CN114591976A

CN114591976A - Gene for encoding GA2 ox-oxidase and application thereof in judging dwarf mango variety

Info

Publication number: CN114591976A
Application number: CN202210361231.XA
Authority: CN
Inventors: 张宇; 赵志常; 黄国弟; 张继; 荣涛; 郑剑; 欧克纬; 莫永龙; 赵英; 唐玉娟; 郭丽梅; 覃昱茗; 庞新华; 郝晓铃; 韦绍龙
Original assignee: Guangxi Subtropical Crops Research Institute
Current assignee: Guangxi Subtropical Crops Research Institute
Priority date: 2022-04-07
Filing date: 2022-04-07
Publication date: 2022-06-07

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a gene for encoding GA2 ox-oxidase and application thereof in judging a mango dwarfing variety. A gene for encoding GA2 ox-oxidase, wherein the nucleotide sequence of the gene is shown as SEQ ID No. 1. The GA2 ox-oxidase gene can provide support for early prejudging whether the test material has dwarfing characteristics.

Description

Gene for encoding GA2 ox-oxidase and application thereof in judging dwarf mango varieties

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a gene for encoding GA2 ox-oxidase and application thereof in judging a mango dwarfing variety.

Background

Dwarf planting is a development trend of unchanged crop production. Plant dwarfing is largely divided into genetic dwarfing and physiological dwarfing. The single gene mutation belongs to genetic dwarfing, the dwarfing stimulus of the single gene mutation is mostly gene synthesis protein, the protein influences hormone, and the hormone influences phenotype; the plant phenotype is changed due to the influence of environmental factors, the plant phenotype is physiologically dwarfed, and the dwarfed mechanism is mainly that the environmental change influences the secretion of plant hormones, and finally the plant hormones are changed. However, regardless of the dwarfing mode, the phenotype is influenced by hormones, and the hormones in the plant growth process are mostly related to gibberellin.

The plant has more enzymes participating in gibberellin synthesis and decomposition, and GA2ox (gibberellin 2 beta oxidase) is one of the enzymes, is an enzyme for inhibiting the synthesis of active gibberellin, and can reduce the content of the active gibberellin, so that the aim of regulating the plant height of the plant is fulfilled. The regulation of GA metabolic pathways by GA2ox has been demonstrated in Arabidopsis.

By comparing the expression quantity of the GA2ox gene of the plant material with the characteristics of arbor and dwarfing, whether the plant material has the dwarfing characteristic can be judged in advance, and time and labor are saved.

Disclosure of Invention

In order to overcome the defects of the technology, the invention provides a gene for encoding GA2 ox-oxidase and application thereof in judging the dwarf mango varieties, which can be used for pre-judging whether the mango varieties have dwarf characteristics.

In order to achieve the purpose, the invention provides the following technical scheme:

the first purpose of the invention is to provide a gene for encoding GA2 ox-oxidase, wherein the nucleotide sequence of the gene is shown as SEQ ID No. 1.

The second purpose of the invention is to provide the application of the gene coding GA2 ox-oxidase, and the gene coding GA2 ox-oxidase is used for prejudging whether the mango variety has dwarfing characteristics.

The third objective of the present invention is to provide a GA2 ox-oxidase, wherein the GA2 ox-oxidase is encoded by the above-mentioned gene.

Preferably, the amino acid sequence of the GA2 ox-oxidase is shown as SEQ ID No. 2; the GA2 ox-oxidase consists of 341 amino acids, has a molecular weight of 38729.40Da and a theoretical isoelectric point of 6.56.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention obtains GA2 ox-oxidase gene sequence from test material, analyzes the physical and chemical activity of the gene, and uses the gene as the index for regulating and controlling dwarfing character.

(2) The invention relates to the GA2 ox-oxidase gene clone and the corresponding amino acid sequence analysis, and the GA2 ox-oxidase gene expression level is determined by a semi-fluorescence quantitative method, so that support can be provided for early prejudging whether a test material has a dwarfing character.

Drawings

FIG. 1 is a diagram showing the result of PCR amplification of an intermediate fragment of GA2 ox-oxidase gene;

FIG. 2 is a diagram showing the results of PCR amplification of the 3' -end of GA2 ox-oxidase gene;

FIG. 3 is a diagram showing the results of PCR amplification of the 5' -end of GA2 ox-oxidase gene;

FIGS. 4-6 are schematic diagrams showing homology alignments of mango GA2ox with other plants GA2 ox;

wherein, Dz: durian; hb: a rubber tree; hu: mallow; jc: jatropha curcas; me: cassava; mg: mango; rc: castor bean; tc: cocoa;

FIG. 7 is an analysis diagram of the signal peptide and transmembrane domain of the protein encoded by GA2 ox;

FIG. 8 is a diagram showing the analysis of the secondary structure of the protein encoded by GA2 ox;

FIG. 9 is a graph of the hydrophobicity analysis of a protein encoded by GA2 ox;

FIG. 10 is a mango GA2ox phylogenetic tree;

FIG. 11 is the expression amount diagram of GA2ox gene in the British mango and the Guiqi mango at different phenological stages.

Detailed Description

In the following, the technical solutions of the present invention will be described clearly and completely, and it is obvious that the described embodiments are some, not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In example 1, the main instruments and reagents were: a PCR instrument: takara; electrophoresis apparatus: Bio-Rad;

2000 spectrophotometer: thermo; a constant temperature shaking table;

ultra-fidelity DNA polymerase: NEB; FirstChoice RLM-RACE Kit: thermo; 2 × Taq Master Mix (Dye Plus): vazyme.

In example 2, the main instruments and reagents were: ABI 7900-Fast Real-Time PCR Detection System：ABI；

2000 spectrophotometer: thermo; an ordinary PCR instrument: takara; All-in-One^TMFirst-Strand cDNA Synthesis Kit：GeneCopoeia(Cat.No.AORT-0050)；All-in-One^TMqPCR Mix：GeneCopoeia(Cat.No.AOPR-0200)；Epcentre^TM RNase R。

Example 1: cloning and sequence analysis of mango GA2 ox-oxidase Gene

First, RNA extraction and extraction

1. Total RNA extraction (Trizol method)

(1) Sample treatment: putting 80-100mg mango leaves into a freezing mortar, adding liquid nitrogen, grinding into powder, transferring into a 1.5mL centrifuge tube filled with 1mL Trizol, shaking, mixing uniformly, and standing at room temperature for about 5 min.

(2) Phase separation: 0.2mL of chloroform was added to 1mL of Trizol, and the mixture was shaken and mixed for 15 seconds, and then allowed to stand at room temperature for about 3min, 4 ℃ and 12000rpm, and centrifuged for 15 min.

(3) And (3) precipitation: transferring the water phase into a new 1.5mL centrifuge tube, adding 0.5mL isopropanol into each 1mL Trizol, mixing, standing at room temperature for 10min, standing at 4 ℃, 12000rpm, and centrifuging for 10 min.

(4) Washing: the supernatant was discarded, 1mL of 75% ethanol was added to 1mL of Trizol, and the mixture was mixed well, centrifuged at 7500rpm at 4 ℃ for 5 min.

(5) Dissolving: discard the supernatant, air-dry the RNA precipitate for about 5min (care should not dry completely, only precipitate whitens), add DEPC treated water to dissolve the RNA precipitate.

2. Purification of extracted RNA

(1) The following reaction solution, 50ul system, was prepared in a microcentrifuge tube:

(2) after 25min at 37 ℃ the Recombinant DNase I was inactivated.

(3) 10ul of 3M sodium acetate and 250ul of cold ethanol were added and left at-80 ℃ for 20 min.

(4) The mixture was centrifuged at 12000rpm for 10min at 4 ℃ and the supernatant was discarded.

(5) Adding 70% cold ethanol, cleaning, centrifuging at 4 deg.C and 12000rpm for 5min, removing supernatant, and drying precipitate.

Cloning target gene intermediate segment

2.1 according to the GA2ox gene cDNA sequence of the plant published in GenBank, after DNAMAN software alignment, a pair of degenerate primers is designed according to the conserved region.

GA2ox-F：AAYGGYGATR TBGGHTGGRTYGAATA

GA2ox-R：TGCYTYACRCTYTTA AAYCTYCCATTWGTCA

2.2 reverse transcription of intermediate fragments

(1) Unfreezing the reagent of the First-Strand cDNA Synthesis Kit, slightly inverting and uniformly mixing, centrifuging for a short time, and placing on ice for later use.

(2) Preparing RNA-Primer Mix on ice, and adding the following reagents into a precooled RNase-free reaction tube until the total volume is 13 mu L;

(3) mixing RNA-Primer Mix, centrifuging for a short time, denaturing at 65 ℃ for 10min, and immediately placing on ice;

(4) the following reagents were added to a total volume of 25 μ L in an RNA-Primer Mix reaction tube;

(5) mixing reaction Mix evenly, centrifuging for a short time, and incubating for 1h at 37 ℃;

(6) after the reaction is finished, inactivating at 85 ℃ for 5min, and storing the reverse transcription product at-20 ℃ for later use.

2.3 intermediate fragment analysis and isolation

Intermediate fragment PCR reaction system:

the PCR reaction program is: 5min at 94 ℃; at 94 ℃ for 40s, at 58 ℃ for 40s, at 72 ℃ for 1min, for 35 cycles; 10min at 72 ℃; 10min at 4 ℃; after the reaction, the PCR reaction product was analyzed by agarose gel electrophoresis with a mass fraction of 1%, and the result is shown in FIG. 1, and the target fragment was recovered by cutting the gel.

As can be seen from FIG. 1, the size of the GA2ox intermediate fragment cloned from mango leaves was approximately 900 bp.

2.4 recovery of fragments of mesh

(1) 50 μ L of the reaction was added in its entirety to a 1% agarose gel well and DNA Marker DM 2000 was spotted on one side of the sample.

(2) Constant voltage 120V, electrophoresis for about 30min, according to the moving position of the strip, judging when to stop glue running.

(3) Under an ultraviolet lamp, cutting and recovering the target strip according to the position of a Marker, and purifying the full-length segment of the target gene by using a DNA agarose gel recovery kit.

(4) The cut gel was quickly placed into a centrifuge tube and the volume was evaluated based on the weight of the gel.

(5) Adding Binding Buffer with the same volume into the gel at 65 ℃ for 5min until the gel is completely melted.

(6) The melt was pipetted into a HiBind DNA Mini column, which was then placed into a centrifuge tube at 10000g, room temperature, and centrifuged for 1 min. Discard waste solution and leave column until all the thawing solution completes the procedure.

(7) mu.L of XP2, 10000g, was added to the column and centrifuged for 1min at room temperature. Discard the waste liquid and leave the column.

(8) Add 700. mu.L of SPW Wash buffer to the column at 10000g, room temperature, centrifuge for 1min, and discard the waste. This step was repeated once.

(9) 13000g at room temperature, idling for 2min, and removing residual waste liquid.

(10) The column was placed in a new centrifuge tube, and 30. mu.L of Elution Buffer was dropped into the center of the membrane at room temperature for 2min, followed by 13000g, centrifugation for 2min, and DNA Elution.

2.5T vector ligation and transformation

I. Connection of

mu.L of PCR product and 1. mu.L of PEASY-T1 cloning vector were mixed gently and reacted at room temperature for 5 min. After the reaction was completed, the centrifuge tube was placed on ice.

II. Transformation of

(1) Coli competent cell DH5a was removed from the freezer at-80 deg.C, placed in ice until completely dissolved and gently mixed.

(2) 50 μ L of competent cells were pipetted into a sterilized centrifuge tube, and 5 μ L of recombinant plasmid was added to the centrifuge tube, gently mixed, and allowed to stand on ice for 30 min.

(3) Placing the centrifuge tube after ice bath in 42 deg.C water bath for 90s, quickly transferring into ice box containing ice for 2min, adding 600ul LB culture solution, placing on 37 deg.C constant temperature shaking table, and culturing at 220rpm for 1 h.

(4) 50ul of the culture solution was spread on solid LB medium containing Amp, dried and placed in an incubator at 37 ℃ for inverted culture. After 12h-16h, the culture dish is checked whether colonies appear.

(5) Selecting single clone for streak culture, and performing colony PCR amplification detection by using upstream and downstream primers of the gene.

(6) Positive single clones were picked for shake and sequenced.

Thirdly, cloning the 3' end of the target gene

3.1 designing a pair of 3' RACE primers according to the obtained target gene intermediate segment.

GA2ox 3'GSP-1:CCGCCATGTCCGGATATTCA

GA2ox 3'GSP-2:ATCCAACAACACTTCCGGCT

3.2 obtaining 3' cDNA

(1) The following ingredients were added to the rnase-free centrifuge tubes and worked on ice:

(2) mixing, incubating at 42 deg.C for 1h, and storing at-20 deg.C.

3.3 Outer 3' PCR reaction System

The PCR reaction program is: 3min at 94 ℃; at 94 ℃ for 40s, at 60 ℃ for 40s, at 72 ℃ for 1min, for 35 cycles; 10min at 72 ℃; 10min at 4 ℃.

3.4 Inner 3' PCR reaction System

The PCR reaction program is: 3min at 94 ℃; at 94 ℃ for 40s, at 60 ℃ for 40s, at 72 ℃ for 1min, for 35 cycles; 10min at 72 ℃; 10min at 4 ℃; after the PCR reaction product is analyzed by agarose gel electrophoresis with the mass fraction of 1%, the result is shown in figure 2, the gel is cut, the target fragment is recovered and is connected to a PEASY-T1 cloning vector to transform competent cells, and the positive bacteria are identified and sequenced.

As can be seen from FIG. 2, the DNA fragments of about 900bp in size were obtained by PCR amplification of GA2ox 3'GSP-1 and GA2ox 3' GSP-2, which were designed based on the sequence information of the obtained GA2ox gene intermediate fragment, and the respective universal primers.

Cloning the 5' end of the target gene

4.1. A pair of 5' RACE primers is designed according to the obtained target gene intermediate segment.

GA2ox 5'GSP-1:GCACCTGCATAATCAACCACC；

GA2ox 5'GSP-2:GCTTGGGTACTGGCAAAAGC。

4.2 Outer 5' PCR reaction System

The PCR reaction program is: 3min at 94 ℃; at 94 ℃ for 40s, at 60 ℃ for 40s, at 72 ℃ for 1min, for 35 cycles; 10min at 72 ℃; 10min at 4 ℃;

4.3 Inner 5' PCR reaction System

The PCR reaction program is: 3min at 94 ℃; at 94 ℃ for 40s, at 60 ℃ for 40s, at 72 ℃ for 1min, for 35 cycles; 10min at 72 ℃; 10min at 4 ℃; after the PCR reaction product is analyzed by agarose gel electrophoresis with the mass fraction of 1%, the result is shown in figure 3, the gel is cut, the target fragment is recovered and is connected to a PEASY-T1 cloning vector to transform competent cells, and the positive bacteria are identified and sequenced.

As can be seen from FIG. 3, the DNA fragments of about 500bp were obtained by PCR amplification of GA2ox 5'GSP-1 and GA2ox 5' GSP-2, which were designed based on the sequence information of the obtained GA2ox gene intermediate fragment, and the respective universal primers.

Fifth, gene sequence splicing and analysis

5.1 splicing the 5 'end, the 3' end and the intermediate segment by using DNAMAN to obtain a full-length sequence of the GA2ox gene, and translating the full-length sequence into a protein sequence; amino acid similarity search was performed using BLAST, and the results are shown in FIGS. 4-6.

As can be seen from fig. 4-6: the GA2ox sequence has 79% consistency with orange (XM _ 006467404.3);

identity with citrus (XM _006449629.2) was 79%;

identity with cassava (XM _021738345.1) is 78%;

the identity with hevea brasiliensis (XM _021820571.1) is 78%;

consistency with upland cotton (XM _016896568.1) was 76%;

the consistency with medium cotton (XM _012597405.1) was 75%.

5.2 analysis of the physicochemical Properties of the amino acid sequence

The GA2ox sequence contained the complete open reading frame and the stop codon was TGA, respectively, as analyzed by protparam (http:// web. expasy. org/protparam /). GA2ox encodes 341 amino acids, and the molecular weight of the product is 38729.40Da, and the theoretical isoelectric point is 6.56. The number of acidic amino acids (Asp + Glu) is 41, and the number of basic amino acids (Arg + Lys) is 39. The protein is unstable and has a fat index of 80.32; the overall average hydrophilicity was-0.301.

The results of the analysis are shown in FIG. 7, using an on-line analysis with Phobius (http:// Phobius. sbc. su. se /).

As can be seen from FIG. 7, the protein encoded by GA2ox may have a signal peptide. Meanwhile, the transmembrane domain of the GA2ox protein sequence is predicted, and the result shows that the whole peptide chain is positioned outside the cell membrane, and the prediction of the signal peptide is combined to conclude that the protein is possible to be synthesized in a cytoplasmic matrix and then directly anchored to a specific position outside the cell membrane for functioning through protein transport.

Prediction of the secondary structure of GA2ox using DNAMAN is shown in fig. 8.

As can be seen from FIG. 8, in the GA2 ox-encoded protein, the helical structure accounted for 21.14%, the folding accounted for 26.67%, and the random coil accounted for 52.19%.

The hydrophobicity of GA2ox was predicted using DNAMAN and the prediction results are shown in figure 9.

As can be seen in FIG. 9, the glutamic acid at position 91 of the GA2ox polypeptide chain had the lowest value of-3.18, which is the most hydrophilic, while the methionine at position 1 has the highest value of 2.84, which is the most hydrophobic.

To study the evolution of Mango GA2ox, a phylogenetic tree of GA2ox amino acid sequences was constructed, as shown in FIG. 10.

As shown in fig. 10, mango GA2ox is closer to jute GA2ox (PON72625.1), jute GA2ox (PON57518.1), mulberry GA2ox (XP _010110967.1), bread GA2ox1(ALS88210.1), bread GA2ox2(ALS88211.1), and bread GA2ox3(ALS 88212.1); the next are castor Ga2ox (XP _015574955.1), jatropha Ga2ox (NP _001292962.1), hevea brasiliensis Ga2ox (XP _021649315.1), cassava Ga2ox (XP _021594037.1), hevea brasiliensis Ga2ox (XP _ 021676263.1).

Example 2: gene relative quantity PCR detection

The Beacon Designer is utilized to design a primer, and the expression conditions of the GA20ox gene in different periods of 'Jinhuang mango' (vigorous characteristic) and 'Guiqi mango' (dwarfing characteristic) are explored.

And designing actin-F and actin-R primers by using mango actin internal reference genes. Specific primers GA20ox-YGF and GA20ox-YGR were designed based on the cloned GA20ox sequence, and the cDNA was obtained as in example 1.

The PCR quantitative reaction steps are as follows:

1) subjecting All-in-One^TMThe qPCR Mix was thawed at room temperature, gently inverted and mixed and centrifuged briefly.

2) PCR Reaction Mix, including 2 × All-in One, was prepared on ice^TMqPCR Mix 10. mu.L, qPCR Primer (1. mu. mol/L) 2. mu.L, cDNA Template 2. mu.L, ddH was added₂O brought the final volume to 20. mu.L. NTC (negative control) was designed in the experiment, i.e., water was used to replace the template cDNA, and the other reagents were not changed, thereby ensuring that the system was free from contamination.

3) The reaction tube was centrifuged briefly to ensure that all the reaction solution was at the bottom of the reaction tube.

The PCR reaction program is 95 ℃ for 10 min; 10s at 95 ℃, 20s at 60 ℃, 15s at 72 ℃ and 40 cycles; 10min at 72 ℃. After the PCR reaction is finished, the analysis of the dissolution curve is carried out, namely the temperature is controlled at 72-95 ℃, the temperature is increased by 0.5 ℃ every 10s, and finally the temperature is kept at 25 ℃ for 30 s. The experimental set-up was 3 replicates.

Using 7300System software and 2^-ΔΔCtThe method performs data analysis, and the results are shown in FIG. 11.

As can be seen from FIG. 11, the GA20ox gene expression level of the test material with dwarfing property is much higher than that of the germplasm with dwarfing property in 5 phenological periods, which indicates that the GA2ox gene has the function of regulating the dwarfing phenotype of plants.

In conclusion, the method for determining the expression level of the GA2 ox-oxidase gene by using the semifluorescent quantitative analysis method can provide support for early prejudging whether the test material has the dwarfing trait.

[1] SEQUENCE LISTING

[2] <110> research institute for subtropical crops in autonomous region of Guangxi Zhuang nationality

[3] <120> a gene encoding GA2 ox-oxidase and application thereof in judging dwarf mango varieties

[4] <130> 2020

[5]

[6] <160> 2

[7]

[8] <170> PatentIn version 3.3

[9] <210> 1

[10] <211> 2075

[11] <212> DNA

[12] <213> Artificial sequence

[13]

[14] <400> 1

[15] acatggggga gagtggtact agtatagggc gattgggccc gacgtcgcat gctcccggcc 60

[16] gccatggcgg ccgcgggaat tcgattaatg gcgatgttgg atggattgaa tacctcctct 120

[17] tcaccgccaa tcaagattct aatctccaac gattcgttta tcctctcgga aaaaacccgg 180

[18] aaaagtttcg gtataaacct gcagaaaaaa tcattccttt tttattttcg tttgcatagc 240

[19] tgtgttttat agtcttgttt ggtgttgctg cagtcgtggc aatttctgcc cgttctttcc 300

[20] cttcctcaat ggcataaata ccaggcagac acacaaagct tctttatagt tagcaacgcc 360

[21] tctctctgcc tctgtcttca tgcatcagca gcaaatcctc tcatttgccc atataaaccc 420

[22] ttcaatctct tcagaccctt ctcaattctc accttctcct cttttccact ctcaatttct 480

[23] ctctctgtct ctctctgtgt tttgtcagaa aaaactcggc caccatggtg gttctgtcac 540

[24] aaccagcatt agaacatttc tctataatcg aaacttacca gccttcaagc tgcttatact 600

[25] caggaattcc agttgtagac atgagacacc ctgaagccaa gttccatgta gtggaagcct 660

[26] gtgaaaaata cggcttcttc aagctcatta accatgatgt tccgttggag ttcatggcca 720

[27] atttagaagc cgaagctgtc aacttcttta acctccctca gtctgagaaa gacaaagctg 780

[28] gaccccctga cccttatggc tatggcagca aaagcattgg ccccaatggt gatgttggtt 840

[29] ggattgaata tctcctcctc aactccaacc ctcaaatcac ttcacaaaaa actctcgcca 900

[30] ttttcaaaca cagccctcat gatttccgga gtgctgtgga gaagtacata acagaaatga 960

[31] agaaacttgc atatgaagtt cttgaattaa tggccgatgg gctaagaata gagccaagga 1020

[32] acattttcag tagattcata agggatgaaa aaagtgactc ctgtttcagg ctgaaccact 1080

[33] acccaccatg tccagagctt caaacattga agaaaggaag caatttgatt gggttcggag 1140

[34] aacacacaga ccctcagatt ctttctgttc taagatctaa caatacttca ggacttgaaa 1200

[35] tttgcttaag agatggcact tgggtttctg tccctgctga tcattcttcc tttttcctca 1260

[36] atgttggtga tgctctgcag gtaatgacta atgggagatt ccaaagtgtg aagcacagag 1320

[37] tgttggctga cagagtgaaa tcaagaattt caatgatata ttttggaggg ccaccattga 1380

[38] atgaaaagat tgcacctttg ccatgcctag tttcaaaaga agaagattgc ttgtacaagg 1440

[39] agttcacttg gtgtgaatac aagtgctctg cctataagtc caagttggct gattataggc 1500

[40] ttgggcagtt tgagaaatga aaaccaaaag gacacttccg taatttagca aaaattaaac 1560

[41] tagccataca tggagaagaa tataaaatag agcctcaact tctcttgttt atttatcaac 1620

[42] aactctgtac tagttaactg cagctacaat gttattatcc actagatctt gatcatgtta 1680

[43] aaataaatta taattttcaa cttattttca tattttattt tatgttaacc acagtttcaa 1740

[44] ttcccccttt gggtttctca ttgtcctgga atttaggcat tcaatttgag tatttgactt 1800

[45] ggcatacatg cacgtcattc ttatctataa ctatcgaata tagctgccat caaaagcttt 1860

[46] tgccagtacc caagcatgaa atgttttttc ctatctgtat tgatgatcaa tagagttgaa 1920

[47] ttactgttcg ttaatggaga tttctgtgtt gggtggttga ttatgcaggt gctgacaaat 1980

[48] ggaagattta agagcgtaag gcaaaatgtt ggagactcat tacaggtata caatttgtac 2040

[49] agtttttacc aaaaaaaaaa aaaaaaaaaa aaaaa 2075

[50]

[51] <210> 2

[52] <211> 341

[53] <212> PRT

[54] <213> Artificial sequence

[55]

[56] <400> 2

[57] Met Cys Phe Val Arg Lys Asn Ser Ala Thr Met Val Val Leu Ser Gln

[58] 1 5 10 15

[59] Pro Ala Leu Glu His Phe Ser Ile Ile Glu Thr Tyr Gln Pro Ser Ser

[60] 20 25 30

[61] Cys Leu Tyr Ser Gly Ile Pro Val Val Asp Met Arg His Pro Glu Ala

[62] 35 40 45

[63] Lys Phe His Val Val Glu Ala Cys Glu Lys Tyr Gly Phe Phe Lys Leu

[64] 50 55 60

[65] Ile Asn His Asp Val Pro Leu Glu Phe Met Ala Asn Leu Glu Ala Glu

[66] 65 70 75 80

[67] Ala Val Asn Phe Phe Asn Leu Pro Gln Ser Glu Lys Asp Lys Ala Gly

[68] 85 90 95

[69] Pro Pro Asp Pro Tyr Gly Tyr Gly Ser Lys Ser Ile Gly Pro Asn Gly

[70] 100 105 110

[71] Asp Val Gly Trp Ile Glu Tyr Leu Leu Leu Asn Ser Asn Pro Gln Ile

[72] 115 120 125

[73] Thr Ser Gln Lys Thr Leu Ala Ile Phe Lys His Ser Pro His Asp Phe

[74] 130 135 140

[75] Arg Ser Ala Val Glu Lys Tyr Ile Thr Glu Met Lys Lys Leu Ala Tyr

[76] 145 150 155 160

[77] Glu Val Leu Glu Leu Met Ala Asp Gly Leu Arg Ile Glu Pro Arg Asn

[78] 165 170 175

[79] Ile Phe Ser Arg Phe Ile Arg Asp Glu Lys Ser Asp Ser Cys Phe Arg

[80] 180 185 190

[81] Leu Asn His Tyr Pro Pro Cys Pro Glu Leu Gln Thr Leu Lys Lys Gly

[82] 195 200 205

[83] Ser Asn Leu Ile Gly Phe Gly Glu His Thr Asp Pro Gln Ile Leu Ser

[84] 210 215 220

[85] Val Leu Arg Ser Asn Asn Thr Ser Gly Leu Glu Ile Cys Leu Arg Asp

[86] 225 230 235 240

[87] Gly Thr Trp Val Ser Val Pro Ala Asp His Ser Ser Phe Phe Leu Asn

[88] 245 250 255

[89] Val Gly Asp Ala Leu Gln Val Met Thr Asn Gly Arg Phe Gln Ser Val

[90] 260 265 270

[91] Lys His Arg Val Leu Ala Asp Arg Val Lys Ser Arg Ile Ser Met Ile

[92] 275 280 285

[93] Tyr Phe Gly Gly Pro Pro Leu Asn Glu Lys Ile Ala Pro Leu Pro Cys

[94] 290 295 300

[95] Leu Val Ser Lys Glu Glu Asp Cys Leu Tyr Lys Glu Phe Thr Trp Cys

[96] 305 310 315 320

[97] Glu Tyr Lys Cys Ser Ala Tyr Lys Ser Lys Leu Ala Asp Tyr Arg Leu

[98] 325 330 335

[99] Gly Gln Phe Glu Lys

[100] 340

Claims

1. A gene encoding GA2 ox-oxidase, characterized in that: the nucleotide sequence of the gene is shown as SEQ ID No. 1.

2. Use of a gene encoding GA2 ox-oxidase according to claim 1, wherein the gene encoding GA2 ox-oxidase is used to predict whether a mango variety has dwarfing properties.

3. A GA2 ox-oxidase, wherein the GA2 ox-oxidase is encoded by the gene of claim 1.

4. A GA2 ox-oxidase according to claim 3, wherein the amino acid sequence of GA2 ox-oxidase is shown in SEQ ID No. 2.

5. A GA2 ox-oxidase as claimed in claim 3, wherein GA2 ox-oxidase consists of 341 amino acids, molecular weight is 38729.40Da, theoretical isoelectric point is 6.56.