Gene for coding KO protein and application of gene in early prediction of dwarf character of unknown mango species

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a gene for coding KO protein and application of the gene in early prejudgment of dwarf characters of unknown mango species. The invention discloses a gene for coding KO protein. The nucleotide sequence of the gene is shown as SEQ ID No. 1. The gene for coding the KO protein can provide support for pre-judging whether a test material has the dwarf characteristic or not in the early stage.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering technology, and specifically relates to a gene encoding KO protein and its application in early prediction of dwarf traits of unknown mango species Background Art

[0002] Dwarfing cultivation is an unchanging development trend of large-scale intensive production of perennial woody fruit trees. Plant dwarfing is mainly divided into hereditary dwarfing and physiological dwarfing. Single gene mutation belongs to hereditary dwarfing, and its dwarfing mechanism is mostly gene synthesis protein, protein affects hormone, and hormone affects phenotype; affected by environmental factors, the plant phenotype changes, which belongs to physiological dwarfing, and its dwarfing mechanism is mostly environmental changes affecting plant hormone secretion, which ultimately leads to changes. However, no matter which dwarfing mode, the phenotype must be affected by hormones, and the hormones in the plant growth process are mostly related to gibberellins (GAs).

[0003] Ent-kaurene oxidase (KO) is a type of monooxidase located upstream of the gibberellin synthesis pathway. It oxidizes ent-kaurene to ent-kaurenic acid, regulating the synthesis of gibberellins. KO exists in various tissues of plants. Its expression level is higher in the inflorescence, stems, leaves and embryos during the vigorous growth period of the plant body, and its expression level is lower in old leaves and stems. At present, the KO gene has been isolated and cloned in many plants such as peas, corn, cucumbers, pears, apples, pecans, pomegranates, etc. Studies have shown that blocking the KO gene site will lead to the loss of KO function, thereby blocking the synthesis process of gibberellins, shortening the internodes of the plant and shortening the plant height. For example, the pea PsKO1 dwarf mutant has a lack of GA in the plant due to a mutation in the KO gene, resulting in shortened internodes and roots, weakened root system, and short plants; the Arabidopsis ga3-1 dwarf mutant has a mutation in a base site of the KO allele, which weakens the activity of KO and inhibits the growth and development of the plant; the rice GA-deficient semi-dwarf mutant d35 has a semi-dwarf phenotype due to a nucleotide site mutation in the exon of OsKO2, which causes the loss of OsKO2 function; a rice dwarf mutant was obtained by EMS induction, and the study found that due to two consecutive mutation sites on the OsKO1 gene, threonine (Thr) in the cytochrome P450 domain was converted to methionine (Met), blocking the biosynthesis of GA, and the plant showed a delayed germination and semi-dwarf phenotype. At the same time, changes in the expression level of the KO gene will also affect the morphological construction of the plant body. For example, the dwarfing rootstock of apple M26 has less KO synthesis, which weakens the synthesis of gibberellins in the plant body and leads to the dwarfing of the apple rootstock. In pecan, due to the overexpression of CcKO, the expression levels of GA20ox and GA2ox genes downstream of the GA synthesis pathway are changed. The expression levels of the three genes are synergistically regulated, which promotes the elongation of plant height.

[0004] By comparing the expression levels of KO genes in plant materials with tall growth characteristics and dwarf growth characteristics, it is possible to predict whether a plant has dwarf growth characteristics, which saves both time and effort. Currently, there are no reports on using KO genes to predict whether a mango variety has dwarf growth characteristics. Summary of the invention

[0005] The present invention provides a gene encoding a KO protein and an application of the gene in early prediction of the dwarf trait of an unknown mango variety, which can predict whether the mango variety has the dwarf characteristic, saving time and labor.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The first object of the present invention is to provide a gene encoding KO protein, the nucleotide sequence of the gene is shown as SEQ ID No.1.

[0008] More specifically, the open reading frame of the gene is 1554 bp.

[0009] The second object of the present invention is to provide the application of the gene encoding the KO protein, and the application of the gene encoding the KO protein in early prediction of the dwarf traits of unknown mango varieties.

[0010] The third object of the present invention is to provide a KO protein, wherein the KO protein is encoded by the gene.

[0011] More specifically, the amino acid sequence of the KO protein is shown in SEQ ID No.2.

[0012] More specifically, the KO protein consists of 517 amino acids, has a molecular weight of 58.65 KD, and a theoretical isoelectric point of 8.56.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] (1) The present invention obtains the KO protein gene sequence from the test material, analyzes the physicochemical activity of the gene, and uses the gene as an indicator for regulating the dwarf trait.

[0015] (2) The present invention relates to cloning of KO protein genes and analysis of the corresponding amino acid sequences. The expression level of KO protein genes is determined by fluorescence quantitative method, which can provide support for early prediction of whether the test material has dwarf traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a schematic diagram of the homology comparison results between mango KO and other plant KO;

[0017] Figure 2 This is the amino acid composition diagram of mango KO protein;

[0018] Figure 3 This is the signal peptide and transmembrane domain analysis diagram of mango KO protein;

[0019] Figure 4 This is the predicted secondary structure of mango KO protein;

[0020] Figure 5 This is the predicted tertiary structure of mango KO protein;

[0021] Figure 6 This is the prediction diagram of the hydrophilicity and hydrophobicity of mango KO protein;

[0022] Figure 7This is the phylogenetic tree of mango KO proteins;

[0023] Figure 8 The graph shows the expression level of KO gene in different tree varieties and dwarf varieties at different phenological stages. DETAILED DESCRIPTION

[0024] The following is a clear and complete description of the technical solution of the patent of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by technicians in this field without creative work are within the scope of protection of the present invention.

[0025] In the following examples, the main consumables and instruments are as follows:

[0026] 1. Consumables

[0027] III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNAdigesterplus), YEASEN; 2×HS Taq Universal SYBR Green QPCR Master Mix, SAIPUBIO; 1.5ml centrifuge tubes, Shanghai Sangon Biotechnology Co., Ltd., F601620-9001; 0.2ml 8StripReal-time PCR Tubes, Shanghai Sangon Biotechnology Co., Ltd., F603001-0001; gun tips, Axygen, USA.

[0028] 2. Instruments

[0029] ND5000 ultra-micro volume UV-visible spectrophotometer; QuantStudio(TM)7Flex SystemThermoFisherScientific; high-speed refrigerated centrifuge, Hangzhou Aosheng Biotechnology Co., Ltd., iCEM-24R; mini centrifuge, American Jingqi Co., Ltd., 4016006; pipette, German Eppendorf.

[0030] Example 1 :Cloning and sequence analysis of mango KO gene

[0031] 1. RNA Extraction

[0032] The total RNA of the leaves of Psoralea corylifolia was extracted using RNA prep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.).

[0033] Weigh 3g of leaf tissue of Psoralea cochinchinensis into a sterilized mortar, quickly grind it into powder in liquid nitrogen, and then extract total RNA according to the instructions of the plant total RNA extraction kit. All consumables in the experiment need to be soaked in DEPC stock solution for 1.5 days, and then sterilized in a high-temperature steam sterilizer at 121℃ for 0.5h to ensure the inactivation of RNase.

[0034] Immediately after RNA extraction, the quality of RNA was tested by 1% agarose gel electrophoresis, and the concentration and purity of RNA were tested by UV spectrophotometer at 260 / 280nm absorbance. When OD260 / 280nm reached about 2.0, it indicated that the extracted RNA had high purity. The successfully extracted RNA was stored in a -80℃ refrigerator for later use.

[0035] 2. cDNA Synthesis

[0036] The first-strand cDNA was synthesized from the extracted mango high-quality RNA using the M-MuL first-strand cDNA synthesis kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.). The specific steps are as follows:

[0037] (1) Add the following reaction mixture to an RNase-free PCR tube in an ice bath: 5 μL total RNA, 1 μL Oligo (DT) (0.5 μg / μL), 6 μL RNase free ddH2O;

[0038] (2) After centrifuging and mixing the above reaction solution, incubate it at 65°C in a PCR instrument for 5 min, immediately cool it in an ice bath for 30 s, centrifuge it for 3-5 s, and then add 4 μL of 5x Reaction Buffer, 1 μL of RNase Inhibitor (40 U / μL), 2 μL of DNTP Mix (10 mmom / L), and 1 μL of M-MuLV RT (200 U / μL);

[0039] (3) The mixed solution was placed in a PCR instrument and incubated at 42°C for 60 min, and then at 70°C for 10 min. After the test, the integrity of the cDNA was detected by 1% agarose gel electrophoresis and the mixture was stored in a -20°C refrigerator for later use.

[0040] 3. Primer Design

[0041] According to the sequence of the KO encoding gene of pistachio (pistacia vera L..) published in the NCBI database (accession number: XP_031283332.1), specific primers were designed using Primer5 software (see Table 1 for details).

[0042] Table 1 Specific primer names and sequences

[0043]

[0044] Using cDNA from the leaves of Psoralea cochinchinensis as a template, PCR amplification was performed using the specific primers in Table 1 and 2x high-fidelity PCR Mix premix. The PCR amplification system is shown in Table 2.

[0045] Table 2 PCR amplification system

[0046]

[0047] The PCR reaction program was as follows: pre-denaturation at 95°C for 5 min; 39 cycles of 95°C for 30 s, 58°C for 120 s, and 72°C for 90 s; and extension at 72°C for 10 min.

[0048] 4. Target fragment recovery

[0049] The target fragment was recovered using the SanPrep column DNA gel recovery kit (Sangon Biotech (Shanghai) Co., Ltd.), and the specific operation was performed according to the instruction manual.

[0050] 5. Target fragment connection

[0051] Use the zero-background TOPO-Blunt blunt-end cloning kit for ligation. The specific steps are as follows:

[0052] (1) Prepare the ligation system in Table 3 in a PCR tube on ice.

[0053] Table 3 Connection system

[0054]

[0055] (2) Connect at room temperature of 20-30℃ for 5 minutes.

[0056] 6. Transformation of recombinant plasmid into competent E. coli DH5α

[0057] The recombinant plasmid was transformed into E. coli DH5α competent cells using the heat shock method. The specific operation is as follows:

[0058] (1) Take out the competent E. coli DH5α cells from the -80°C refrigerator, put them on ice to thaw, and immediately dispense them into cold 1.5 mL centrifuge tubes after they have just thawed;

[0059] (2) Add 4 μL of the ligation reaction solution to 50 μL of E. coli competent DH5α cells, flick the bottom of the tube several times to mix, and quickly place on ice to incubate the reaction for 30 min;

[0060] (3) Heat shock the centrifuge tube in a 42°C water bath for 90 seconds and then quickly place it on ice for 5 minutes;

[0061] (4) After the reaction, add 500 μL of sterile LB liquid medium without any antibiotics and incubate at 37°C and 200 rpm in a shaker for 60 min;

[0062] (5) After the shaking is finished, centrifuge at 4000 rpm for 3 min to collect the bacteria, remove a portion of the supernatant, retain 100 μL of the bacterial solution and mix well, and evenly spread the remaining bacterial solution on a plate containing 25 μg / mL of Kana resistance;

[0063] (6) Incubate the plate upright at 37°C until the bacterial solution is completely absorbed, then invert the plate and incubate for 12-16 h.

[0064] (7) Pick 8-10 white single colonies from each plate and add them to 1 mL of LB liquid medium containing Kana antibiotics. Continue shaking and culturing at 225 rpm in a shaker at 37°C for 3 h.

[0065] (8) The obtained bacterial solution is subjected to bacterial solution PCR detection, and the PCR reaction results are detected by 1% agarose gel electrophoresis. 3-5 bacterial solutions with the same size as the target band are selected for sequencing.

[0066] 7. Analysis of physicochemical properties of KO genes and amino acid sequences

[0067] 7.1 The amino acid sequences were analyzed for sequence similarity using BLAST on the NCBI website.

[0068] Results Figure 1 .

[0069] Depend on Figure 1 It can be seen that by comparing the KO protein sequences of plants such as ryegrass, cotton, and Amomum villosum registered on NCBI, the obtained gene was determined to be the mango KO gene.

[0070] 7.2 Use protparam to analyze the KO sequence and use PROSITE Scan to predict and analyze the conserved regions of the gene.

[0071] The open reading frame of the KO gene is 1554 bp; the KO gene encodes 517 amino acids, has a molecular weight of 58.65 KD, and a theoretical isoelectric point of 8.56.

[0072] 7.3 Use DNAMAN software to analyze the structural characteristics of gene amino acids.

[0073] The amino acid composition of the KO protein is shown in Figure 2 .

[0074] Depend on Figure 2It can be seen that the amino acid composition of KO protein is mainly composed of leucine, isoleucine, lysine, glutamic acid and alanine, including 60 acidic amino acids (Asp+Glu) and 65 basic amino acids (Arg+Lys).

[0075] 7.4 Use Phobius (http: / / phobius.sbc.su.se / ) to analyze the transmembrane region of the protein sequence online. The analysis results are shown in Figure 3 .

[0076] Depend on Figure 3 It can be seen that the KO protein is a cytoplasmic protein, and the proteins encoded by KO have no signal peptide. The prediction results of the transmembrane domain of the KO protein sequence show that the KO transmembrane region is obvious.

[0077] 7.5 Prediction of the secondary structure of KO protein using DNAMAN. The results are shown in Figure 4 .

[0078] Depend on Figure 4 It can be seen that among the KO-encoded proteins, helical structures accounted for 43.13%, folding accounted for 10.44%, and random coiling accounted for 46.42%.

[0079] 7.6 Prediction of KO protein tertiary structure. The prediction results are shown in Figure 5 .

[0080] Depend on Figure 5 It can be seen that the KO protein is composed of several different α-helices, β-turns and random coils to form the protein peptide chain, and the prediction results are highly consistent with the secondary structure analysis results.

[0081] 7.7 Using DNAMAN to predict the hydrophilicity and hydrophobicity of KO, the prediction results are shown in Figure 6 .

[0082] Depend on Figure 6 It can be seen that the arginine at position 164 of the KO polypeptide chain has the lowest value of -3.53 and is the most hydrophilic, while the leucine at position 31 has the highest value of 3.94 and is the most hydrophobic.

[0083] 7.8 In order to study the evolution of Mango KO protein, MEGA7.0 was used to construct a phylogenetic tree of the KO protein amino acid sequence. The results are shown in Figure 7 .

[0084] Depend on Figure 7It can be seen that mango KO is closely related to pistachio KO (XP_031279374.1); followed by Xanthoceras sorbifolia (KAH7567353.1), Acer yangbiensis (TXG52327.1), Melia azedarach (KAJ4701830.1), Satsuma mandarin (GAY65602.1), Citrus sweet orange (XP_006492563.2), Clementine (XP_024040161.1), Vitex tricolor (TQD97754.1), Oleaster oleraceus (KAF3446236.1) and O. longicornis (OMO74862.1).

[0085] Example 2 : Gene relative quantity PCR detection

[0086] Mango leaves from the mango resource garden of Guangxi Subtropical Crops Research Institute were taken to explore the expression of KO genes in different tree varieties and dwarf varieties at different stages. Among them, the tree varieties are Jinhuang Mango, Huang Xiangya and Hong Xiangya; the dwarf varieties are Guiqi Mango, Aiwen Mango and Qiu Mango; mature leaves were taken at the budding stage, flowering stage, 3-4 weeks of fruit setting, 7-8 weeks of fruit setting and fruit maturity for analysis.

[0087] Real-time fluorescence quantitative PCR was performed using the SYBR GREEN dye method and mango-GAPDH as an internal reference for relative quantitative detection. The specific operation is as follows:

[0088] 1. RNA Extraction

[0089] Same as Example 1.

[0090] 2. RNA quality detection and concentration determination

[0091] Measure the sample OD value and observe its absorbance at 260nm and 280nm to determine the quality of RNA. Perform agarose gel electrophoresis to determine the integrity of the extracted RNA and the contamination of DNA.

[0092] 3. RNA Reverse Transcription

[0093] 3.1 Removal of residual genomic DNA

[0094] Take an appropriate amount of RNA (300 ng in this experiment), add RNase-free ddH2O to 12 μL, add 3 μL 5×g DNA digester Mix, mix gently with a pipette, and incubate at 42°C for 5 min;

[0095] 3.2 Reverse transcription reaction

[0096] Add 5 μL of the mixture in the previous step III SuperMix plus, and mix gently with a pipette to obtain a 20 μL reaction system. The PCR program was set to: 27°C for 4 min, 55°C for 12 min, and 85°C for 5 min. The reaction product was cDNA, which was immediately used for PCR reaction.

[0097] 4. Real-time fluorescence quantification

[0098] Prepare the reaction system shown in Table 4.

[0099] Table 4 Reaction system

[0100]

[0101] Setting of two-step fluorescence quantitative PCR amplification conditions:

[0102] Amplification curve: 94°C for 5 min, 1 cycle; 94°C for 12 s, 60°C for 35 s, 40 cycles.

[0103] Melting curve: 94°C for 20 s, 60°C for 65 s, 94°C for 20 s, continuous detection signal.

[0104] Results Figure 8 .

[0105] Depend on Figure 8 It can be seen that the expression level of KO gene in dwarf varieties of mango in the five key phenological periods is below 2 and the expression level does not change much in each period; in the arborescent mango varieties, the expression level of KO gene is above 10, and the highest can reach 20. The expression level of KO gene in different arborescent mango varieties has no regular change in the five key phenological periods, but the expression level of KO gene in arborescent mango varieties is significantly higher than that in dwarf mango varieties in any mango phenological period. The expression of mango KO gene can be used as an early predictive indicator of whether mango has dwarf traits.

[0106] In summary, the present invention can provide support for early prediction of whether the test material has a dwarf trait by measuring the expression amount of the KO gene through the fluorescence quantitative analysis method.

[0107] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A gene encoding a KO protein, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID No.

1.

2. The use of the gene encoding KO protein according to claim 1, characterized in that The open reading frame of the gene is 1554 bp.

3. Use of the gene encoding KO protein according to claim 1, characterized in that: The application of the gene encoding the KO protein in early prediction of the dwarf traits of unknown mango varieties.

4. A KO protein, characterized in that The KO protein is encoded by the gene according to claim 1.

5. The KO protein according to claim 4, characterized in that The amino acid sequence of the KO protein is shown in SEQ ID No.

2.

6. The KO protein according to claim 4, characterized in that The KO protein consists of 517 amino acids, has a molecular weight of 58.65 KD, and a theoretical isoelectric point of 8.56.

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