Gene for coding GA20ox2 oxidase and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a gene for coding GA20ox2 oxidase and application thereof. The invention relates to a gene for coding GA20ox2 oxidase. The nucleotide sequence of the gene is shown as SEQ ID No. 1. The gene for coding the GA20ox2 oxidase 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 present invention belongs to the field of genetic engineering technology, and specifically relates to a gene encoding GA20ox2 oxidase and its application 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] GA20 oxidase (GA20ox) converts the inactive GA into 53 and GA 12 Catalyzed into GA9 and GA 20, and then after continuous oxidation reactions, active GA1 and GA4 are generated, which control the synthesis content of active gibberellins. At present, the GA20ox gene has been isolated and cloned in many plants such as Arabidopsis, chestnut, cotton, pear and barley. There are many studies on the function of GA20ox in regulating plant height. It has been found in many plants that overexpression of GA20ox gene can promote the elongation of plant height, while mutation or downregulation of GA20ox expression will inhibit the growth of plant height (Huang et al., 1998; Qiao Feng et al., 2013). In grass plants, such as rice, overexpression of OsGA20ox2 and OsGA20ox3 genes leads to increased levels of active gibberellins GA1 and GA4 in the plant, thereby promoting the elongation of plant height. The absence of rice OsGA20ox2 sequence leads to a decrease in the level of active gibberellins in the plant, and the plant exhibits a semi-dwarf phenotype, thereby enhancing the plant's resistance to lodging. Overexpression of the GA20ox1 gene in corn increases the leaf length and plant height. The expression pattern of wheat GA20ox is different at different growth and development stages. TaGA20ox1 is highly expressed in leaves and stems in the early and rapid elongation stages of development, while TaGA20ox2 is highly expressed in mature tissues. Studies have found that the HvGA20ox2 gene is an important factor in regulating the plant height trait of the barley semi-dwarf mutant Risono.9265. The deletion of the DNA fragment of GA20ox2 leads to the appearance of its dwarf phenotype, and overexpression of HvGA20ox2 can increase the content of GAs, thereby restoring the plant to normal height; the internode length of transgenic Arabidopsis plants overexpressing sugarcane ScGA20ox1 is significantly higher than that of the wild type, indicating that overexpression of ScGA20ox1 can promote the elongation of sugarcane plant height; the castor GA20ox gene also plays an important role in regulating the height of the plant stem. The study found that the low expression level of GA20ox in dwarf castor varieties is an important cause of dwarfing. In fruit trees, the GA20ox gene is an important factor in regulating citrus dwarfing. Overexpression of the citrus GA20ox gene will increase plant height, while inhibition of the gene's expression will reduce plant height. The dwarfing of the pear dwarf mutant 'Zhongai 1' is mainly due to the decreased expression level of GA20ox, proving that the height of the plant is regulated by the expression level of GA20ox. In apple dwarfing rootstocks, due to the low activity of the GA20ox1 gene, the activity of GA20 oxidase is inhibited, the gibberellin content in the plant is low, and the dwarfing trait is exhibited. In watermelon, GA20ox is the main regulatory gene of the watermelon dwarf mutant dsh. Due to the mutation of the ClaGA20ox2 gene promoter, the active GA3 content is reduced, making the watermelon plant show dwarfing characteristics. In tomatoes, inhibiting the expression of SlGA20ox1 or SlGA20ox2 will lead to shortened stems of tomato plants and smaller and dark green leaves.

[0004] By comparing the expression levels of the GA20ox2 oxidase gene in plant materials with tall growth characteristics and dwarf growth characteristics, it is possible to predict whether the plant has dwarf growth characteristics, which saves time and effort. Currently, there are no reports on using the GA20ox2 oxidase gene to predict whether a mango variety has dwarf growth characteristics. Summary of the invention

[0005] The present invention provides a gene encoding GA20ox2 oxidase and application thereof, which can predict whether a mango variety has a 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 GA20ox2 oxidase, the nucleotide sequence of the gene is shown in SEQ ID No.1.

[0008] More specifically, the open reading frame of the gene is 1044 bp.

[0009] The second object of the present invention is to provide an application of the gene encoding GA20ox2 oxidase in early prediction of the dwarf traits of unknown mango species.

[0010] The third object of the present invention is to provide a GA20ox2 oxidase, wherein the GA20ox2 oxidase is encoded by the gene.

[0011] More specifically, the amino acid sequence of the GA20ox2 oxidase is shown in SEQ ID No.2.

[0012] More specifically, the GA20ox2 oxidase consists of 347 amino acids, has a molecular weight of 39.97 KD, and a theoretical isoelectric point of 6.13.

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

[0014] (1) The present invention obtains the GA20ox2 oxidase 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 GA20ox2 oxidase gene and the analysis of its corresponding amino acid sequence. The expression level of the GA20ox2 oxidase 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 1This is a schematic diagram of the homology comparison results between mango GA20ox2 oxidase gene and other plant GA20ox2 oxidase genes;

[0017] Figure 2 The amino acid composition diagram of mango GA20ox2 oxidase;

[0018] Figure 3 This is the signal peptide and transmembrane domain analysis diagram of mango GA20ox2 oxidase;

[0019] Figure 4 This is the predicted secondary structure of mango GA20ox2 oxidase;

[0020] Figure 5 This is the predicted tertiary structure of mango GA20ox2 oxidase;

[0021] Figure 6 The prediction diagram of hydrophilicity and hydrophobicity of mango GA20ox2 oxidase;

[0022] Figure 7 This is the phylogenetic tree of mango GA20ox2 oxidase;

[0023] Figure 8 This is a graph showing the expression levels of the GA20ox2 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 mango GA20ox2 oxidase 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 200 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 50 s, centrifuge it for 6-10 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_031283093.1), 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; 35 cycles of 95°C for 20 s, 58°C for 20 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 the physicochemical properties of the GA20ox2 gene and amino acid sequence

[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 GA20ox2 oxidase sequences of plants such as ryegrass, cotton, and Amomum villosum registered on NCBI, it was determined that the obtained gene was the mango GA20ox2 gene.

[0070] 7.2 Use protparam to analyze the GA20ox sequence and use PROSITE Scan to predict and analyze the conserved regions of the gene.

[0071] The open reading frame of GA20ox2 gene is 1044bp; GA20ox2 gene encodes 347 amino acids, with a molecular weight of 39.97KD and a theoretical isoelectric point of 6.13.

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

[0073] The amino acid composition of GA20ox2 oxidase is shown in Figure 2 .

[0074] Depend on Figure 2 It can be seen that the amino acid composition of GA20ox2 protein is mainly composed of serine, leucine, phenylalanine, glutamic acid, alanine and other amino acids, including 44 acidic amino acids (Asp+Glu) and 41 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 GA20ox2 oxidase is a non-cytoplasmic protein, and the proteins encoded by GA20ox2 have no signal peptide. The prediction results of the transmembrane domain of the GA20ox2 oxidase sequence show that GA20ox2 has no transmembrane region.

[0077] 7.5 Using DNAMAN to predict the secondary structure of GA20ox2 oxidase, the results are shown in Figure 4 .

[0078] Depend on Figure 4 It can be seen that in the protein encoded by GA20ox2, the helical structure accounts for 45.70%, the folding accounts for 8.47%, and the random coil accounts for 45.83%.

[0079] 7.6 Prediction of the tertiary structure of GA20ox2 oxidase. The prediction results are shown in Figure 5 .

[0080] Depend on Figure 5 It can be seen that GA20ox2 oxidase 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 GA20ox2, the prediction results are shown in Figure 6 .

[0082] Depend on Figure 6 It can be seen that the arginine at position 247 of the GA20ox2 polypeptide chain has the lowest value of -2.94 and is the most hydrophilic, while the leucine at position 347 has the highest value of 4.03 and is the most hydrophobic.

[0083] 7.8 In order to study the evolution of Mango GA20ox2 oxidase, a phylogenetic tree of the amino acid sequence of GA20ox2 oxidase was constructed using MEGA7.0. The results are shown in Figure 7 .

[0084] Depend on Figure 7 It can be seen that mango GA20ox2 is closely related to pistachio GA20 (XP_031283093.1); followed by China neem (KAJ4726140.1), Wenzhou mandarin (GAY52177.1), clementine (XP_006452181.1), sweet orange (KAH9651410.1), cassava (XP_021593735.1), European cork oak (XP_023876953.1), rubber wood (KAJ9153719.1), castor (XP_002522843.1), and Acer truncatum (TXG67388.1).

[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 GA20ox2 gene in different tree varieties and dwarf varieties at different stages. Among them, the tree varieties are Jinhuang Mango, Mache Mango and Red Ivory; 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 stage for analysis.

[0087] Real-time fluorescence quantitative PCR was performed using the SYBR GREEN dye method and the mango actin gene 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 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.

[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: 95°C for 5 min, 1 cycle; 94°C for 10 s, 60°C for 30 s, 40 cycles.

[0103] Melting curve: 95℃15s, 60℃60s, 95℃15s, continuous detection signal.

[0104] Results Figure 8 .

[0105] Depend on Figure 8It can be seen that in the five key phenological periods of mango, the expression levels of GA20ox2 gene in three dwarf mango varieties and three arbor mango varieties are significantly different. The GA20ox2 gene in dwarf mango varieties shows a trend of first rising and then falling in the five key phenological periods of mango. The highest expression level can reach 15 in the 7-8 weeks of fruit setting, and even the lowest expression level of GA20ox2 gene in the fruit ripening period is above 8; while the expression level of GA20ox2 gene in the three arbor mango varieties not only has no obvious changes among the dwarf varieties, but also has no obvious changes in different phenological periods. The expression level of GA20ox2 gene in the three arbor mango varieties is about 1, and the highest does not exceed 2. Therefore, the expression of GA20ox2 gene can be used as an early predictive indicator of whether the unknown mango species has dwarf traits.

[0106] In summary, the present invention can provide support for early prediction of whether the test material has dwarf traits by measuring the expression amount of GA20ox2 gene through fluorescence quantitative analysis.

[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 GA20ox2 oxidase, characterized in that The nucleotide sequence of the gene is shown in SEQ ID No.

1.

2. The gene encoding GA20ox2 oxidase according to claim 1, characterized in that The open reading frame of the gene is 1044 bp.

3. Use of the gene encoding GA20ox2 oxidase according to claim 1 in early prediction of dwarf traits of unknown mango species.

4. A GA20ox2 oxidase, characterized in that The GA20ox2 oxidase is encoded by the gene according to claim 1.

5. The GA20ox2 oxidase according to claim 4, characterized in that The amino acid sequence of the GA20ox2 oxidase is shown in SEQ ID No.

2.

6. The GA20ox2 oxidase according to claim 4, characterized in that The GA20ox2 oxidase consists of 347 amino acids, has a molecular weight of 39.97 KD, and a theoretical isoelectric point of 6.13.

Images