Functional gene paaco2 for regulating apricot fruit ripening and application thereof

Abstract

The application discloses a functional gene PaACO2 for regulating apricot fruit ripening and an application thereof, and the nucleotide sequence of the PaACO2 gene is shown as SEQ ID NO. 1. The PaACO2 gene is used for regulating apricot fruit ripening, the apricot fruit ripening is promoted by increasing the expression amount of the PaACO2 gene, or the fruit ripening is delayed by inhibiting the expression of the PaACO2 gene. The functional gene PaACO2 for regulating apricot fruit ripening is used for promoting tomato fruit ripening, or for regulating the content of abscisic acid and / or amino cyclopropane carboxylic acid in plant tissues. The application verifies that the PaACO2 gene regulates the ripening of apricot fruits by regulating the content of ABA and ACC of apricots, and therefore, the PaACO2 gene can be used for apricot breeding to regulate the ripening of apricot fruits.

Description

Technical Field

[0001] This invention relates to the field of apricot fruit ripening regulation technology. Specifically, it relates to a functional gene PaACO2 that regulates apricot fruit ripening and its applications. Background Technology

[0002] Apricot (Prunus armeniaca L.) is one of the oldest cultivated fruit trees in my country. Its fruit is rich in soluble sugars, proteins, carotenoids, vitamin C, and minerals such as calcium and phosphorus. Typical main cultivated apricot varieties, such as 'Xiaobaixing', 'Liguangxing', 'Shushanggan', and 'Zhenzhuyouxing', have particularly vibrant colors, high sweetness, excellent flavor, and rich nutritional value, fetching a market price of 20-60 yuan / kg. In recent years, they have gradually gained attention and recognition from consumers. However, due to the strong seasonality of apricot fruit, the supply period of the aforementioned main cultivated varieties is only 20 days, which is insufficient to meet daily consumer demand and greatly restricts the market share and healthy development of the apricot industry. Therefore, breeding apricot varieties with different ripening periods has become one of the important goals of apricot breeding. Elucidating the molecular mechanism of apricot fruit ripening is of great significance for developing functional molecular markers and creating new varieties. Summary of the Invention

[0003] Therefore, the technical problem to be solved by this invention is to provide a functional gene PaACO2 that regulates the ripening of apricot fruits, and to verify that the PaACO2 gene regulates the ripening of apricot fruits by regulating the content of ABA and ACC in apricots. Therefore, the PaACO2 gene can be used in apricot breeding to promote the ripening of apricot fruits.

[0004] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0005] A functional gene PaACO2 that regulates apricot fruit ripening, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0006] PaACO2 gene CDS sequence (SEQ ID NO.1):

[0007] ATGGAGAACTTCCCAATCATCAACTTGGAGGGCCTCAATGGAGAGGGAAGGAAAGCAACAATGGAAAAAATCAAAGATGCCTGTGAGAACTGGGGCTTCTTTGAGCTTGTGAGTCATGGGATACCAACTGAGTTTTTGGACACAGTGGAGAGGTTGACTAAAGAACACTACAGGCAGTGCTTGGAGCAGAGGTTCAAGGAGCTGGTGGCCAGCAAGGGCCTTGAGGCTGTCAAGACAGAGGTCAATGATATGGACTGGGAAAGCACCTTCTACTTGCGCCATCTTCCAAAATCTAACATATCTGAAGTTCCAGATCTTGAGGATCAGTACAGGAATGTGATGAAGGAATTTGCATTGAAGTTGGAGAAATTAGCAGAGCAGCTCCTAGACTTGCTCTGTGAGAATCTTGGACTTGAACAAGGGTACCTCAAGAAGGCCTTCTATGGAACAAATGGACCAACTTTTGGCACCAAGGTTAGCAACTACCCTCCTTGTCCCAACCCTGAGCTGATCAAGGGTCTCCGGGCTCACACCGATGCCGGCGGCCTCATCCTGCTCTTCCAGGATGACAAGGTCAGTGGTCTGCAGCTCCTCAAGGATGGCCAATGGATTGATGTGCCCCCCATGCGCCACTCCATTGTTATCAACCTTGGTGACCAACTTGAGGTGATCACTAACGGGAAGTACAAGAGCGTGGAGCACAGAGTGATTGCCCAAACTGATGGCACCAGAATGTCAATAGCTTCCTTCTACAACCCTGGCAGTGATGCTGTCATCTACCCTGCACCAACACTGGTGGAGAAAGAAGCAGAGGAGAAGAATCAAGTGTACCCGAAATTCGTGTTCGAAGACTACATGAAGCTCTATGCTGGCCTCAAGTTCCAGCCCAAAGAGCCAAGATTTGAAGCCATGAAAGCAGTGGAAACCAATATCAGTTTGGGTCCAATTGCAACAGCTTAA。

[0008] The above-mentioned functional gene PaACO2, which regulates the ripening of apricot fruit, has an amino acid sequence of the protein encoded by the PaACO2 gene as shown in SEQ ID NO.2.

[0009] The amino acid sequence of the protein encoded by the PaACO2 gene (SEQ ID NO.2):

[0010] MENFPIINLEGLNGEGRKATMEKIKDACENWGFFELVSHGIPTEFLDTVERLTKEHYRQCLEQRFKELVASKGLEAVKTEVNDMDWESTFYLRHLPKSNISEVPDLEDQYRNVMKEFALKLEKLAEQLLDLLCENLGLEQGYLKKAFYGTNGPTFGTKVS NYPPCPNPELIKGLRAHTDAGGLILLFQDDKVSGLQLLKDGQWIDVPPMRHSIVINLGDQLEVITNGKYKSVEHRVIAQTDGTRMSIASFYNPGSDAVIYPAPTLVEKEAEEKNQVYPKFVFEDYMKLYAGLKFQPKEPRFEAMKAVETNISLGPIATA.

[0011] An application of the functional gene PaACO2 that regulates apricot fruit ripening: The aforementioned functional gene PaACO2 that regulates apricot fruit ripening is used to regulate apricot fruit ripening. Apricot fruit ripening is promoted by increasing the expression level of the PaACO2 gene, or fruit ripening is delayed by inhibiting the expression of the PaACO2 gene.

[0012] The above-mentioned functional gene PaACO2, which regulates apricot fruit ripening, was applied to the breeding of early-maturing apricots.

[0013] The application of the aforementioned functional gene PaACO2, which regulates apricot fruit ripening, involves introducing the recombinant overexpression vector of PaACO2 into apricot fruits via Agrobacterium tumefaciens, thereby increasing the expression level of the PaACO2 gene in apricot fruits and promoting apricot fruit ripening.

[0014] In the application of the functional gene PaACO2 that regulates apricot fruit ripening, when constructing the recombinant overexpression vector, PCR amplification was performed using primers shown in SEQ ID NO.3 and SEQ ID NO.4; the original vector used was the PHG plasmid, and the CDS sequence of the PaACO2 gene was located between the BamHI and PstI restriction sites of the PHG plasmid; the primers used for quantitative PCR of the PaACO2 gene were shown in SEQ ID NO.5 and SEQ ID NO.6.

[0015] SEQ ID NO.3:

[0016] CTCTCTCTCAAGCTTGGATCCATGGAGAACTTCCCAATCATCA;

[0017] SEQ ID NO.4:

[0018] ACGGGTCATGAGCTCCTGCAGAGCTGTTGCAATTGGACC;

[0019] SEQ ID NO.5: TGCTGTCATCTACCCTGCAC;

[0020] SEQ ID NO. 6: CTGGAACTTGAGGCCAGCAT.

[0021] An application of the functional gene PaACO2 that regulates apricot fruit ripening: The aforementioned functional gene PaACO2 that regulates apricot fruit ripening is used to promote tomato fruit ripening.

[0022] The above-mentioned functional gene PaACO2, which regulates apricot fruit ripening, was applied by using a recombinant overexpression vector of PaACO2 to genetically transform 'Micro-Tom' tomatoes via Agrobacterium tumefaciens, thereby promoting the ripening of 'Micro-Tom' tomatoes.

[0023] In the application of the functional gene PaACO2 that regulates apricot fruit ripening, when constructing the recombinant overexpression vector, the primers shown in SEQ ID NO.3 and SEQ ID NO.4 were used for PCR amplification. The original vector used was the PHG plasmid, and the CDS sequence of the PaACO2 gene was located between the BamHI and PstI restriction sites of the PHG plasmid. The primers used for quantitative PCR of the PaACO2 gene are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0024] An application of the functional gene PaACO2 that regulates apricot fruit ripening: The aforementioned functional gene PaACO2 that regulates apricot fruit ripening is used to regulate the content of abscisic acid (ABA) and / or aminocyclopropane carboxylic acid (ACC, a precursor for ethylene synthesis) in plant tissues.

[0025] The technical solution of the present invention achieves the following beneficial technical effects:

[0026] This invention verifies the function of the PaACO2 gene in apricots and finds that the PaACO2 gene promotes fruit ripening by increasing the content of abscisic acid (ABA) in apricot fruits and decreasing the content of aminocyclopropane carboxylic acid (ACC), a precursor of ethylene synthesis. Therefore, the PaACO2 gene can be used in the breeding of early-maturing apricots to shorten the ripening period of apricot fruits and promote ripening. Attached Figure Description

[0027] Figure 1 The spectrum of the PHG plasmid used in the embodiments of this invention;

[0028] Figure 2 In this embodiment of the invention, the phenotype of 'Sungold' apricot fruits was induced by injecting a growth medium containing PHG-PaACO2 and a control PHG empty vector into the bacterial permeate.

[0029] Figure 3 In this embodiment of the invention, the changes in ABA content in 'Sungold' apricot fruits after injecting growth slurry containing PHG-PaACO2 and control PHG empty vector into the bacterial permeate solution were described.

[0030] Figure 4 In this embodiment of the invention, the changes in ACC content in 'Sungold' apricot fruits were observed after injecting growth slurry containing PHG-PaACO2 and control PHG empty vector into the bacterial permeate solution.

[0031] Figures 5a to 5e The figures show the relative expression of PaPDS, PaPSY, PaSWEET, PaSUS and PaACO2 genes in 'Sungold' apricot fruits after injection of bacterial permeate growth solution containing PHG-PaACO2 and control PHG empty vector in the embodiments of the present invention (p<0.001).

[0032] Figure 6 In this embodiment of the invention, the PCR results of PHG-PaACO2 genetically transformed seedlings (target fragment size approximately 485 bp) show that 8 lines in the figure are positive.

[0033] Figure 7 The fruit development cycle of tomatoes overexpressing the PaACO2 gene in this embodiment of the invention (the first row is the fruit development cycle of WT wild-type tomato lines, and the second to fourth rows are the fruit development cycles of tomato lines overexpressing the PaACO2 gene (OE-1, OE-2, OE-3)).

[0034] Figure 8 ACC content of mature fruits from various tomato lines overexpressing the PaACO2 gene in this embodiment of the invention;

[0035] Figures 9a to 9e The figures represent the relative expression levels of maturation-related genes PaACO2, SIACS, SIPDS, SIPSY, and SICNR during the development of each tomato line overexpressing the PaACO2 gene in the embodiments of the present invention. Detailed Implementation

[0036] 1. Materials and Methods

[0037] 1.1 Total RNA and Quality Detection of Samples

[0038] The pulp of the apricot germplasm 'Sungold' was flash-frozen in liquid nitrogen and ground into powder. Buffer SPL was first added to lyse the sample. The lysate was then added to a genomic DNA adsorption column to remove gDNA. Anhydrous ethanol was added to the filtrate to adjust the column loading environment. The entire mixture was then transferred to an RNA adsorption column, impurities were washed away, and finally, RNA was eluted. 1.00 μL of RNA was analyzed for RNA purity (OD260 / 280 and OD260 / 230 ratios) using a Thermo Fisher Scientific NanoDrop ONEC analyzer. 1.00 μg of RNA was then subjected to gel electrophoresis to check for integrity and the presence of RNA degradation.

[0039] 1.2 RNA reverse transcription to synthesize cDNA

[0040] The reaction system was prepared by adding Enzyme Mix, 5×All-in-one qRT Super Mix (kit), template RNA, and RNase-free ddH2O; a reverse transcription negative control reaction was prepared by adding 5 μL of No RT Control Mix 5×All-in-one qRT Super Mix, 1 ng of template RNA, and 20 μL of RNase-free ddH2O to test for the presence of residual genomic DNA in the RNA template. The PCR reaction conditions were: 50℃ for 15 min, followed by 85℃ for 5 sec. The PCR reaction system for reverse transcription to synthesize first-strand cDNA is shown in Table 1.

[0041] Table 1. PCR reaction system for reverse transcription synthesis of first-strand cDNA

[0042]

[0043] 1.3 Construction of overexpression vectors

[0044] The overexpression vector PHG was double-digested with BamHI and PstI restriction enzymes under the following conditions: 37℃ water bath for 1 hour. The PHG digestion system is shown in Table 2, where BamHI and PstI are the original restriction enzyme solutions provided with the kit.

[0045] Table 2 PHG enzyme digestion system

[0046]

[0047] 1.4 Cloning of the PaACO2 gene

[0048] Using the 'Sungold' cDNA synthesized via reverse transcription as a template, PCR amplification of the candidate gene was performed using Phanta Flash Super-Fidelity DNA Polymerase (Vazyme). The reaction system and standard amplification procedure are shown in Tables 4 and 5, and the amplification primers are detailed in Table 3 below.

[0049] Table 3 Primer sequence listing

[0050]

[0051] Table 4 PCR reaction system

[0052]

[0053] Table 5 Standard PCR Amplification Procedure

[0054]

[0055] After PCR amplification, the samples were spotted onto a 0.8 wt% agarose gel and placed in 1× TAE buffer. Electrophoresis was performed at 160 V, 360 A, and 25 min. The correctly amplified target band was extracted and recovered using the Omega Gel Extraction Kit (D2500). First, the desired target fragment was excised from the agarose gel and placed in a 1.5 mL centrifuge tube for weighing. Then, the corresponding weight of dissolving buffer was added to completely dissolve the gel. All liquid was then transferred to a binding column, washed, dried, and finally eluted to obtain the extracted gel product.

[0056] The linearized PHG overexpression vector was mixed with the corresponding PaACO2 gene fragment with the vector adapter, and 2×EasyGeno Assembly Mix (Tiangen Biotech) was added for recombination. The mixture was then incubated in a 50℃ water bath for 30 min.

[0057] The 10 μL recombination system consisted of 5 μL of 2×EasyGeno Assembly Mix, 2.5 μL of enzyme-digested vector DNA, and 2.5 μL of DNA fragment (150 ng / μL).

[0058] The reaction system was added to a 250 μL EP tube, placed in a 50°C water bath for 30 minutes, and then transformed into E. coli and plated. After incubating at 37°C for 16 hours, bacteria were picked and sent for sequencing. The plasmid with the correct sequence was transformed into Agrobacterium GV3101 for subsequent transient expression of apricot and genetic transformation of tomato.

[0059] 2. Transient expression in apricot fruit

[0060] 2.1 Buffer solution preparation

[0061] (1) Preparation of 100 mmol / L acetylsylgenone (stored at -20℃): Dissolve 0.3924g of acetylsylgenone powder directly in 20 mL of DMSO, sterilize by filtration membrane, and then dispense and freeze.

[0062] (2) Preparation of 1mol / L MES: Dissolve 4.265g of MES powder in 20 mL of ultrapure water, and adjust the pH to 5.6-5.7 with 1mol / L NaOH.

[0063] (3) Preparation of 1 mmol / L MgCl2: Dissolve 1.9042 g of MgCl2 powder in 20 mL of ultrapure water.

[0064] Table 6 Preparation of Osmotic Buffer

[0065]

[0066] 2.2 Instantaneous injection of apricot fruit

[0067] Take 20 μL of Agrobacterium tumefaciens bacterial suspension containing the overexpression vector PHG-PaACO2 and the PHG control vector, add 5 mL of liquid LB medium containing Kan (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride), and shake at 28℃ and 220 rpm for about 6 hours. Then, take 1 mL of the bacterial suspension and add it to 50 mL of liquid LB medium containing Kan, and incubate overnight at 28℃ and 220 rpm until the OD600 of the bacterial suspension is about 0.6-1.0. Then, centrifuge at 6000 rpm at room temperature for 10 min to precipitate the bacterial cells, discard the supernatant, and resuspend the bacterial cells in 50 mL of prepared osmotic buffer to achieve an OD600 between 0.6 and 1.0. Incubate the prepared bacterial suspension osmotic buffer at room temperature for 4 hours. Select healthy, disease-free fruits for injection experiments. Inject the bacterial suspension osmotic buffer at 3-5 different locations on each fruit and mark the injection sites with a marker. Seven days later, the fruit phenotype was observed, and the phenotyped portions of apricot fruits were cut off and flash-frozen in liquid nitrogen for subsequent experiments.

[0068] 3. Genetic transformation of tomatoes and identification of transformed phenotypes

[0069] 3.1 Genetic transformation of 'Micro-Tom' tomatoes

[0070] 3.1.1 Obtaining sterile vaccines

[0071] Select plump, uniformly sized, and fresh tomato seeds, rinse them repeatedly with sterile water several times, then disinfect them with 70% alcohol for 30 seconds, rinse them three times with sterile water, then disinfect them with 10wt% sodium hypochlorite for 10 minutes, and finally rinse them 4-5 times with sterile water. After drying with sterile filter paper, inoculate them into seed germination medium. Incubate in the dark until most seeds have germinated and shown white sprouts, then place them in a culture environment with 16 hours of light per day, a light intensity of 1600-1800 lx, and a temperature of (24±2)℃.

[0072] 3.1.2 Agrobacterium tumefaciens culture

[0073] Using a sterile pipette tip, pick up a labeled Agrobacterium tumefaciens monoclonal sample and inoculate it into 5 mL of LB liquid medium containing the appropriate antibiotic (using a 50 mL blue cap centrifuge tube). Incubate at 28°C and 200 rpm for 24 hours with shaking. Centrifuge at 20°C and 4,000 rpm for 15 min to collect the bacterial cells. Resuspend the bacterial cells in transformation buffer until the OD600 is approximately 0.5.

[0074] 3.1.3 Explant preparation, inoculation, and co-culture

[0075] True leaves, cotyledons, and hypocotyls were selected as explants for transformation. The tips and petioles of the true leaves and cotyledons were removed, and the remaining parts were cut into leaf pieces measuring 0.5 cm × 0.5 cm. Hypocotyls were cut into segments approximately 0.5–0.6 mm in length and placed horizontally on pre-culture medium, 15–20 segments per dish. Culture conditions were the same as above, with pre-culture for 1 day.

[0076] Remove the explants from the pre-med medium and place them in a culture dish containing Agrobacterium tumefaciens diluted with transformation buffer to approximately OD600=0.5. Transform for 30 min, remove the explants, blot them dry on sterile paper, and return them to the pre-med medium. Culture for a total of 1-2 days.

[0077] 3.1.4 Selection, hardening-off, and transplanting culture

[0078] The co-cultured explants were transferred to a selection medium for selective culture. After a few days of selection culture, the cotyledons began to thicken, and the hypocotyls began to thicken. The transformed explants formed callus and adventitious shoots on the selection medium, and were subcultured every two weeks. The callus with bud primordia was cut into small pieces and transferred to a stem elongation medium for subculture, again every two weeks, or even more frequently if necessary. When the adventitious shoots reached about 1 cm in length, healthy regenerated shoots were selected, and the basal callus and medium were completely removed. The shoots were then transferred to a rooting medium to develop into complete plants. After the seedlings developed lateral roots, the bottle caps were removed, and a small amount of sterile water (covering the medium by 3-5 mm) was poured into the culture bottles. The bottles were then placed in a cool, well-ventilated place for hardening off. After 3 days, the medium was washed off the roots, and the seedlings were transferred to soil. For the first 7 days, the seedlings were covered with a transparent plastic film and cultured under low light to allow the regenerated seedlings to adapt to the transition from the medium to the nutrient substrate.

[0079] 4. Phenotypic determination of transient expression in apricot and overexpression in tomato, and analysis by real-time quantitative PCR (qRT-PCR).

[0080] 4.1 Validation by Real-Time Quantitative PCR of Genes

[0081] As shown in Table 7, PaUBQ was used as the internal reference gene for the relative expression analysis of apricot genes in this embodiment, SIACT was used as the internal reference gene for tomatoes, and HYR gene was used to detect whether the overexpression lines of tomatoes were positive.

[0082] Table 7 Primers used for PCR and qRT-PCR

[0083]

[0084] The ChamQ Universal SYBR qPCR Master MiX (Vazyme) real-time PCR kit was used for this experiment. The qRT-PCR reaction system and procedure are shown in Tables 8 and 9.

[0085] Table 8 qRT-PCR reaction system

[0086]

[0087] Table 9 qRT-PCR amplification program

[0088]

[0089] In this embodiment, all qRT-PCR experiments were repeated four times, and the relative expression levels of each gene were calculated using the 2^-ΔΔCt method. Finally, the results were presented in graph form using GraphPad Prism 8.0.2.

[0090] 5. Determination of hormone ABA and ACC content

[0091] 5.1 Solid Samples

[0092] (1) Take out the apricot fruits and overexpressed tomato fruits that were cryopreserved and injected at ultra-low temperature (no special requirements, the default is fresh samples), and grind them in liquid nitrogen with a grinder (30 Hz, 1 min) until they are powdery.

[0093] (2) Weigh 50 mg of the ground sample in liquid nitrogen, add 10 μL of internal standard mixed solution with a concentration of 100 ng / mL and 1 mL of methanol / water / formic acid (15:4:1, v / v / v) extractant, and mix well;

[0094] (3) Vortex for 10 min, centrifuge for 5 min at 4°C and 12000 r / min, and transfer the supernatant to a new centrifuge tube for concentration;

[0095] (4) After concentration, redissolve in 100 mL of 80% methanol / water solution, filter through a 0.22 μm filter membrane, and place in a sample vial for LC-MS / MS analysis.

[0096] 5.2 Chromatography-Mass Spectrometry Acquisition Conditions

[0097] The data acquisition instrument system mainly includes ultra-high performance liquid chromatography (UPLC) (ExionLCT AD, https: / / sciex.com.cn / ) and tandem mass spectrometry (MS / MS) (OTRAP® 6500+, https: / / sciex.com.cn / ).

[0098] Liquid phase conditions mainly include:

[0099] 1) Column: Waters ACQUITY UPLC HSS T3 C18 column (1.8 μm, 100 mm x 2.1 mm); 2) Mobile phase: Phase A, ultrapure water (with 0.04% acetic acid added, i.e., 4 mL acetic acid added to 1000 mL ultrapure water); Phase B, acetonitrile (with 0.04% acetic acid added, i.e., 4 mL acetic acid added to 1000 mL acetonitrile); 3) Gradient elution program: 0 min A / B 95:5 (V / V), 1.0 min A / B 95:5 (V / V), 8.0 min 5:95 (V / V), 9.0 min 5:95 (V / V), 9.1 min 95:5 (V / V), 12.0 min 95:5 (V / V); 4) Flow rate 0.35 mL / min; column temperature 40°C; injection volume 2 μL.

[0100] Mass spectrometry conditions mainly include:

[0101] Electrospray ionization (ESI) was performed at a temperature of 550°C. The mass spectrometry voltage was 5500 V in positive ion mode and -4500 V in negative ion mode. The curtain gas (CUR) pressure was 35 psi. In the Q-Trap 6500+, each ion pair was scanned and detected based on optimized declustering potential (DP) and collision energy (CE).

[0102] 6 Results Analysis

[0103] 6.1 Phenotypic Analysis of Apricot Fruits Transiently Overexpressing PaACO2

[0104] To determine the regulatory role of the PaACO2 gene in fruit ripening, the CDS sequence of the cloned PaACO2 gene was constructed into the linearized overexpression vector PHG using the method described above, and subsequent transient expression experiments were conducted. Prepared bacterial permeate solutions containing PHG-PaACO2 and the empty PHG vector were injected into apricot fruits in the late stage of slow growth, and the fruits were labeled. The transient expression phenotype of the PaACO2 gene in apricots was observed 5-7 days later.

[0105] Depend on Figure 2 It was found that apricot fruits injected with PHG-PaACO2 bacterial solution (PaACO2-OE) turned orange, promoting ripening; while apricot fruits injected with the blank control PHG bacterial solution remained green and did not change color. This indicates that the PaACO2 gene can promote the ripening process of apricot fruits. The ABA and ACC contents of fruits from the injected areas were measured, and the results are as follows... Figure 3 and Figure 4 As shown, the ABA content of apricot fruits injected with PHG-PaACO2 bacterial solution was 499.70 ng / g, while the ABA content of apricot fruits injected with the blank control was 327.90 ng / g. The ABA content in PaACO2-OE was significantly higher than that in the control group. Conversely, the ACC content of PaACO2-OE fruits was 26.46 ng / g, while the ACC content of the control group fruits was 36.68 ng / g, significantly higher than that of PaACO2-OE fruits. These hormone assay results indicate that overexpression of the PaACO2 gene can promote an increase in ABA content, inhibit ACC accumulation and its rapid conversion into ethylene, and promote fruit ripening.

[0106] Subsequently, the expression of genes related to maturation and ABA and ethylene synthesis was measured, such as... Figures 5a to 5e As shown, the expression of the PaACO2 gene in fruits injected with PaACO2-OE was significantly higher than that in the control group. In addition, the expression levels of genes related to apricot ripening, such as the carotenoid synthesis (color-related) genes PaPDS and PaPSY, and the sugar transporter genes PaSWEET and PaSUS, were significantly higher than those in the control group.

[0107] In summary, the PaACO2 gene can promote fruit color change and the conversion of sugar transport proteins, increase ABA content and accumulation, and enable ACC to be rapidly converted into ethylene, thereby promoting apricot fruit ripening.

[0108] 6.2 Phenotypic Analysis of Tomatoes Overexpressing PaACO2

[0109] To verify the function of PaACO2 in regulating fruit ripening and ABA synthesis, PaACO2 was expressed ectopically in micro-TOM tomatoes. The micro-TOM tomato transformation ultimately yielded eight genetically transformed tomato lines with the PaACO2 gene. Figure 6 The tomato genetic transformation lines with the PaACO2 gene (numbered 3-10) were used. Wild-type (WT) tomatoes, sown at the same time, served as a control. Both were transplanted into a constant-temperature incubator with a temperature of 25℃, humidity of 75%, and light intensity of 16 Hz and darkness of 8 Hz for subsequent experimental observation. During the tomato growth process, attention was paid to watering and the prevention and control of pests, diseases, and fungal infections. Appropriate pesticides were sprayed after flowering for timely prevention and control to ensure normal growth of the tomatoes in the later stages.

[0110] The entire growth and development process from flowering to maturity was monitored and statistically analyzed in tomato lines and WT lines overexpressing the PaACO2 gene (see...). Figure 7 The study found that the overall fruit development cycle of tomatoes overexpressing the PaACO2 gene was shorter than that of WT tomato lines. The WT tomato lines took 77 days from flowering to maturity, while the PaACO2-overexpressing tomato lines took 68 days. Therefore, we selected three independent transgenic tomato lines with the most pronounced late-maturing traits and a fruit development cycle of 68 days (OE-1, OE-2, and OE-3) for subsequent ABA and ACC content determination and expression analysis of maturity-related genes.

[0111] Based on the mature phenotypes of the fruit growth and development cycle of tomato lines (OE-1, OE-2, OE-3) and WT lines that overexpress the PaACO2 gene, the content of ABA and ACC and the expression of the mature gene were obtained using LC-MS / MS and qRT-PCR as experimental materials. Figure 8 The results showed that the PaACO2 gene was overexpressed in tomato lines (OE-1, OE-2, and OE-3), with significantly higher expression levels than the control group WT. The ACC content in WT was 144.7 ng / g, in OE-1 it was 240.39 ng / g, in OE-2 it was 237.88 ng / g, and in OE-3 it was 256.29 ng / g, demonstrating that overexpression of the PaACO2 gene promotes fruit ripening by increasing the ACC content in the fruit.

[0112] also, Figures 9a to 9eThe results showed that the ethylene synthesis gene SlACS, and the carotenoid and lycopene synthesis (color-related) genes SlPDS and SlPSY in PaACO2-overexpressing tomato lines were significantly higher than those in the control group. The expression level of the ripening-related transcription factor SlCNR in PaACO2-overexpressing tomato lines was lower than that in the control group at maturity because its expression was high during the color-breaking stage but almost non-existent during the red-ripe stage. In conclusion, the PaACO2 gene can promote tomato fruit color change, and the accumulation of ACC content promotes ethylene production, thereby promoting tomato fruit ripening.

[0113] PaACO2 gene CDS sequence (SEQ ID NO.1):

[0114] ATGGAGAACTTCCCAATCATCAACTTGGAGGGCCTCAATGGAGAGGGAAGGAAAGCAACAATGGAAAAAATCAAAGATGCCTGTGAGAACTGGGGCTTCTTTGAGCTTGTGAGTCATGGGATACCAACTGAGTTTTTGGACACAGTGGAGAGGTTGACTAAAGAACACTACAGGCAGTGCTTGGAGCAGAGGTTCAAGGAGCTGGTGGCCAGCAAGGGCCTTGAGGCTGTCAAGACAGAGGTCAATGATATGGACTGGGAAAGCACCTTCTACTTGCGCCATCTTCCAAAATCTAACATATCTGAAGTTCCAGATCTTGAGGATCAGTACAGGAATGTGATGAAGGAATTTGCATTGAAGTTGGAGAAATTAGCAGAGCAGCTCCTAGACTTGCTCTGTGAGAATCTTGGACTTGAACAAGGGTACCTCAAGAAGGCCTTCTATGGAACAAATGGACCAACTTTTGGCACCAAGGTTAGCAACTACCCTCCTTGTCCCAACCCTGAGCTGATCAAGGGTCTCCGGGCTCACACCGATGCCGGCGGCCTCATCCTGCTCTTCCAGGATGACAAGGTCAGTGGTCTGCAGCTCCTCAAGGATGGCCAATGGATTGATGTGCCCCCCATGCGCCACTCCATTGTTATCAACCTTGGTGACCAACTTGAGGTGATCACTAACGGGAAGTACAAGAGCGTGGAGCACAGAGTGATTGCCCAAACTGATGGCACCAGAATGTCAATAGCTTCCTTCTACAACCCTGGCAGTGATGCTGTCATCTACCCTGCACCAACACTGGTGGAGAAAGAAGCAGAGGAGAAGAATCAAGTGTACCCGAAATTCGTGTTCGAAGACTACATGAAGCTCTATGCTGGCCTCAAGTTCCAGCCCAAAGAGCCAAGATTTGAAGCCATGAAAGCAGTGGAAACCAATATCAGTTTGGGTCCAATTGCAACAGCTTAA;

[0115] Amino acid sequence of the protein encoded by the PaACO2 gene (SEQ ID NO.2):

[0116] MENFPIINLEGLNGEGRKATMEKIKDACENWGFFELVSHGIPTEFLDTVERLTKEHYRQCLEQRFKELVASKGLEAVKTEVNDMDWESTFYLRHLPKSNISEVPDLEDQYRNVMKEFALKLEKLAEQLLDLLCENLGLEQGYLKKAFYGTNGPTFGTKVSNYPPCPNPELIKGLRAHTDAGGLILLFQDDKVSGLQLLKDGQWIDVPPMRHSIVINLGDQLEVITNGKYKSVEHRVIAQTDGTRMSIASFYNPGSDAVIYPAPTLVEKEAEEKNQVYPKFVFEDYMKLYAGLKFQPKEPRFEAMKAVETNISLGPIATA。

Claims

1. Functional genes PaACO2 The application is characterized by, PaACO2 Gene overexpression is used to promote tomato fruit ripening; among them, PaACO2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The functional gene according to claim 1 PaACO2 The application is characterized by, use PaACO2 The recombinant overexpression vector of the gene was genetically transformed into Micro-Tom tomatoes through Agrobacterium tumefaciens mediation, promoting the ripening of Micro-Tom tomato fruits.

3. The functional gene according to claim 2 PaACO2 The application is characterized by, When constructing the recombinant overexpression vector, PCR amplification was performed using primers shown in SEQ ID NO.3 and SEQ ID NO.4, and the original vector used was the PHG plasmid. PaACO2 The CDS sequence of the gene is located between the BamHI and PstI restriction sites in the PHG plasmid. PaACO2 The primers used for quantitative PCR of the gene are shown in SEQ ID NO.5 and SEQ ID NO.

6.

4. Functional genes PaACO2 The application is characterized by, PaACO2 Gene overexpression was used to increase the content of abscisic acid and / or aminocyclopropanecarboxylic acid in tomato tissues; among which, PaACO2 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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