Application of TabZIP4 in improving drought resistance of wheat
By knocking out the TabZIP4 gene, wheat was improved using CRISPR-Cas9 technology, enhancing its drought resistance and addressing the impact of drought stress on wheat yield. This approach achieved the goal of improving wheat's drought resistance without increasing water consumption.
Patent Information
- Application Number
- CN202511592986.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-03
- Publication Date
- 2025-12-02
AI Technical Summary
Wheat yields are affected by drought stress, and global climate change has led to water scarcity. Existing technologies are insufficient to reduce water use to cope with drought stress without affecting yields.
TabZIP4 knockout lines were prepared by knocking out or reducing the expression level of TabZIP4, and wheat genetic transformation was carried out using CRISPR-Cas9 technology to improve the drought resistance of wheat.
It significantly improves the drought resistance of wheat, enhances its growth potential and survival rate under drought conditions, reduces the rate of water loss, and reduces dependence on water resources.
Smart Images
Figure CN121046444A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant gene technology, specifically relating to a... TabZIP4 Application in improving wheat drought resistance. Background Technology
[0002] Wheat, as one of the three staple grains, feeds nearly one-fifth of the world's population, and its production plays a crucial role in ensuring global food security. Statistics show that the current global average annual wheat production is approximately 785 million tons, and it is projected that by 2050, global wheat production will need to reach 858 million tons to meet the needs of the rapidly growing population. Wheat yields are typically affected by various abiotic stresses, and global climate change will further exacerbate these effects. Drought is a major abiotic stress affecting wheat yields, and to ensure food security, an additional 1 million hectares of land and doubled agricultural water use are expected. However, due to climate change, freshwater availability is projected to decrease by 50%. How to reduce water use without affecting yields has become a critical scientific challenge for breeders. Summary of the Invention
[0003] The purpose of this invention is to provide a TabZIP4 Its application in improving the drought resistance of wheat has solved the problems existing in the current technology.
[0004] The technical solution adopted in this invention is: This invention provides TabZIP4 Application in improving wheat drought resistance, the aforementioned TabZIP4 The base sequence is shown in SEQ ID NO.9, and the amino acid sequence is shown in SEQ ID NO.10; The application refers to knocking out TabZIP4 or reduce TabZIP4 We used the expression level of [a specific substance] to prepare knockout lines in order to improve the drought resistance of wheat.
[0005] Preferably, the method for preparing the knockout strain is as follows: Using pMT1T2 as a template, PCR amplification was performed using the primers shown in SEQ ID NO.1~SEQ ID NO.4 to obtain PCR products; The PCR product was ligated with the CRISPR-Cas9 vector by enzyme digestion to obtain the knockout vector; The knockout vector was transformed with Agrobacterium to obtain recombinant Agrobacterium; Using wheat as the recipient, recombinant Agrobacterium was used to genetically transform wheat to obtain knockout lines.
[0006] Preferably, the CRISPR-Cas9 vector is pBUE411.
[0007] Preferably, the steps for preparing the PCR product are as follows: Take 1 μL each of 100 μM SEQ ID NO.1, 100 μM SEQ ID NO.4, 2 μM SEQ ID NO.2, and 2 μM SEQ ID NO.3, mix them thoroughly, and obtain the mixed primers; PCR amplification was performed using 2 μL of mixed primers, 10 μL of high-fidelity enzyme, 1 μL of pMT1T2, and 7 μL of ddH2O to obtain the PCR product.
[0008] Preferably, the conditions for PCR amplification are as follows: Pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 s, annealing at 58℃ for 15 s, extension at 72℃ for 1 min, 35 cycles; final extension at 95℃ for 5 min.
[0009] Preferably, the PCR product is ligated with the CRISPR-Cas9 vector via enzyme digestion as follows: 2 μL of PCR product, 2 μL of CRISPR-Cas9 vector, 1.5 μL of 10×NEB T4 Buffer, 1.5 μL of CutSmart Buffer, 1 μL of BsaI, 1 μL of T4 Ligase, and 6 μL of ddH2O.
[0010] Preferably, the enzyme digestion and ligation conditions are 37°C for 12 hours.
[0011] Preferably, the Agrobacterium used for Agrobacterium transformation is EHA105.
[0012] Compared with the prior art, the beneficial effects of the present invention are: This invention provides TabZIP4 Application in improving wheat drought resistance, the aforementioned TabZIP4 The base sequence is shown in SEQ ID NO. 9, and the amino acid sequence is shown in SEQ ID NO. 10; the application refers to knocking out TabZIP4 or reduce TabZIP4 The expression level of [a specific gene] was used to prepare knockout lines to improve the drought resistance of wheat. Drought stress has become a significant factor limiting wheat yield among various abiotic stresses. This invention utilizes a transgenic method to obtain [a specific gene]. TabZIP4 The knockout strains were identified, and their functions were analyzed and identified in depth, further clarifying their... TabZIP4 The role of genes in wheat's response to drought stress. The cloned gene in this invention... TabZIP4 The gene analysis provides new genes related to wheat drought resistance, offering new clues for wheat drought-resistant breeding. Attached Figure Description
[0013] Figure 1 for TabZIP4 Gene structure and target knockout type of transgenic lines.
[0014] Figure 2 Representative images of wild-type and knockout lines subjected to drought stress or normal irrigation treatment.
[0015] Figure 3 This is a statistical analysis of the survival rate and fresh weight of wild-type and knockout lines after drought stress. A: Survival rate; B: Fresh weight.
[0016] Figure 4 To detect the water loss rate of wild-type and knockout lines after drought stress.
[0017] Figure 5 Representative images of leaf temperature before and after drought stress for wild-type and knockout lines.
[0018] Figure 6 This is a statistical result of leaf temperature before and after drought stress in wild-type and knockout lines. Detailed Implementation
[0019] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.
[0020] The inventive concept of this invention is as follows: Drought, as the most prominent abiotic stress affecting wheat yield, harms wheat throughout its entire growth cycle: drought at sowing leads to uneven emergence and reduced tillering; drought during the jointing and heading stages shortens the photosynthetic duration of the flag leaf and reduces the number of florets; and drought during the grain-filling stage directly inhibits starch and protein synthesis, causing a 10%–40% decrease in thousand-grain weight. Prolonged drought induces oxidative stress, damaging PSII reaction centers and causing a sharp drop in net photosynthetic rate of over 50%. Simultaneously, root water absorption is impaired, and ABA concentration rises dramatically, further closing stomata and inhibiting cell division, resulting in a double decrease in both the number of grains per ear and the number of ears per unit area. Against the backdrop of global warming, the frequency and intensity of droughts are significantly increasing. It is estimated that drought will reduce global wheat yields by an average of 7%–20% over the next 30 years, and in semi-arid rainfed agricultural areas, the reduction could be as high as 60%, directly threatening global food security. Therefore, a new strategy to address drought stress is urgently needed.
[0021] Based on this, the present invention provides a TabZIP4 Application in improving wheat drought resistance, the aforementioned TabZIP4 The base sequence is shown in SEQ ID NO. 9, and the amino acid sequence is shown in SEQ ID NO. 10; the application refers to knocking outTabZIP4 or reduce TabZIP4 We used the expression level of [a specific substance] to prepare knockout lines in order to improve the drought resistance of wheat.
[0022] SEQ ID NO.9:
[0023] SEQ ID NO.10: MQQPKPADPPGRPFPPPSPSMAAAAATAMRGAHHRRARSEVAFHLPDDLDIGGGADGDGSAGFDEIGSEDDLFSTFMDIEKISSGPAAGSDRDRDRAAETSSPPRPKHRYSSSVDGSGLFSAAGSAARRDAAAAQALADVLEAKKAMSPEQLAELAAIDPKRAKRILANRQSAARSKER KARYMTELERKVQTLQTEATTLSAQLTLFQRDTTGLSSENAELKIRLQAMEQQAQLRDALNDALKQEVERLKMATGEMSNSSDAYSVGLQHVLYNSSFFPQSQQNTAQHQGGARFPPPFHPPHPNVPNHQMLSHPNTLSDIMQQDHLARLQGLDISKGHPVVKSESSSISASESSSTF.
[0024] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.
[0025] Example 1 TabZIP4 Its application in improving wheat drought resistance is as follows: 1. TabZIP4 Obtaining genetically modified knockout wheat.
[0026] 1.1 TabZIP4 The knockout primers are shown in Table 1.
[0027] Table 1 TabZIP4 Knockout primers 1.2 Using the vector pMT1T2 as a template, the DNA fragment containing the vector adapter and target site was amplified using the primers shown in SEQ ID NO.1~SEQ ID NO.4, and then purified by gel extraction to obtain the DNA fragment for knockout. TabZIP4 PCR products.
[0028] Take 100μM TabZIP4 -p414-F, 100μM TabZIP4 -p414-R, 2μM TabZIP4 -p414-F0, 2μM TabZIP4Mix 1 μL each of p414-R0 to obtain a mixed primer. Take 2 μL of the mixed primer, 10 μL of high-fidelity enzyme, 1 μL of pMT1T2, and 7 μL of ddH2O for PCR amplification. After amplification, perform agarose gel electrophoresis and purification to obtain the primer for knockout. TabZIP4 PCR products.
[0029] The PCR amplification conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 15 s, 72℃ extension for 1 min, 35 cycles; 95℃ final extension for 5 min.
[0030] The pMT1T2 plasmid is described in the following literature: Xing, HL, Dong, L., Wang, ZP, Zhang, HY, Han, CY, Liu, B., Wang, XC, & Chen, QJ (2014). A CRISPR / Cas9toolkit for multiplex genome editing in plan ts. BMC plant biology, 14, 327.3, where it is named pCBC-MT1T2.
[0031] 1.3. The CRISPR-Cas9 vector pBUE411 and the PCR product were digested with BsaI and ligated overnight using T4 ligase, followed by transformation into E. coli. The ligation system of pBUE411 vector and PCR product is shown in Table 2.
[0032] Table 2 Enzyme digestion and ligation system After mixing by suction and stirring, the sample was reacted in a metal bath at 37°C for 12 hours to obtain the ligation product, which was then stored at 4°C.
[0033] The ligation product was added to 100 μL of TransT1 E. coli competent cells using a pipette, incubated on ice for 30 min, then heat-shocked at 42 °C for 90 s, incubated on ice for 2 min, 800 μL of LB was added, and the cells were incubated on a shaker at 37 °C for 1 h. After centrifugation at 4000 rpm for 1 min, the supernatant was discarded. The remaining supernatant and precipitate were mixed by pipetting and spread onto the corresponding antibody solid LB plate, and incubated overnight at 37 °C.
[0034] The CRISPR-Cas9 vector pBUE411 is described in the following literature: Xing, HL, Dong, L., Wang, ZP, Zhang, HY, Han, CY, Liu, B., Wang, XC, & Chen, QJ (2014). ACRISPR / Cas9 toolkit for multiplex genome editing in plants. BMC plant biology , 14 , 327. https: / / doi.org / 10.1186 / s12870-014-0327-y, the title of this document is pBUE411 .
[0035] 1.4. Positive clones were screened by PCR reaction, sequenced, and simultaneously the knockout vector was extracted, transformed into Agrobacterium, and sent to a wheat transgenic platform for wheat genetic transformation to obtain... TabZIP4 Knockout strains.
[0036] Single clones from the solid LB agar plates were selected for PCR testing. Clones testing positive by PCR were cultured and sent to a sequencing company for sequencing. Corresponding antibiotic-resistant liquid LB medium was added to the correctly sequenced strains, and the cultures were incubated overnight at 37°C. Plasmids were then extracted to obtain knockout vectors, which were used to transform Agrobacterium.
[0037] The steps for transforming Agrobacterium with a knockout vector are as follows: 1) Add 1 μg of the knockout vector to 100 μL of EHA105 Agrobacterium and incubate on ice for 20 min.
[0038] 2) Quick freeze in liquid nitrogen for 5 minutes, bathe in water at 37°C for 5 minutes, and then quickly place on ice for 5 minutes.
[0039] 3) Add 600 μL of antibiotic-free LB liquid medium and incubate at 28°C with shaking for 3 h.
[0040] 4) Centrifuge at 4000 rpm for 5 min, keep a small amount of supernatant and resuspend the precipitate, and spread it on LB agar containing 50 mg / L Rif + 50 mg / L Kan.
[0041] 5) Incubate at 28℃ for 3 days, and then perform PCR identification to obtain positive colonies.
[0042] 6) Sequencing and sequence alignment of the bacterial solutions corresponding to positive colonies; positive colonies with correct sequencing are recombinant Agrobacterium.
[0043] Add the recombinant Agrobacterium bacterial suspension and v / v 50% glycerol in a 1:1 ratio to a sterile 2mL EP tube, mix thoroughly by inverting, and then store in an ultra-low temperature freezer at -80℃.
[0044] Recombinant Agrobacterium was sent to a wheat transgenic platform to perform genetic transformation of wheat using Fielder as the recipient, resulting in... TabZIP4 Knockout strains.
[0045] 1.5. The obtained T0 generation knockout lines were identified by PCR and sequencing.
[0046] 1.6 After harvesting the identified T0 generation positive knockout lines, they were further processed in a greenhouse, and the T3 generation plants were screened using PCR and sequencing to obtain a homozygous knockout line, TabZIP KO.
[0047] For TabZIP4 The primers used for detecting knockout strains are shown in Table 3.
[0048] Table 3 TabZIP4 Detection primers for knockout strains 2. TabZIP4 Drought stress experiment on wheat lines after knockout.
[0049] To clarify TabZIP4 To understand the biological functions of CRISPR-Cas9, a CRISPR-Cas9 vector was constructed and transformed into Fielder. TabZIP4 The target was designed into three conserved regions of homologous genes in exon 1, specifically 10bp–28bp and 257bp–27bp. Ultimately, one target was obtained. TabZIP-B, TabZIP-A, TabZIP-D The homozygous triple knockout strain TabZIP-KO, mutation type as follows Figure 1 The knockout strains were modified by inserting an A base at position 25 bp in genome A, inserting a G base at position 272 in genomes B and D respectively, and deleting the G base at position 272 in genomes A, B, and D. The seedling drought resistance of the knockout lines was then assessed.
[0050] To investigate TabZIP4To achieve the desired function, this invention constructed a transgenic knockout vector and transformed it using wheat Fielder as the recipient, obtaining an independent homozygous knockout line. One line of the identified homozygous knockout wheat, TabZIP KO, and seeds of the control wild-type Fielder were taken, disinfected with 1% hydrogen peroxide (v / v) for 10 min, washed four times with distilled water, and placed in petri dishes lined with two layers of filter paper and a small amount of distilled water. The dishes were incubated at room temperature for 48 h. Seeds with consistent germination were selected and transplanted into culture boxes containing nutrient soil. Each culture box was divided into two parts: one half planted with wild-type Fielder seeds (WT), and the other half planted with the homozygous knockout line TabZIP KO, for a total of four replicates. After transplanting the seeds into the culture boxes, all culture boxes were randomly divided into a drought stress group and a control group. The drought stress group was watered once and then not watered again until the soil moisture content reached approximately 2.5%, which was considered a critical time point. Five days later, the group was re-watered and allowed to recover for another five days. The control group was watered normally throughout the entire process. After the event, phenotypic patterns were observed, photos were taken, survival rates were recorded, and the fresh weight of the aboveground parts was tallied.
[0051] The results are as follows Figure 2 The results showed that under normal conditions, there was no difference in growth status between the knockout line and the wild-type Fielder; however, after drought stress, the seedlings of the knockout line exhibited significantly stronger growth vigor than the wild-type Fielder. Figure 3 As shown, survival rate statistics revealed that the survival rate of TabZIP KO after drought stress was 82%, compared to 44% for the wild type. Fresh weight statistics also showed that the fresh weight of TabZIP KO after drought stress was significantly higher than that of the wild-type Fielder. These results indicate... TabZIP It negatively participates in regulating wheat drought resistance.
[0052] To further verify the drought resistance of TabZIP KO, this invention conducted a water loss rate test, specifically by subjecting wheat seedlings at the two-leaf stage to in vitro dehydration treatment. The results are as follows: Figure 4 As shown, the leaf water loss rate of TabZIP KO was significantly lower than that of the wild type. This invention also included leaf temperature measurements before and after drought stress, with results as follows: Figure 5 and Figure 6 As shown, without drought stress, the leaf temperature of both the knockout line and the wild type remained consistent at approximately 24.4℃. However, after drought stress, the leaf temperature of both the knockout line and the wild type increased, with the knockout line reaching 25℃ and the wild type reaching 24.7℃. This further indicates that the water loss rate of the knockout line was significantly lower than that of the wild-type Fielder after drought stress. These experimental results demonstrate that... TabZIP4 It negatively participates in regulating the drought resistance of wheat.
[0053] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0054] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.
Claims
1. TabZIP4 Its application in improving wheat drought resistance is characterized by, The TabZIP4 The base sequence is shown in SEQ ID NO.9, and the amino acid sequence is shown in SEQ ID NO.10; The application refers to knocking out TabZIP4 or reduce TabZIP4 We used the expression level of [a specific substance] to prepare knockout lines in order to improve the drought resistance of wheat.
2. The application as described in claim 1, characterized in that, The method for preparing the knockout strain is as follows: Using pMT1T2 as a template, PCR amplification was performed using the primers shown in SEQ ID NO.1~SEQ ID NO.4 to obtain PCR products; The PCR product was ligated with the CRISPR-Cas9 vector by enzyme digestion to obtain the knockout vector; The knockout vector was transformed with Agrobacterium to obtain recombinant Agrobacterium; Using wheat as the recipient, recombinant Agrobacterium was used to genetically transform wheat to obtain knockout lines.
3. The application as described in claim 2, characterized in that, The CRISPR-Cas9 vector is pBUE411.
4. The application as described in claim 2, characterized in that, The steps for preparing the PCR product are as follows: Take 1 μL each of 100 μM SEQ ID NO.1, 100 μM SEQ ID NO.4, 2 μM SEQ ID NO.2, and 2 μM SEQ ID NO.3, mix them thoroughly, and obtain the mixed primers; PCR amplification was performed using 2 μL of mixed primers, 10 μL of high-fidelity enzyme, 1 μL of pMT1T2, and 7 μL of ddH2O to obtain the PCR product.
5. The application as described in claim 4, characterized in that, The conditions for the PCR amplification are as follows: Pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 s, annealing at 58℃ for 15 s, extension at 72℃ for 1 min, 35 cycles; final extension at 95℃ for 5 min.
6. The application as described in claim 2, characterized in that, The system for ligating the PCR product with the CRISPR-Cas9 vector via enzyme digestion is as follows: PCR product 2 μL, CRISPR-Cas9 vector 2 μL, 10×NEB T4 Buffer 1.5 μL, CutSmart Buffer 1.5 μL, BsaI 1 μL, T4 Ligase 1 μL, and ddH2O 6 μL.
7. The application as described in claim 2, characterized in that, The enzyme digestion and ligation conditions were 37℃ for 12 hours.
8. The application as described in claim 2, characterized in that, The Agrobacterium used for Agrobacterium transformation is EHA105.