVTKFLEEHPGGE EVLIESAGKDATKEFEDIGHSKAAKNFLLKYQIGYLQGYKIQDDDDNLFSDSYKEPIKAKE MEAFVIKEDSKPKYLVFVEYFVPFLAAAFFLYYQYPTGALQI, as shown in SEQ ID NO.2.

[0055] 3. Gel recovery of PCR products

[0056] After electrophoresis, use a gel imager to take pictures, select the length of the target band for gel recovery, and refer to the FastPure Gel DNA Extraction Mini Kit (Vazyme) DNA gel recovery kit. The specific operation steps are as follows:

[0057] (1) Cut the single target band with good specificity under ultraviolet light (operate quickly to prevent base mutations under ultraviolet light), and place it in a clean 1.5 ml centrifuge tube;

[0058] (2) Add an equal volume of Buffer GDP. Incubate in a water bath at 50 - 55 °C for 7 - 10 min, and adjust the time appropriately according to the gel size to ensure that the gel block is completely dissolved. Invert and mix 2 times during the water bath to accelerate the solubilization;

[0059] (3) Briefly centrifuge to collect the droplets on the tube wall. Place the FastPure DNA Mini Columns - G adsorption column in a 2 ml Collection Tubes collection tube, transfer the sol solution to the adsorption column, and centrifuge at 12000 rpm for 30 s;

[0060] (4) Discard the filtrate, add 300 μl of Buffer GDP, let stand for 1 min, and centrifuge at 12000 rpm for 30 s;

[0061] (5) Discard the filtrate, add 700 μl of Buffer GW, and centrifuge at 12000 rpm for 30 s;

[0062] (6) Repeat the previous step;

[0063] (7) Place the adsorption column in a sterilized 1.5 ml centrifuge tube, add 25 μl of Elution Buffer to the center of the adsorption column, and let stand for 2 min.

[0064] (8) Centrifuge at 12000 rpm for 1 min. Obtain the DNA solution and store it at -20 °C.

[0065] 4. Ligation of the cloning vector

[0066] Use -Blunt Simple Cloning Kit (TRANS) reagent to ligate the PCR gel extraction product to the cloning vector -Blunt Simple Cloning Vector as follows. The cloning reaction system is shown in Table 2.

[0067] Table 2

[0068]

[0069] Gently mix the above reaction solution and incubate at 20 °C - 37 °C for 30 min (the reaction time is determined according to the fragment size). After the reaction, place the centrifuge tube on ice.

[0070] 5. Transformation of the cloning vector and screening of positive clones

[0071] (1) Place the ligated product and Escherichia coli competent cells on ice. Take 50 μl of the thawed Escherichia coli competent cells and add them to 10 μl of the recombinant product.

[0072] (2) Gently mix and place on ice for 30 min. Turn on the water bath at 42 °C and prepare 600 μl of liquid LB in the laminar flow hood.

[0073] (3) Heat shock in a 42 °C water bath for 45 s, then immediately transfer to ice for 2 min.

[0074] (4) Add to 600 μl of liquid LB medium and mix well.

[0075] (5) Incubate at 37 °C with shaking at 250 rpm for 1 h to allow the bacteria to recover. Prepare the laminar flow hood and spreader.

[0076] (6) Centrifuge at 5000 rpm for 30 s and discard the supernatant.

[0077] (7) Pipette up and down repeatedly in the laminar flow hood and spread evenly on LB agar medium containing kanamycin antibiotic using a sterilized spreader.

[0078] (8) Incubate upside down overnight in a 37 °C constant temperature incubator in the dark.

[0079] (9) Use a sterilized toothpick to pick a single colony and streak it on a plate and transfer it to a PCR tube for centrifugation. Use 2×TaqMaster Mix (Dye Plus) (Vazyme) reagent for PCR verification. The reaction system is shown in Table 3 below.

[0080] Table 3

[0081]

[0082] PCR reaction program: Pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, extension at 72 °C for 1 min for a total of 35 cycles; final extension at 72 °C for 5 min.

[0083] (10) After the reaction is completed, perform electrophoresis detection. Send the positive clones to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Perform BLAST analysis on the sequencing results.

[0084] Example 2

[0085] Construction of SmCytb5 gene overexpression vector

[0086] 1. Plasmid extraction

[0087] In this experiment, a PurePlasmid Mini Kit (CWBIO) high-purity plasmid mini-prep kit was used to extract the plasmid:

[0088] (1) Add 1 ml of the overnight cultured bacterial solution into a 1.5 ml centrifuge tube, centrifuge at 13,000 rpm for 30 s to collect the bacterial cell pellet, and discard the supernatant as much as possible.

[0089] (2) Repeat the above steps twice.

[0090] (3) Add 250 μl of Buffer P1 (RNase A has been added) to the centrifuge tube containing the bacterial cell pellet, and use a vortex oscillator to mix well to suspend the bacterial cell pellet (if not thoroughly mixed, it will affect the lysis effect, resulting in low extraction yield and purity).

[0091] (4) Add 250 μl of Buffer P2 to the centrifuge tube, gently invert the tube 4 - 6 times (vigorous shaking will break the genomic DNA, causing genomic DNA fragments to be mixed in the extracted plasmid), to fully lyse the bacteria. At this time, the solution should become clear and viscous. The time used for this step should not exceed 5 min to avoid plasmid damage.

[0092] (5) Add 350 μl of Buffer N3 to the centrifuge tube, immediately invert the tube 8 - 10 times. At this time, a white flocculent precipitate appears. Centrifuge at 13,000 rpm for 12 min.

[0093] (6) Transfer the obtained supernatant to the adsorption column (Spin Columns DM) in the collection tube in batches, centrifuge at 13,000 rpm for 30 s, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0094] (7) Add 150 μl of Buffer PB to the adsorption column, centrifuge at 13,000 rpm for 30 s, and pour out the waste liquid in the collection tube.

[0095] (8) Add 400 μl of Buffer PW (anhydrous ethanol has been added) to the adsorption column, centrifuge at 13,000 rpm for 1 min, and pour out the waste liquid in the collection tube.

[0096] (9) Put the adsorption column back into the collection tube, centrifuge at 13,000 rpm for 1 min, and pour out the waste liquid in the collection tube.

[0097] (10) Place the adsorption column in a new 1.5 ml centrifuge tube, add 60 μl of Buffer EB to the adsorption membrane, let it stand at room temperature for 3 min, centrifuge at 13,000 rpm for 1 min to obtain the plasmid solution, and store it at -20 °C.

[0098] 2. Plasmid digestion

[0099] The pHB plasmid was from the research group of Professor Yang Hongquan at Fudan University (Kang, C.Y., Lian, H.L., Wang, F.F., Huang, J.R., and Yang, H.Q. 2009. Cryptochromes, Phytochromes, and COP1 Regulate Light-Controlled Stomatal Development in Arabidopsis. The Plant Cell 21, 2624-2641). The enzyme digestion reaction system is shown in Table 4.

[0100] Table 4

[0101]

[0102] Digest at 37°C for 30 min, and detect the enzyme digestion products and the control by agarose gel electrophoresis. Recover the linearized fragment from the gel.

[0103] 3. Ligation of expression vector

[0104] Use the ClonExpress Ultra One Step Cloning Kit V2 (Vazyme) product. Based on the principle of special recombinase and homologous recombination, efficient seamless splicing is achieved. The system is as follows. Table 5 shows the establishment of the expression reaction system.

[0105] Table 5

[0106]

[0107] Gently mix the above system (Linearized vector is the product recovered from the gel in step 2 of this example, and Inserts is the product recovered from the gel in step 4 of Example 1), and react at 50°C for 15 min. After the reaction, place the centrifuge tube on ice to cool for several seconds, and then use the product for transformation into Escherichia coli (the same as step 5 in Example 1). Then extract the plasmid from the positive clone and transform it into Agrobacterium.

[0108] 4. Transformation of Agrobacterium by freeze-thaw method

[0109] (1) Place the competent cells of Agrobacterium LBA4404 on ice to thaw. Take 10 μl of the expression vector and 50 μl of the competent cells of Agrobacterium into a 1.5 ml centrifuge tube, gently mix and place on ice for 5 min. Turn on the water bath at 37°C and prepare the LB liquid medium.

[0110] (2) Take it out and quickly freeze it in liquid nitrogen for 5 min, heat shock it in a 37°C water bath for 5 min, and then immediately place it on ice for 5 min.

[0111] (3) Add all of it to 600 μl of LB liquid medium and incubate it with shaking at 220 rpm at 28 °C for 2 - 3 h. Prepare a laminar flow hood and a spreader.

[0112] (4) Centrifuge at 4000 rpm for 1 min and discard the supernatant.

[0113] (5) Pipette the precipitated bacterial solution repeatedly, and use a sterilized spreader in the laminar flow hood to spread it on an LB medium containing 25 mg / L rifampicin and 50 mg / L kanamycin for resistance screening. Incubate it upside down in an incubator at 28 °C until single colonies grow.

[0114] (6) Pick single colonies for PCR verification of positive clones and preservation of the bacterial solution.

[0115] 5. Obtaining transgenic seedlings by Agrobacterium infection

[0116] Sow the seeds of 'Jinyangwang No. 8' in the laminar flow hood. After dark incubation until germination, transfer them to light for about 7 d until the eggplant cotyledons are flattened. Cut the cotyledons and lay them flat on the pre - culture medium. Shake the Agrobacterium tumefaciens LBA4404 containing the recombinant plasmid pHB - SmCytb5 overnight. The next day, perform large - scale shaking culture. When the OD value of the bacterial solution is 0.6, centrifuge at 3000 g for 10 min. Discard the supernatant in the laminar flow hood, dissolve the Agrobacterium precipitate attached to the bottom of the centrifuge tube with MS liquid medium to make an Agrobacterium suspension. Pipette an appropriate amount and add it to 20 ml of MS liquid medium to make the infection concentration OD value about 0.2. Gently immerse the cotyledon explants on the pre - culture medium in the Agrobacterium suspension for 15 - 20 min, shaking once every 5 min during this period. After the infection, take out the cotyledons with forceps, try not to damage the cotyledons, dry the residual bacterial solution on the surface of the explants with sterile paper, and then inoculate the cotyledons on the co - culture medium and place them in the dark for co - culture for 4 d. The infected cotyledons are placed on the differentiation and screening medium to grow. The medium is changed every about 15 d, and small seedlings are differentiated from the callus in about 4 months. Cut the small seedlings in the laminar flow hood and place them in the rooting medium for rooting culture. Obtain transgenic plants.

[0117] Example 3

[0118] Verification of transgenic plants

[0119] 1. Extraction of eggplant DNA

[0120] Extract the total DNA of eggplant by the CTAB method. The steps are as follows:

[0121] (1) Turn on the water bath at 65 °C, add 1 ml of 2% CTAB extraction solution to a centrifuge tube, and place it in the water bath to pre - heat for 15 min.

[0122] (2) Take 0.2 g of eggplant tissue material, grind it into powder with liquid nitrogen, transfer it to preheated CTAB, mix well, incubate at 65 °C for 15 - 30 min, and shake it several times during this period;

[0123] (3) Centrifuge at 10000 rpm at room temperature for 7 min, and take about 700 μl of the supernatant;

[0124] (4) Add an equal volume of chloroform / isoamyl alcohol (24 / 1) for extraction, and shake vigorously to mix well;

[0125] (5) Centrifuge at 12000 rpm at room temperature for 10 min, and take about 650 μl of the supernatant;

[0126] (6) Add an equal volume of isopropanol, mix gently, and place at -20 °C for 20 min;

[0127] (7) Centrifuge at 12000 rpm at room temperature for 5 min, and discard the supernatant;

[0128] (8) Wash the precipitate twice with 75% ethanol, and air dry;

[0129] (9) Add 40 μl of ddH2O to dissolve, detect by electrophoresis, and store at -20 °C.

[0130] Use SmCytb5 - F (5'-ATGGACACAAAAAATATGTTC-3', as shown in SEQ ID NO.5;) and pHB - R (5'-GCATTGAACTTGACGAACGTTGTCGA-3', as shown in SEQ ID NO.6) for PCR transgenic verification. The reaction conditions and procedures refer to the PCR amplification process in Table 1. The results show that the plant is a transgenic SmCytb5 plant (as Figure 3 shown).

[0131] 2. Determination of anthocyanin content in transgenic plants

[0132] (1) Weigh 0.2 g of eggplant leaf material, grind it into powder with liquid nitrogen, add 300 μl of methanol acidified with 1% HCl to submerge it, and incubate at 4 °C overnight;

[0133] (2) Add 200 μl of deionized water and 200 μl of chloroform, shake well, and centrifuge at 14000 rpm at room temperature for 10 min;

[0134] (3) Pipette 300 μl of the supernatant into a new centrifuge tube, and add an equal volume of 60% methanol acidified with 1% HCl;

[0135] (4) The relative amount of anthocyanin in eggplant leaves was calculated according to the formula (A530 - A657) / g by measuring A530 and A657 using a UV spectrophotometer (UV-2600, Shimadzu).

[0136] The results showed that the leaves of OE-SmCytb5 transgenic plants changed from green to purple, and the anthocyanin content increased significantly (as Figure 5 shown).

[0137] 3. RNA was extracted from the leaves of wild-type and transgenic plants respectively (the steps were the same as step 1 in Example 1), and fluorescence quantitative verification was carried out. The ChamQ SYBR qPCR Master Mix (Low Rox Premixed) (Vazyme) reagent was used in the experiment, and the instrument was Applied BiosystemsTM 7500 Fast Dx Real-Time PCR. The operation was as follows:

[0138] The primer sequences used for fluorescence quantification are shown in Table 6.

[0139] Table 6

[0140]

[0141]

[0142] The following system was added to a 0.2 ml fluorescence quantitative optical flat-cap 8-tube strip, see Table 7:

[0143] Table 7

[0144]

[0145] Program: Pre-denaturation at 95 °C for 3 min; 95 °C for 10 s, 60 °C for 30 s, 40 cycles; Melt curve, End.

[0146] The results showed that the expression level of SmCytb5 in SmCytb5 transgenic plants was significantly up-regulated (as Figure 4 shown). Overexpression of SmCytb5 could significantly up-regulate the expression levels of SmF3’5’H and the downstream genes SmDFR, SmMYB113 and Sm5GT related to anthocyanin synthesis in the anthocyanin synthesis pathway, and down-regulate the expression levels of the early anthocyanin synthesis genes SmCHI, SmCHS, SmF3H and SmF3’H upstream of SmF3’5’H (as Figure 6 shown). These results indicate that the SmCytb5 gene can regulate the synthesis of anthocyanin and significantly increase the anthocyanin content in plants.

[0147] The various embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. For the same or similar parts among the various embodiments, reference can be made to each other.

[0148] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. SmCytb5 gene, characterized in that The amino acid sequence encoded by it is shown in SEQ ID NO.

2.

2. The SmCytb5 gene according to claim 1, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

1.

3. SmCytb5 protein, characterized in that Its amino acid sequence is shown in SEQ ID NO.

2.

4. A biomaterial, characterized in that: The biological material is any one of the following: A: an expression cassette capable of overexpressing the SmCytb5 gene with a nucleotide sequence as shown in SEQ ID NO.1; B: a recombinant vector containing the expression cassette described in A; C: a recombinant microorganism containing the expression cassette described in A or the recombinant vector described in B; D: A non-regenerable plant part containing the expression cassette described in A, the recombinant vector described in B, or the recombinant microorganism described in C.

5. Use of the gene according to claim 1 or 2, or the protein according to claim 3, or the biomaterial according to claim 4, characterized in that: The application is to regulate the anthocyanin content in eggplant or cultivate high anthocyanin eggplant varieties.

6. A method for cultivating high anthocyanin eggplant germplasm, characterized in that: Increase the expression level of the SmCytb5 gene; wherein the nucleotide sequence of the SmCytb5 gene is shown in SEQ ID NO.

1.

7. A method for increasing the anthocyanin content of eggplant, characterized in that: Increase the expression level of the SmCytb5 gene; wherein the nucleotide sequence of the SmCytb5 gene is shown in SEQ ID NO.

1.

8. A method for identifying a plant, characterized in that Whether the plant comprises the eggplant of the biological material of claim 4, or the eggplant obtained by the method of claim 6, it comprises the following step: determining whether the plant comprises the gene SmCytb5 of claim 1.

9. The method for identifying plants according to claim 8, characterized in that: The determination method is PCR verification, and the required primers include SmCytb5-F and vector primer pHB-R, whose nucleotide sequence is: SmCytb5-F: 5′-ATGGACACAAAAAATATGTTC-3′, as shown in SEQ ID NO. 5; pHB-R: 5′-GCATTGAACTTGACGAACGTTGTCGA-3′, as shown in SEQ ID NO.

6.

10. The method for identifying plants according to claim 9, characterized in that: PCR reaction procedure: Pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 s, annealing at 55°C for 5 s, extension at 72°C for 10 s, 35 cycles; final extension at 72°C for 5 min.

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