Eggplant SmNPC2 gene and its application

Abstract

The invention discloses an eggplant SmNPC2 gene and an application thereof, and belongs to the technical field of genetic engineering. The CDS sequence of the eggplant SmNPC2 gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. The invention discloses the eggplant SmNPC2 gene for the first time, and discloses that under normal phosphorus concentration treatment, overexpression of the eggplant SmNPC2 gene can significantly increase the number of lateral roots, main root length and plant height of the eggplant, and knocking out the eggplant SmNPC2 gene can significantly reduce the number of lateral roots, main root length, plant height, stem thickness and root tip auxin content of the eggplant; after low phosphorus concentration treatment, the stem thickness, number of lateral roots and main root length of the SmNPC2 gene overexpression plant and the knockout plant are not significantly different from those under normal phosphorus treatment, but the plant height is significantly higher than that under low phosphorus concentration treatment; the eggplant SmNPC2 gene can be used to cultivate new eggplant varieties with efficient phosphorus utilization.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, in particular to an eggplant SmNPC2 gene and an application thereof. Background Art

[0002] Eggplant (Solanum melongena L.) is rich in protein, fat, carbohydrates, vitamins, and nutrients such as calcium, phosphorus, and iron. It has the effects of lowering cholesterol, preventing microvascular rupture and bleeding, and inhibiting the proliferation of digestive tract tumor cells, and is therefore deeply loved by consumers.

[0003] The absorption of phosphorus by Solanum mainly occurs during the flowering and fruiting periods. Insufficient phosphorus affects the development of eggplant flowers and fruit setting, so phosphorus is one of the important factors that determine the formation of eggplant yield. In traditional agricultural production, increasing the amount of fertilizer is often used to increase eggplant yield. However, problems such as excessive fertilizer application and low land nutrient utilization rate will seriously affect the healthy development of the eggplant industry. Therefore, the use of modern biotechnology to promote the efficient use of phosphorus fertilizers plays an important role in promoting the cost-saving, quality-enhancing and efficiency-enhancing production of eggplant and the high-quality development of the industry.

[0004] Studies have shown that under low-phosphorus stress, plants will increase their absorption of phosphorus by promoting the development and elongation of lateral roots and expanding the root absorption area. Auxin signals are involved in the development of primary and lateral roots. Plant nonspecific phospholipase C (NPC) is involved in a series of abiotic stresses and plays an important role in root and lipid remodeling. Whether NPC and its hydrolysis product diacylglycerol (DAG) are involved in auxin-regulated root development has not been reported.

[0005] Therefore, how to provide an eggplant root development regulating gene to increase eggplant nutrient absorption is a problem that technicians in this field need to solve urgently. Summary of the invention

[0006] The purpose of the present invention is to provide an eggplant SmNPC2 gene and its application, so as to provide a gene that has a significant effect on the root development, plant height and stem thickness of eggplant, and to provide relevant genes for gene-assisted breeding.

[0007] To achieve the above object, the present invention provides an eggplant SmNPC2 gene, the CDS sequence of the SmNPC2 gene is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO.2.

[0008] The biological material comprises the above-mentioned SmNPC2 gene, wherein the biological material is an overexpression vector, a gene editing vector, a cloning vector, an engineering bacterium comprising the SmNPC2 gene, or a plant that overexpresses or knocks out / silences the SmNPC2 gene.

[0009] An application of the eggplant SmNPC2 gene as described above in regulating the height of eggplant plants, overexpression of the eggplant SmNPC2 gene significantly increases the plant height of eggplant, and knockout of the eggplant SmNPC2 gene significantly reduces the plant height of eggplant.

[0010] An application of the eggplant SmNPC2 gene as described above in regulating the development of the eggplant root system, wherein overexpression of the eggplant SmNPC2 gene significantly increases the number of lateral roots and the length of the main root of the eggplant, and knockout of the eggplant SmNPC2 gene significantly reduces the number of lateral roots and the length of the main root of the eggplant.

[0011] An application of the eggplant SmNPC2 gene as described above in regulating the stem thickness of eggplant, wherein knocking out the eggplant SmNPC2 gene significantly reduces the stem thickness of eggplant.

[0012] An application of the eggplant SmNPC2 gene as described above in eggplant breeding, wherein the eggplant breeding is eggplant breeding for efficient phosphorus utilization.

[0013] Therefore, the eggplant SmNPC2 gene and its application provided by the present invention have the following specific technical effects:

[0014] (1) The present invention discloses the eggplant SmNPC2 gene for the first time, and finds that under normal phosphorus concentration treatment, overexpression of the eggplant SmNPC2 gene can significantly increase the number of lateral roots, main root length and plant height of eggplant, and knockout of the eggplant SmNPC2 gene can significantly reduce the number of lateral roots, main root length, plant height, stem diameter and root tip auxin content of eggplant; after low phosphorus concentration treatment, the stem diameter, number of lateral roots and main root length of the SmNPC2 gene overexpression plants and knockout plants were not significantly different from those under normal phosphorus concentration treatment, and the plant height was significantly higher than that under low phosphorus concentration treatment;

[0015] (2) The eggplant SmNPC2 gene can be used to breed new eggplant varieties with efficient phosphorus utilization.

[0016] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0018] Figure 1 This is a gel electrophoresis diagram of the PCR amplification product of the SmNPC2 gene in Example 2 of the present invention;

[0019] Figure 2This is the verification result of the overexpression transgenic plant in Example 3 of the present invention;

[0020] Figure 3 The results of investigating the relative expression levels of the overexpressing transgenic plants in Example 3 of the present invention are as follows;

[0021] Figure 4 The sequencing comparison results of the plants with SmNPC2 gene knocked out in Example 4 of the present invention;

[0022] Figure 5 The photographs are of wild-type plants (WT), overexpressing transgenic plants (OE), and SmNPC2 gene knockout mutants (npc2) after normal phosphorus concentration treatment (A) and low phosphorus concentration treatment (B) in Example 5 of the present invention;

[0023] Figure 6 The statistical results of plant height (A), stem diameter (B), number of lateral roots (C), and main root length (D) of the wild-type plant (WT), the overexpression transgenic plant (OE), and the SmNPC2 gene knockout mutant (npc2) in Example 5 of the present invention are shown;

[0024] Figure 7 The handheld fluorescence identification result in Example 6 of the present invention, wherein OE is the overexpression plant and WT is the wild-type plant;

[0025] Figure 8 This is the root auxin distribution diagram of the knockout mutant npc2 plant (npc2) and the wild type (WT) in Example 7 of the present invention, green is the location of auxin distribution, and the scale is 50 microns;

[0026] Fig. 9 This is a diagram of the root cell morphology of the knockout mutant npc2 plant and the wild type in Example 7 of the present invention, with a scale of 80 microns. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0028] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0029] The instruments, reagents, kits and carriers used in the examples can all be obtained through commercial channels, and the methods not mentioned are all conventional experimental methods.

[0030] The components of the culture medium used in the examples are shown in the following table.

[0031]

[0032] Embodiment 1

[0033] The SmNPC2 gene was cloned. The CDS sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. The method is as follows:

[0034] 1.1. Total RNA was extracted from leaves of 'Changza No. 8' eggplant using the Trizol method. The specific steps are as follows:

[0035] (1) Add 1 mL of Trizol reagent to an RNase-free centrifuge tube;

[0036] (2) Grind 100 mg of young eggplant leaves into powder in liquid nitrogen, add to 1 mL of Trizol, vortex and mix, and let stand at room temperature for 5 min;

[0037] (3) Centrifuge at 12,000 rpm, 4°C for 10 min;

[0038] (4) Take the supernatant, transfer it to a new centrifuge tube, add 200 μL of chloroform, shake vigorously by hand for 15 seconds to remove the protein, and let it stand at room temperature for 5 minutes;

[0039] (5) Centrifuge at 12,000 rpm, 4°C for 15 min;

[0040] (6) The solution is now divided into three layers. Take the top layer (about 500 μL) and place it in a new centrifuge tube. Add an equal volume of isopropanol, mix by inverting, and let stand at room temperature for 10 min.

[0041] (7) Centrifugation at 12,000 rpm and 4°C for 10 min;

[0042] (8) Discard the supernatant, add 1 mL of 75% ethanol, thoroughly wash the tube cap and tube wall, and gently tap the bottom of the tube to suspend the precipitate. Let it stand for 5 min.

[0043] (9) Centrifugation at 12000 rpm and 4°C for 5 min;

[0044] (10) Wash the precipitate again with 1 mL of 75% ethanol, discard the supernatant, and dry the precipitate in a clean bench;

[0045] (11) Add 30 μL of RNase-free ddH2O to the dried precipitate and allow it to dissolve.

[0046] (12) After the precipitate is completely dissolved, take 1 μL of RNA solution and measure the RNA concentration. Take 5 μL and use 1% agarose to detect RNA integrity.

[0047] 1.2. PCR amplification of the full length of SmNPC2 gene

[0048] Take 5 μL of total RNA solution and Reverse transcription was performed according to the instructions of the One-Step gDNA Removal and cDNA Synthesis SuperMix (TRANS) kit, and the cDNA solution diluted to a final concentration of was used as a template for PCR amplification, SmNPC2F (sequence as shown in SEQ ID NO.3) and SmNPC2R2 (sequence as shown in SEQ ID NO.4) were used as primers, and PCR was performed using the amplification system shown in Table 1. The PCR reaction program was: pre-denaturation at 95°C for 3min / 30sec; denaturation at 95°C for 15s, annealing at 58°C for 15s, denaturation + annealing for 32 cycles; extension at 72°C for 1min; and complete extension at 72°C for 5min.

[0049] Table 1

[0050] Element Volume (μL) <![CDATA[ddH2O]]> Up to 25 Template cDNA 1 SmNPC2-F (10 μM) 1 SmNPC2-R (10 μM) 1 2*PrimerStarMix 12.5

[0051] SEQ ID NO.1

[0052]

[0053] SEQ ID NO.2

[0054] MSSKNHTTKSPATVFVAGLLLSLILVDNHHHNWAAAAAAASSPIKTVVVLVMENRSFDHMLGWMKKLNPEINGVDGSESNPISTGDPKSRRIFFGDQSHYVDPDPGHSFQAMREQIFGSNDSSKNPAPMNGFAQ QALSMDSNMPEQVMNGFQPDMVAVYKTLVSEFAVFDRWFASVPASTQPNRLYVHSATSHGATSNIASLLAKGYPQRTIFENLDDAGINFGIYYQNIPATLFYLNLRKLKYVGKFHPYDLTFKSDAKSGKLPGYV VVEQRYMDSKLQPANDDHPSHDVYQGQMFVKEVYETLRSSPQWNETLFIITYDEHGGFFDHVPTPVSGVPSPDGIVGPDPFDFKFDRLGVRIPTIMISPWIEKGTVVHGPNGSPFPTSEYEHSSIPATVKKIF DLPSPFLTKRDAWAGTFEQILQIRKEPRTDCPEKLPTPTKIRKGEANEDANISEFQQELVQLAAVLKGDHLLTSYPEKIGKGMTVRQGKAYMEDAVRRFFEAGFAAKRIGVDEEHIVQMRPSLTTRSSSTNLHP

[0055] SEQ ID NO.3: TCTTCACTGTTGATACATATGTCATCCAAAAATCACACA

[0056] SEQ ID NO.4: GCCCTTGCTCACCATCATATGAGGGTGCAAATTTGTAGATGA

[0057] Embodiment 2

[0058] Connect the PCR product to the cloning vector as follows:

[0059] 2.1. Gel recovery of the PCR product obtained in Example 1

[0060] The PCR products were subjected to agarose gel electrophoresis ( Figure 1 After that, a gel imager was used to take pictures, and the length of the target band (about 1605 bp) was selected for gel recovery. Quick Gel Extraction Kit (TRANS) DNA gel recovery kit, the specific operation steps are as follows:

[0061] (1) Quickly cut the target band under ultraviolet light and place the cut target band into a clean 1.5 mL centrifuge tube;

[0062] (2) Add 600 μL of GSB solution to the centrifuge tube and place in a 55°C water bath for about 10 min until the gel is completely melted. Shake several times during this period to ensure complete melting. After complete melting, the solution is yellow.

[0063] (3) After the melted gel solution is cooled to room temperature, it is placed in a centrifuge tube and allowed to stand for 1 min;

[0064] (4) Centrifuge at 10,000 rpm for 5 min at room temperature, discard the flow-through, and add 650 μL of WB solution;

[0065] (5) Centrifuge at 10,000 rpm for 1 min at room temperature and discard the waste liquid;

[0066] (6) Repeat the previous step;

[0067] (7) Place the centrifuge column in a new 1.5 mL centrifuge tube and leave it uncapped for 2 min to allow the remaining ethanol to evaporate. Add 40 μL of ddH2O preheated to 60°C to the center of the column and leave it at room temperature for 2 min.

[0068] (6) Centrifuge at 10,000 rpm for 1 min at room temperature to obtain a DNA solution, which was then stored at -20°C.

[0069] 2.2. Connect the cloning vector

[0070] use -Blunt Simple Cloning Kit (TRANS) reagent, connect the PCR gel recovery product to the cloning vector -Blunt Simple Cloning Vector, prepare the cloning reaction system according to the composition shown in Table 2, gently mix the above reaction solution, connect at 25℃ for 15 minutes, and place the centrifuge tube on ice after the reaction.

[0071] Table 2

[0072]

[0073] 2.3. Cloning vector transformation and positive clone screening. The specific steps are as follows:

[0074] (1) Place the ligated product and competent E. coli on ice, take 50 μL of the thawed competent E. coli and add it to 10 μL of the ligated product obtained in step 2.2; (2) Mix gently, place on ice for 30 min, turn on the water bath and set the temperature to 42°C, and prepare 500 μL of sterile liquid LB in the clean bench;

[0075] (3) Heat shock in a 42°C water bath for 30 seconds, then immediately transfer to ice for 2 minutes;

[0076] (4) Add to 600 μL of sterile liquid LB medium and mix well;

[0077] (5) Incubate at 250 rpm and 37°C for 1 h to allow the bacteria to recover;

[0078] (6) Centrifuge at 4000 rpm for 30 s and discard the supernatant;

[0079] (7) Repeatedly pipette and spread the solution evenly on LB agar medium containing kanamycin antibiotics using a sterilized applicator;

[0080] (8) Place upside down in a dark 37°C constant temperature incubator and culture overnight;

[0081] (9) A single clone was picked with a sterile toothpick and streaked on LB solid medium supplemented with 50 mg / L kanamycin sulfate. A single clone was picked for PCR using the system shown in Table 3. The PCR reaction program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s (denaturation + annealing); extension at 72°C for 1 min, for a total of 32 cycles. The DNA polymerase used was DNA Polymerase (TRANS) fidelity enzyme.

[0082] Table 3

[0083]

[0084] (10) After the reaction is completed, add 5 μL 6× DNA Loading Buffer and perform agarose electrophoresis detection. The clones with clear bands are positive clones. The positive clones are sent to Shanghai Biotechnology Co., Ltd. for sequencing.

[0085] Embodiment 3

[0086] The method for constructing plants overexpressing the SmNPC2 gene is as follows:

[0087] 3.1. Construction of SmNPC2 overexpression vector

[0088] 3.1.1. Extraction of plasmid

[0089] Use PurePlasmid Mini Kit (CWBIO) to extract plasmids. The specific steps are as follows:

[0090] (1) Pick the positive clones determined in Example 2 and place them in LB liquid medium, culture them at 37°C and 200 rpm overnight, then take 1 mL of the bacterial solution and add it to a 1.5 mL centrifuge tube, centrifuge at 13000 rpm for 30 seconds to collect the bacterial precipitate, and discard the supernatant;

[0091] (2) Repeat the above steps twice;

[0092] (3) Add 250 μL of Buffer P1 (with RNase A added) to the centrifuge tube containing the bacterial pellet and mix thoroughly using a vortex oscillator to suspend the bacterial pellet;

[0093] (4) Add 250 μL of Buffer P2 to the centrifuge tube and gently mix by inverting it 4 to 6 times to fully lyse the bacteria. The solution should become clear and viscous. This step should not take more than 5 minutes to avoid damage to the plasmid.

[0094] (5) Add 350 μL of Buffer N3 to the centrifuge tube and immediately mix by inverting the tube 8 to 10 times. A white flocculent precipitate will appear. Centrifuge at 13,000 rpm for 12 min.

[0095] (6) The obtained supernatant was transferred to the adsorption column (Spin Columns DM) in the collection tube in batches, centrifuged at 13000 rpm for 30 s, the waste liquid in the collection tube was discarded, and the adsorption column was placed back into the collection tube;

[0096] (7) Add 150 μL of Buffer PB to the adsorption column, centrifuge at 13,000 rpm for 30 s, and discard the waste liquid in the collection tube;

[0097] (8) Add 400 μL of Buffer PW (with anhydrous ethanol added) to the adsorption column, centrifuge at 13,000 rpm for 1 min, and discard the waste liquid in the collection tube;

[0098] (9) Place the adsorption column back into the collection tube, centrifuge at 13,000 rpm for 1 min, and discard the waste liquid in the collection tube;

[0099] (10) Place the adsorption column in a new 1.5 mL centrifuge tube, add 60 μL of Buffer EB to the adsorption film, place at room temperature for 3 min, centrifuge at 13,000 rpm for 1 min to obtain a plasmid solution, and store at -20 °C.

[0100] 3.1.2 Plasmid linearization

[0101] The enzyme digestion system was prepared according to the components shown in Table 4, and the digestion was carried out at 37°C for 30 min. The digestion products were detected by agarose gel electrophoresis. The pRI-201-mCherry plasmid was used as a control. The results showed that the size of the digestion fragment was in line with expectations. The linearized fragment was recovered by gel excision using a kit.

[0102] Table 4

[0103] Element Volume (μL) pRI-201-mCherry plasmid 10 10×FastDigestGreenBuffer 2 NdeⅠ 1 <![CDATA[ddH2O]]> 7

[0104] 3.1.3. Ligation of expression vector

[0105] use -Uni Seamless Cloning and Assembly Kit (TRANS) homologous recombination kit, using special recombinase and the principle of homologous recombination to achieve efficient seamless splicing, prepare the system shown in Table 5, gently mix the above system, and react at 50°C for 15 minutes. After the reaction, place the centrifuge tube on ice for a few seconds. The template is the gel recovery product of the linearized fragment obtained in step 3.1.2, and the SmNPC2 gene product is the gel recovery product after electrophoresis of the PCR product in step 2.1 of Example 2.

[0106] Table 5

[0107] Element Volume (μL) 2×AssemblyMix 5 template 4 SmNPC2 gene product 1 <![CDATA[ddH2O]]> Up to 10

[0108] 3.1.4 Transformation of Escherichia coli

[0109] The product obtained in step 3.1.3 was transformed into Escherichia coli (the steps were the same as step 1.5 in Example 1), and then the plasmid was extracted from the positive clone (clear bands verified by PCR) to obtain the recombinant plasmid pRI-SmNPC2-mCherry.

[0110] 3.1.5. The positive recombinant plasmid obtained in step 3.1.4 was transformed into Agrobacterium using the freeze-thaw method. The specific steps are as follows:

[0111] (1) Thaw the LBA4404 Agrobacterium competent cells on ice, take 10 μL of the obtained recombinant plasmid pRI-SmNPC2-mCherry and 50 μL of Agrobacterium competent cells into a 1.5 mL centrifuge tube, mix gently and place on ice for 30 min;

[0112] (2) Then, the mixture was quickly frozen in liquid nitrogen for 5 min, heat-shocked in a 37°C water bath for 5 min, and immediately placed on ice for 2 min;

[0113] (3) Add all of it to 600 μL of sterile LB liquid culture medium and shake culture at 220 rpm and 28°C for 2 to 3 h;

[0114] (4) Centrifuge at 4000 rpm for 1 min and discard the supernatant;

[0115] (5) The precipitated bacterial solution was repeatedly aspirated and spread on LB medium containing 25 mg / L rifampicin and 50 mg / L kanamycin using a sterilized applicator in a clean bench for resistance screening, and then inverted and cultured in a 28°C constant temperature incubator until a single colony grew;

[0116] (6) Pick a single colony for PCR verification (the steps are the same as step 2.3 in Example 2). The single colony that can amplify a clear band is a positive clone, and the bacterial solution of the positive clone is saved.

[0117] 3.2. Obtaining transgenic seedlings by Agrobacterium infection

[0118] Sow the 'Changza 8' eggplant seeds in the germination medium in the clean bench, culture in the dark until germination, and then culture in the light for about 7 days until the eggplant cotyledons are flattened, and cut the cotyledons and spread them on the pre-culture medium. Shake the positive clone Agrobacterium LBA4404 obtained in step 3.1.5 at 220rpm and 28℃ overnight, centrifuge at 3000g for 10min the next day, discard the supernatant in the clean bench, dissolve the Agrobacterium precipitate attached to the bottom of the centrifuge tube with MS liquid culture medium, prepare Agrobacterium suspension, and pipette an appropriate amount into 20mL MS liquid culture medium to make the infection concentration OD value about 0.2.

[0119] Use sterilized tweezers to gently immerse the cotyledon explants on the pre-culture medium in the Agrobacterium suspension for 20 minutes, shaking every 5 minutes. After the infection is completed, use tweezers to gently remove the cotyledons, try not to damage the cotyledons, use sterile paper to absorb the residual bacterial liquid on the surface of the cotyledon explants, and then inoculate the co-culture medium and place it in the dark at 18°C ​​for co-culture for 2 days. The infected cotyledons are placed on the screening culture medium for growth. The culture medium is replaced every 15 days. After about 4 months, the callus differentiates into seedlings. The seedlings are cut in the clean bench and placed in the rooting culture medium for rooting culture.

[0120] 3.3. Transgenic plant verification

[0121] 3.3.1. Extraction Step 3.2: Obtain DNA from regenerated eggplant seedlings using the CTAB method. The specific steps are as follows:

[0122] (1) Turn on the water bath at 65°C, add 1 mL of 2% CTAB extract into the centrifuge tube, and place it in the water bath to preheat for 15 min;

[0123] (2) Take 0.2 g of young leaves of regenerated eggplant seedlings and grind them into powder with liquid nitrogen. Transfer them to preheated CTAB and mix them evenly. Keep the temperature at 65°C for 30 min, shaking several times during the process.

[0124] (3) Centrifuge at 10,000 rpm for 7 min at room temperature and collect about 700 μL of the supernatant;

[0125] (4) Add an equal volume of chloroform / isoamyl alcohol (volume ratio of 24:1) for extraction and shake vigorously to mix;

[0126] (5) Centrifuge at 12000 rpm for 10 min at room temperature and collect about 650 μL of supernatant;

[0127] (6) Add an equal volume of isopropanol, mix gently, and place at -20°C for 20 min;

[0128] (7) Centrifuge at 12000 rpm for 5 min at room temperature and discard the supernatant;

[0129] (8) Wash the precipitate twice with 75% ethanol and dry it;

[0130] (9) Add 40 μL ddH2O to dissolve the precipitate, detect by agarose gel electrophoresis, and store at -20°C.

[0131] Using npc2-F (sequence as shown in SEQ ID NO.5) and npc2-R (sequence as shown in SEQ ID NO.6) as primers, PCR was performed (the steps were the same as step 2.3 in Example 2), and the PCR product was subjected to agarose gel electrophoresis. The results were as follows: Figure 2 As shown in the figure, the transgenic plants were those that could amplify the bands, and a total of 2 transgenic seedlings were obtained. The young leaves of the transgenic seedlings were taken for qPCR, and the wild-type plants were used as controls. The results are shown in the figure below. Figure 3 shown.

[0132] SEQ ID NO.5:

[0133] TCTTCACTGTTGATACATATGTCATCCAAAAATCACACA

[0134] SEQ ID NO.6:

[0135] GCCCTTGCTCACCATCATATGAGGGTGCAAATTTGTAGATGA

[0136] Embodiment 4

[0137] The method for constructing a SmNPC2 knockout plant is as follows:

[0138] The gene sequence of SmNPC2 was obtained from the eggplant database, and different exon sequences of the SmNPC2 gene were analyzed. A sequence was randomly selected in the first exon and uploaded to the website http: / / www.genome.arizona.edu / crispr / CRISPRsearch.html. A total of 2 target sites were screened. After the selected target sites were aligned to the genome sequence and verified to be correct, primers were designed. The primers for target site 1 were DT1-BsF (sequence as shown in SEQ ID NO.7) and DT1-F0 (sequence as shown in SEQ ID NO.8), and the primers for target site 2 were DT2-R0 (sequence as shown in SEQ ID NO.9) and DT2-BsR (sequence as shown in SEQ ID NO.10). Then, the sequence of the guide RNA+U6 promoter target site was amplified and connected to the pHSE401 transformation vector, and then Escherichia coli and Agrobacterium (Kan resistance) were transformed for genetic transformation, and DNA was extracted for PCR to recover the amplified product by gel. The specific steps were the same as those in Examples 2 and 3.

[0139] SEQ ID NO.7: ATATATGGTCTCGATTGAGCAGTCCGATAAAGACGGGTT

[0140] SEQ ID NO.8: TGAGCAGTCCGATAAAGACGGGTTTTAGAGCTAGAAATAGC

[0141] SEQ ID NO.9: AACACGGGATTTCGGATCTCCGCAATCTCTTAGTCGACTCTAC

[0142] SEQ ID NO.10: ATTATTGGTCTCGAAACACGGGATTTCGGATCTCCGC

[0143] The product was recovered and connected to the cloning vector. After the positive clones (those not screened by Kan) grew out, 9 were randomly selected for sequencing to verify whether the plant was a gene-edited plant. The sequencing results were as follows: Figure 4 As shown, the first target site lacks three bases and the second target site lacks one base, resulting in premature termination of protein expression, wherein 900 is a 900-base sequence including the target site, 1 and 2 represent two different target sequences, respectively, and [M13+] represents different positive clone sequences.

[0144] Embodiment 5

[0145] The phenotypic traits of the SmNPC2 gene overexpression plant (OE) obtained in Example 3 and the SmNPC2 gene knockout plant (npc2) obtained in Example 4 were investigated as follows:

[0146] S5.1. Germinate wild-type seeds (WT), OE seeds, and npc2 seeds respectively, then sow in 50-hole seedling trays and place in a solar greenhouse for seedling cultivation (growth conditions: 25°C during the day / 16°C at night, 16h light and 8h dark, 40d growth). After the cotyledons are flattened, use 1 / 2 concentration eggplant nutrient solution instead of tap water for watering; after the first true leaf is flattened, use full nutrient solution for watering. The nutrient solution used is Hoagland 1 / 2 concentration nutrient solution.

[0147] S5.2, hydroponic treatment, select two-leaf and one-heart eggplant seedlings with consistent growth, carefully clean the vermiculite and substrate mixture at the root, wrap the bottom of the stem with a sponge pad and stuff it into a foam board with holes, put it into a hydroponic pot, add 5L 1 / 2 concentration nutrient solution to slow the seedlings for 2 days, and take a sample at the end of 2 days as a 0d control sample. The remaining seedlings are treated with low phosphorus and normal phosphorus, and samples are taken on the 10th day after the low phosphorus treatment. The phosphorus concentration of the normal phosphorus treatment is 500μM, and the phosphorus concentration of the low phosphorus treatment is 10μM, and K2SO4 is used to supplement the missing potassium element. Other nutrients are exactly the same.

[0148] S5.3. Inspect the plant height, stem diameter, number of lateral roots and taproot length of seedlings of different strains, place a ruler and take photos. See the photos for details. Figure 5 ImageJ software was used to measure plant height and taproot length, the number of lateral roots was counted to count the number of root primordia and lateral roots, and the stem diameter was measured using a vernier caliper. Figure 6 As shown in the figure, compared with wild-type eggplant, plant height and lateral root number of OE strain increased by 12.3% and 60.4%, respectively; plant height, stem diameter, taproot length and lateral root number (all lateral roots, including obvious root primordia) of npc2 strain decreased by 22.6%, 13.4%, 33.4% and 55.0%, respectively. Ten days after low phosphorus treatment, there was no significant difference between OE strain and wild-type eggplant, while plant height, stem diameter, taproot length and lateral root number of npc2 strain decreased by 18.3%, 13.9%, 48.2% and 83.5%, respectively.

[0149] Embodiment 6

[0150] The root system of the transgenic plants obtained in Example 3 was observed using a handheld fluorescent lamp and red lens glasses. The results are as follows: Figure 7 As shown, WT eggplant seedlings do not produce red fluorescence and have fewer lateral roots, while the red fluorescence of the OE strain is distributed throughout the eggplant seedlings, that is, high-intensity fluorescence is produced and the number of lateral roots is large. Statistics show that the fluorescence intensity and the number of lateral roots between non-transgenic plants (WT) and overexpression plants are significantly different.

[0151] Embodiment 7

[0152] The auxin level of the transgenic plants obtained in Example 3 was investigated using the auxin whole body in situ localization technology, and the steps were as follows:

[0153] Eggplant seeds were sown on MS medium. After the seedlings grew on MS medium for 14-20 days, they were transplanted into planting soil and cultured under a photoperiod of 16h:8h (light:dark) and a constant temperature of 22°C. The 14-day-old seedlings were taken for hormone whole body immunolocalization.

[0154] Pick up the seedlings with tweezers and place them in a 4°C pre-cooled EDC / NHS buffer containing 0.1% Tween-20, and vacuum to help the EDC / NHS solution penetrate until all samples sink to the bottom. This process needs to be done in the dark. Replace with a new EDC / NHS buffer and continue to fix the sample in a 4°C refrigerator for 60 minutes. EDC / NHS can cross-link small molecules IAA and ABA to form haptens. All solutions should be kept ice-cold throughout the process. Pour out the EDC / NHS solution and wash the sample 3 times with 1×PBST, 10 minutes each time. Since DMSO and NP-40 have the ability to regulate cell membrane permeability, add 4% PFA / PBST and 10% DMSO, 3% NP-40 fixative to the sample and vacuum until all materials sink to the bottom of the fixative and no more bubbles emerge. Replace with a new 4% PFA / PBST, place in a 4°C refrigerator overnight, and continue to fix.

[0155] During permeabilization, pour off 4% PFA / PBST, rinse the sample with 1×PBST, then pour off 1×PBST, rinse the sample with 25% methanol / PBST, 50% methanol / PBST, 75% methanol / PBST and 100% methanol. 20 minutes each time, repeat twice or until chlorophyll disappears. Next, rinse the sample with 75% methanol / 25% ethanol, 50% methanol / 50% ethanol, 25% methanol / 75% ethanol and 100% ethanol for 20 minutes respectively. Finally, pour off the ethanol, add the sample to the newly prepared permeabilization solution, incubate at 65°C for at least 2 hours, and the sample will be completely transparent after permeabilization. This step makes the tissue more permeable and accessible to antibodies.

[0156] When blocking, pour off the permeabilization solution, add 4% formaldehyde / PBST fixative, and fix the sample for 30 minutes. Then, wash the sample 3 times with 1×PBST at room temperature, 10 minutes each time. Then, add proteinase K solution (final concentration of 100μg / mL in 1×PBST) to the sample, incubate at 37℃ with gentle shaking for 1 hour, and add 5% glycine / PBST to terminate the reaction. Remove the reaction buffer and continue to treat the sample with 10% DMSO / PBST solution at room temperature for 1 hour (DMSO is used to adjust membrane permeability). Pour off the 10% DMSO / PBST solution, add 4% formaldehyde / PBST, fix again for 30 minutes, and then wash the sample 3 times with 1×PBST, 10 minutes each time. Finally, place the sample in 3% (mass / volume) BSA / PBST blocking solution and incubate at room temperature for 4 hours to block non-specific binding sites.

[0157] Pour off the 3% (mass / volume) BSA / PBST blocking solution, wash the tissue three times with 1×PBST, gently shake at room temperature, 10 minutes each time. Next, place the sample in IAA antibody (A0855Sigma-Aldrich) (1:100) / 3% (mass / volume) BSA / PBST hybridization solution, gently shake at room temperature, and incubate for 6 hours or 4°C overnight (no negative control for IAA antibody is set in this step). Pour off the hybridization solution and wash the sample three times in PBST, 10 minutes each time. Finally, place the sample in 1:500 secondary antibody (AF488 goat anti-mouse IgG heavy and light chain, TRANS) 3% (mass / volume) BSA / PBST and incubate for 4 hours at room temperature, or gently shake overnight at 4°C.

[0158] At room temperature, the sample was washed three times with 1×PBS, 0.1% (volume ratio) Tween-20, each time for 20 min. Then, the sample was placed on a glass slide, a few drops of chloral hydrate transparent solution were added to the glass slide, and the sample was examined under a microscope.

[0159] The results are as follows Figure 8 As shown in the figure, under normal phosphorus treatment, auxin was evenly distributed from the WT root apex meristem to the elongation zone, and polar transport was not affected, while auxin in the npc2 strain was concentrated below the quiescent center, and polar transport was blocked, indicating that SmNPC2 regulates root development by affecting auxin polar transport. After low phosphorus treatment, auxin was evenly distributed from the WT root apex meristem to the elongation zone, but the auxin distribution was closer to the root tip than under normal phosphorus treatment, while auxin in the npc2 strain was concentrated at the root tip.

[0160] Embodiment 8

[0161] The number of cells in the root meristem of wild-type plants and npc2 strains was investigated as follows:

[0162] The roots of different strains were taken for paraffin sections. Fig. 9 As shown, the number of cells in the root meristem of the npc2 line was less than that of the wild-type plant.

[0163] The results showed that compared with wild-type seedlings, the number of lateral roots of overexpressing transgenic seedlings increased after low phosphorus stress, while the lateral roots of mutant plants were significantly reduced, the main root length was shortened, and the growth and development were hindered (such as Figure 1 , 6 , 7). This is because it shows that SmNPC2 can regulate the distribution of auxin, thereby affecting the development of the root system. Under the condition of sufficient nutrition, overexpression of SmNPC2 causes plants to produce more lateral roots (such as Figure 1 ), increase its root surface area, improve the plant's nutrient absorption efficiency, and help alleviate the harm caused by nutrient deficiency.

[0164] Therefore, the present invention discloses the eggplant SmNPC2 gene for the first time, and discloses that overexpressing the eggplant SmNPC2 gene can significantly increase the number of lateral roots, main root length and plant height of eggplant, and knocking out the eggplant SmNPC2 gene can significantly reduce the number of lateral roots, main root length, plant height, stem diameter and root tip auxin content of eggplant; the eggplant SmNPC2 gene can be used to cultivate high-resistance and low-phosphorus eggplant varieties.

[0165] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. Eggplant SmNPC2 The application of genes in regulating eggplant strains is characterized by: Overexpression of eggplant SmNPC2 Gene significantly increases eggplant plant height; SmNPC2 The CDS sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.

2.

2. An eggplant as claimed in claim 1 SmNPC2 The application of the gene in regulating the root development of eggplant is characterized by: Overexpression of eggplant SmNPC2 The gene significantly increases the number of lateral roots and the length of the taproot in eggplant.

3. An eggplant as claimed in claim 1 SmNPC2 The application of the gene in eggplant breeding is characterized by: The eggplant breeding is eggplant breeding for efficient phosphorus utilization; overexpression of the eggplant SmNPC2 The number of lateral roots of seedlings with the gene increased after low-phosphorus stress.

Images