Functional gene pa spl8 for regulating apricot fruit ripening and application thereof

Abstract

The application discloses a functional gene PaSPL8 for regulating apricot fruit ripening, and the nucleotide sequence of the PaSPL8 gene is shown as SEQ ID NO. 1. The functional gene PaSPL8 for regulating apricot fruit ripening is used for regulating apricot or tomato fruit ripening, and the fruit ripening is delayed by increasing the expression amount of the PaSPL8 gene; or the functional gene PaSPL8 for regulating apricot fruit ripening is used for regulating the content of abscisic acid in plant tissues. The application verifies that the PaSPL8 gene regulates the ripening of apricot fruits by regulating the content of ABA in apricots, and therefore, the PaSPL8 gene can be used for apricot breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of apricot fruit ripening regulation. Specifically, it is a functional gene PaSPL8 for regulating apricot fruit ripening and application thereof. BACKGROUND

[0002] Apricot belongs to the plant of the genus Armeniaca in the Rosaceae family, and is believed to originate from the northern, northwestern and northeastern provinces of China. Apricot fruit is rich in apricot dietary fiber, protein, sugar, fatty acids, trace nutrients, volatile compounds, carotenoids, phenolic substances and lignans. Almond contains cyanogenic glycosides, and the main component (up to 4.9%) is amygdalin. At the same time, apricot is an easy-to-grow tree species with ecological, economic and social benefits. As the saying goes, "apricot trees are easy to grow into forests", and vigorously developing apricot industry helps to restore the ecological environment of the land, stimulate the regional economy and create a better human environment. Everything should follow the laws of nature and follow the growth laws of plants as much as possible. Off-season apricot fruit can meet the curiosity of the public, but it still lacks in taste, nutrition and health, and is expensive. Apricot fruit has very high discriminability, and almost one variety has one shape and taste. In recent years, more and more excellent varieties have appeared in front of the public, opening up the sales market of apricot fruit, but due to its strong seasonality, most of the main varieties mature in May-July, which is difficult to meet the daily needs of consumers. Therefore, cultivating early or late varieties and configuring mid-mature varieties can extend the supply period of apricot fruit as a whole, which is beneficial to apricot to occupy a larger market share and meet the eating needs of consumers. Maturity is accompanied by the formation of a series of fruit quality traits, so the study of the traits of the ripening period has great scientific research significance and application value. SUMMARY

[0003] Therefore, the technical problem to be solved by the present application is to provide a functional gene PaSPL8 for regulating apricot fruit ripening, and it is verified that the PaSPL8 gene regulates the ripening of apricot fruit by inhibiting the expression amount of ABA in apricot, so the PaSPL8 gene can be used in the breeding of apricot to delay the ripening of apricot fruit.

[0004] To solve the above technical problems, the present application provides the following technical solutions:

[0005] A functional gene PaSPL8 for regulating apricot fruit ripening, the nucleotide sequence of the PaSPL8 gene is shown in SEQ ID NO. 1. SEQ ID NO. 1:

[0006] ATGGAATCAAACAGAGCTCATGGGAAGAGGAGCTTGAACTACAAGATGGAGGAAGAGGAGGAGGAGGAGGATGAAGAAGAAGAGCAGGATGAAGAGGAGGATGATGAAGATACTAACAGATCATCACTAGTGTACGGTGAGGATGAGAGGAGGAAAAGAGTGTTGATGGTGAATAGTGCAACTACTACTCCTAACAAGAGAGGATCTGGTGCAGGAGGGTCTATGGCAAGGCCATCTTGTCAAGCAGATGATTGCAATGCTGACTTGAGTGATGCAAAGCAATACTATCGCCGCCATAAGGTCTGCGGTGTTCATGCCAAGGCTCCGGCGGTGCGTGTGGGTGGAGTTCAACAGCGCTTCTGCCAGCAATGTAGCAGTGCAGAGAAAATAGCTAAAGAAAAAGAGAAAAACAAGATGAAGGAGAGAAAACCATTTACCCCAAAAAAGAAAGAGAGAGAGGAAGAAGAAGAAGAAAAAGAAGTGGTTTAA.

[0007] The functional gene PaSPL8 for regulating apricot fruit ripening, the amino acid sequence of the protein encoded by the PaSPL8 gene is shown in SEQ ID NO. 2. SEQ ID NO. 2:

[0008] MESNRAHGKRSLNYKMEEEEEEEEDEEEEQDEEEDDEDTNRSSLVYGEDERRKRVLMVNSATTTPNKRGSGAGGSMARPSCQADDCNADLSDAKQYYRRHKVCGVHAKAPAVRVGGVQQRFCQQCSSAEKIAKEKEKNKMKERKPFTPKKKEREEEEEEKEVV*.

[0009] The application of the functional gene PaSPL8 for regulating apricot fruit ripening, the functional gene PaSPL8 for regulating apricot fruit ripening is used to regulate apricot fruit ripening, and the fruit ripening is delayed by increasing the expression amount of the PaSPL8 gene.

[0010] The application of the functional gene PaSPL8 for regulating apricot fruit ripening, the functional gene PaSPL8 for regulating apricot fruit ripening is used to cultivate late-maturing apricots.

[0011] The application of the functional gene PaSPL8 for regulating apricot fruit ripening improves the expression amount of the PaSPL8 gene in apricot fruit by mediating the recombination overexpression vector of the functional gene PaSPL8 for regulating apricot fruit ripening into apricot fruit through Agrobacterium tumefaciens, and delays the ripening of apricot fruit.

[0012] In the application of the functional gene PaSPL8 for regulating apricot fruit ripening, the primers shown in SEQ ID NO. 3 and SEQ ID NO. 4 are used for PCR amplification when the recombination overexpression vector is constructed; the original vector used is PHG plasmid, and the CDS sequence of the PaSPL8 gene is located between the BamHI and PstI enzyme cutting sites of the PHG plasmid; the primers used for quantitative PCR of the PaSPL8 gene are shown in SEQ ID NO. 5 and SEQ ID NO. 6.

[0013] PCR amplification primer SEQ ID NO. 3:

[0014] CTCTCTCTCAAGCTTGGATCCATGGAATCAAACAGAGCTCATG;

[0015] PCR amplification primer SEQ ID NO. 4:

[0016] ACGGGTCATGAGCTCCTGCAGAACCACTTCTTTTTCTTCTTCTTCT;

[0017] Quantitative PCR primer SEQ ID NO. 5:

[0018] GCAGGAGGGTCTATGGCAAG;

[0019] Quantitative PCR primer SEQ ID NO. 6:

[0020] TGTTGAACTCCACCCACACG.

[0021] The application of the functional gene PaSPL8 for regulating apricot fruit ripening is used for delaying the ripening of tomato fruit.

[0022] The application of the functional gene PaSPL8 for regulating apricot fruit ripening delays the ripening of ‘Micro-Tom’ tomato fruit by mediating the recombination overexpression vector of the functional gene PaSPL8 for regulating apricot fruit ripening through Agrobacterium tumefaciens to genetically transform ‘Micro-Tom’ tomato.

[0023] In the application of the functional gene PaSPL8 for regulating apricot fruit ripening, the recombinant overexpression vector is constructed by using the primers shown in SEQ ID NO. 3 and SEQ ID NO. 4 for PCR amplification, and the original vector used is PHG plasmid, and the CDS sequence of the PaSPL8 gene is located between the BamHI and PstI enzyme cutting sites of the PHG plasmid; the primers used for quantitative PCR of the PaSPL8 gene are shown in SEQ ID NO. 5 and SEQ ID NO. 6.

[0024] The application of the functional gene PaSPL8 for regulating apricot fruit ripening is used for regulating the content of abscisic acid in plant tissues.

[0025] The technical scheme of the present application has the following beneficial technical effects:

[0026] The present application verifies the function of the PaSPL8 gene in apricots, and for the first time finds that the PaSPL8 gene delays fruit ripening by inhibiting the synthesis of abscisic acid (ABA) in apricot fruits, so the PaSPL8 gene can be used in the breeding of late-maturing apricots to prolong the fruit ripening period and delay the ripening of apricot fruits. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The PHG plasmid map used in the embodiments of the present application;

[0028] Figure 2 The bacterial liquid permeation liquid growth liquid containing PHG-PaSPL8 and the control PHG empty load is injected into the 'XYZS' apricot fruits, and the phenotypes of the fruits are shown in the following table:

[0029] Figure 3 The bacterial liquid permeation liquid growth liquid containing PHG-PaSPL8 and the control PHG empty load is injected into the 'XYZS' apricot fruits, and the gene expression in the ABA metabolic pathway in the fruits after injection is shown in the following table (p<0.001):

[0030] Figure 4 The bacterial liquid permeation liquid growth liquid containing PHG-PaSPL8 and the control PHG empty load is injected into the 'XYZS' apricot fruits, and the changes in the contents of ABA and ACC in the fruits after injection are shown in the following table:

[0031] Figure 5 The PCR results of the PHG-PaSPL8 genetic transformation seedlings in the embodiments of the present application (the target fragment size is about 595 bp, and 16 lines are determined to be positive):

[0032] Figure 6The fruit development cycle of the tomato strain overexpressing the PaSPL8 gene (the first row is the fruit development cycle of the WT wild-type tomato strain, and the second to fourth rows are the fruit development cycles of the tomato strains OE-8, OE-9 and OE-16 overexpressing the PaSPL8 gene);

[0033] Figure 7 The expression level of PaSPL8 in the development process of the WT wild-type tomato strain overexpressing PaSPL8 according to an embodiment of the present application;

[0034] Figure 8 The ABA and ACC contents of the mature fruit of the WT wild-type tomato strain overexpressing PaSPL8 according to an embodiment of the present application. DETAILED DESCRIPTION

[0035] 1. Materials and methods

[0036] 1.1 Total RNA sample and quality detection

[0037] The fruit pulp of the apricot germplasm 'LS-11' was rapidly frozen in liquid nitrogen and ground into powder. Buffer SPL was added for sample lysis, and then the filtrate was added to a genomic adsorption column to remove gDNA (genomic DNA). Then, anhydrous ethanol was added to the filtrate to adjust the column environment, and the entire mixture was transferred to an RNA adsorption column to rinse the impurities. Finally, the RNA was eluted, and 1.00 μL of RNA was taken for RNA purity detection (OD260 / 280 and OD260 / 230 ratio) using a Thermo Fisher NanoDrop ONEC ultramicro nucleic acid protein detector. 1.00 μg of RNA was taken for gel electrophoresis to check the integrity and whether there was RNA degradation. The apricot germplasm 'LS-11' used in this embodiment was collected from the National Forest Tree Germplasm Resource Library in the north and is a late-maturing apricot variety.

[0038] 1.2 RNA reverse transcription to synthesize cDNA

[0039] Enzyme Mix, 5×All-in-one qRT Super Mix, template RNA and RNase-free ddH2O were added to configure the reaction system. No RT Control Mix 5×All-in-one qRT Super Mix 5ul, template RNA (1 ng) and RNase-free ddH2O (to 20uL) were added to configure the reverse transcription negative control reaction, which was used to check whether there was residual genomic DNA in the RNA template. The PCR reaction system was 50℃ for 15 min and 85℃ for 5 sec.

[0040] The reverse transcription to synthesize the first strand cDNA was performed according to the PCR reaction system in Table 1.

[0041] Table 1 Reverse transcription synthesis of first strand cDNA PCR reaction system

[0042]

[0043] 1.3 Construction of overexpression vector

[0044] Select BamHI and PstI endonuclease to double enzyme cut overexpression vector PHG, 37°C water bath for 1h, PHG enzyme cutting system as shown in Table 2.

[0045] Table 2 PHG enzyme cutting system

[0046]

[0047]

[0048] 1.4 Cloning of PaSPL8 gene

[0049] The above reverse transcription synthesis of 'LS-11' cDNA as template, using Phanta Flash Super-Fidelity DNA Polymerase (Vazyme) for PCR amplification of candidate genes. Reaction system and standard amplification program as shown in Table 3 and 4, primer details see Table 5.

[0050] Table 3 PCR reaction system

[0051]

[0052] Table 4 PCR standard amplification program

[0053]

[0054] Table 5

[0055]

[0056] After PCR amplification, the sample was spotted on a 0.8wt% agarose gel, placed in 1xTAE buffer, and electrophoresis conditions were voltage 160V, current 360A, and time 25min. Using Omega gel recovery kit Gel Extraction Kit (D2500), the correct target band was cut and recovered. First, the desired fragment was cut from the agarose gel and placed in a 1.5mL centrifuge tube, and weighed; second, the corresponding weight of the dissolution solution was added to dissolve the gel, so that the gel was completely dissolved. Then transfer all the liquid to the binding column, wash, dry, and finally elute the corresponding gel recovery product.

[0057] Mix the linearized PHG overexpression vector with the corresponding PaSPL8 fragment with vector adapter, add 2x EasyGeno Assembly Mix (Tiangen) for recombination, 50℃ water bath for 30min.

[0058] 10uL recombination system: 5uL 2x EasyGeno Assembly Mix; 2.5uL digested vector DNA; 2.5uL fragment DNA.

[0059] Put the reaction system into 250uL EP tube, 50℃ water bath for 30min, then transform E. coli and plate, 37℃ incubator for 16h, pick bacteria and send for sequencing. Transform the plasmid with correct sequence into Agrobacterium GV3101 for subsequent apricot variety transient expression and tomato genetic transformation.

[0060] 2Transient expression of PaSPL8 gene in apricot fruit

[0061] 2.1 Buffer preparation

[0062] 100mmol / L acetosyringone preparation (-20℃ storage): 0.3924g acetosyringone powder directly dissolved in 20mL DMSO, filter sterilization and aliquot for storage.

[0063] 1mol / L MES preparation: 4.265g MES powder dissolved in 20mL ultrapure water, adjust pH to 5.6-5.7 with 1mol / L naOH solution.

[0064] 1mmol / L MgCl2 preparation: 1.9042g MgCl2 powder dissolved in 20mL ultrapure water.

[0065] Table 6 Preparation of osmotic buffer

[0066]

[0067] 2.2 Transient injection of apricot fruit

[0068] Agrobacterium liquid containing overexpression vector PHG-SPL8 and PHG control vector was taken 20uL into 5mL liquid LB medium containing Kan, and cultured in a 28℃, 220rpm shaker for about 6h. Then 1mL bacterial liquid was taken into 50mL liquid LB medium containing Kan, and cultured in a 28℃, 220rpm shaker overnight. The culture was incubated until the OD 600 of the bacterial liquid was about 0.6-1.0. Then the bacterial pellet was obtained by centrifugation at 6000rpm for 10min at room temperature, and the supernatant was discarded. The bacterial pellet was resuspended in 50mL prepared buffer to make the OD 600between 0.6-1.0. The prepared bacterial solution buffer was incubated at room temperature for 4h. Healthy and disease-free fruits (‘XYZS’ apricot fruits were used for the transient expression test of PaSPL8 gene in this example, and the apricot variety ‘XYZS’ was collected from the Northern Major Economic Tree Species National Forest Germplasm Bank, which is an early-maturing apricot variety) were selected for injection test, and 3-5 different positions of each fruit were injected with buffer, and marked with a marker pen to mark the injection site. After 7 days, the fruit phenotype was observed, and part of the apricot fruits with phenotype were cut and stored in liquid nitrogen for quick freezing for subsequent tests.

[0069] 3. Tomato genetic transformation and transformation phenotype identification

[0070] 3.1 Obtaining of sterile seedlings

[0071] Full, uniform, and fresh ‘Micro-Tom’ tomato seeds were repeatedly washed with sterile water several times, then the tomato seeds were sterilized with 70% alcohol for 30s, washed with sterile water for 3 times, then sterilized with 10wt% sodium hypochlorite solution for 10min, washed with sterile water for 4-5 times, and then dried with sterile filter paper, and then inoculated in seed germination medium. After dark culture until most of the seeds germinate and turn white, they were placed in a condition of 16h light per day, light intensity of 1600-1800lx, and temperature of (24±2)℃.

[0072] 3.2 Agrobacterium tumefaciens culture

[0073] The overexpression vector constructed as described above was used to transform Agrobacterium tumefaciens, and the marked Agrobacterium monoclonal was inoculated into 5mL LB liquid medium containing the corresponding antibiotic (in a 50mL blue cap centrifuge tube) at 28℃, 200rpm shaking culture for 24 hours, 20℃, 4000rpm, centrifugation for 15min, and the bacterial body was collected; the bacterial body was uniformly blown with transformation Buffer and resuspended to OD 600 =0.5 or so.

[0074] 3.3 Preparation of explants, inoculation and co-culture

[0075] True leaves, cotyledons and hypocotyls were selected as explants for transformation. The true leaves and cotyledons were cut off the leaf tips and petioles, and the rest were cut into leaf blocks of 0.5cm×0.5cm in size; the hypocotyls were cut into segments of about 0.5-0.6mm in length and placed horizontally on the pre-culture medium, 15-20 pieces per dish. The culture conditions were the same as above, and the pre-culture was 1 day.

[0076] The explants were taken from the pre-culture medium and placed in the transformation Buffer diluted to OD 600Transform the explants in a culture dish containing approximately 0.5% Agrobacterium for 30 minutes. Remove the explants, blot them dry on sterile paper, and then place them back into the pre-culture medium. Culture for a total of 1-2 days.

[0077] 3.4 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 will form callus and adventitious buds on the selection medium, and 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. If necessary, subculture at a shorter interval. When the adventitious buds grew to about 1 cm, healthy regenerated buds were selected, and the basal callus and medium were completely removed. The buds were then transferred to a rooting medium to develop into complete plants. After the seedlings developed lateral roots, the bottle cap was removed, and a small amount of sterile water was poured into the culture bottle (covering the medium by 3-5 mm). The culture bottle was placed in a cool, 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 change from the medium to the nutrient substrate.

[0079] 4. Transient expression of apricot and phenotypic determination of tomato overexpression positive plants and analysis by real-time quantitative PCR (qRT-PCR).

[0080] The primers used for transient expression of apricot and phenotypic determination of tomato overexpression positive plants and for real-time quantitative PCR (qRT-PCR) analysis are shown in Table 7.

[0081] Table 7 Primers used for PCR and qRT-PCR

[0082]

[0083] 4.1 Validation of Genes by Real-Time Quantitative PCR

[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] The relative expression analysis of apricot genes involved in this example all selected PaUBQ as the internal reference gene (its primer is shown in Table 7), and SlACT was used as the internal reference gene in tomato (its primer is shown in Table 7), and the HYR gene was used to detect whether the overexpression strain of tomato was positive; all qRT-PCR tests were repeated 4 times, and the relative expression of each gene was calculated by the 2^-ΔΔCt method. Finally, GraphPad Prism 8.0.2 was used to present in the form of charts.

[0090] 4.2 Hormone ABA and ACC content determination

[0091] 4.2.1 Solid sample

[0092] (1) Take out the ultra-low temperature preserved transient injection apricot fruits and overexpression tomato fruits (no special requirements, default fresh sample), and grind in liquid nitrogen with a grinder (30Hz, 1min) to powder;

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

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

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

[0096] 4.2.2 Chromatography mass spectrometry collection conditions

[0097] The data collection instrument system mainly includes ultra-high performance liquid chromatography (Ultra Performance Liquid Chromatography, UPLC) (ExionLCT AD, https: / / sciex.com.cn / ) and tandem mass spectrometry (Tandem Mass Spectrometry, MS / MS) (6500+, https: / / sciex.com.cn / ).

[0098] The liquid phase conditions mainly include:

[0099] 1) Chromatographic column: Waters ACQUITY UPLC HSS T3 C18 column (1.8μm, 100mmx2.1mm i.d.);

[0100] ​2) Mobile phase: Phase A, ultrapure water (with 0.04% acetic acid added); Phase B, acetonitrile (with 0.04% acetic acid added);

[0101] 3) Gradient elution program: 0 min A / B is 95:5 (V / V), 1.0 min A / B is 95:5 (VN), 8.0 min is 5:95 (V / ), 9.0 min is 5:95 (V / ), 9.1 min is 95:5 (V / ), 12.0 min is 95:5 (VV);

[0102] 4) Flow rate 0.35 mL / min; column temperature 40℃; injection volume 2 μL.

[0103] Mass spectrometry conditions mainly include:

[0104] Electrospray ionization (ESI) temperature was 550℃, mass spectrometry voltage was 5500V in positive ion mode and -4500V in negative ion mode, and curtain gas (CUR) was 35psi. In the Q-Trap6500+, each ion pair was scanned and detected based on optimized declustering potential (DP) and collision energy (CE).

[0105] 5 Results Analysis

[0106] 5.1 Phenotypic Analysis of Apricot Fruits with Transient Overexpression of PaSPL8

[0107] To determine the function of the PaSPL8 gene in fruit ripening, the CDS sequence of the cloned PaSPL8 gene was constructed into the linearized overexpression vector PHG according to the prescribed method, and subsequent transient expression experiments were conducted. Following the method, apricot fruits in the late stage of slow growth were injected with prepared bacterial permeate containing PHG-PaSPL8 and the empty PHG vector, and the fruits were labeled. The phenotype of transient PaSPL8 gene expression in apricots was observed after 5-7 days.

[0108] Depend on Figure 2 It was found that the apricot fruit areas injected with PHG-PaSPL8 bacterial solution retained their green peel, thus delaying fruit ripening; while the apricot fruit injected with the blank control PHG bacterial solution normally faded and ripened. This indicates that the PaSPL8 gene can delay the ripening process of apricot fruit. The ABA and ACC contents of fruits from the injected areas were measured (see...). Figure 4The apricot fruits injected with PHG-PaSPL8 bacterial solution had an ABA content of 330.12 ng / g and an ABA-GE content of 482.85 ng / g, while the apricot fruits injected with the blank control had an ABA content of 493.39 ng / g and an ABA-GE content of 1093.17 ng / g. The experimental group was significantly lower than the control group. Conversely, the ACC content of the experimental group was 27.71 ng / g, while the overripe ACC content of the control group was 33.57 ng / g. Hormone assays showed that overexpression of the PaSPL8 gene could inhibit the increase of ABA content, leading to ACC accumulation and delaying fruit ripening. The expression of genes related to the ABA and ethylene metabolic pathways was then measured, such as... Figure 3 As shown, the expression of the PaSPL8 gene in fruits injected with PHG-PaSPL8 bacterial solution was significantly higher than that in the control group, approximately five times higher. Furthermore, the expression levels of other genes related to the abscisic acid metabolism pathway, such as PaNCED5, were decreased, while PaPY707A expression was increased. However, there was no significant difference in the expression of PaCtrz and PaACO genes between the two groups. In conclusion, the PaSPL8 gene can inhibit the increase and accumulation of ABA content in fruits, leading to the accumulation of ACC content, thereby delaying the ripening of apricot fruits.

[0109] 2. Phenotypic analysis of tomato overexpression PaSPL8

[0110] To verify the function of PaSPL8 in regulating fruit ripening and ABA synthesis, PaSPL8 was expressed ectopically in micro-TOM tomatoes. The transformation process in micro-TOM tomatoes followed the prescribed method, ultimately resulting in 16 genetically transformed tomato lines with the PaSPL8 gene. Figure 5 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 hours / day and darkness of 8 hours / day for subsequent experimental observation. During the tomato growth process, attention was paid to watering and prevention of pests, diseases, and fungal infections. Appropriate pesticides were sprayed after flowering for timely control to ensure normal growth of the tomatoes in the later stages.

[0111] The entire growth and development process from flowering to maturity was monitored and statistically analyzed in tomato lines and WT lines overexpressing the PaSPL8 gene (see...). Figure 6), it was found that the overall fruit growth and development cycle of tomato overexpressing PaSPL8 gene was longer than that of WT tomato. The period from flowering to tomato maturity of WT tomato was 62d, while the period from flowering to fruit maturity of tomato overexpressing PaSPL8 gene was 74d. Therefore, we selected three independent transgenic tomato lines with the most obvious late-maturing traits, and the fruit development cycle was 74d (OE-8, OE-9, OE-16) to determine the ABA content and analyze the expression of related genes in the ABA metabolic pathway.

[0112] On the basis of obtaining the mature phenotype of the fruit growth and development cycle of tomato overexpressing PaSPL8 gene lines (OE-8, OE-9, OE-16) and WT lines, the last mature tomato fruit was used as the test material, and the ABA content and the expression of related genes in the ABA metabolic pathway were obtained by LC-MS / MS and qRT-PCR methods. As shown in Figure 7 , the PaSPL8 gene overexpression in tomato lines overexpressing PaSPL8 gene (OE-8, OE-9, OE-16) was significantly higher than that in the control group WT. As shown in Figure 8 , the ABA content in WT was 966.31 ng / g, the ABA content in OE-8 was 122.62 ng / g, the ABA content in OE-9 was 111.83 ng / g, the ABA content in OE-16 was 144.71 ng / g, and the ABA-GE (abscisic acid ester) content in WT was 6434.00 ng / g, and in OE three lines was 3607.35 ng / g, 3448.98 ng / g and 3636.64 ng / g, respectively, which was opposite to the expression of ABA content. At the same time, the ACC content in WT was 84.62 ng / g, the ACC content in OE-8 was 181.17 ng / g, the ACC content in OE-9 was 228.56 ng / g, and the ACC content in OE-16 was 271.96 ng / g, which proved that overexpression of PaSPL8 gene delayed fruit ripening by inhibiting the ABA content in the fruit.

[0113] CDS sequence of PaSPL8 gene (SEQ ID NO. 1):

[0114] ATGGAATCAAACAGAGCTCATGGGAAGAGGAGCTTGAACTACAAGATGGAGGAAGAGGAGGAGGAGGAGGATGAAGAAGAAGAGCAGGATGAAGAGGAGGATGATGAAGATACTAACAGATCATCACTAGTGTACGGTGAGGATGAGAGGAGGAAAAGAGTGTTGATGGTGAATAGTGCAACTACTACTCCTAACAAGAGAGGATCTGGTGCAGGAGGGTCTATGGCAAGGCCATCTTGTCAAGCAGATGATTGCAATGCTGACTTGAGTGATGCAAAGCAATACTATCGCCGCCATAAGGTCTGCGGTGTTCATGCCAAGGCTCCGGCGGTGCGTGTGGGTGGAGTTCAACAGCGCTTCTGCCAGCAATGTAGCAGTGCAGAGAAAATAGCTAAAGAAAAAGAGAAAAACAAGATGAAGGAGAGAAAACCATTTACCCCAAAAAAGAAAGAGAGAGAGGAAGAAGAAGAAGAAAAAGAAGTGGTTTAA

[0115] The amino acid sequence of the protein encoded by the PaSPL8 gene (SEQ ID NO. 2) is:

[0116] MESNRAHGKRSLNYKMEEEEEEEEDEEEEQDEEEDDEDTNRSSLVYGEDERRKRVLMVNSATTTPNKRGSGAGGSMARPSCQADDCNADLSDAKQYYRRHKVCGVHAKAPAVRVGGVQQRFCQQCSSAEKIAKEKEKNKMKERKPFTPKKKEREEEEEEKEVV

[0117] It is apparent that the above-described embodiments are merely illustrative in nature and are not intended to limit the embodiments in any respect. Various modifications and changes can be made by those skilled in the art which fall within the scope of the embodiments. It is not necessary to set forth in detail all embodiments of the embodiments. Obviously, modifications and changes can be made in the embodiments based on the description as set forth herein without departing from the scope of the claims of the present patent application.

Claims

1. A functional gene for regulating the maturation of apricot fruits PaSPL8 characterized in that, PaSPL8 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The functional gene for regulating apricot fruit ripening according to claim 1 PaSPL8 characterized in that, PaSPL8 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

3. Use of a functional gene for regulating the ripening of apricot fruits, characterized in that, PaSPL8 The functional gene for regulating apricot fruit ripening according to claim 1 PaSPL8 for regulating apricot fruit ripening by increasing the expression amount of the gene PaSPL8 to delay fruit ripening. ​ 4. A functional gene for regulating apricot fruit ripening according to claim 3, characterized in that, PaSPL8 The application is characterized in that, Functional gene for regulating apricot fruit ripening as claimed in claim 1 PaSPL8 Overexpression in apricot fruits for late maturing apricot breeding.

5. A functional gene for regulating apricot fruit ripening according to claim 4 PaSPL8 for use, characterized in that, The recombinant overexpression vector of the functional gene for regulating apricot fruit ripening according to claim 1 is introduced into apricot fruit by Agrobacterium tumefaciens mediation to increase the expression amount of the gene in apricot fruit and delay apricot fruit ripening. PaSPL8 The recombinant overexpression vector of the functional gene for regulating apricot fruit ripening according to claim 1 is introduced into apricot fruit by Agrobacterium tumefaciens mediation to increase the expression amount of the gene in apricot fruit and delay apricot fruit ripening. PaSPL8 The recombinant overexpression vector of the functional gene for regulating apricot fruit ripening according to claim 1 is introduced into apricot fruit by Agrobacterium tumef 6. A functional gene for regulating apricot fruit ripening according to claim 5 PaSPL8 for use, characterized in that, In constructing the recombinant overexpression vector, the primers shown in SEQ ID NO. 3 and SEQ ID NO. 4 are used for PCR amplification; the original vector used is PHG plasmid, PaSPL8 The CDS sequence of the gene is located between the BamHI and PstI enzyme cutting sites of the PHG plasmid; PaSPL8 The primers used for quantitative PCR of the gene are shown in SEQ ID NO. 5 and SEQ ID NO.

6.

7. Use of a functional gene for regulating the ripening of apricot fruits, characterized in that, PaSPL8 Functional gene for regulating apricot fruit ripening as claimed in claim 1 PaSPL8 Overexpression of the gene in tomato for delaying ripening of tomato fruits. ​ 8. A functional gene for regulating apricot fruit ripening according to claim 7 PaSPL8 for use, characterized in that, Recombinant overexpression vector of the functional gene for regulating apricot fruit ripening according to claim 1 PaSPL8 Genetic transformation of 'Micro-Tom' tomato by Agrobacterium tumefaciens-mediated transformation of the recombinant overexpression vector of the functional gene for delaying fruit ripening of 'Micro-Tom' tomato.

9. A functional gene for regulating apricot fruit ripening according to claim 8 PaSPL8 for use, characterized in that, In constructing the recombinant overexpression vector, the primers shown in SEQ ID NO. 3 and SEQ ID NO. 4 are used for PCR amplification, and the original vector used is PHG plasmid, PaSPL8 The CDS sequence of the gene is located between the BamHI and PstI enzyme cutting sites of the PHG plasmid; PaSPL8 The primers used for quantitative PCR of the gene are shown in SEQ ID NO. 5 and SEQ ID NO.

6.

10. Use of a functional gene for regulating the ripening of apricot fruits, characterized in that, PaSPL8 Functional genes for regulating apricot fruit ripening as claimed in claim 1 PaSPL8 Overexpression for inhibiting the increase in abscisic acid content in apricot fruits or tomatoes. ​

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