Gene for coding DELLA1 protein and application thereof
By analyzing and determining the expression amount of mango DELLA1 protein gene, the problem of difficulty in early prediction of dwarf characteristics in the prior art is solved, and efficient and accurate dwarf trait prediction is achieved.
Patent Information
- Application Number
- CN202410325476.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-03-21
- Publication Date
- 2025-03-28
AI Technical Summary
The prior art is difficult to predict whether mango varieties have dwarf characteristics in the early stages, and there is a lack of efficient methods to analyze the expression of the DELLA1 protein gene.
By obtaining and analyzing the gene sequence encoding the DELLA1 protein, cloning and measuring its expression, the expression of the DELLA1 gene was determined by fluorescence quantitative PCR method to predict the dwarf traits of mango varieties.
The ability to early predict whether mango varieties have dwarf characteristics is achieved, the analysis efficiency and accuracy are improved, and the dwarf characteristics of plants can be quickly judged without long-term breeding experiments.
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Figure CN119709768A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of genetic engineering technology, and specifically relates to a gene encoding DELLA1 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] Gibberellins are central regulators in the growth and development of plants, regulating processes such as seed germination, seedling growth, leaf development, root and stem growth, floral organ development, and fruit ripening. DELLA proteins are negative regulators in the GA metabolic pathway and belong to the plant-specific GRAS family. DELLA proteins not only mediate GA responses, but are also integrators of plant responses to environmental signals (light, temperature, water, and salt, etc.) and hormone signals (GAs, IAA, ABA, ET, JA, and BR, etc.). The inhibitory effect of DELLA proteins on plants is beneficial when plants are subjected to adverse stress. At present, DELLA protein genes have been cloned from dozens of plants such as Arabidopsis, rice, wheat, soybean, rapeseed, cotton, plum, apple, pear, and strawberry, and their functions and regulatory mechanisms have been studied in depth. Model plants such as Arabidopsis and rice have been studied more thoroughly. The functions of DELLA proteins are highly conserved among different species, but the number of DELLA protein gene members and the mode of action are different in different species. During the "Green Revolution" of the 1950s, wheat used beneficial mutations of DELLA proteins to achieve semi-dwarf breeding, increase grain yields, and solve the problem of food and clothing.
[0004] By comparing the expression levels of the DELLA1 gene in plant materials with tree-like and dwarf characteristics, it is possible to predict whether a plant has dwarf characteristics, which saves both time and effort. Currently, there are no reports on using the DELLA1 gene to predict whether a mango variety has dwarf characteristics. Summary of the invention
[0005] The present invention provides a gene encoding a DELLA1 protein and an application thereof, which can predict whether a mango variety has a dwarf characteristic, thus 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 DELLA1 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 1734 bp.
[0009] The second object of the present invention is to provide an application of the gene encoding the DELLA1 protein in early prediction of the dwarf traits of unknown mango species.
[0010] The third object of the present invention is to provide a DELLA1 protein, wherein the DELLA1 protein is encoded by the gene.
[0011] More specifically, the amino acid sequence of the DELLA1 protein is shown in SEQ ID No.2.
[0012] More specifically, the DELLA1 protein consists of 577 amino acids, has a molecular weight of 62.83 KD, and a theoretical isoelectric point of 4.95.
[0013] Compared with the prior art, the present invention has the following beneficial effects:
[0014] (1) The present invention obtains the DELLA1 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 the cloning of the DELLA1 protein gene and the analysis of its corresponding amino acid sequence. The expression level of the DELLA1 protein gene is determined by a fluorescence quantitative method, which can provide support for early prediction of whether the test material has a dwarf trait. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 This is a schematic diagram of the homology comparison between mango DELLA1 and other plant DELLA1;
[0017] Figure 2 This is the amino acid composition diagram of mango DELLA1 protein;
[0018] Figure 3 This is the signal peptide and transmembrane domain analysis diagram of mango DELLA1 protein;
[0019] Figure 4 This is the predicted secondary structure of mango DELLA1 protein;
[0020] Figure 5 This is the predicted tertiary structure of mango DELLA1 protein;
[0021] Figure 6 This is the prediction map of the hydrophilicity and hydrophobicity of mango DELLA1 protein;
[0022] Figure 7 This is the phylogenetic tree of mango DELLA1 protein;
[0023] Figure 8 This is a graph showing the expression levels of the DELLA1 gene in different tree-growing 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 DELLA1 gene from mango
[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 150 mg 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 day, and then sterilized in a high-temperature steam sterilizer at 121°C for 20 minutes 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 DELLA encoding gene of pistachio (pistacia vera L..) published in the NCBI database (accession number: XP_0312257899.1), 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; 40 cycles of 95°C for 10 s, 60°C for 30 s, and 72°C for 60 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]
[0056] (2) Connect at room temperature of 20-30℃ for 5 minutes.
[0057] 6. Transformation of recombinant plasmid into competent E. coli DH5α
[0058] The recombinant plasmid was transformed into E. coli DH5α competent cells using the heat shock method. The specific operation is as follows:
[0059] (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;
[0060] (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;
[0061] (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;
[0062] (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;
[0063] (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;
[0064] (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.
[0065] (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.
[0066] (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.
[0067] 7. Analysis of the physicochemical properties of the DELLA1 gene and amino acid sequence
[0068] 7.1 The amino acid sequences were analyzed for sequence similarity using BLAST on the NCBI website.
[0069] Results Figure 1 .
[0070] Depend on Figure 1 It can be seen that by comparing the DELLA1 protein sequences of plants such as ryegrass, cotton, and Amomum villosum registered on NCBI, it was determined that the obtained gene was the mango DELLA1 gene.
[0071] 7.2 Use protparam to analyze the DELLA1 sequence and use PROSITE Scan to predict and analyze the conserved regions of the gene.
[0072] The open reading frame of the DELLA1 gene is 1734bp; the DELLA1 gene encodes 577 amino acids, has a molecular weight of 62.83KD, and a theoretical isoelectric point of 4.95.
[0073] 7.3 Use DNAMAN software to analyze the structural characteristics of gene amino acids.
[0074] The amino acid composition of DELLA1 protein is shown in Figure 2 .
[0075] Depend on Figure 2 It can be seen that the amino acid composition of DELLA1 protein is mainly composed of alanine, leucine, glycine, serine, glutamic acid and other amino acids, including 72 acidic amino acids (Asp+Glu) and 46 basic amino acids (Arg+Lys).
[0076] 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 .
[0077] Depend on Figure 3 It can be seen that DELLA1 protein is a non-cytoplasmic protein, and the proteins encoded by DELLA1 do not have a signal peptide. The prediction results of the transmembrane domain of the DELLA1 protein sequence show that the transmembrane region of DELLA1 is not obvious.
[0078] 7.5 Using DNAMAN to predict the secondary structure of DELLA1 protein, the results are shown in Figure 4 .
[0079] Depend on Figure 4 It can be seen that in the DELLA1-encoded protein, the helical structure accounts for 34.84%, the folding accounts for 10.92%, and the random curling accounts for 54.25%.
[0080] 7.6 Prediction of the tertiary structure of DELLA1 protein. The prediction results are shown in Figure 5 .
[0081] Depend on Figure 5 It can be seen that the DELLA1 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.
[0082] 7.7 Using DNAMAN to predict the hydrophilicity and hydrophobicity of DELLA1, the prediction results are shown in Figure 6 .
[0083] Depend on Figure 6It can be seen that the arginine at position 169 of the DELLA1 polypeptide chain has the lowest value of -3.89 and is the most hydrophilic, while the alanine at position 528 has the highest value of 2.96 and is the most hydrophobic.
[0084] 7.8 In order to study the evolution of Mango DELLA1 protein, a phylogenetic tree of DELLA1 protein amino acid sequence was constructed using MEGA7.0. The results are shown in Figure 7 .
[0085] Depend on Figure 7 It can be seen that mango DELLA1 is closely related to pistachio DELLA (XP_031257899.1); followed by sweet orange (KAH9739056.1), clementine (XP_006430617.1), sweet orange (XP_006482132.1), Xanthoceras sorbifolia (KAH7556977.1), Acer truncatum (TXG69935.1), Acer rubrum (KAI9162322.1), longan (AUF71967.1), Columbia mallow (XP_021296557.1), and cocoa (XP_017975440.1).
[0086] Example 2 : Fluorescence PCR
[0087] Mango leaves from the mango resource garden of Guangxi Subtropical Crops Research Institute were taken to explore the expression of DELLA1 gene in different tree varieties and dwarf varieties at different stages. Among them, the tree varieties are Sannian Mang, Yellow Ivory and Red Ivory; the dwarf varieties are Marika, Aiwen Mang and Qiu Mang; mature leaves were taken at the budding stage, flowering stage, 3-4 weeks of fruit setting and fruit maturity stage for analysis.
[0088] Real-time fluorescence quantitative PCR was performed using the SYBR GREEN dye method and mango GAPDH as an internal reference. The specific operation is as follows:
[0089] 1. RNA Extraction
[0090] Same as Example 1.
[0091] 2. RNA quality detection and concentration determination
[0092] 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.
[0093] 3. RNA Reverse Transcription
[0094] 3.1 Removal of residual genomic DNA
[0095] 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;
[0096] 3.2 Reverse transcription reaction
[0097] Add 5 μL of 4× III SuperMix plus, and mix gently with a pipette to obtain a 20 μL reaction system. The PCR program was set to: 25°C for 5 min, 55°C for 15 min, and 85°C for 5 min. The reaction product was cDNA, which was immediately used for PCR reaction.
[0098] 4. Real-time fluorescence quantification
[0099] Prepare the reaction system shown in Table 4.
[0100] Table 4 Reaction system
[0101]
[0102] Setting of two-step fluorescence quantitative PCR amplification conditions:
[0103] Amplification curve: 95°C for 5 min, 1 cycle; 95°C for 10 s, 60°C for 30 s, 40 cycles.
[0104] Melting curve: 95℃15s, 60℃60s, 95℃15s, continuous detection signal.
[0105] Results Figure 8 .
[0106] Depend on Figure 8 It can be seen that the expression level of DELLA1 gene in dwarf mango varieties is much greater than that in arborescent mango varieties in any key phenological period of mango; the expression level of DELLA1 in arborescent mango varieties in any key phenological period of mango is basically around 1, with no obvious changes in different periods, while the expression level of DELLA1 gene in dwarf mango varieties is basically above 6, and is even higher at 12 during the fruit ripening period. Therefore, determining the expression level of mango DELLA1 gene can be used as an important indicator for early prediction of whether unknown mango species have dwarf traits.
[0107] In summary, the present invention can provide support for early prediction of whether the test material has a dwarf trait by measuring the expression level of the DELLA1 gene through fluorescent quantitative analysis.
[0108] 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 DELLA1 protein, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID No.
1.
2. The gene encoding DELLA1 protein according to claim 1, characterized in that The open reading frame of the gene is 1734 bp.
3. A use of the gene encoding DELLA1 protein according to claim 1, characterized in that: The application of the gene encoding DELLA1 protein in early prediction of dwarf traits of unknown mango species.
4. A DELLA1 protein, characterized in that The DELLA1 protein is encoded by the gene according to claim 1.
5. The DELLA1 protein according to claim 4, characterized in that The amino acid sequence of the DELLA1 protein is shown in SEQ ID No.
2.
6. The DELLA1 protein according to claim 4, characterized in that The DELLA1 protein consists of 577 amino acids, has a molecular weight of 62.83 KD, and a theoretical isoelectric point of 4.95.