MaGA20ox2f gene capable of regulating the flowering time and fruit yield of bananas and its application

Abstract

The present invention discloses the MaGA20ox2f gene that can regulate the flowering time and fruit yield of bananas and its applications. The MaGA20ox2f gene that can regulate the flowering time and fruit yield of bananas has a nucleotide sequence as shown in SEQ ID NO.1. The present invention studied the biological function of MaGA20ox2f, used the CRISPR / Cas9 system to mutate Cavendish bananas, compared the differences in phenotypes, gibberellin content, protein expression profiles, etc. between wild-type and mutant MaGA20ox2f, and found that mutating the MaGA20ox2f gene can delay the flowering time of bananas and reduce the fruit yield of bananas.

Description

Technical Field

[0001] The invention belongs to the field of fruits, and particularly relates to a MaGA20ox2f gene capable of regulating the flowering time and fruit yield of bananas and an application thereof. Background Art

[0002] Triploid bananas (Cavendish) often have plant heights exceeding 2 meters and a reproductive period exceeding 12 months. Most diploid bananas are low-yielding. These characteristics make bananas vulnerable to natural stresses such as typhoons, pests and diseases. The identification of genes that contribute to high yield, early flowering, and ideal plant architecture, and their application in genetic improvement, is a critical need for the current banana industry.

[0003] Gibberellins (GAs) are plant hormones that play a key role in regulating plant biological processes, many of which are agriculturally important, such as root and stem elongation, flowering, fruit morphology, and responses to biotic and abiotic stresses. Gibberellin 20 oxidase (GA20 oxidase2) is a key enzyme that catalyzes sequential steps in the late stages of GA biosynthesis. To date, several GA20ox genes have been successfully cloned and characterized in Arabidopsis, rice, and other plants. AtGA20ox1 and AtGA20ox2 are redundant in promoting hypocotyl, internode, and anther filament elongation, flowering, and seed number, with AtGA20ox1 contributing more to internode and filament elongation and AtGA20ox2 contributing more to flowering time and filament length. The cytoplasmic-localized genes OsGA20ox1 (OsGNP1) and OsGA20ox2 (OsSD1) have been identified as affecting plant height and yield. Summary of the Invention

[0004] The first object of the present invention is to provide a MaGA20ox2f gene that can regulate the flowering time and fruit yield of banana.

[0005] The nucleotide sequence of the MaGA20ox2f gene capable of regulating the flowering time and fruit yield of banana is shown in bases 2023-4005 of SEQ ID NO.1.

[0006] The second object of the present invention is to provide a protein encoded by the MaGA20ox2f gene.

[0007] The third object of the present invention is to provide the use of the MaGA20ox2f gene in regulating the flowering time and fruit yield of banana.

[0008] Preferably, the sequence shown in SEQ ID NO. 1 comprising a promoter + MaGA20ox2f gene + a terminator is used to regulate the flowering time and fruit yield of banana.

[0009] Preferably, the mutant MaGA20ox2f gene or its promoter is used to delay banana flowering time, reduce banana fruit yield, and reduce chlorophyll a and / or b content.

[0010] Preferably, the reduction of banana fruit yield is to reduce the number of banana combs per bunch, the average fruit index per bunch and the average fruit weight.

[0011] The fourth object of the present invention is to provide a method for regulating the flowering time and fruit yield of banana, which comprises mutating the banana MaGA20ox2f gene or its promoter or overexpressing the MaGA20ox2f gene.

[0012] Preferably, the mutant MaGA20ox2f gene, or its promoter or the overexpressed MaGA20ox2f gene is used to advance the flowering time of banana and increase the yield of banana fruit.

[0013] This study investigated the biological function of MaGA20ox2f, using the CRISPR / Cas9 system to mutate Cavendish bananas. The differences in phenotype, gibberellin content, and protein expression profile between the wild-type and mutant MaGA20ox2f were compared. The results showed that the mutant MaGA20ox2f gene could delay banana flowering time and reduce banana fruit yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The relative mRNA levels of MaGA20ox2f and MaGA20ox2d genes were determined in different banana tissues (Musa Cavendish, AAA, cv. Grand Nain) using RT-qPCR in four biological replicates per tissue. Data are mean ± SD (n = 4).

[0015] Figure 2 Construction of a CRISPR / Cas9 editing vector targeting ΔMaGA20ox2f, and the phenotypes of mature plants of WT and ΔMaGA20ox2f mutants. A: Location and sequence of the sgRNA within the MaGA20ox2f gene; B. Vector map of the pYLCRISPR / Cas9-MaGA20ox2f; C. Growth phenotypes of WT and ΔMaGA20ox2f mutants; D. Mutation types at the target site in two homozygous ΔMaGA20ox2f banana mutants.

[0016] Figure 3 are the concentrations of GA15, GA9, GA19, and GA20 in WT and ΔMaGA20ox2f mutant seedlings.

[0017] Figure 4 Figure 3 is the GO function (A) and KEGG pathway (B) enrichment analysis of differentially expressed proteins in ΔMaGA20ox2f#1 and #2 compared with WT.

[0018] Figure 5 Chlorophyll a and b contents in leaves of WT and ΔMaGA20ox2f mutant. Values ​​are the means of three replicates, representing three independent experiments. Bars indicate standard errors. Lowercase letters above columns indicate significant differences at p < 0.05 by Duncan's test. DETAILED DESCRIPTION

[0019] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0020] Example 1:

[0021] 1. Materials and Methods

[0022] 1. Plant materials

[0023] Transgenic bananas were obtained in the genetic background of the Cavendish banana variety GrandNain and cultivated in the experimental field of the Fruit Research Institute of Guangdong Academy of Agricultural Sciences.

[0024] 2. RNA extraction and real-time quantitative PCR (RT-qPCR) analysis

[0025] RT-qPCR primers for the MaGA20ox2f gene were designed from the NCBI website (Table 1). Total RNA was extracted from the roots, pseudostems, leaves, stalks, peels, and pulps of Cavendish bananas according to the instructions of the Tiangen RNA Isolation Kit (Tiangen, Beijing, China). RT-qPCR was performed using the Applied Biosystems QuantStudio 3 Real-time PCR System (Thermo Fisher Scientific Inc.), using MaActin for normalization of target gene transcripts using the 2-ΔΔCt method.

[0026] 3. Plasmid construction and plant transformation

[0027] We used the CRISPOR tool (http: / / crispor.tefor.net / crispor.py / ) to design a sgRNA (ccttcatggtacgtaacctgctg) targeting the MaGA20ox2f gene. The 20-bp sgRNA sequence was cloned into pYLCRISPR-Cas9Pubi-H using the Golden Gate cloning method. The positive plasmid was introduced into Agrobacterium tumefaciens EHA105 and then transformed into banana (Musa Cavendish, AAA) to generate transgenic bananas. Transformation of banana (Musa Cavendish, AAA) was performed using a banana embryogenic cell suspension system. Transformed cell clusters were screened on semisolid selection medium (SSM) containing hygromycin B. Cell clusters with strong GFP fluorescence were selected for somatic embryo induction and germination on SSM, resulting in transgenic bananas.

[0028] 4. Deep Amplicon Sequencing

[0029] Genomic DNA from wild-type and transgenic bananas was extracted using the Tiangen DNA Extraction Kit (Tiangen Biotechnology Co., Ltd.). Deep amplicon sequencing primers were designed (Table 1) and PCR was performed to amplify a fragment approximately 200 bp in length. After gel purification, the PCR product was sent to Sangon Biotechnology Co., Ltd. for deep amplicon sequencing to determine the mutation type and efficiency.

[0030] 5. Agronomic trait determination

[0031] Growth-related data were collected during the two flowering seasons, and yield-related data were collected during the fruit ripening period. Agronomic traits of bananas were analyzed according to the "Banana Germplasm Resource Description Specifications and Data Standards."

[0032] 6. Determination of gibberellin content

[0033] Grind the plant into dry powder in liquid nitrogen, take an appropriate amount of sample and place it in a glass test tube. Add 10ml of isopropanol-water-hydrochloric acid extract and 8μL of 1μg / mL internal standard solution, shake on a shaker at 4℃100r / min for 30min, then add 20ml of dichloromethane, shake at 4℃ for 30min, centrifuge at 4℃, 13000g for 5min, obtain the lower organic phase, and dry it with nitrogen. Dissolve the organic phase with methanol-water solution (0.1% formic acid) and centrifuge at 4℃ for 10min (13000g). The supernatant is detected by HPLC-MS / MS using a 0.22μm filter membrane. After extracting the hormone, prepare a standard solution and analyze the sample using high performance liquid chromatography and tandem mass spectrometry.

[0034] 7. Quantitative proteomics determination

[0035] Take an appropriate amount of sample and place it in a pre-cooled mortar and grind it thoroughly into powder with liquid nitrogen. Add 4 volumes of phenol extraction buffer (containing 10mM dithiothreitol and 1% protease inhibitors) to each sample and perform sonication. Then, add an equal volume of Tris to neutralize the phenol and centrifuge at 5,500g for 10 minutes at 4°C. Add 5 volumes of 0.1M ammonium acetate / methanol to the supernatant and precipitate overnight. Wash the precipitate with methanol and acetone. Redissolve the precipitate with 8M urea, determine the protein concentration, and digest the protein with trypsin. The digested peptides are desalted using StrataXC18 and then freeze-dried in vacuum. The peptides are separated by high-performance liquid chromatography (HPLC) and analyzed by liquid chromatography-mass spectrometry after vacuum freeze-drying.

[0036] 8. Determination of chlorophyll ab content

[0037] Total chlorophyll content was determined using the ethanol-acetone extraction method. Leaf tissue samples (0.3 g fresh weight [FW]) were soaked in 10 mL of a 1:1 ethanol-acetone mixture for approximately 24 h. The absorbance of the extract was measured at 663 nm, 645 nm, and 440 nm using a spectrophotometer (Liang et al., 2022; S. Wu et al., 2021). Chlorophyll content was calculated using the following formula:

[0038] Chlorophyll a (mg g-1FW) = (12.70 × A663 - 2.69 × A645) × V / W × 1000

[0039] Chlorophyll b (mg g-1FW) = (22.88 × A645 - 4.67 × A663) × V / W × 1000

[0040] Total chlorophyll (mg g-1FW) = (8.04×A663+20.29×A645)×V / W×1000,

[0041] Where A is absorbance, V is volume, and W is weight.

[0042] Table 1 Primers used in this application

[0043]

[0044] 2. Results

[0045] 1. Expression of MaGA20ox2f and MaGA20ox2d genes in different banana tissues

[0046] The functional differences between MaGA20ox2f (nucleotide sequence shown in bases 2023-4005 of SEQ ID NO.1, where the sequence shown in SEQ ID NO.1 is the promoter + MaGA20ox2f gene + terminator) and MaGA20ox2d (nucleotide sequence shown in SEQ ID NO.2) may be due to differences in their expression patterns. Real-time quantitative PCR (RT-qPCR) was used to monitor transcript levels and study their expression in different tissues. MaGA20ox2f was highly expressed in leaves, stalks, peels, and flesh, while MaGA20ox2d was expressed at a lower level, but its expression pattern was similar to that of MaGA20ox2f ( Figure 1 This highlights the importance of MaGA20ox2f in banana fruit development. Therefore, this homolog of OsSD1 was selected for functional analysis.

[0047] 2. Obtaining MaGA20ox2f knockout mutants using CRISPR / Cas9 technology

[0048] A loss-of-function mutant of MaGA20ox2f (ΔMaGA20ox2f) was generated in the genetic background of the Cavendish banana variety Grand Nain using a single guide RNA (sgRNA) driven by the rice U6b promoter, which specifically targets the second exon of MaGA20ox2f. Two independent homozygous lines were obtained from third-generation suckers. Deep sequencing confirmed that the mutation rate of ΔMaGA20ox2f#1 and #2 in the target site (loss-of-function mutant of MaGA20ox2f) was 99.99%, indicating homozygosity. Both mutants flowered later than the wild type (WT) and had lower yields ( Figure 2 Therefore, considering the “abnormal flowering and fruiting” phenotype, Ma04g15900 was named MaGA20ox2f.

[0049] 3. Phenotypic Analysis of the ΔMaGA20ox2f Mutant

[0050] As shown in Table 2, the ΔMaGA20ox2f#1 and #2 mutants exhibited late flowering and low yield during reproductive growth. Plants exhibited significantly shorter stem lengths during both vegetative and reproductive stages compared to the WT. Compared to the WT, the average days to flowering were delayed by 61 and 58 days for ΔMaGA20ox2f#1 and #2, respectively, representing significant reductions of 24.30% and 23.11%, respectively. In particular, the average fruit yield of ΔMaGA20ox2f#1 and #2 was significantly reduced by 81.14% and 76.23%, respectively (20.78 kg vs. 3.92 and 4.94 kg), which was closely associated with significant decreases in the average number of combs per ear, average fruit index per ear, and average fruit weight. The average number of combs per ear in ΔMaGA20ox2f#1 and #2 decreased by 62.50% and 50%, respectively (8 combs vs. 3 and 4 combs / ear). The average fruit index per ear decreased by 78.78% and 66.44%, respectively (146 vs. 31 and 49 per ear). The average fruit index per comb decreased by 56.90% and 46.55%, respectively (29 vs. 12.5 and 15.5 per comb). The average fruit index per third comb decreased by 50.00% and 44.44%, respectively (18 vs. 9 and 10). The average fruit weight per fruit decreased by 33.33% and 26.67%, respectively (0.15 kg vs. 0.10 and 0.11 kg). However, the plant size of ΔMaGA20ox2f#1 and #2 was proportionally smaller than that of the WT, and the decrease in related parameters was smaller. The average plant height decreased by 23.19% and 22.33% (219.9 cm vs 168.9 and 170.8 cm), the average pseudostem circumference decreased by 23.41% and 20.23% (45.8 cm vs 59.8 and 47.7 cm), the average leaf length decreased by 27.60% and 25.93% (142.7 cm vs 197.1 and 146.0 cm), the average leaf width decreased by 11.27% and 9.72% (57.5 cm vs 64.8 and 58.5 cm), the average fruit length decreased by 19.86% and 18.29% (13.80 cm vs 17.22 and 14.07 cm), and the average fruit width decreased by 6.40% and 4.56% (10.67 cm vs 11.40 and 10.88 cm). In addition, the average number of suckers in ΔMaGA20ox2f#1 and #2 was reduced by 44.44% and 33.33%, respectively (9 vs. 5 and 6 suckers).

[0051] Table 2 Comparison of agronomic traits of the wild type (Musa Cavendish, AAA, cv. GrandNain) and its ΔMaGA20ox2f mutant

[0052]

[0053] The values ​​in the table are mean ± standard deviation (± sd.), n = 5. Duncan multiple range test was used (p < 0.05). (a), pseudostem circumference: pseudostem circumference at 30 cm height of the plant

[0054] 4. Analysis of GAs Content in WT and ΔMaGA20ox2f Mutant

[0055] GA20ox can catalyze GA12 to produce GA15, GA24, and GA9 (GA4 metabolic pathway), and can also catalyze GA53 to produce GA44, GA19, and GA20 (GA1 metabolic pathway). The effects of reduced expression of the ΔMaGA20ox2f gene on the endogenous GA content in 2-month-old banana plants were studied, ensuring that all plants were analyzed at the same developmental stage. Compared with the WT, the concentrations of GA15 and GA9 in ΔMaGA20ox2f#1 were reduced by 99.9% and 93.4%, respectively, and the concentrations of GA15 and GA9 in ΔMaGA20ox2f#2 were reduced by 98.5% and 73.6%, respectively. The levels of GA19 and GA20 in ΔMAGA20ox2f#1 and ΔMAGA20ox2f#2 were reduced by 33.0% and 100%, respectively. Figure 3 ).

[0056] Table 3 Summary of differentially expressed proteins in ΔMaGA20ox2f#1 and ΔMaGA20ox2f#2 compared with WT

[0057]

[0058] All differentially expressed proteins identified from the two mutants were functionally classified by GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) (Table 3). In the Gene Ontology classification, differentially expressed proteins were classified according to cellular components. Most of the up-regulated proteins found in the study belonged to the GO categories of cell wall and mitochondrial respiratory enzymes, while the down-regulated proteins belonged to chloroplasts and chloroplast thylakoids. KEGG analysis results showed that the up-regulated proteins were mainly enriched in biosynthetic proteins such as phenylpropanoids and flavonoids, while the down-regulated proteins were mainly enriched in biosynthetic proteins such as photosynthesis, photosynthetic antenna proteins, porphyrin and chlorophyll metabolism, and ribosomes ( Figure 4 ).

[0059] 5. Effect of mutation on chlorophyll a and b content

[0060] To confirm the effect of the ΔMaGA20ox2f mutation on photosynthesis, the chlorophyll a and b contents of leaves of the WT and two ΔMaGA20ox2f mutants were measured. Compared with the WT, the chlorophyll a and b contents of ΔMaGA20ox2f#1 and #2 were significantly reduced by 32.6% and 32.1%, 31.9% and 27.1%, respectively. Figure 5 ).

[0061] MaGA20ox2f (SEQ ID NO. 1)

[0062] ATAATAATTATTTCTTGAGAGACCTAAACGTGATATACTGTTCTCCTCCACAATCAATTTTG

[0063] TTGCCTTCCATGTTTTGGACGTGTCTGAGACAAGAGGATATTTATGGGCAGGCCCTGGCC

[0064] AATTGGAGATATATATAGATATATCTCTTGAAAAGTGTTCTTATTTTTAGAATTTTCTGATG

[0065] ATGTCATTTTCTTCCTAATACTTGTAATACTCTTGATTTATTATTGATTTCCACCTCTTTTTT

[0066] TTTCTTCTCTTTTTCTATTGATTGGTCTGAGCGAACTGGTCCAGTTATAATGATTTTACATC

[0067] GTGTTTTCGACAATACAAGAAAAGCACTATAACGTAATACAAAGATATTAAATTATAATAT

[0068] AATATTTTTTTAATATTATATCATTATAATATTTTTTTAATAATATTAAAAAAATATATTGTAACA

[0069] TAGCATAAAAATAGTTATATAATTTTTTTATTACTAAAAGTATTATAAATAGATCAACTC

[0070] CAAAAGAGAGAGAAAAGACGTATGTGGATGACTATGATATTCCAAGTATTATGCAACAA

[0071] GGAATATTCTAAAAATATGGGCAATTTTTGAAAAAGCTAGAAAAGATGAGTTTTTCTG

[0072] GAACTCATGTTATATATAATAGATATCACTTTACAAGATGGGTATTTAAAATAAAAAAAATTA

[0073] CTTACTTTATTGCGATATGGAAATATAATTGACTAACTTCACTGTTTGAAGTATTAATTTT

[0074] TTTCTTTAATATATTTAATTAAATTAATAATCATGCAATAAACCTATTCTTACTTTGATCTTA

[0075] CAATAACAAGAAAAGATTGTTCTTTCTCTAGTAGAAGCATTTCATAAGTTCTTCTCTTG

[0076] AGGATATTATAAGTTACAACCATTTCTTCCTACGCATACATATACAAGAGAGAAAAAATTA

[0077] TTTACTATTCCTAATTGTTTAATTCATAAGTTGCTTCTTTTTAGGAAAAAGTTTACATTGCCA

[0078] CTGAACACTTAAAGTAGAATGAGATCAAGCTAGCAATAGGGTTTTCATTTCTTCCATCCA

[0079] TCCGATCATTATGAGTCCAATAAATATGCTATAAGATTAAAGGCAAAACCTAGTAAATTAT

[0080] CAATCCATAATTGATTTTAGGCTCACTCTAGTTGAGCATCTAGTGAAAACAATCTATAAGT

[0081] TGCTGCATGACAAACAACATTACTCTTAATAGAAAAATATATAGCATAAAATCAAATTAT

[0082] TGTTTATTATATATATATATATATATATATATATATATATATATATATATATATATATTATGATAAAG

[0083] GATACACATGACTCTACTCATGATTATGGTATTCTTCAGATATCCATTCAATAGGAAGAGT

[0084] ATTCTCTCAATAAAATAAGATAATTAAAAAAATTATGTATATATCTTTCATTTAAATACTCTA

[0085] AGCATCATGAGATATGTTTGGATCTATAAATTTACCGGGATTGGATACATATCCAATTAGGA

[0086] TATGTTGTGGTTTCTTTATTACTAATATCAGCAATGTTACTATACCATGAATTACATATAAAT

[0087] TTTAGTTGACTTTTCACATCCTTTGAAAGCCATCGCTATCATGAACGGATGGCTATAAAAT

[0088] TTATCTATTGTCTTACTACAATAATTCATAGATTGATCTTTTAATTTAGAATGACTCGAACT

[0089] ATTTATCTAATTTAAAATTTTTTTGGTGATAAAGAAGGTGTACACCCAAAAGAACAGAAT

[0090] TTGAGTTAAGGGACTCTAATCCTTCATCTCAATTTTGTGTTAACATGTTATAGAAATATTCT

[0091] AATAATTAGGCGTTGTATGTACCCTTCGCCCTCCAAAGGCTATCACTGGCGGCGTTTGCC

[0092] CTTTCTCCCAAACCCAAACTATGCTCCATACCCTTTCTATCCCTTCCTTTGTCCTTATCCTGC

[0093] CTCGCACCCTTACAAATACCTCATCTTGACGCCACATCCTTCTTGTCTCTCTCCTGCGCTC

[0094] CCTCCAATCACGCACCCAGAAACTACATTCTGCTGTCTCCTCCTCCTCATGGACTGCAAT

[0095] CCCACCTCCATCCTCCTGCGCACTCCCTTGGACCTCAACAAGGAGCGAGACGCTGGCGC

[0096] CGGCGGCGTCGTCTTCAACTCTTCGGTCCTCGGGAACCAAGCCAGCATACCCAAGGCAT

[0097] TCATCTGGCCGCAGTGCCACAGACCCGCCACCATCGACGATCTCGACGCCCCGGTGGTG

[0098] GACCTCGGCGGCTTTCTCCGCGGCGACGAAGCGTCGACGGCGCGCGCGGTGGAGTACG

[0099] TCAGGGCCGCCTGCTCCACCCACGGCTTCTTCCAGGTCGCCAACCATGGCGTCGACGCG

[0100] TCGCTGGCCCGAGAGGCGATGGACTGCGTTGACGGGTTCTTTAAGCTCCCTCTCTGTCA

[0101] CAAGCTACGCGCCCGGCGGAAGCCCGGTAGCGTGTGGGGCTACACCGGGGCGCACGCC

[0102] GACCGCTTCTCCTCCGAGCTGCCATGGAAGGAGACCCTCTCCTTTGGCTACCACGAGGC

[0103] CGGCGGCGGCGACCGCGTCGTCGTCGACTACTTCGCTTCCATCTTAGGCCCTGATTTCGA

[0104] TAGGACGGGGTAAGCCATCGCACCATCTTACGATAAGCACGAATCGGCGAAATCCACTG

[0105] GCATTGCCGCTGAAGCTTTCTTGTACACCAGGCTGGTTCTTCAGAGGTACTGCGAGGCG

[0106] ATGAAGAAGCTATCGCTGGTGATCATGGAGCTCCTGGCAATAAGCTTAGGAGTGGAAAG

[0107] AAGCCACTACAGAGACTTCTTCGAGGACAGTTGCTCAATAATGAGATGCAACTACTATC

[0108] CTCCATGTCAGGAGCCGGAGCTGACGCTCGGCACCGGCCCGCACTGCGACCCCACCTC

[0109] GCTGACCATCCTTCAACAGGACCAGGTGGAGGGCCTCGAGGTCTTCTCCGCCAACAAG

[0110] TGGCGGTCGGTCCGGCCGATTCGCGACGCTTTGGTGATCAACATTGGTGACACCTTCAT

[0111] GGTACGTAACCTGCTGCCTCTCTTTCTTTCTATAGACAAGACTAATAGTAGTGGAGACGC

[0112] CAAAGGGCAAACACCATGGAGTAACAGTGCAGAGTTGCTGGTCGCTGTCTCCTCTTCTT

[0113] TACTGTTTGCTTGATCACGGTAGGACCAGATGGAAACATTAGCATCAAATAATTAAGCCA

[0114] GGATTAAGCTCAAAAAAAAGAAAAAAGAAAAAAAAAGACACATACATACATACATACAT

[0115] ACATACATCATCAACTACACCAATGCTTGTCTTAGGTTCACAGGATTGAGTATGTGCTAG

[0116] ACGACAGGGTTCGATGTGCAGCGCTCAAAATGATGTGGGTGCGTCCCCGAGAGGGACA

[0117] GCTGCTTATGTATTTTACGTACCCGTTTTATATGTGCTATGCTATAAAACGTGTTTGGCCCA

[0118] ATTTTACTACGAGTGCACTCAGATGCATATATCCTTTTCTAAGTGATTGAAGCGTCCGACT

[0119] CCAAAACACTATCGAACAGGACGTGTGTTTTGTATTTAATATCTAATGTCTCAGATATTGT

[0120] ACACTAAAATTCGATCTTTGCAAGTTGGTGTACCTATTCAAAGTTTGCTTTTAGATTTTAT

[0121] AGGAAGCTTCCAAAGAAATTAAAGAGAAAAGAGAGAGAGAGAGAGAGAGAGAGAGA

[0122] GAGAGAACCAAGGAAGAACCTGAATGTTCTATTGCGATGATGAAGAATGGCTCCCATGT

[0123] CTAATTATTGTATGCCCCCCTGTCTTTGTTCTGCAACATAGGCGTTGTCCAATGGAAGGTA

[0124] CAAAAGCTGCCTGCACCGGGCTGTGGTGAACAGGCATCGGGAACGGAAGTCGCTGGCT

[0125] TTCTTCGTGTGCCCCAGGGAGGACCGCACCATCCGGCCACCGCCGGGGGACCTCGGCG

[0126] GCTCGCGGCTGTACCCGGACTTCACGTGGGCGGAGTTCATGCAGTTCACGCAGCGCCAC

[0127] TACCGGTCTGACACGAGGACACTCCACAGCTTCACCAATTGGCTCTCCTCCTCTAACTC

[0128] CCTCCGCCAGTCCACCTAGAGGTGCAGATCAGGAGGTCACCTGATGCTCTCCGTTCATG

[0129] TCTTTTCTTTCTTCGTGTACAGCGCATGACAGCAGCGGTAAAGACGAGGGTGATGAACT

[0130] CATGATTGCATGCAGCACCCATTCTGCATCACATATGTATGGAGAGCTCGATATGATTGGT

[0131] CGCTTCATAGTGTTTTATGCTTCTGTGTTACAAGTTGAGTTACGCAACTGGATCTGAGGA

[0132] GGTAAA

[0133] MaGA20ox2d(SEQ ID NO.2)

[0134] ATGGACTGCAACCCCACCTCCGTTGTCCTGCGCCCTTCCTTGGCCCTCGATAAAGAGAG

[0135] AGAAGCCGGCTCCGGCGGCGTCGTCTTCAGCTCTGCGCTCCTCCCGAACCAAGCCACCA

[0136] TCCCCAAGGCCTTCATCTGGCCTCAGTGCGACAGGCCGACCGCCATCGAGGAGCTGGA

[0137] CTCCCCTGTCGTAGACCTCGATGGCTTTCTTCGTGGCGACGAAGCGTCCACGGCGCGGG

[0138] CGGTCGACTGCGTCAGGGCCGCCTGCTCCACCCACGGCTTCTTCCAAGTCGCCAACCAC

[0139] GGCGTGGACGCGTCGCTCGGCCGGGACGCGCTTGGCTGCATGGACGAGTTCTTCGGGC

[0140] TCCCGCTCTGCCACAAGTTACGCGCCCGGAGGAAGCCCGGAAACATGTGGGGCTACGT

[0141] CGGCGCTCACGCCGACCGCTTCTCCTCGGAGCTGCCCTGGAAGGAGACCCTCTCCTTCG

[0142] GCTATCATGAGGCCGGCGACGACCGCGTCGTTATCGACTACTTCACGTCCACCTTGGGC

[0143] AATGATTTCCAGAGGATGGGGTGAGCCATTAGATTACCCTACAATCGACATGCATCGAGA

[0144] CCCAAAGGGAATGGCATTGCCATTCACGTCGCATTCTAACCTCTTCGGTGCTTGATTAGG

[0145] TTGGTCTTCCAGAGATACTGCGAGGCCATGAAGAAGCTATCTCTGGTGATCATGGAGCTC

[0146] CTGGCGATGAGCCTGGGAGTGGACAGAACCCACTACAGAGACTTCTTCGAGGACAGTC

[0147] GGTCGATAATGAGATGCAACTACTACCCTCCATGTCCGGAGCCGGAGCTGACGCTCGGC

[0148] ACCGGCCCACACTGCGATCCCACCTCGCTCACCATTCTTCGACAGGACCAAGTGGACGG

[0149] GCTCGAGGTGTTCGACGGCAACAGATGGCGGTCCGTCCGGCCTATTCGCGACGCCCTTG

[0150] TGATCAACATCGGTGATACGTTCATGGTACGTGAGCTGTCTCTGCGCCTACTGATTACTC

[0151] AAAAGTTCTCTTTCTATTAGGTGTACATGCAACATAAAATGTTCGGTAATTTAGACACTGC

[0152] AAAACTGAGATTACGTACTTAGAAGACAATTCCACTGCTGGGAACCTTAATTTCCTTGAT

[0153] GAAGTGAATCCATGTATGGCATGTCGGTGAAGTGCATCAAATCCAAGATTTAGCAGCAAT

[0154] ATTTAGGATGATTTGCTGCATGCCATATCATGTCAGCCACAAGCTCATCATCTTCAACATT

[0155] CTGACTAACTCTTCCGCAAAGTAATCCACTAAAAGAGAGATGAGAGGACACACAAGAT

[0156] GTAAAGATTCAGGAGCATGGTTGTCTGGTTTGTCATCTTGCAGAAAGAAACAGATCTGC

[0157] ATTGTCTTATTGCAACAGTGAAATCACCTGCAATTTCTGATAGCTTCTGTGCCTTTGATTT

[0158] TGGTTTTGTTTGGGAACCAAGGCACTGTCCAACGGGAGGTACAAAAGCTGCCTGCACC

[0159] GGGCCGTGGTGAACAGGCGCCGGCAACGAAAGTCGCTGGCCTTCTTCCTCTGCCCCAG

[0160] GGAGGACCGCGTGGTCCGGCCGCCGCCGGGGGAGCTCGGCGGCGCGAGGCTGTACCCG

[0161] GACTTCACGTGGGCGGAGCTGTTGGAGTTCACGCAGCGCCACTACCGGGCCGACATGA

[0162] AGACGCTCCACAGCTTCACCGACTGGCTTCTCTCCTCCTCTTCCCCATCCCCTCTCCGGT

[0163] CCACCTGA

Claims

1. A device capable of adjusting the flowering time and fruit yield of bananas MaGA20ox2f A gene characterized by The nucleotide sequence is shown in bases 2023 to 4005 of SEQ ID NO.

1.

2. The method according to claim 1 MaGA20ox2f Protein encoded by a gene.

3. Knock out the protein described in claim 1 MaGA20ox2f Genes delay flowering time, reduce fruit yield and lower chlorophyll in bananas a and / or b Application in content.

4. The use according to claim 3, characterized in that: The reduction of banana fruit yield is to reduce the number of banana combs per bunch, the average fruit index per bunch and the average fruit weight.

5. A method for delaying the flowering time and reducing the fruit yield of bananas, characterized in that: The knockout banana of claim 1 MaGA20ox2f Gene.

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