Plant drought resistance related protein tacep5 and coding gene and application thereof

Abstract

The present application relates to a plant drought resistance related protein TaCEP5 and its coding gene and application, the TaCEP5 protein is derived from wheat (Triticum aestivum L.), and the gene for coding TaCEP5 protein is named as TaCEP5 gene.The present application inhibits the expression of TaCEP5 gene by using genetic engineering technology, so that the root length of wheat is significantly increased and the drought resistance is significantly improved.The present application has important significance for the research and application of plant root development and plant drought resistance.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the plant drought resistance-related protein TaCEP5, its encoding gene, and its applications. Background Technology

[0002] Roots are vital organs for absorbing water and nutrients. Under conditions of soil moisture deficit, large and deep root systems are generally an ideal trait, providing a growth advantage for the above-ground parts of the plant and thus influencing crop yield. In particular, the ability of roots to extract soil moisture stored in deeper soil layers is considered the most important factor determining the yield of dryland crops such as wheat.

[0003] Wheat is the world's second-largest food crop, providing 20% ​​of global dietary calories and playing a vital role in agricultural production. With the intensification of global climate change, the frequency, intensity, and duration of disasters such as droughts are showing a significant increasing trend. Drought has become one of the major abiotic stresses limiting crop yield increases.

[0004] Therefore, in-depth exploration of new drought-resistant genes and research into new mechanisms of crop drought resistance are of great significance for improving crop drought resistance. Currently, no polypeptide hormone genes involved in wheat root growth development and drought resistance mechanisms have been reported. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide the application of the plant drought resistance-related protein TaCEP5 and its encoding gene.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] 1. TaCEP5, a plant drought resistance-related protein, selected from any of the following:

[0008] (a1) A protein with the amino acid sequence shown in SEQ ID NO.1;

[0009] (a2) Proteins with the amino acid sequence shown in SEQ ID NO.1 are modified by substitution and / or deletion and / or addition of one or more amino acid residues to produce proteins derived therefrom that are associated with plant root length.

[0010] (a3) Proteins with the amino acid sequence shown in SEQ ID NO.1 are modified by substitution and / or deletion and / or addition of one or more amino acid residues to produce proteins derived therefrom that are associated with plant drought resistance.

[0011] (a4) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein described in (a1);

[0012] (a5) is a protein derived from wheat and has more than 96% identity with (a1) and is related to plant root length;

[0013] (a6) is a protein derived from wheat that shares more than 96% identity with (a1) and is associated with plant drought resistance.

[0014] As one of the preferred technical solutions, the tag sequence described in (a4) is shown in Table 1;

[0015] Table 1. Label Sequence

[0016]

[0017]

[0018] 2. The gene TaCEP5 encoding the aforementioned protein is selected from any of the following:

[0019] (1) The coding region is as shown in SEQ ID NO.2 of the DNA molecule.

[0020] (2) A DNA molecule with a nucleotide sequence as shown in SEQ ID NO.3;

[0021] (3) A DNA molecule that hybridizes under stringent conditions with a DNA molecule defined in (1) or (2) and encodes the protein thereon;

[0022] As one of the preferred technical solutions, the strict conditions described in (3) are as follows: hybridization and washing of the membrane twice at 68°C in a solution of 2×SSC (nucleic acid hybridization washing buffer) and 0.1% SDS (sodium dodecyl sulfate) for 5 min each time, and then hybridization and washing of the membrane twice at 68°C in a solution of 0.5×SSC and 0.1% SDS for 15 min each time.

[0023] (4) A DNA molecule derived from wheat that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the DNA molecule defined in (1) or (2) and that encodes the protein.

[0024] 3. Recombinant vectors, expression cassettes, or recombinant bacteria containing the TaCEP5 gene sgRNA.

[0025] 4. Knockout expression vectors containing TaCEP5 gene sgRNA.

[0026] 5. The aforementioned method for constructing the knockout expression vector involves designing any single-stranded guide RNA (sgRNA) targeting the TaCEP5 gene on the sequence shown in SEQ ID NO.2, and constructing a plant binary expression vector PUbi414-TaCEP5 containing this sgRNA to knock out the TaCEP5 gene in wheat. To facilitate the identification and screening of transgenic plants, the expression vector can be modified, such as by adding genes that express enzymes or luminescent compounds that produce color changes in plants, antibiotic resistance markers, or chemical reagent resistance marker genes. From a transgenic safety perspective, no selective marker genes may be added, and transformed plants can be directly screened based on phenotype.

[0027] As one of the preferred technical solutions, the specific steps are as follows:

[0028] (1) Use the online software E-CRISPR (http: / / www.e-crisp.org / E-CRISP / ) to design TaCEP5 knockout target primer pairs, and use Plants ensembl (http: / / plants.ensembl.org / index.html) to perform BLAST and select specific targets for knocking out the TaCEP5 gene;

[0029] (2) The target primers are linked to the wheat TaU6 promoter to form an sgRNA expression cassette; this promoter drives the expression of the CRISPR / Cas9 system, ensuring the correct guidance of the primers;

[0030] (3) Prepare PUbi414 plasmid: Prepare a plasmid suitable for the CRISPR / Cas9 system, which includes Cas9 protein and corresponding RNA components;

[0031] (4) Restriction endonuclease digestion: The sgRNA expression cassette and PUbi414 plasmid were digested with restriction endonucleases respectively in order to ligate them together;

[0032] (5) Ligation: The enzyme-digested sgRNA expression cassette is ligated to the PUbi414 plasmid to form the PUbi414-TaCEP5 knockout expression vector.

[0033] Construction method references: Kumar, R., Mamrutha, HM, Kaur, A., Venkatesh, K., Sharma, D., & Singh, GP (2019). Optimization of Agrobacterium-mediated transformation inspring bread wheat using mature and immature embryos. Molecular biology reports, 46(2), 1845–1853.

[0034] As a further preferred technical solution, the sgRNA primer pair for the TaCEP5 gene in step (1) is as follows:

[0035] TaCEP5-414F:5′-GCGTCCTCCCTGCACTGGAAGGG-3′, as shown in SEQ ID NO.4;

[0036] TaCEP5-414R:5′-GACTATTATGGCCAATGCTAGGG-3′, as shown in SEQ ID NO.5.

[0037] 6. Application of the aforementioned TaCEP5 protein in regulating plant root length and / or regulating plant drought resistance.

[0038] 7. Application of the aforementioned gene TaCEP5, the aforementioned recombinant vector, expression cassette or recombinant bacteria, and the aforementioned knockout expression vector in regulating plant root length and / or regulating plant drought resistance.

[0039] As one of the preferred technical solutions, the specific details are as follows (b1) and / or (b2):

[0040] (b1) Cultivating transgenic plants with increased root length;

[0041] (b2) Breeding transgenic plants with improved drought resistance.

[0042] 9. Application of the aforementioned gene TaCEP5, the aforementioned recombinant vector, expression cassette or recombinant bacteria, and the aforementioned knockout expression vector in the cultivation of transgenic plants.

[0043] 10. A method for cultivating transgenic plants, comprising the following steps: introducing the aforementioned recombinant plasmid PUbi414-TaCEP5 into a recipient plant, and obtaining transgenic plants with increased root length and / or improved drought resistance through induction, subculture, pre-culture, co-culture, resistance screening, differentiation, rooting culture, hardening, and transplanting.

[0044] As one of the preferred technical solutions, the recipient plant is a monocotyledonous plant or a dicotyledonous plant, more preferably a grass plant, even more preferably a wheat plant, even more preferably a hexaploid wheat, and even more preferably wheat Fielder.

[0045] 11. A plant breeding method, comprising the following steps: reducing the content and / or activity of the aforementioned TaCEP5 protein in the target plant, thereby increasing the root length and / or improving the drought resistance of the plant.

[0046] The beneficial effects of this invention are as follows:

[0047] This invention provides the plant drought-resistance-related protein TaCEP5, its encoding gene, and its applications. The TaCEP5 protein is derived from wheat (Triticum aestivum L.). The gene encoding the TaCEP5 protein is also within the scope of protection of this invention. The gene encoding the TaCEP5 protein is named the TaCEP5 gene. This invention protects methods for knocking out TaCEP5 using gene editing. This invention also protects the application of the TaCEP5 protein in regulating plant root length and regulating plant drought resistance. This invention also protects the applications of the TaCEP5 gene, namely (b1) and / or (b2): (b1) cultivating transgenic plants with increased root length; (b2) cultivating transgenic plants with improved drought resistance. This invention is of great significance for the research and application of plant root development and plant drought resistance.

[0048] This invention utilizes genetic engineering technology to suppress the expression of the TaCEP5 gene, thereby significantly increasing the root length and drought resistance of wheat. This invention is of great significance for the research and application of plant root development and drought resistance. Attached Figure Description

[0049] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0050] Figure 1 The results show the expression levels of the TaCEP5 gene under drought treatment conditions.

[0051] Figure 2 Electrophoresis diagram for PCR identification of transgenic plants.

[0052] Figure 3 This is a comparison diagram of sequencing results for transgenic plants.

[0053] Figure 4 This is the result of a root scan.

[0054] Figure 5 This is the statistical result of root length.

[0055] Figure 6Phenotypic photographs of the drought-treated group.

[0056] Figure 7 The survival rate statistics for the drought-treated group are shown. Detailed Implementation

[0057] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0058] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. Unless otherwise specified, the quantitative experiments in the following examples were all performed in triplicate, and the results were averaged.

[0059] I. Discovery of TaCEP5 protein and its encoding gene

[0060] A novel protein was discovered in the roots of wheat fielder plants grown hydroponically for 8 days. Its amino acid sequence is shown in SEQ ID NO. 1, and it has been named TaCEP5 protein. The gene encoding TaCEP5 protein has been named the TaCEP5 gene. The TaCEP5 gene is located on wheat chromosome 7AL. The coding sequence of the TaCEP5 gene in the cDNA of wheat fielder is shown in SEQ ID NO. 2.

[0061] II. TaCEP5 gene expression exhibits tissue specificity.

[0062] The test material was wheat Fielder roots subjected to drought treatments of 0 hours, 4 hours, 6 hours, and 8 hours.

[0063] Total RNA was extracted from the test material and reverse transcribed to obtain cDNA. Real-time quantitative PCR (polymerase chain reaction) was then performed using the cDNA as a template.

[0064] The primer pairs used to detect the TaCEP5 gene (which contains homologous genes from the B and D genomes) are as follows:

[0065] qTaCEP5-F: 5′-CTCCCTGCACTGGAAGGG-3′;

[0066] qTaCEP5-R: 5′-GGGCCATCTCAGTTTGGTG-3′.

[0067] The primer pairs used for detecting the internal reference gene (β-Actin gene) are as follows:

[0068] β-Actin-F: 5′-GGAATCCATGAGACCACCTAC-3′;

[0069] β-Actin-R: 5′-GACCCAGACAACTCGCAAC-3′.

[0070] The reaction system for real-time quantitative PCR (10 μl) is as follows: 5 μl of 2× Green Master Mix, 1 μl of primer F (2 μM), 1 μl of primer R (2 μM), 1 μl of cDNA template, and 2 μl of ddH2O.

[0071] The reaction procedure for real-time quantitative PCR is as follows: pre-denaturation at 94℃ for 5 min; 40 cycles of 94℃ for 20 s, 60℃ for 20 s, and 72℃ for 25 s; 72℃ for 5 min; melting curve analysis at 60℃-95℃, with readings every 0.2℃.

[0072] C = 2 -△CT ΔCt=Ct 目标基因 –Ct 内参基因 The average C-value of the three replicates was calculated as the relative expression level of the target gene. A two-tailed t-test with equal variances was used for significance testing (**p<0.01, ***p<0.001, ****p<0.0001).

[0073] See results Figure 1 .

[0074] III. Construction of Recombinant Plasmids

[0075] Referring to the reference "Optimization of Agrobacterium-mediated transformation in spring bread wheat using mature and immature embryos" (Kumar R, Mamrutha HM, Kaur A, Venkatesh K, Sharma D, Singh GP. 2019, Molecular Biology Reports 46:1845–1853), the sgRNA of the TaCEP5 gene was ligated to the wheat U6 promoter to form an sgRNA expression cassette; the sgRNA expression cassette was then ligated to the PUbi414 plasmid to obtain the recombinant plasmid PUbi414-TaCEP5. The recombinant plasmid PUbi414-TaCEP5 has been sequenced and verified.

[0076] The sgRNA primer pair for the TaCEP5 gene is as follows:

[0077] TaCEP5-414F:5′-GCGTCCTCCCTGCACTGGAAGGG-3′, as shown in SEQ ID NO.4;

[0078] TaCEP5-414R:5′-GACTATTATGGCCAATGCTAGGG-3′, as shown in SEQ ID NO.5.

[0079] IV. Preparation of Transgenic Plants

[0080] 1. The recombinant plasmid PUbi414-TaCEP5 was introduced into Agrobacterium tumefaciens EHA105 (WEIDI,CAT#:AC1013) to obtain recombinant Agrobacterium.

[0081] 2. The recombinant Agrobacterium obtained in step 1 was used to infect the embryogenic callus of wheat Fielder, and then differentiation culture, rooting culture and herbicide resistance screening (screening concentration of 250 mg / L) were carried out in sequence to obtain 4 T0 generation regenerated plants.

[0082] 3. The four T0 generation regenerated plants obtained in step 2 were identified by PCR.

[0083] PCR identification method: Take plant leaves, extract genomic DNA, and perform PCR amplification using primer pair composed of cas9-F and cas9-R. If the amplification product is obtained, the identification result is positive, and the plant is a transgenic plant.

[0084] cas9-F: 5′-CTAAGCGGAACAGCGACAAG-3′;

[0085] cas9-R: 5′-GGCCAGGTAGAGGAAGTTCAC-3′.

[0086] Of the four T0 generation regenerated plants, four were transgenic plants.

[0087] Electrophoresis images of transgenic plants for PCR identification are shown below. Figure 2 . Figure 2 In the diagram, M represents the molecular weight marker, #1 to #4 represent different transgenic plants, + represents the recombinant plasmid PUbi414-TaCEP5 (positive control), H2O represents sterile water (negative control), and WT represents wheat Fielder plants (negative control).

[0088] 4. The TaCEP5 gene editing status of the four transgenic plants screened in step 3 was identified by sequencing the sgRNA knockout target.

[0089] (1) Take plant leaves and extract DNA.

[0090] (2) Using DNA as a template, the amplified products were sequenced for verification.

[0091] The primer pair used to identify the TaCEP5A gene (a homologous gene in the A genome) is as follows:

[0092] TaCEP5A-F: 5′-GACTGGAACATGTCACTT-3′;

[0093] TaCEP5A-R: 5′-GTCGATCGCATGCCTTATG-3′.

[0094] The coding region of the TaCEP5B gene is shown in SEQ ID NO.6. The primer pair used to identify the TaCEP5B gene (a homologous gene in the B genome) is as follows:

[0095] TaCEP5B-F: 5′-CCTGCTTCCATTTTTCAAGC-3′;

[0096] TaCEP5B-R: 5′-CAATAGGCCCTCCTCAGAGA-3′.

[0097] The coding region of the TaCEP5D gene is shown in SEQ ID NO.7. The primer pair used to identify the TaCEP5D gene (a homologous gene in the D genome) is as follows:

[0098] TaCEP5D-F: 5′-GCGCACAATAGACCCAAAGT-3′;

[0099] TaCEP5D-R: 5′-CAATAGGCCCTCCTCAGAGA-3′.

[0100] PCR reaction system (10 μl): 2×Mix 5 μl, primer F (2 μM) 1 μl, primer R (2 μM) 1 μl, DNA template 1 μl, ddH2O 2 μl.

[0101] PCR reaction procedure: 94℃ pre-denaturation for 5 min; 94℃ for 20 s, 58℃ for 20 s, 72℃ for 30 s, 35 cycles; 72℃ for 5 min; 12℃ for 5 min.

[0102] The amplified products were sequenced for verification, and the sequencing results were analyzed using SnapGene software.

[0103] Some results can be found Figure 3 . Figure 3The study presented two TaCEP5 transgenic lines (Tacep5-4 and Tacep5-7), with TaCEP5-4A and TaCEP5-7A having a 1bp deletion; TaCEP5-4B and TaCEP5-7B having a 1bp deletion; and TaCEP5-4D having a 38bp deletion and TaCEP5-7D having a 1bp deletion.

[0104] 5. Produce offspring through self-pollination.

[0105] Transgenic plants are self-pollinated to obtain seeds, which are called T1 generation seeds. Plants grown from T1 generation seeds are called T1 generation plants. T1 generation plants are self-pollinated to obtain seeds, which are called T2 generation seeds. Plants grown from T2 generation seeds are called T2 generation plants. Leaves of T2 generation plants are taken, and DNA is extracted for PCR identification (method as in step 4). For a given T2 generation plant, if the PCR sequencing result is positive, the T2 generation plant and its offspring constitute a homozygous transgenic line.

[0106] Two transgenic lines (Tacep5-4 and Tacep5-7) were randomly selected for identification in step five.

[0107] V. Phenotypic Identification

[0108] Seeds tested: T3 generation seeds of Tacep5-4, T3 generation seeds of Tacep5-7, and seeds of wheat Fielder.

[0109] 1. Identification of root length characteristics during the seedling stage

[0110] Culture conditions: 22℃, 16h light / 8h darkness.

[0111] (1) Take the test seeds, soak them in a 1% sodium hypochlorite aqueous solution for 15 minutes for disinfection, and then wash them 6 times with distilled water.

[0112] (2) Take the seeds from step (1) and place them at 4℃ away from light for 3 days.

[0113] (3) Take the seeds from step (2) and cultivate them until the seedlings germinate for 2 days.

[0114] (4) Take seedlings with uniform growth and transfer them to Hogland nutrient solution with pH 6.0 for hydroponics (change the nutrient solution every 2 days). After 6 days of hydroponics, observe the root growth status of the plants, scan the root system with a scanner, and count the root length of the plants.

[0115] Three experiments were conducted, with 20 biological replicates for each type of seed in each replicate.

[0116] Root length scan results are shown Figure 4 Root length statistics are shown in [link to statistics]. Figure 5 Compared to wheat Fielder, the root length of both the Tacep5-4 and Tacep5-7 lines was significantly increased.

[0117] 2. Drought resistance assessment

[0118] A rectangular culture pot (10cm*10cm*10cm) contains 60g of soil.

[0119] Sow 9 seeds in each culture pot.

[0120] Normal group: The test seeds were sown in the culture pot (at time 0), and then watered until the soil was saturated with moisture, and then watered once every 3 days.

[0121] Drought treatment group: The test seeds were sown in culture pots and then watered until the soil was saturated. After that, the seedlings were watered once every 3 days for 2 weeks. Then, the seedlings were not watered for 2 weeks and the growth status of the wheat was observed until they were on the verge of wilting. Then, the seedlings were re-watered for one week.

[0122] Culture conditions: 22℃, 16h light / 8h darkness.

[0123] The number of days is counted starting from time 0, with each 24-hour period considered as one day. The survival rate on the 28th day is then calculated.

[0124] Three replicate experiments were conducted, with 54 biological replicates for each type of seed in each replicate experiment.

[0125] In the normal group, there were no significant differences in the growth status of Tacep5-4, Tacep5-7 and wheat Fielder plants.

[0126] Drought treatment group: a large number of wheat Fielder plants wilted and died, while the survival rate of Tacep5-4 and Tacep5-7 lines was significantly higher than that of wheat Fielder plants.

[0127] On day 28, photos of the drought treatment team can be found here. Figure 6 Survival rate see Figure 7 . Figure 6 and Figure 7 Among them, the survival rate of wheat Fielder plants was 53.8%, the survival rate of Tacep5-4 line plants was 80.8%, and the survival rate of Tacep5-7 line plants was 77.9%.

[0128] The results showed that knocking out the TaCEP5 gene significantly improved the drought resistance of wheat.

[0129] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. Gene knockout TaCEP5 Its application in increasing wheat root length and / or improving wheat drought resistance is characterized by, The aforementioned gene TaCEP5 The coding region sequence is shown in SEQ ID NO.

2.

2. The application of reducing TaCEP5 protein expression in the breeding of transgenic wheat with increased root length and / or improved drought resistance, characterized in that... The TaCEP5 protein is: (a1) A protein with an amino acid sequence as shown in SEQ ID NO.1; or (a4) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein described in (a1).

3. A wheat breeding method, characterized in that, The steps include: reducing the content and / or activity of TaCEP5 protein in the target wheat, thereby increasing wheat root length and improving drought resistance; The TaCEP5 protein is: (a1) A protein with an amino acid sequence as shown in SEQ ID NO.1; or (a4) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein described in (a1).

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