Gibberellin 13-hydroxylase gene ZmGA13ox1 and application thereof

Abstract

The present application is suitable for the field of molecular biology and biotechnology, and provides a maize gibberellin 13-hydroxylase gene ZmGA13ox1 and application thereof. The maize gibberellin 13-hydroxylase gene ZmGA13ox1 has a nucleotide sequence shown as SEQ ID NO:1 in the sequence listing. The maize gibberellin 13-hydroxylase protein is encoded by the maize gibberellin 13-hydroxylase gene ZmGA13ox1, and has an amino acid sequence shown as SEQ ID NO:2 in the sequence listing. The present application provides the nucleotide sequence of the ZmGA13ox1 gene in the maize inbred line B73 and the amino acid sequence information of the protein encoded thereby, and uses the agrobacterium-mediated transformation technology to introduce a plant expression vector containing the gene into Arabidopsis thaliana, and cultivates a homozygous T3 generation transgenic Arabidopsis thaliana plant. The experimental results show that the overexpression of the ZmGA13ox1 gene in Arabidopsis thaliana has a promoting effect on the root growth.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology and biotechnology, and particularly relates to a maize gibberellin 13-hydroxylase gene ZmGA13ox1 and application thereof. BACKGROUND

[0002] Improving the root length of crops has multiple practical significance for agricultural production: 1. Enhancing stress resistance and stability. The improved root system is more developed (root length increased by more than 30%), which can significantly improve the adaptability of plants to drought, high temperature and other stress. Deep root system can absorb soil deep water, reducing the impact of drought stress. 2. Improving yield and resource utilization efficiency. Root optimization can expand the range of nutrient absorption and improve the utilization rate of key elements such as nitrogen, phosphorus and potassium. Studies have shown that the combination of dense planting technology and root system improvement technology can increase corn yield by 20%-30%. In addition, the developed root system promotes the translocation of photosynthetic products to the grain, reduces the bare tip phenomenon, and improves the thousand-grain weight. 3. Promoting soil health and ecological protection. The root system can effectively fix soil and reduce soil erosion after deepening into the soil, and its secretions can also improve the community structure of soil microorganisms and enhance soil organic matter accumulation. Through root configuration optimization, the amount of chemical fertilizer can be reduced, and the risk of agricultural non-point source pollution can be reduced. It can be seen that crop root length improvement not only directly improves crop productivity, but also has far-reaching significance for resource efficient use, ecological environment protection and agricultural sustainable development.

[0003] GA13ox (gibberellin 13-hydroxylase) is one of the key enzymes in gibberellin (GAs) biosynthesis, belonging to the 2-oxoglutarate-dependent dioxygenase family (2-OGD). Its main function is to catalyze the gibberellin precursor GA12 to GA53, opening the 13-hydroxylation pathway. In plants, the synthesis pathway of gibberellin is divided into non-13-hydroxylation pathway (generating GA4, GA7) and 13-hydroxylation pathway (generating GA1, GA3). Whether GA13ox exists or not determines which path the plant adopts, for example, in rice and other plants, GA13ox has high activity, mainly through the 13-hydroxylation pathway to generate GA53; while pumpkin, Arabidopsis and other plants tend to non-13-hydroxylation pathway.

[0004] The coding 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 proportion of active gibberellins. The decrease of GA13ox activity leads to the decrease of 13-hydroxylation pathway products, and the increase of the proportion of GA4 of non-13-hydroxylation pathway, thereby affecting stem elongation; GA13ox can also participate in the regulation of alpha-amylase activity in seeds, affecting seed germination; the high expression of GA13ox is also related to the gibberellin peak at the early stage of bolting, and promotes reproductive growth. Although GA13ox plays an important role in the above processes, there is no research report on directly improving the root length of plants by using GA13ox gene. Therefore, the present application provides a corn gibberellin 13-hydroxylase gene ZmGA13ox1 and its application. SUMMARY

[0005] The purpose of the present application is to provide a corn gibberellin 13-hydroxylase gene ZmGA13ox1 and its application, which aims to solve the problems raised in the background art.

[0006] The purpose of the present application is achieved by the following technical solutions:

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

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

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

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

[0011] Compared with the prior art, the present application has the following beneficial effects:

[0012] The present application not only provides the nucleotide sequence of the ZmGA13ox1 gene in the corn inbred line B73 and the amino acid sequence information of the protein encoded by the ZmGA13ox1 gene, but also uses the Agrobacterium-mediated transformation technology to introduce a plant expression vector containing the gene into Arabidopsis thaliana, and cultivate a homozygous T3 generation transgenic Arabidopsis thaliana plant. By observing the phenotype of the transgenic Arabidopsis thaliana, it is found that the root length of the transgenic Arabidopsis thaliana is significantly increased compared with the wild type Arabidopsis thaliana, which shows that the overexpression of the ZmGA13ox1 gene in Arabidopsis thaliana has a promoting effect on the root growth, and provides valuable gene resources and theoretical basis for future crop genetic improvement. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 Figure 3 is a multiple sequence comparison diagram of the amino acids of the corn ZmGA13ox1 and other plant GA13oxs; wherein: OsGA13ox1 is rice; GmGA13ox1 is soybean.

[0014] Figure 2 Figure 4 is a schematic diagram of the phylogenetic tree analysis of the GA13ox class genes; wherein: OsGA13ox class is rice; AtGA2ox class is Arabidopsis thaliana; GmGA13ox class is soybean; BnGA13ox is rape; SlGA13ox class is tomato; TwGA13ox class is Tripterygium wilfordii.

[0015] Figure 3 Figure 5 is a comparison diagram of the growth of Arabidopsis thaliana; wherein: a is a comparison diagram of the growth of the T3 generation material of the Arabidopsis thaliana overexpressing the ZmGA13ox1 gene (two plants, respectively, ZmGA13ox1 overexpression Arabidopsis thaliana-1 and ZmGA13ox1 overexpression Arabidopsis thaliana-2) and the wild type material rosette leaf; b is a comparison diagram of the root length of the T3 generation material of the Arabidopsis thaliana overexpressing the ZmGA13ox1 gene and the wild type material. DETAILED DESCRIPTION

[0016] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present application, the technical solutions of the present application will be described in detail below, but it cannot be understood as limiting the scope of the implementation of the present application.

[0017] The specific implementation of the present application will be described in detail below in combination with specific examples.

[0018] Example 1: Cloning of Gibberellin 13-hydroxyase gene ZmGA13ox1

[0019] 1. Extraction of RNA

[0020] The corn inbred line B73 was cultured to the three-leaf stage, and the total RNA of the leaf was extracted using the ultrapure RNA extraction kit (CW0581) of Kangweishiji. The specific steps are as follows:

[0021] (1) Take fresh corn leaves and grind them thoroughly in liquid nitrogen. Add 1 ml 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 allow the sample to be fully lysed.

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

[0024] (4) Centrifuge at 12000 rpm for 10 min at 4°C. Take 550 μl of the upper aqueous phase and transfer it to a new RNase-Free centrifuge tube.

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

[0026] (6) Add the solution obtained in the previous step to the Spin Columns RM loaded in the collection tube. If the solution cannot be added at one time, it can be added in several times. Centrifuge at 12000 rpm for 20 s, discard the waste in the collection tube, and place the Spin Columns RM back in the collection tube.

[0027] (7) Add 700 μl Buffer RW1 to the Spin Columns RM, centrifuge at 12000 rpm for 20 s, discard the waste in the collection tube, and place the Spin Columns RM back in the collection tube.

[0028] (8) Add 500 μl Buffer RW2 (check whether anhydrous ethanol has been added before use) to the Spin Columns RM, centrifuge at 12000 rpm for 20 s, discard the waste in the collection tube, and place the Spin Columns RM back in the collection tube.

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

[0030] (10) Centrifuge at 12000 rpm for 2 min, discard the waste in the collection tube. Let the Spin Columns RM stand at room temperature for several minutes and dry them completely.

[0031] (11) Place the Spin Columns RM in a new RNase-Free centrifuge tube, add 30-50 μl RNase-Free Water to the middle part of the Spin Columns RM, let it stand at room temperature for 1 min, centrifuge at 12000 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 Reverse Transcriptase from Takara Bio Inc. TMThe extracted RNA was reverse transcribed using the RT reagent kit with gDNA Eraser (RR047A).

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

[0035] Table 1. gDNA removal reaction system

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

[0037] After mixing the premixed solution, incubate it at 42°C for 2 minutes, and then keep it at 4°C for 5 minutes.

[0038] The reverse transcription reaction was performed using 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] The entire system was placed in a PCR instrument, and the program was set as follows: 37℃, 15 min; 85℃, 5 s.

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

[0044] Based on the ORF gene sequences of the GA13ox1 gene in other species published by NCBI, similar sequence information was retrieved from the maize genome, and specific cloning primers for this gene were designed using the bioinformatics software Primer 5.0 (following primer design principles).

[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 cDNA obtained from reverse transcription as a template, PCR amplification was performed using the high-fidelity, thermostable DNA polymerase PrimeSTAR GXL. The PCR reaction system is shown in Table 3, and the PCR reaction procedure is shown in Table 4. If the cDNA concentration is too low, the amount of cDNA can be increased appropriately. If the target band is too faint in the first PCR, the product can be recovered and the same PCR amplification steps can be performed again.

[0048] Table 3. PCR reaction system

[0049]

[0050]

[0051] Table 4. PCR reaction program

[0052]

[0053] 4. DNA fragment recovery and point mutation of ZmGA13ox1.

[0054] The SanPrep column DNA gel recovery kit of Shengwo was used to recover the ZmGA13ox1 target fragment in the PCR product. The specific steps are as follows:

[0055] (1) After electrophoresis, the gel block containing the ZmGA13ox1 target fragment was cut and weighed, and placed in a 1.5 ml centrifuge tube. According to the weight of the gel block, about 100 mg of gel corresponds to the addition of 300 μl Buffer B2.

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

[0057] (3) The obtained solution was placed in the adsorption column and centrifuged at 8000 rpm for 30 s. If the total volume of the solution is greater than 750 μl, 750 μl is added each time, and the operation is repeated multiple times.

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

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

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

[0061] (7) Add 30 ul TE buffer or ddH2O to the center of the adsorption membrane, stand at room temperature for 2 min, centrifuge at 9000 rpm for 60 s, collect the obtained DNA solution, and store at -20℃ for standby.

[0062] 5. ZmGA13ox1 is connected into pMD18-T Vector.

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

[0064] (1) The 3' end of the gel-recovered DNA fragment of the three target fragments was subjected to "A" tailing reaction (A-Tailing).

[0065] ① The ligation reaction system shown in Table 5 was prepared in a 200 μl centrifuge tube:

[0066] Table 5. Ligation reaction system

[0067] Component Volume 10x A-Tailing Buffer 5 μl dNTP Mixture 4 μl A-Tailing Enzyme 0.5 μl ZmGA13ox1 recovery segment 25 μl ddH2O 15.5 μl

[0068] ② Reaction at 72°C for 20 min.

[0069] ③ Stand in ice for 2 min.

[0070] (2) After the A-Tailing reaction was completed, the DNA fragment with A tail was ligated with 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 procedure: reaction at 16°C for 1 h.

[0074] 6. Top10 E. coli competent transformation and PCR detection.

[0075] (1) 50 μl of TOP10 E. coli competent was thawed on ice.

[0076] (2) 5 μl of ligation product or recombinant plasmid was taken with a pipette and added to 50 μl of TOP10 E. coli competent.

[0077] (3) After ice bath for 30 min, heat shock at 42°C for 90 s, ice bath for 5 min. Then add 800 μl of LB liquid medium.

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

[0079] (5) Pick single colony into 800 μl LB liquid medium containing corresponding antibiotics, put the centrifuge tube into the shaker, 37℃, 180 rpm, shake culture for about 10 hours.

[0080] (6) Use Ex Taq enzyme to perform PCR molecular detection on the bacterial solution. The bacterial solution with positive PCR results is expanded, 15% glycerol is added to the bacterial solution, and it is sent to Huada Gene Company for sequencing. The original bacterial solution is stored in a -80 refrigerator.

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

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

[0083] The transmembrane region of the protein was preliminarily predicted using the TMHMM Server v.2.0 online website, and it was found that there was no transmembrane domain in the protein. It was preliminarily speculated that ZmGA13ox1 was a non-transmembrane protein.

[0084] As shown in Figure 1 and Figure 2 , the corn gibberellin 13-hydroxylase ZmGA13ox1 protein belongs to the 2-oxoglutarate-dependent dioxygenase family (2-OGD). Through phylogenetic tree analysis of GA13ox proteins in corn, rice, soybean, Arabidopsis, tomato, rape, and Tripterygium, etc. Different species were found to have several branches, indicating that the functions of these proteins may differ. 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 proportion of active gibberellins, but there has been no report of using GA13ox gene to improve plant root length.

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

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

[0087] 2. After amplifying the gene using high-fidelity enzyme, a series of steps including recovery, adding "A", connecting T vector, transforming E. coli, PCR enzyme digestion identification, and sequencing are used to ensure the correctness of the expression gene, including the complete open reading frame, and no mismatch or frame shift phenomenon occurs.

[0088] 3. For the genes that are sequenced correctly, use the designed restriction sites for enzyme digestion. In this example, the restriction enzymes Xho I and Eco RI are chosen to cut the correctly sequenced target fragments from the intermediate vector. The enzyme digestion system is shown in Table 7. The enzyme digestion reaction is performed in a water bath at 37°C for 1 h. The enzyme digestion products are recovered by electrophoresis to obtain small fragments (genes).

[0089] Table 7. Enzyme digestion system

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

[0091] 4. The expression vector part-cam-flag is digested using the same two restriction enzymes Xho I and Eco RI, and the large fragments (vectors) are recovered.

[0092] 5. The recovered large fragments are ligated with the small fragments using DNA ligase to recombine into a plant expression vector for the ZmGA13ox1 gene. The ligation reaction is performed at 16°C for 3 h. The ligation system is shown in Table 8.

[0093] Table 8. Ligation system

[0094] Component Volume Desired fragment 5.5 μl Vector large fragment 2.5 μl 10x T4 ligase buffer 1 μl T4 DNA ligase 1 μl

[0095] 6. After the ligation is completed, the recombinant vector is transformed into E. coli competent cells, and then the plasmid is extracted for PCR and enzyme digestion identification. It is ensured 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 Arabidopsis thaliana transformed with ZmGA13ox1 gene

[0097] The Arabidopsis thaliana is transformed using the dipping method, and the specific steps are as follows:

[0098] 1. The flowering Arabidopsis thaliana is inverted with the flower buds facing down, and it is immersed in the Agrobacterium liquid containing the target plasmid for 2 min.

[0099] 2. The transformed Arabidopsis thaliana plant is placed flat and covered with plastic wrap. It is grown under low light intensity for 24 h and then cultured under normal light conditions. The above infection step is repeated once a week.

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

[0101] 4. The harvested part of the T0 generation seeds are screened by kanamycin and identified by PCR to obtain T1 generation transgenic plants. After two generations, T3 generation Arabidopsis thaliana plants are obtained, which can be used for subsequent phenotype screening.

[0102] Example 5: Determination of root growth of T3 generation of Arabidopsis thaliana overexpressing ZmGA13ox1 gene.

[0103] T3 generation seeds of wild type Arabidopsis thaliana and transgenic Arabidopsis thaliana of the application were randomly sowed respectively, and then were moved to 22℃ under light condition for culture, and the morphology and growth of the plants were observed and recorded. The results showed that the plant morphology of the Arabidopsis thaliana progeny material overexpressing ZmGA13ox1 gene did not change obviously. The two transgenic materials overexpressing ZmGA13ox1 gene were not seriously affected in phenotype, and the rosette leaves had no significant difference compared with the wild type, and could develop and set seeds normally. Figure 3 However, it was observed on MS medium that the root length of Arabidopsis thaliana overexpressing ZmGA13ox1 gene was significantly longer than that of the wild type. Figure 3 This result showed that the overexpression of ZmGA13ox1 gene significantly affected the GA biosynthesis in the root of Arabidopsis thaliana, and further affected the root phenotype of Arabidopsis thaliana.

[0104] In summary, a gibberellin 13-hydroxylase gene was obtained from corn in the application, which was named as ZmGA13ox1. The plant expression vector containing ZmGA13ox1 was successfully transformed into Arabidopsis thaliana by Agrobacterium-mediated transformation method, and homozygous T3 generation of transgenic Arabidopsis thaliana plants were obtained. The results of growth observation of T3 generation of transgenic Arabidopsis thaliana plants showed that compared with wild type Arabidopsis thaliana, the root morphology of transgenic Arabidopsis thaliana plants changed significantly. Specifically, the root length of transgenic Arabidopsis thaliana plants increased significantly, which indicated that the overexpression of ZmGA13ox1 gene in Arabidopsis thaliana could significantly affect the growth of its root.

[0105] The above is only the preferred embodiment of the present application, and it should be noted that for those skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which should be considered as the protection scope of the present application, and these will not affect the effect and practicality of the patent.

Claims

1. A gene for Gibberellin 13-hydroxylase ZmGA13ox1 or a Gibberellin 13-hydroxylase protein in promoting root length in Arabidopsis thaliana, characterized in that, Nucleotide sequence of a maize gibberellin 13-hydroxylase gene ZmGA13ox1 is set forth in SEQ ID NO: 1 of the Sequence Listing; the maize gibberellin 13-hydroxylase protein is encoded by the maize gibberellin 13-hydroxylase gene ZmGA13ox1 is set forth in SEQ ID NO: 2 of the Sequence Listing.

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