Corn gibberellin 13-hydroxylase gene ZmGA13ox1 and application thereof

Abstract

The invention is suitable for the technical fields of molecular biology and biology, and provides a corn gibberellin 13-hydroxylase gene ZmGA13ox1 and application thereof. The nucleotide sequence of the corn gibberellin 13-hydroxylase gene ZmGA13ox1 is as shown in SEQ ID NO: 1 in a sequence table. The corn gibberellin 13-hydroxylase protein is coded by a corn gibberellin 13-hydroxylase gene ZmGA13ox1, and the amino acid sequence of the corn gibberellin 13-hydroxylase protein is shown as SEQ ID NO: 2 in a sequence table. The invention provides a nucleotide sequence of a ZmGA13ox1 gene in a corn inbred line B73 and amino acid sequence information of an encoded protein of the ZmGA13ox1 gene, and a plant expression vector containing the gene is introduced into arabidopsis thaliana by utilizing an agrobacterium-mediated transformation technology to cultivate a homozygous T3-generation transgenic arabidopsis thaliana plant. Experimental results show that overexpression of the ZmGA13ox1 gene in arabidopsis thaliana has a promoting effect on root growth of the arabidopsis thaliana.

Description

Technical Field

[0001] The present invention belongs to the fields of molecular biology and biotechnology, and particularly relates to the maize gibberellin 13-hydroxylase gene ZmGA13ox1 and its applications. Background Art

[0002] Improving the root length of crops has multiple practical significances for agricultural production: 1. Enhancing stress resistance and stability. The improved root system is more developed (the root length increases by more than 30%), which can significantly improve the adaptability of plants to adverse environments such as drought and high temperature. The deep roots can absorb the deep soil water and reduce the impact of drought stress. 2. Increasing yield and resource utilization efficiency. Root system optimization can expand the nutrient absorption range and improve the utilization rates of key elements such as nitrogen, phosphorus, and potassium. Research shows that the combination of close planting technology and root system improvement technology can increase the maize yield per unit area by 20%-30%. In addition, the developed root system promotes the transportation of photosynthetic products to grains, reduces the bald tip phenomenon, and increases the 1000-grain weight. 3. Promoting soil health and ecological protection. After the roots penetrate deep into the soil, they can effectively fix the soil and reduce soil erosion. At the same time, their secretions can improve the community structure of soil microorganisms and enhance the accumulation of soil organic matter. By optimizing the root architecture, the amount of chemical fertilizers used can be reduced, and the risk of agricultural non-point source pollution can be lowered. It can be seen that improving the root length of crops not only directly improves crop productivity, but also has far-reaching significance for efficient resource utilization, ecological environment protection, and sustainable agricultural development.

[0003] GA13ox (gibberellin 13-hydroxylase) is one of the key enzymes in the biosynthesis of gibberellins (GAs) and belongs to the 2-oxoglutarate-dependent dioxygenase family (2-OGD). Its main function is to catalyze the gibberellin precursor GA12 into GA53, initiating the 13-hydroxylation pathway. In plants, the gibberellin synthesis pathway is divided into the non-13-hydroxylation pathway (generating GA4 and GA7) and the 13-hydroxylation pathway (generating GA1 and GA3). The presence or absence of GA13ox determines which pathway the plant adopts. For example, in plants such as rice, the GA13ox activity is relatively high, and GA53 is mainly generated through the 13-hydroxylation pathway; while in pumpkins, Arabidopsis thaliana, etc., they tend to adopt the non-13-hydroxylation pathway.

[0004] The encoding gene of GA13ox belongs to a multi-gene family, and its expression is regulated by developmental stages, environmental signals (such as photoperiod, temperature) and endogenous hormone feedback. GA13ox affects multiple physiological processes by regulating the ratio of active gibberellins. A decrease in GA13ox activity leads to a reduction in the products of the 13-hydroxylation pathway and an increase in the proportion of GA4 in the non-13-hydroxylation pathway, thereby affecting stem elongation; GA13ox can also participate in regulating the activity of α-amylase in seeds and affect seed germination; the high expression of GA13ox is also related to the gibberellin peak at the initial stage of bolting and promotes reproductive growth. Although GA13ox plays an important role in the above processes, there is currently no research report on directly improving plant root length by using the GA13ox gene. Therefore, the present invention provides the maize gibberellin 13-hydroxylase gene ZmGA13ox1 and its application. Summary of the Invention

[0005] The purpose of the present invention is to provide the maize gibberellin 13-hydroxylase gene ZmGA13ox1 and its application, aiming to solve the problems raised in the above background technology.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The maize gibberellin 13-hydroxylase gene ZmGA13ox1 has an ORF full length of 1539 bp, with the start codon being ATG and the stop codon being TAG, and its nucleotide sequence is as shown in SEQ ID NO:1 in the sequence listing.

[0008] The maize gibberellin 13-hydroxylase protein is encoded by the maize gibberellin 13-hydroxylase gene ZmGA13ox1 as described above, and its amino acid sequence is as shown in SEQ ID NO:2 in the sequence listing.

[0009] The recombinant plant expression vector contains the maize gibberellin 13-hydroxylase gene ZmGA13ox1 as described above.

[0010] The application of the maize gibberellin 13-hydroxylase gene ZmGA13ox1 as described above or the maize gibberellin 13-hydroxylase protein as described above in improving the root length of Arabidopsis thaliana.

[0011] Compared with the prior art, the beneficial effects of the present invention are:

[0012] The present invention not only provides the nucleotide sequence of the ZmGA13ox1 gene in the maize inbred line B73 and the amino acid sequence information of the protein encoded by it, but also uses the Agrobacterium-mediated transformation technology to introduce the plant expression vector containing this gene into Arabidopsis thaliana to cultivate homozygous T 3The first generation of transgenic Arabidopsis plants. By observing the phenotypes of transgenic Arabidopsis, it was found that compared with wild-type Arabidopsis, the root length of transgenic Arabidopsis increased significantly. This result indicates that the overexpression of the ZmGA13ox1 gene in Arabidopsis promotes its root growth, providing valuable gene resources and theoretical basis for future crop genetic improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is a multiple sequence comparison diagram of maize ZmGA13ox1 and GA13ox amino acids of other plants; among them: OsGA13ox1 is rice; GmGA13ox1 is soybean.

[0014] Figure 2 It is a schematic diagram of the phylogenetic tree analysis of GA13ox genes; among them: OsGA13ox genes are rice; AtGA2ox genes are Arabidopsis; GmGA13ox genes are soybean; BnGA13ox is rapeseed; SlGA13ox genes are tomato; TwGA13ox genes are Tripterygium wilfordii.

[0015] Figure 3 It is a comparison diagram of the growth of Arabidopsis; among them: a is the comparison diagram of the rosette leaf growth of the T generation materials of Arabidopsis overexpressing the ZmGA13ox1 gene (two plants, namely Arabidopsis overexpressing ZmGA13ox1 - 1 and Arabidopsis overexpressing ZmGA13ox1 - 2) and wild-type materials; 3 b is the comparison diagram of the root length of the T generation materials of Arabidopsis overexpressing the ZmGA13ox1 gene and wild-type materials. 3 3 DETAILED DESCRIPTION OF THE INVENTION

[0016] For a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the technical solutions of the present invention are described in detail below, but it should not be construed as a limitation on the implementable scope of the present invention.

[0017] The following describes the specific implementation of the present invention in detail with reference to specific embodiments.

[0018] Example 1: Cloning of gibberellin 13-hydroxylase gene ZmGA13ox1.

[0019] 1. Extraction of RNA.

[0020] The maize inbred line B73 was cultured until the three-leaf stage, and the total RNA of the leaves was extracted using the UltraPure RNA Extraction Kit (CW0581) from ComWin Biotech. The specific steps are as follows:

[0021] (1) Take fresh maize leaves and grind them thoroughly in liquid nitrogen. Add 1 ml of TRIzon reagent to every 30 - 50 mg of tissue and mix well.

[0022] (2) Gently invert the mixed sample several times and let it stand at room temperature for 5 min to fully lyse the sample.

[0023] (3) Add 200 μl of chloroform, cover the centrifuge tube cap, shake vigorously for 15 s, and let it stand at room temperature for 2 min.

[0024] (4) Centrifuge at 12,000 rpm for 10 min at 4 °C, aspirate 550 μl of the upper aqueous phase, and transfer the upper aqueous phase to a new RNase-Free centrifuge tube.

[0025] (5) Add 550 μl of 70% ethanol (prepared with RNase-Free water) to the aqueous solution and invert to mix well.

[0026] (6) Add all the solution obtained in the previous step to the adsorption column (Spin Columns RM) placed in the collection tube. If the solution cannot be added all at once, it can be transferred in multiple portions. Centrifuge at 12,000 rpm for 20 s, pour out the waste liquid in the collection tube, and place the adsorption column back into the collection tube.

[0027] (7) Add 700 μl of Buffer RW1 to the adsorption column, centrifuge at 12,000 rpm for 20 s, pour out the waste liquid in the collection tube, and place the adsorption column back into the collection tube.

[0028] (8) Add 500 μl of Buffer RW2 (check whether absolute ethanol has been added before use) to the adsorption column, centrifuge at 12,000 rpm for 20 s, pour out the waste liquid in the collection tube, and place the adsorption column back into the collection tube.

[0029] (9) Repeat step (8).

[0030] (10) Centrifuge at 12,000 rpm for 2 min, pour out the waste liquid in the collection tube. Place the adsorption column at room temperature for several minutes to completely dry.

[0031] (11) Place the adsorption column in a new RNase-Free centrifuge tube, add 30 - 50 μl of RNase-Free Water to the middle part of the adsorption column, let it stand at room temperature for 1 min, centrifuge at 12,000 rpm for 1 min, collect the RNA solution, and store the RNA at -80 °C to prevent degradation.

[0032] 2. Reverse transcription.

[0033] Use PrimeScript TM RT reagent Kit with gDNA Eraser (RR047A) from Takara Bio Inc. to perform reverse transcription on the extracted RNA.

[0034] The reaction system for removing gDNA is shown in Table 1:

[0035] Table 1. Reaction system for removing gDNA

[0036] Reagent Volume 5×gDNA Eraser Buffer 2 μl gDNA Eraser 1 μl Total RNA 1 μg RNase Free Water up to 10 μl

[0037] After mixing the premix, incubate at 42 °C for 2 min first, and then keep at 4 °C for 5 min.

[0038] The reverse transcription reaction uses the SYBR Green qPCR method. The specific reverse transcription reaction system is shown in Table 2:

[0039] Table 2. Reverse transcription reaction system

[0040]

[0041]

[0042] Put the whole system into a PCR instrument, and the program is set as: 37 °C, 15 min; 85 °C, 5 s.

[0043] 3. Amplification of the full-length ORF of the ZmGA13ox1 gene.

[0044] According to the ORF gene sequences of the GA13ox1 genes of other species published by NCBI, retrieve the similar sequence information in the maize genome, and use the bioinformatics software Primer 5.0 (following the primer design principles) to design the specific cloning primers for this gene:

[0045] ZmGA13ox1-FP: 5’-ATGGAGACGACGGAGATGGCGGCGGC-3’ (as shown in SEQ ID NO:3 in the sequence listing);

[0046] ZmGA13ox1-RP: 5’-CTAGCTAGCACCCCTGGGCTCCACGT-3’ (as shown in SEQ ID NO:4 in the sequence listing).

[0047] Using the reverse-transcribed cDNA as a template, perform PCR amplification using the high-fidelity heat-resistant DNA polymerase PrimeSTAR GXL DNA polymerase. The PCR reaction system is shown in Table 3, and the PCR reaction program is shown in Table 4. If the cDNA concentration is too low, the amount of cDNA can be appropriately increased. If the target band of the first PCR is too light, the product can be recovered and the PCR amplification of the same steps can be performed again.

[0048] Table 3. PCR reaction system

[0049]

[0050]

[0051] Table 4. PCR reaction program

[0052]

[0053] 4. Recovery and site-directed mutagenesis of the DNA fragment of ZmGA13ox1.

[0054] Use the SanPrep Column DNA Gel Extraction Kit from Sangon Biotech to recover the target fragment of ZmGA13ox1 in the PCR product. The specific steps are as follows:

[0055] (1) After electrophoresis, cut out the gel block containing the target fragment of ZmGA13ox1, weigh it, and place it in a 1.5 ml centrifuge tube. According to the weight of the gel block, add 300 μl of Buffer B2 for every about 100 mg of gel.

[0056] (2) Put the centrifuge tube into a 50 °C metal bath for 10 min. During this period, the centrifuge tube can be inverted several times to mix the melted liquid and the unmelted gel block to accelerate melting.

[0057] (3) Place the obtained solution in an adsorption column and centrifuge at 8000 rpm for 30 s. If the total volume of the solution is greater than 750 μl, add 750 μl each time and repeat the operation multiple times.

[0058] (4) Add 300 μl of Buffer B2 to the adsorption column, set the rotation speed to 9000 rpm, centrifuge for 30 s, and pour out the waste liquid.

[0059] (5) Add 500 μl of Buffer B2 to the adsorption column, set the rotation speed to 9000 rpm, centrifuge for 30 s, and pour out the waste liquid. Repeat once more.

[0060] (6) Put the empty adsorption column and the collection tube together into the centrifuge and centrifuge at 9000 rpm for 60 s. Take a new 1.5 mL centrifuge tube, place the adsorption column in it, and let it stand for 10 min.

[0061] (7) Add 30 μl of TE buffer or ddH 2 O to the center of the adsorption membrane, let it stand at room temperature for 2 min, centrifuge at 9000 rpm for 60 s, collect the obtained DNA solution, and store it at -20 °C for later use.

[0062] 5. Ligate ZmGA13ox1 into the pMD18-T Vector.

[0063] The target fragment was ligated with the pMD18-T vector using TaKaRa's DNA A-Tailing Kit and pMD18-T Vector Cloning Kit to obtain a recombinant vector for gene sequencing.

[0064] (1) Perform an A-Tailing reaction on the 3' ends of the three target fragment gel-extracted DNA fragments.

[0065] ① Prepare the ligation reaction system as shown in Table 5 in a 200 μl centrifuge tube:

[0066] Table 5. Ligation reaction system

[0067] Component Volume 10×A-Tailing Buffer 5 μl dNTP Mixture 4 μl A-Tailing Enzyme 0.5 μl ZmGA13ox1 Recovery Segment 25 μl <![CDATA[ddH 2 O]]> 15.5 μl

[0068] ② React at 72 °C for 20 min.

[0069] ③ Incubate on ice for 2 min.

[0070] (2) After completing the A-Tailing reaction, ligate the DNA fragment with an A tail to the pMD18-T vector. The pMD18-T ligation reaction system is shown in Table 6:

[0071] Table 6. pMD18-T ligation reaction system

[0072] Component Volume pMD18-T Vector 1 μl A-Tailing ZmGA13ox1 DNA 4 μl Solution I 5 μl

[0073] Reaction program: React at 16 °C for 1 h.

[0074] 6. Transformation of Top10 Escherichia coli competent cells and PCR detection.

[0075] (1) Place 50 μl of Top10 Escherichia coli competent cells on ice to thaw.

[0076] (2) Use a pipette to aspirate 5 μl of the ligation product or recombinant plasmid and add it to 50 μl of Top10 Escherichia coli competent cells.

[0077] (3) After ice-bathing for 30 min, perform heat shock at 42 °C for 90 s and then ice-bathe for 5 min. Subsequently, add 800 μl of LB liquid medium.

[0078] (4) Incubate with shaking at 37 °C for 1 h, centrifuge at 8000 rpm for 5 min. Discard the supernatant, leaving approximately 50 μl of medium in the centrifuge tube. After pipetting and mixing evenly, spread it on an LB solid medium containing the corresponding antibiotic, incubate at 37 °C, inverted, for 12 - 16 h.

[0079] (5) Pick a single colony and inoculate it into 800 μl of LB liquid medium containing the corresponding antibiotic. Place the centrifuge tube in a shaker and incubate it at 37°C with a shaking speed of 180 rpm for about 10 h.

[0080] (6) Use Ex Taq enzyme to perform PCR molecular detection on the bacterial solution. Amplify and shake the bacterial solution with a positive PCR result, add 15% glycerol to the bacterial solution, send it to BGI for sequencing, and store the original bacterial solution in a -80°C refrigerator.

[0081] Example 2: Bioinformatics analysis of maize genes.

[0082] The ZmGA13ox1 gene encodes a maize gibberellin oxidase, and its open reading frame contains 1539 bp of nucleotides, encoding 512 amino acids.

[0083] Use the TMHMM Server v.2.0 online website to preliminarily predict the transmembrane region of this protein, and it is found that no transmembrane domain appears in this protein. It is preliminarily speculated that ZmGA13ox1 is a non-transmembrane protein.

[0084] As Figure 1 and Figure 2 shown, the maize gibberellin 13-hydroxylase ZmGA13ox1 protein belongs to the 2-oxoglutarate-dependent dioxygenase family (2-OGD). By performing phylogenetic tree analysis on GA13ox proteins in different species such as maize, rice, soybean, Arabidopsis thaliana, tomato, rapeseed, and Tripterygium wilfordii, it is found that they have several branches, indicating that the functions of these proteins may be different. Among them, ZmGA13ox1 has the closest genetic relationship with OsGA13ox1 (rice). GA13ox can affect multiple physiological processes such as plant stem elongation, seed germination, and bolting by regulating the ratio of active gibberellins, but there is no report on improving plant root length using the GA13ox gene.

[0085] Example 3: Construction of a plant expression vector for the ZmGA13ox1 gene.

[0086] 1. According to the relevant information of the multiple cloning site of the intermediate vector pCHF3300, add the corresponding restriction enzyme sites when designing primers to amplify the ZmGA13ox1 gene.

[0087] 2. After amplifying the gene using a high-fidelity enzyme, ensure the correctness of the expressed gene through a series of steps including recovery, adding "A", ligating to a T vector, transforming Escherichia coli, PCR restriction enzyme digestion identification, and sequencing, including a complete open reading frame and no mismatches or frameshift phenomena.

[0088] 3. For genes with correct sequencing, use the designed restriction sites for digestion. In this example, restriction endonucleases Xho I and Eco RI are selected to cut the correctly sequenced target fragment from the intermediate vector. The digestion system is shown in Table 7. The digestion reaction is carried out in a water bath at 37 °C for 1 h, and the digested product is electrophoretically recovered to obtain small fragments (genes).

[0089] Table 7. Digestion system

[0090] Component Volume Xho I 2.5 μl Eco RI 2.5 μl 10×T Buffer 5 μl 0.1%BSA 5 μl DNA or vector 20 μl Sterilized Water 15 μl

[0091] 4. Use the same two restriction endonucleases Xho I and Eco RI to digest the expression vector part-cam-flag, and recover the large fragment (vector).

[0092] 5. Use DNA ligase to ligate the recovered large fragment with the small fragment to recombine into a plant expression vector of the ZmGA13oc1 gene. The ligation reaction is carried out at 16 °C for 3 h, and the ligation system is shown in Table 8:

[0093] Table 8. Ligation system

[0094] Component Volume Target Fragment 5.5 μl Vector Large Fragment 2.5 μl 10×T4 ligase buffer 1 μl T4 DNA ligase 1 μl

[0095] 6. After completion of ligation, transform the recombinant vector into Escherichia coli competent cells, and then extract the plasmid for PCR and digestion identification. Ensure that the target gene has been successfully inserted into the expression vector and maintains the correct reading frame and sequence integrity.

[0096] Example 4: Obtaining and molecular detection of ZmGA13ox1 transgenic Arabidopsis thaliana.

[0097] Use the floral dip method to transform Arabidopsis thaliana with the gene. The specific steps are as follows:

[0098] 1. Invert the flowering Arabidopsis thaliana so that the flower buds face down, and immerse it in the Agrobacterium tumefaciens solution containing the target plasmid for 2 min.

[0099] 2. Lay the transformed Arabidopsis thaliana plants flat, cover them with plastic wrap, grow them under low light intensity for 24 h, and then place them under normal light conditions for cultivation. Repeat the above infection step after one week.

[0100] 3. The transformed Arabidopsis thaliana plants can grow and flower normally. When the siliques are completely yellow and about to crack, the seeds can be harvested.

[0101] 4. Some of the harvested T 0 -generation seeds are screened by kanamycin and identified by PCR to obtain T 1 -generation transgenic plants. After two generations of selfing, T 3Arabidopsis thaliana plants can be used for subsequent phenotypic screening.

[0102] Example 5: T 3 Measurement of the root growth of Arabidopsis thaliana plants transformed with the ZmGA13ox1 gene.

[0103] Randomly sow the seeds of Arabidopsis thaliana wild type and the T 3 generation seeds of the transgenic Arabidopsis thaliana of the present invention, and transfer them to a light condition of 22 °C for cultivation, and observe and record the morphology and growth of the plants. The results show that there is no obvious change in the plant morphology of the progeny materials of Arabidopsis thaliana overexpressing the ZmGA13ox1 gene. The phenotypes of the two transgenic materials overexpressing the ZmGA13ox1 gene are not severely affected, and there is no significant difference in the rosette leaves compared with the wild type, and they can develop and set seeds normally ( Figure 3 as shown in A). However, it was observed on the MS medium that the root length of Arabidopsis thaliana overexpressing the ZmGA13ox1 gene was significantly longer than that of the wild type ( Figure 3 as shown in B). This result indicates that the overexpression of the ZmGA13ox1 gene significantly affects the biosynthesis of GA in the roots of Arabidopsis thaliana, and further affects the root phenotype of Arabidopsis thaliana.

[0104] In summary, the present invention obtains a gibberellin 13-hydroxylase gene from maize, named ZmGA13ox1. Through the Agrobacterium-mediated transformation method, the plant expression vector containing ZmGA13ox1 was successfully transformed into Arabidopsis thaliana, and homozygous T 3 generation transgenic Arabidopsis thaliana plants were obtained. The results of observing the growth of the T 3 generation transgenic Arabidopsis thaliana plants show that compared with wild-type Arabidopsis thaliana, the root morphology of the transgenic Arabidopsis thaliana plants has changed significantly. Specifically, the root length of the transgenic Arabidopsis thaliana has increased significantly, indicating that the overexpression of the ZmGA13ox1 gene in Arabidopsis thaliana can significantly affect its root growth.

[0105] The above is only the preferred embodiment of the present invention. It should be noted that for those skilled in the art, without departing from the concept of the present invention, several deformations and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicability of the patent.

Claims

1. A maize gibberellin 13-hydroxylase gene ZmGA13ox1, characterized in that: The nucleotide sequence is shown in SEQ ID NO: 1 in the sequence listing.

2. A corn gibberellin 13-hydroxylase protein, characterized in that: It is encoded by the maize gibberellin 13-hydroxylase gene ZmGA13ox1 as claimed in claim 1, and its amino acid sequence is shown in SEQ ID NO: 2 in the sequence list.

3. A recombinant plant expression vector, characterized in that: Contains the maize gibberellin 13-hydroxylase gene ZmGA13ox1 as claimed in claim 1.

4. Use of the maize gibberellin 13-hydroxylase gene ZmGA13ox1 according to claim 1 or the maize gibberellin 13-hydroxylase protein according to claim 2 in improving the root length of Arabidopsis thaliana.

Images