FaABI3 gene and application thereof in regulation and control of strawberry fruit maturity and strawberry flavonoid content

Abstract

The invention discloses a FaABI3 gene and application thereof in regulation and control of strawberry fruit maturity and strawberry flavonoid content, and relates to the field of gene engineering, the nucleotide sequence of the FaABI3 gene is shown as SEQ ID NO.1, and the amino acid sequence of protein encoded by the FaABI3 gene is shown as SEQ ID NO.2. A FaABI3 gene silencing vector is constructed and is used for infecting octaploid strawberry fruits in a small green period, so that the strawberry fruit maturity and strawberry flavonoid content can be regulated and controlled, and the strawberry fruit maturity and strawberry flavonoid content can be regulated and controlled. Strawberry fruits with the FaABI3 gene silenced are obtained through screening according to the gene expression quantity in the fruits, it is indicated that by changing the expression quantity of the FaABI3 gene in strawberries, the growth and development of the strawberries can be regulated and controlled, the FaABI3 gene can be applied to strawberry genetic breeding, an important foundation is laid for strawberry variety improvement, and the method has a wide application prospect. The application of the FaABI3 gene in increasing the content of strawberry flavonoid spinosin, sophoricoside, formononin and delphinidin is found for the first time, and further understanding of a strawberry flavonoid regulation mechanism is facilitated, so that the strawberry flavonoid content can be regulated by utilizing a biotechnological means, and an important foundation is laid for strawberry variety improvement.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, in particular to a FaABI3 gene and application thereof in regulating strawberry fruit ripening and strawberry flavonoid content. Background Art

[0002] Strawberry (Fragaria×ananassa Duch.) is a perennial herbaceous plant of the genus Fragaria in the family Rosaccae. It has excellent flavor and high nutritional value. It is deeply loved by consumers and has considerable economic value.

[0003] In the actual production of strawberries, the short supply season is one of the main problems. Fruit ripening is a complex physiological and biochemical process, which is regulated by plant hormones, transcription factors, etc. How to discover important quality regulatory genes and use genetic engineering to improve strawberry varieties is an urgent problem to be solved in future production.

[0004] The flavonoids rich in strawberries are involved in many cellular activities, such as signal transduction, auxin transport and pigmentation. One of the most prominent functions is to protect plants from abiotic (such as drought, salinity, ultraviolet radiation and heat) and biotic (such as insect and pathogen attacks) stresses, play a role in regulating reactive oxygen species (ROS), and have the function of shielding ultraviolet rays. They are important defense metabolites for plants. Therefore, increasing the level of flavonoid metabolism will help enhance the plant chemical defense system. There are many kinds of flavonoid compounds, which are divided into six categories due to their different structures: flavonoids, flavonols, dihydroflavonoids, flavanols, isoflavones and anthocyanins. The flavonoid substances contained in strawberries include spinosin, sophoroside, formononetin and delphinidin. Among them, spinosin is a C-glycoside flavonoid with neuroprotective effects, which has multiple pharmacological effects such as sedation and hypnosis, anti-anxiety, antioxidant, protection of brain nerves, and enhancement of learning and memory; sophoroside is an isoflavone glycoside with multiple biological activities, including anti-inflammatory, anti-cancer, prevention and treatment of osteoporosis, anti-cardiovascular disease, antioxidant, anti-inflammatory, contraceptive, nerve protection, anti-radiation, immune enhancement and anti-asthma; formononetin is a type of isoflavone glycoside that has the effects of promoting skin growth and enhancing immunity; delphinidin is an important active monomer in anthocyanin, with anti-inflammatory, antioxidant and anti-cancer properties.

[0005] How to increase the content of flavonoids spinosin, sophoroside, formononetin and delphinidin in strawberries will help to further improve the nutritional and economic value of strawberries. Summary of the invention

[0006] The present invention aims to provide a FaABI3 gene and its application in regulating the ripening of strawberry fruits and the content of strawberry flavonoids, so as to regulate the growth, development and ripening of strawberries by controlling the expression amount of the FaABI3 gene in strawberries, and regulate the content of strawberry flavonoids by controlling the FaABI3 gene.

[0007] In order to achieve the above object, the specific scheme adopted by the present invention is: a FaABI3 gene, the nucleotide sequence of the FaABI3 gene is shown in SEQ ID NO.1,

[0008] SEQ ID NO.1: atggatggagtgcaagttcaagtccacggtaaccaccaccaagatctgcatgcagcggat caccacggccagcatctcatcggcaaagaagagatgcgaaacgatttcggagaagaagat ggagacgccgctgatctatggcttgataatgaccaagactctcttctcgctgacggcgcc ggtgaagacatgtcgacgtcaccctcggcgtcttcttcttcctccgccgcttcctgggct gttctcaagtccgatgctgaggataatcattctcatgattatcagcagcaacaagaccac aacaacaacactcactactctcataacgattctgctgatgcatatccggctggggcgttg tcccccaccccctctatggagatctctcagcagtcggatctcggaatggagtgcatggac atgatggagacttttgggtacatagatctgttcgaaggcaacgagttcttcgacccttct tccattttccaaaacgagaacccaataatggaccagtttcaaccgcaggaacaaccttct caggcgcatgaagagaatcaaaccccacacccacaagagaacatgatcatggggagatcag agcatgaaggtcctagaggggggatccttataccaacagagcaacaagatgccggaagac gacatggcgtctgtgttcttggagtggcttaggtctaaccgagagacggtttctgcggag gatttgaggagcgtgaagatcaagaagtcaacaatcgagtgcgctgccaggcgtttgggt ggaggaaaagaggcgatgaagcagttactcaaactagtgcttgaatgggttcaaactaat catctccaaaagaggcgcggtagtagtaccaaagactcggcgatacaacactatgctgta gacccatttcaaagcgccaaccctaaccctaaccctagtcttaaccctacgcaaaatcct cccatatcatcgccgtggatggcgtctccccagtatgatgcggcggcgcctgtattagtc ccgactccgtcaactccgatgatggggttcatgggtcaggacccgtttggaagcgggccg ggttaccagcaaccgaattcagagcaataccagcatcaaatgctggagactgcaccaacc tggccaccttcatctccatttatgggcaacaattatgggtctttcccggatactaatgtc caactagcaccgacgagtcagcatcagcaacagttttccggttacggagggcagtatggc cagtatcagtattttggagggcattcaggtgagcctcagctggtgaggttagcctcttca gccactaaagaggctaggaagaagaggatgcaaaggcaaagaaggactttgtcacaccat ggaaggcatcacggccatcaacagaatcagcatcctaatcagatgctggatcagaggcta gttgggaatattgatcctaattgcactactgccgcaatgggcaatccctctgcagctaac tggttttattggcccacagcggctgccggtggtcctgctcctgctgccaccccggcaatg gtacagaatatgactcccgaggcagcaccatcagtgcttccggttgatcgtccggccagt caagggcagaattataatctgggccggattaatgcgcaggaaaggcgacagggatggaaa cctgatcagaataagaatttgaggttccttcttcagaaagtgttgaagcaaagtgatgta ggcaaccttggaagaattgttttgccaaagaaagaagctgaaacccatctgcctgaacta gaggcaagggatggaatttcgattgcaatggaagacatcgggacttctcgtgtatggaat atgcgctataggtactggcccaacaacaaaagcaggatgtatctccttgaaaacacagga gattttgtgagggcaaatgggcttcaagaaggggatttcatagtcatctattcagacatc aagtgtaacaaatatatgatacgaggagtgaaggtacggcaagctgggacgaaatcagag accaaaaggccaggaaagtcacaaaggaaccagcatgcaagcaccccatctggcaacaat ggctcttcatctaagaaacagtaa

[0009] A protein encoded by the FaABI3 gene, wherein the amino acid sequence of the protein is as shown in SEQ ID NO.2

[0010] SEQ ID NO.2: MDGVQVQVHGNHHQDLHAADHHGQHLIGKEEMRNDFGEEDGDAADLWLDNDQDSLLADGA GEDMSTSPSASSSSSAASWAVLKSDAEDNHSHDYQQQQDHNNNTHYSHNDSADAYPAGAL SPTPSMEISQQSDLGMECMDMMETFGYIDLFEGNEFFDPSSIFQNENPIMDQFQPQEQPS QAHEENQTPHPQENMIMGDQSMKVLEGGSLYQQSNKMPEDDMASVFLEWLRSNRETVSAE DLRSVKIKKSTIECAARRLGGGKEAMKQLLKLVLEWVQTNHLQKRRGSSTKDSAIQHYAV DPFQSANPNPNPSLNPTQNPPISSPWMASPQYDAAAPVLVPTPSTPMMGFMGQDPFGSGP GYQQPNSEQYQHQMLETAPTWPPSSPFMGNNYGSFPDTNVQLAPTSQHQQQFSGYGGQYG QYQYFGGHSGEPQLVRLASSATKEARKKRMQRQRRTLSHHGRHHGHQQNQHPNQMLDQRL VGNIDPNCTTAAMGNPSAANWFYWPTAAAGGPAPAATPAMVQNMTPEAAPSVLPVDRPAS QGQNYNLGRINAQERRQGWKPDQNKNLRFLLQKVLKQSDVGNLGRIVLPKKEAETHLPEL EARDGISIAMEDIGTSRVWNMRYRYWPNNKSRMYLLENTGDFVRANGLQEGDFIVIYSDI KCNKYMIRGVKVRQAGTKSETKRPGKSQRNQHASTPSGNGNGSSSKKQ

[0011] Application of a FaABI3 gene in regulating strawberry fruit ripening.

[0012] As a further optimization of the above technical solution: the ripening of strawberry fruit is delayed by reducing the expression level of the FaABI3 gene in strawberry.

[0013] Application of FaABI3 gene in regulating flavonoid content in strawberry fruit.

[0014] As a further optimization of the above technical solution: by reducing the expression level of the FaABI3 gene in strawberries, the content of strawberry flavonoids is increased.

[0015] As a further optimization of the above technical solution: Agrobacterium is transformed by the FaABI3 gene silencing vector, and the strawberry is transformed by the Agrobacterium-mediated method to increase the flavonoid content of the strawberry.

[0016] As a further optimization of the above technical solution: the FaABI3 gene was connected to the vector pTRV2 to obtain the FaABI3 gene silencing vector pTRV2-FaABI3, and then transformed with Agrobacterium tumefaciens GV3101, and the strawberry small green period fruit was transformed by the Agrobacterium-mediated method to increase the flavonoid content of the strawberry fruit.

[0017] As a further optimization of the above technical solution: it is characterized in that the flavonoids include spinosin, sophoroside, formononetin and delphinidin.

[0018] The invention discloses an application of FaABI3 gene in regulating the ripening of strawberry fruit and / or regulating the flavonoid content of strawberry fruit. The FaABI3 gene is connected with a vector pTRV2 to obtain a FaABI3 gene silencing vector pTRV2-FaABI3, and then transformed with Agrobacterium tumefaciens GV3101. Finally, the strawberry tissue culture seedlings are immersed in an Agrobacterium bacterial solution carrying the FaABI3 gene for infection, and then subcultured to obtain transgenic strawberry plants.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention constructs a FaABI3 gene silencing vector and infects octoploid strawberry small green period fruits with it, and obtains FaABI3 gene-silenced strawberry fruits according to the gene expression level in the fruits, indicating that the growth, development and maturity of strawberries can be regulated by changing the expression level of the FaABI3 gene in strawberries. The FaABI3 gene can be applied to strawberry genetic breeding, laying an important foundation for strawberry variety improvement.

[0021] The present invention first discovered the application of FaABI3 gene in increasing the content of strawberry flavonoids spinosin, sophora glycoside, formononetin and delphinidin, designed full-length primers based on octoploid strawberry fruit genome data to clone the strawberry FaABI3 gene, constructed a FaABI3 gene silencing vector, and transformed strawberry fruit by Agrobacterium-mediated method, measured gene expression screening to obtain FaABI3 gene silenced strawberry fruit, compared with non-silenced FaABI3 gene fruit, FaABI3 gene silenced strawberry flavonoids spinosin, sophora glycoside, formononetin and delphinidin content significantly increased, indicating that silencing FaABI3 gene can increase the content of strawberry flavonoids. The present invention helps to further understand the regulation mechanism of strawberry flavonoids, so that the content of strawberry flavonoids can be regulated by biotechnology means, laying an important foundation for strawberry variety improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The electrophoresis diagram of the synthesis and enzyme digestion identification of the FaABI3 silencing fragment of octoploid strawberry;

[0023] Figure 2 The phenotypes of FaABI3 gene-silenced strawberry fruit (b) and wild-type control fruit (a);

[0024] Figure 3 is the relative expression level of FaABI3 gene in FaABI3 gene silenced strawberry fruit and wild-type control fruit;

[0025] Figure 4 This is the expression map of genes in the flavonoid biosynthesis pathway of strawberry fruit after FaABI3 gene silencing;

[0026] Figure 5 This is a graph showing the changes in the content of flavonoids spinosin, sophoroside, formononetin and delphinidin in strawberry fruits after FaABI3 gene silencing. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further elaborated in detail below in conjunction with specific embodiments. The parts of the present invention that are not described and disclosed in detail in the following embodiments should be understood as the prior art known or should be known to those skilled in the art.

[0028] Example 1 Cloning of strawberry FaABI3 gene

[0029] Octoploid strawberry small green stage fruit was used as material, and total RNA was extracted using the improved CTAB method. The specific steps are as follows:

[0030] Add 4 mL of CTAB extract and 80 μL of β-mercaptoethanol to a centrifuge tube; grind 1.0 ± 0.1 g of pulp tissue with liquid nitrogen, add it to the 65°C preheated extract, mix well, vortex and shake, and place in a 65°C water bath for 5 min; add 4 mL of chloroform / isoamyl alcohol (V / V = 24:1) and vortex to mix well, centrifuge at 15°C 12,000 × g for 10 min; aspirate the supernatant to a new centrifuge tube, add an equal volume of chloroform / isoamyl alcohol (V / V = 24:1) and vortex to mix well, centrifuge at 15°C 12,000 × g for 10 min; aspirate the supernatant, add 1 / 5 volume of 12M LiCl, stand at 4℃ overnight; centrifuge at 4℃12,000×g for 30min; discard the supernatant, dissolve in 500μL (65℃ preheated) SSTE solution, transfer to 1.5mL centrifuge tube; add 500μL chloroform / isoamyl alcohol (V / V=24:1) and vortex to mix, centrifuge at 15℃12,000×g for 10min; transfer the supernatant to a 1.5mL centrifuge tube, add 2 times volume of anhydrous ethanol (-20℃ precooled) and mix, stand at -80℃ for 30min; centrifuge at 4℃12,000×g for 20min; discard the supernatant and dissolve the precipitate in 20μL DEPC water; Nanodrop 2000 nucleic acid analyzer detects the concentration and OD260 / 280 value of total RNA. If it is between 1.8-2.0, it indicates that the total RNA is of high purity; use 1% agarose gel electrophoresis at 6Vcm -1 Electrophoresis was performed under a voltage of 10.5 V and the RNA integrity was observed after 20 min. The brightness of the 28S rRNA band was nearly twice that of the 18S rRNA band, indicating that the RNA sample was relatively intact and not contaminated or degraded.

[0031] Synthesis of the first strand of cDNA: About 1.0 ± 0.1 μg of the total RNA was taken and the cDNA was synthesized according to PrimeScript TM RT reagent Kit with gDNA Eraser (Takara, Japan) was used to remove genomic DNA and reverse transcribe to synthesize the first-strand cDNA according to the instructions.

[0032] PCR amplification: Using the strawberry cDNA as template, a high-fidelity polymerase system was used Max Master Mix, PCR amplification was performed using primers FaABI3-F (5'-ggatcttccagagatctcgagATGGATGGAGTGCAAGTTCAAGT-3') and FaABI3-R (5'-ctgccgttcgacgatggtaccCTGTTTCTTAGATGAAGAGCCATTGT-3'), and the PCR 25 μL reaction system was as follows: 12.5 μL 2× Phanta Max Master Mix, 1 μL FaABI3-F (10 μM), 1 μL FaABI3-R (10 μM), 1 μL cDNA, 9.5 μL ddH 2 O.

[0033] The PCR reaction program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 1.5 min, 35 cycles; and extension at 72°C for another 5 min.

[0034] The amplified product was detected by 1% agarose gel electrophoresis, and the FaABI3 fragment was recovered and purified according to the instructions of the DNA gel recovery kit (Takara, Japan).

[0035] The vector pC1300s-GFP was double-digested with EcoRI and SmaI. After digestion at 37°C for 4 h, the DNA was recovered and purified using a DNA gel recovery kit (Takara, Japan) and the concentration was determined. The recovered FaABI3 fragment was connected to the pC1300s-GFP vector by homologous recombination. The recombination reaction system was referred to II One Step Cloning Kit (Nanjing Novogene Biotech Co., Ltd., Nanjing, China) was used to obtain the pC1300s-GFP-FaABI3 plasmid, which was transformed into Escherichia coli DH5α competent cells. Positive clones were screened by colony PCR, and the plasmids were extracted and sequenced. The sequencing result is shown in SEQ ID NO.1.

[0036] Example 2 Construction of Strawberry FaABI3 Gene Silencing Vector

[0037] The amino acid sequence of the FaABI3 gene of octoploid strawberry (SEQ ID NO.2) was used as the query sequence, and the homologous gene sequence of Fragaria×ananassa was obtained by BLASTP search on strawberryGARDEN (http: / / strawberry-garden.kazusa.or.jp / ). The amino acid sequence was aligned using Clustal X (version 2.0.12), and the sequences lacking homology were deleted. Then, the CDS sequences of the remaining sequences with high homology were aligned, and the 300bp region with high specificity was selected. The specific sequence is as follows:

[0038] gcaggaaaggcgacagggatggaaacctgatcagaataagaaatttgaggttccttcttcagaaagtgttgaag caaagtgatgtaggcaaccttggaagaattgttttgccaaagaaagaagctgaaacccatctgcctgaactagaggcaagg gatggaatttcgattgcaatggaagacatcgggacttctcgtgtatggaatatgcgctataggtactggcccaacaacaaaa gcaggatgtatctccttgaaaacacaggagattttgtgagggcaaatgggcttcaagaagggga (SEQ ID NO. 3).

[0039] The BamHI restriction site was introduced at the 5' end, and the SacI restriction site was introduced at the 3' end. The sequence was sent to a biological company for sequence synthesis and assembled into the pTRV2 vector to obtain the strawberry FaABI3 gene silencing vector pTRV2-FaABI3 ( Figure 1 ).

[0040] Example 3 Strawberry FaABI3 gene silencing

[0041] 5uL of the FaABI3 gene silencing vector pTRV2-FaABI3 in Example 2 was transformed into 100μL of Agrobacterium tumefaciens GV3101 by freeze-thaw method, gently pipetted and mixed, ice bathed for 30min, frozen in liquid nitrogen for 5min, and heat-shocked in a 37°C water bath for 5min. Add 1mL of liquid LB medium without antibiotics, incubate at 28°C for 2h, and then spread on solid LB medium containing 50μg / mL Kan and 25μg / mL Rif antibiotics, and inverted in a 28°C incubator to grow a single colony. Colony PCR was performed to identify monoclonal positive strains according to the instructions of 2×T5 Super PCR Mix (Basic) (Beijing Qingke Biotechnology Co., Ltd., Beijing, China).

[0042] The octoploid 'Akihime' strawberry fruits in the small green fruit period were selected as test materials and infected by injection. The specific fruit selection method was as follows: multiple fruits of the same size, shape, maturity and no mechanical damage were selected from the strawberry plants, and the strawberry fruits were divided into two groups. One group was injected with an equal volume of mixed Agrobacterium GV3101 transformed with pTRV1 and pTRV2 vectors as the control group, and the other group was injected with an equal volume of mixed Agrobacterium GV3101 transformed with pTRV1 and FaABI3 gene silencing vector pTRV2-FaABI3 as the experimental group. The specific method is as follows:

[0043] 1) Pick the positive clones of Agrobacterium GV3101 transformed with pTRV1, pTRV2 and pTRV2-FaABI3 respectively, inoculate them into 4 mL LB medium (50 μg / mL Kan and 25 μg / mL Rif), and shake the culture at 28°C and 200 rpm for 12 h to obtain bacterial solution I containing pTRV1, bacterial solution I containing pTRV2 and bacterial solution I containing pTRV2-FaABI3;

[0044] 2) The three bacterial solutions I were inoculated into 100 mL LB liquid medium containing Kan (100 μL) and Rif (50 μL) at a ratio of 1:100, and cultured at 28°C, 200 rpm, with shaking until OD 600 =1.0, the bacterial solution was transferred to a 50 mL centrifuge tube, centrifuged at 4000 rpm for 10 min, and the supernatant was discarded to collect the bacterial solution, thereby obtaining bacterial solution II containing pTRV1, bacterial solution II containing pTRV2, and bacterial solution II containing pTRV2-FaABI3;

[0045] 3) Add 20 mL of 4°C pre-cooled sterilized ultrapure water to the three bacterial solutions II respectively, mix well, centrifuge at 4°C, 4000 rpm for 10 min, discard the supernatant, repeat the operation once, and obtain bacterial solution precipitates containing pTRV1, pTRV2, and pTRV2-FaABI3;

[0046] 4) Weigh MgCl 2 6H 2 O 2.033 g, fatty acid methyl ester sulfonate (MES) 2.172 g, dissolved in 1 L H 2 O, adjust pH to 5.7, sterilize at 121℃ for 15min to obtain blank infection solution, add acetosyringone to the blank infection solution to 150μmol / L, precool, and use the precooled blank infection solution to suspend the three bacterial solutions in step 3) to precipitate to OD 600 =1.0-1.5, and placed at 25°C for 3h to obtain an Agrobacterium suspension containing pTRV1, an Agrobacterium suspension containing pTRV2, and an Agrobacterium suspension containing pTRV2-FaABI3;

[0047] The Agrobacterium suspension containing pTRV1 and the Agrobacterium suspension containing pTRV2 were mixed in equal volumes to form the infection solution for the control group, and the Agrobacterium suspension containing pTRV1 and the Agrobacterium suspension containing pTRV2-FaABI3 were mixed in equal volumes to form the infection solution for the test group, and the strawberry fruits were infected by injection.

[0048] The strawberry plants of the control group and the experimental group were placed under the same room temperature and left to grow naturally for 7 days before being picked. Figure 2 The picture shown is of strawberries 7 days later. Figure 2 (a) It can be seen that most of the strawberries in the control group have turned red and are close to ripe. Figure 2 (b) The strawberry fruit infected with the FaABI3 gene silencing vector is green in color, indicating that the ripening of the strawberry can be delayed after being infected with the FaABI3 gene silencing vector, that is, the ripening speed of the strawberry can be regulated by controlling the expression of the FaABI3 gene.

[0049] In order to verify the expression of the silent FaABI3 gene, qRT-PCR was used to further detect the expression of the FaABI3 gene in infected strawberry fruits. The primer sequences used were FaABI3-F / FaABI3-R and FaActin-F / FaActin-R. The results showed that ( Figure 3 ), compared with the control group fruits, the expression level of FaABI3 gene in silenced fruits was significantly lower than that in control group fruits, indicating that FaABI3 gene was silenced in strawberry fruits.

[0050] FaABI3-F: 5'-AGCAAAGTGATGTAGGCAACC-3'

[0051] FaABI3-R: 5'-TAGCGCATATTCCATACACG-3'

[0052] FaActin-F: 5'-GACAGGTGTTGTGGTAGACG-3'

[0053] FaActin-R: 5'-ACGGACTTGATGCTGCTCCC-3'

[0054] Example 4 Effect of strawberry FaABI3 gene silencing on flavonoids

[0055] 1. Strawberry FaABI3 gene silencing affects genes in the flavonoid metabolic pathway

[0056] According to PrimeScript TM The expression levels of genes in the strawberry flavonoid metabolic pathway after FaABI3 gene silencing in Example 3 were detected using the RT-PCR Kit (Takara, Japan). Figure 4 As shown, silencing of the strawberry FaABI3 gene can upregulate the FaF3'5'H, FaF3H1, and FaF3H2 genes in the flavonoid metabolic pathway, and downregulate the FaFLS1, FaFLS3, FaF3GT3, and FaF3GT7 genes, indicating that the FaLABI3 gene can regulate the genes in the strawberry flavonoid metabolic pathway.

[0057] 2. Strawberry FaABI3 gene silencing increases the content of flavonoids spinosin, sophoroside, formononetin and delphinidin

[0058] The strawberry FaABI3 gene silenced fruits and the control group fruits in Example 3 were placed in a freeze dryer (Scientz-100F) for vacuum freeze drying for 63 hours, and ground (30 Hz, 1.5 min) into powder using a grinder (MM 400, Retsch). 50 mg of the powder was weighed respectively, and 1200 μL of -20°C pre-cooled 70% methanol / water (1:1) internal standard extract was added. The internal standard extract was prepared by dissolving 1 mg of the standard in 1 mL of 70% methanol water to prepare a 1000 μg / mL standard stock solution, and the 1000 μg / mL stock solution was further diluted with 70% methanol to prepare a 250 μg / mL internal standard solution; vortexing once every 30 minutes, each time lasting 30 seconds, and vortexing 6 times in total; centrifuging at 12000 rpm for 3 minutes, aspirating the supernatant, filtering the sample with a 0.22 μm pore size microporous filter membrane, and storing it in a sample injection bottle. The separation was performed using an ultra-high performance liquid chromatography system equipped with an ACQUITY Premier HSS T3 Column (1.8 μm, 2.1 mm×100 mm, Waters, UK). Mobile phase A was 0.1% formic acid / water; mobile phase B was 0.1% formic acid / acetonitrile; column temperature was 40°C, flow rate was 0.4 mL / min, injection volume was 4 μL; gradient elution program: 0-2 min, 80% mobile phase A; 2-5 min, 60% mobile phase B; 5-7.5 min, 1-99% mobile phase B; 7.6-10 min, 1-95% mobile phase A. The eluent was collected by tandem mass spectrometry AB TripleTOF (Waters, UK) in positive and negative ion modes. In positive ion mode, the ionization voltage was 5000 V and the ion source temperature was 550 °C. In negative ion mode, the ionization voltage was -4000 V and the ion source temperature was 450 °C. ESI mode was used for data collection, with MS1 mass range of 50-1000 Da and acquisition time of 0.2 s, and MS2 mass range of 25-1000 Da and acquisition time of 0.04 s. The raw data of the mass spectrometer were converted into mzXM format by ProteoWizard, and peak extraction, alignment, and retention time correction were performed using the XCMS program. Peaks with a missing rate of >50% in each group of samples were filtered, and blank values ​​were filled with KNN. The peak area was corrected using the SVR method. The peaks after correction and screening were identified by searching the laboratory's self-built database, integrated public library, prediction library, and metDNA method. Finally, substances with a comprehensive identification score of 0.5 or above and a QC sample CV value of less than 0.5 were extracted and then merged into positive and negative modes. Based on the variable importance projection (VIP) obtained by the orthogonal partial least squares discriminant analysis model, differential metabolites were preliminarily screened, and metabolites with VIP>1 were significantly different.At the same time, the P-value of univariate analysis was combined to further screen out the differential metabolites, and P ≤ 0.01 was considered extremely significant. Figure 5 As shown, compared with the fruits of the control group where the FaABI3 gene was not silenced, the strawberry flavonoids spinosin, sophoroside, formononetin and delphinidin increased significantly in the FaABI3 gene-silenced fruits, indicating that the FaABI3 gene can negatively regulate the metabolism of strawberry flavonoids spinosin, sophoroside, formononetin and delphinidin.

[0059] Example 5

[0060] The invention discloses an application of the FaABI3 gene in regulating the ripening of strawberry fruits and / or regulating the flavonoid content of strawberry fruits. The FaABI3 gene is connected to the vector pTRV2 in the manner of Examples 2 and 3 to obtain the FaABI3 gene silencing vector pTRV2-FaABI3, and the competent state of Agrobacterium tumefaciens GV3101 is transformed. Finally, the strawberry tissue culture seedlings are immersed in an equal volume mixed Agrobacterium GV3101 bacterial solution carrying pTRV1 and pTRV2-FaABI3 for infection, and then the tissue culture seedlings are moved to a regeneration medium for culture; then the tissue culture seedlings are moved to a differentiation medium for culture to obtain resistant buds; finally, the resistant buds are rooted on a rooting medium, and then the seedlings are hardened and transplanted to obtain transgenic strawberry plants. The specific methods of immersion and obtaining transgenic strawberry plants are both technologies, which will not be repeated here.

[0061] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A FaABI3 gene, characterized in that: The nucleotide sequence of the FaABI3 gene is shown in SEQ ID NO.

1.

2. A protein encoded by the FaABI3 gene as claimed in claim 1, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. Use of the FaABI3 gene as claimed in claim 1 in regulating the ripening of strawberry fruits.

4. The use of the FaABI3 gene in regulating the ripening of strawberry fruit as claimed in claim 3, characterized in that: By reducing the expression level of the FaABI3 gene in strawberry, the ripening of strawberry fruit can be delayed.

5. Use of the FaABI3 gene as claimed in claim 1 in regulating the flavonoid content of strawberry fruit.

6. The use of the FaABI3 gene in regulating the flavonoid content of strawberry fruit as claimed in claim 5, characterized in that: By reducing the expression level of FaABI3 gene in strawberry, the content of strawberry flavonoids was increased.

7. The use of the FaABI3 gene in regulating the flavonoid content of strawberry fruit as claimed in claim 5, characterized in that: Agrobacterium was transformed with the FaABI3 gene silencing vector, and the flavonoid content of strawberry was increased after transformation of strawberry via the Agrobacterium-mediated method.

8. The use of the FaABI3 gene in regulating the flavonoid content of strawberry fruit as claimed in claim 5, characterized in that: The FaABI3 gene was connected to the vector pTRV2 to obtain the FaABI3 gene silencing vector pTRV2-FaABI3, which was then transformed into Agrobacterium tumefaciens GV3101. The strawberry small green period fruit was transformed by the Agrobacterium-mediated method to increase the flavonoid content of the strawberry fruit.

9. Use of the FaABI3 gene according to any one of claims 5 to 8 in regulating the flavonoid content of strawberry fruit, characterized in that: The flavonoids include spinosin, sophoroside, formononetin and delphinidin.

10. Use of the FaABI3 gene as claimed in claim 1 in regulating the ripening of strawberry fruits and / or regulating the flavonoid content of strawberry fruits, characterized in that: The FaABI3 gene was connected to the vector pTRV2 to obtain the FaABI3 gene silencing vector pTRV2-FaABI3, and then transformed into Agrobacterium tumefaciens GV3101. Finally, the strawberry tissue culture seedlings were immersed in the Agrobacterium bacterial solution carrying the FaABI3 gene for infection, and subcultured to obtain transgenic strawberry plants.

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