Application of a substance for knocking out a TaSWEET11f gene in improving resistance of a cereal crop to scab

Abstract

The application discloses application of a substance for knocking out a TaSWEET11f gene in improving scab resistance of cereal crops, and relates to the field of agricultural biotechnology; the application finds the correlation between the TaSWEET11f gene and the scab resistance for the first time, and screens three homologous genes TaSWEET11f-A, TaSWEET11f-B and TaSWEET11f-D of the gene; the CRISPR / Cas9 gene editing technology is adopted to specifically knock out the wheat TaSWEET11f gene in a receptor material; the single flower dripping method is used for scab resistance identification; and the result shows that the CR-Tasweet11f gene edited plant has obviously improved scab resistance compared with the receptor material Fielder; the application knocks out the wheat TaSWEET11f gene by using the CRISPR / Cas9 technology, and is expected to provide technical support for directional and rapid creation of new germplasm resistant to scab, and has wide breeding utilization value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural biotechnology, in particular to the application of a substance knocking out TaSWEET11f gene in improving the resistance of cereal crops to scab. BACKGROUND

[0002] Wheat scab (Fusarium Head Blight, FHB) is a disease caused by Fusarium spp., which has caused serious threats to global cereal crops (including wheat, barley, oat, etc.). Scab not only leads to a significant decrease in cereal crop yield, but also contaminates grains, making them contain mycotoxins (such as deoxynivalenol, DON) that are harmful to human and animal health. In recent years, with climate change and adjustment of agricultural planting structure, the frequency and severity of wheat scab have been increasing, which has become a major problem restricting wheat production and food safety.

[0003] Currently, the prevention and control of wheat scab mainly rely on the use of chemical pesticides and the breeding of disease-resistant varieties. Although chemical pesticides can control the occurrence of diseases to some extent, long-term and large-scale use of pesticides will lead to environmental pollution, affect ecological balance, and frequent use of pesticides increases planting costs and reduces the economic benefits of farmers. Although traditional disease-resistant breeding methods have achieved certain results in breeding disease-resistant varieties, due to the diversity and variability of scab pathogens, the breeding cycle is long, the efficiency is low, and it is difficult to meet the production demand.

[0004] SWEET genes (Sugars Will Eventually be Exported Transporters) encode a class of sugar transport proteins, which are involved in the transport of sugars inside and outside the cell, and play a role in the transport of carbohydrates in plants and the process of pathogen infection. Some studies have shown that TaSWEET14 gene is related to wheat stripe rust, and stripe rust can activate the SWEET gene of host plants by secreting effector proteins, increase extracellular sugar, and thus provide energy for the growth and reproduction of pathogenic bacteria (“Functional Research on TaSWEET14 Gene in the Interaction between Wheat and Stripe Rust”, Zheng Peijing, Northwest Agriculture and Forestry University). Some SWEET genes in rice (such as OsSWEET13 and OsSWEET14) have been identified as targets of Xanthomonas oryzae. Currently, the correlation between TaSWEET11f gene and wheat scab has not been reported. SUMMARY

[0005] In view of the above problems, the application takes the wheat TaSWEET11f gene as a gene editing target, and provides an application of a substance for knocking out the TaSWEET11f gene in improving the resistance of cereal crops to scab. That is, a new material with the characteristics of resistance to scab is created by targeting the wheat SWEET gene TaSWEET11f using the CRISPR-Cas9 gene editing technology, so as to provide technical support for the directional and rapid creation of new germplasm resistant to scab, and provide an efficient and environmentally friendly solution for the prevention and control of wheat scab.

[0006] To achieve the above-mentioned object, the application provides the following technical solutions.

[0007] Firstly, the application provides an application of a substance for knocking out the TaSWEET11f gene in improving the resistance of cereal crops to scab.

[0008] The TaSWEET11f gene has three homologous genes TaSWEET11f-A, TaSWEET11f-B and TaSWEET11f-D, the nucleotide sequences of which are shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 in order, and the amino acid sequences of which are shown in SEQ IN NO. 6, SEQ IN NO. 7 and SEQ IN NO. 8 in order.

[0009] The cereal crops include wheat, barley and oats.

[0010] Further, the application refers to introducing a vector containing the substance for knocking out the TaSWEET11f gene or an agrobacterium strain containing the vector into cells, tissues or plant individuals of a host plant to create a gene editing material resistant to scab. The substance for knocking out the TaSWEET11f gene includes a gene editing vector CR-TaSWEET11f. The host plant includes barley, wheat or oats. The vector used is a conventional vector in the art, including but not limited to the vector pBUE411 (a commercially available vector), such as the vector disclosed in the document “Xing, Hui-Li, et al. A CRISPR / Cas9 toolkit for multiplex genome editing in plants. BMC plant biology 14 (2014): 1-12.” In specific implementation, the vectors pYLCRISPR / Cas9Pubi-H, pYLCRISPR / Cas9pUbi-N, pYLCRISPR / Cas9P35S-B, pYLCRISPR / Cas9Pubi-B and the like can also be used; and the agrobacterium used is also a conventional agrobacterium, including but not limited to the conventional commercially available agrobacterium strain agrobacterium EHA105.

[0011] Secondly, the application provides a method for improving the resistance of a cereal crop to scab, comprising knocking out a TaSWEET11f gene using a CRISPR / Cas9 system to change the structure or function of the TaSWEET11f gene; the CRISPR / Cas9 system comprises two sgRNAs that simultaneously target TaSWEET11f-A, TaSWEET11f-B and TaSWEET11f-D genes. The cereal crop includes wheat, barley and oat.

[0012] Further, the specific steps of the method for improving the resistance of a cereal crop to scab are as follows:

[0013] 1) Taking TaSWEET11f-A, TaSWEET11f-B and TaSWEET11f-D as CRISPR / Cas9 gene editing targets, designing sgRNA1 and sgRN2; connecting the sgRNA1 and sgRN2 with a CRISPR / Cas9 gene editing vector (such as pCBC-MT1T2) to obtain a gene editing vector CR-TaSWEET11f;

[0014] The nucleotide sequences of the sgRNA1 and sgRN2 are shown in SEQ ID NO. 4 and SEQ ID NO. 5, respectively, and the gene targets can simultaneously target TaSWEET11f-A, TaSWEET11f-B and TaSWEET11f-D genes located on A, B and C chromosomes of wheat;

[0015] 2) Transforming the gene editing vector CR-TaSWEET11f obtained in step 1) into E. coli, and then extracting the plasmid;

[0016] 3) Transferring the plasmid obtained in step 2) into Agrobacterium to obtain transformed bacteria;

[0017] 4) Introducing the transformed bacteria obtained in step 3) into a host plant by genetic transformation to obtain a gene editing plant T0, continuously self-crossing the T0 generation to obtain a T2 generation editing plant; detecting the genes of the T2 generation editing plant, and selecting a homozygous editing plant therefrom, i.e. obtaining a gene editing plant with improved resistance to scab.

[0018] The gene detection of the T2 generation refers to sequence detection of the first target site (SEQ IN NO. 4) of the transgenic plant T2 using a forward primer SEQ ID NO. 15 and a reverse primer SEQ ID NO. 16, and sequence detection of the second target site (SEQ IN NO. 5) of the transgenic plant T2 using a forward primer SEQ ID NO. 17 and a reverse primer SEQ ID NO. 18.

[0019] In the above step 4), the term "genetic transformation" refers to introducing a vector (such as CR-TaSWEET11f) containing the TaSWEET11f gene target sequence (SEQ INNO.4 and SEQ IN NO.5) or an Agrobacterium strain containing the vector into a host plant by using the genetic transformation method disclosed in the document "Hayta S, Smedley M A, Clarke M, et al. An Efficient Agrobacterium-Mediated Transformation Protocol for Hexaploid and Tetraploid Wheat [J]. Current Protocols, 2021, 1(3)." to create a gene edited plant resistant to scab.

[0020] In the present application, the term "knockout" refers to the deletion, insertion or base substitution of the TaSWEET11f gene sequence, the mutation of the corresponding encoded protein, and the obtained gene edited strain is "gene knockout strain".

[0021] The present application first discovered the correlation between the wheat SWEET gene TaSWEET11f and the resistance to scab, and then used the CRISPR-Cas9 gene editing technology to create new materials with anti-scab characteristics by taking the gene TaSWEET11f as the target. Compared with the prior art, the beneficial effects of the present application are reflected in:

[0022] The present application first edits TaSWEET11f in the receptor material by CRISPR / Cas9 gene editing technology, provides an effective target, thereby enhancing the disease resistance of plants, reducing the occurrence of diseases, and quickly creating new materials resistant to scab. In the present application, the TaSWEET11f gene edited plant CR-Tasweet11f obtained in the examples is identified for resistance to scab by single flower dripping method, and the scab resistance of the gene edited plant CR-Tasweet11f is obviously improved. The editing application based on the TaSWEET11f gene provided by the present application provides technical support for the directional and rapid creation of new germplasm resistant to scab, provides an efficient and environmentally friendly solution for the prevention and control of wheat scab, improves the yield and quality of wheat, and ensures food safety, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The figure is a schematic diagram of the TaSWEET11f gene structure.

[0024] Figure 2Schematic diagram of the target segment DNA sequence of TaSWEET11f gene edited plant;

[0025] Figure 3 Scab resistance phenotype of T2 generation homozygous edited plant of TaSWEET11f gene edited.

[0026] Figure 4 Statistical results of scab spikelet number of T2 generation homozygous edited plant of TaSWEET11f gene edited. DETAILED DESCRIPTION

[0027] The methods and reagents used in this example are all conventional methods and experimental reagents used in the art. The wheat variety Fielder involved in the example is a conventional variety, as disclosed in the literature “He Y, Wu L, Liu X, et al. TaUGT6, a novel UDP-glycosyltransferase gene enhances the resistance to FHB and DON accumulation in wheat [J]. Frontiers in Plant Science, 2020: 1549.” The Fielder wheat used in the example is preserved by the Wheat Genetic Breeding Team of Jiangsu Academy of Agricultural Sciences.

[0028] PCR product recovery kit and plasmid extraction kit were purchased from Nanjing Nvwaizan Biotechnology Co., Ltd.;

[0029] E. coli DH5a and Agrobacterium EHA105 (catalog number: AC1012) were purchased from Shanghai Weidi Biological Company;

[0030] Bsa I restriction endonuclease and T4 DNA ligase were purchased from BioLabs Company;

[0031] Trypsin, agar powder, sodium chloride, kanamycin (Kan, 50 μg / mL) and other chemical reagents were all domestic analytical pure reagents.

[0032] Primer synthesis and sequencing were completed by Sheng Wu Bioengineering (Shanghai) Co., Ltd.

[0033] pCBC-MT1T2 is a common plasmid in the art, as disclosed in the document "Xing, Hui-Li, et al. A CRISPR / Cas9 toolkit for multiplex genome editing in plants", purchased from Addgene (Plasmid #50593) in the examples, and pBUE411 vector purchased from Addgene (Plasmid #62200).

[0034] LB+Kan solid medium: 10 g Tryptone, 5 g Yeast Extract, 10 g NaCl, add water to 1000 mL, adjust pH 7.2; then add agar powder to a final concentration of 1.8%, and Kan to a final concentration of 50 μg / ml.

[0035] Example 1 Creation of CR-Tasweet11f wheat plants by CRISPR / Cas9 gene editing

[0036] Applicants previously found through phosphoproteomics that the TaSWEET11f gene is located on the 5D chromosome of wheat, and there is a difference in wheat scab resistant and susceptible varieties, so further screening obtained TaSWEET11f-A (XM_044529353.1), TaSWEET11f-B (XM_044537462.1), TaSWEET11f-D (XM_044545647.1) three homologous genes, the nucleotide sequences of the three genes are shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, respectively, and the amino acid sequences encoded by them are shown in SEQ IN NO. 6, SEQ IN NO. 7, SEQ IN NO. 8, respectively.

[0037] Figure 1 is a schematic diagram of the gene TaSWEET11f, wherein the blue box represents the exon region, the orange box represents the two conserved functional domains PQ-loop and MtN3_slv of TaSWEET11f, and the black line is the target site of the gene editing sgRNA1 and sgRN2.

[0038] The CRISPR / Cas9 gene editing steps are as follows:

[0039] 1) Design the target site sequence sgRNA1 (SEQ IN NO. 4) and sgRNA2 (SEQ IN NO. 5) of the three homologous genes (TaSWEET11f-A, TaSWEET11f-B, TaSWEET11f-D) of TaSWEET11f simultaneously targeted by the public website http: / / crispr.hzau.edu.cn / CRISPR-Cereal / , and then design the primer sequence containing the two target sites. The forward and reverse primer sequences of target sgRNA1 are shown in SEQ IN NO. 9 and SEQ IN NO. 10, respectively, and the forward and reverse primer sequences of target sgRNA2 are shown in SEQ IN NO. 11 and SEQ IN NO. 12, respectively.

[0040] In the PCR reaction system (Novagen, Catalog No.: P511), 2 ng of pCBC-MT1T2 was used as the template, 1 μM of each of primers SEQ ID NO. 9 and SEQ ID NO. 12, 0.05 μM of each of primers SEQ ID NO. 10 and SEQ ID NO. 11, 2× Master Mix 25 μl.

[0041] The PCR reaction program was set as 25 cycles: 94℃ 10 s, 58℃ 15 s, 68℃ 20 s. The PCR amplification fragment was subjected to gel recovery (Novagen, Catalog No.: DC301), and the recovery product was obtained.

[0042] 2) The recovery product was ligated with the pBUE411 vector, and the ligation system was as follows: 2 μl of the recovery product, 60 ng of the pBUE411 vector, 1.5 μl of 10×CutSmart Buffer, 1.5 μl of 10 mM ATP, 10 U of Bsa I-HF endonuclease, 35 U of T4 ligase, and ddH2O was added to a total volume of 15 μl. The ligation was performed by using a variable temperature cycle for 15 cycles: 37℃ 5 min; 10℃ 5 min, 20℃ 5 min; finally 37℃ 5 min, and the ligation product was obtained.

[0043] 3) The ligation product was used to transform E. coli, and positive clones were screened on LB+Kan solid medium. The plasmid was extracted, and the positive clones were identified by PCR and sent for sequencing. The primer sequences for identification and sequencing are shown in SEQ ID NO. 13 and SEQ ID NO. 14. The plasmid with correct alignment (i.e., the target site sequence in the plasmid is consistent with SEQ IN NO. 4 and SEQ IN NO. 5) was named as the gene editing vector CR-TaSWEET11f.

[0044] 4) Then the gene editing vector CR-Ta TaSWEET11f was transformed into Agrobacterium EHA105 to obtain Agrobacterium EHA105 / CR-Ta TaSWEET11f. The Agrobacterium EHA105 / CR-Ta TaSWEET11f was transformed into the immature embryo of wheat Fielder by using the general immature embryo-based genetic transformation method to obtain the gene editing plants (TO generation).

[0045] The above genetic transformation method is a conventional method in the art, and the method disclosed in the literature "Ishida Y, Tsunashima M, Hiei Y, et al. Wheat (Triticum aestivum L.) transformation using immature embryos [M]. Agrobacterium protocols. Springer, New York, NY, 2015: 189-198." was used in this embodiment.

[0046] 5) The TO generation was selfed to obtain the T1 generation, and the T1 generation was selfed to obtain the T2 generation. For the T2 generation of wheat plants, the first target site (SEQ IN NO. 4) of the transgenic plants T2 was detected by using the forward primer SEQ ID NO. 15 and the reverse primer SEQ ID NO. 16, and then the second target site (SEQ IN NO. 5) of the transgenic plants T2 was detected by using the forward primer SEQ ID NO. 17 and the reverse primer SEQ ID NO. 18.

[0047] Through sequence alignment analysis, the editing plants in which the TaSWEET11f gene was simultaneously knocked out on A, B and D chromosomes were obtained, and the homozygous editing plants were numbered as 7 (CR-TaSWEET11f-7) and 10 (CR-TaSWEET11f-10), respectively. The target segment DNA sequences of the two homozygous editing plants are shown in Figure 2 Figure 2 In the above, the black letters are the target sequences, the green letters are the PAM sequences, the horizontal lines "-” represent base deletions, and the red letters represent base insertions.

[0048] ​Compared with the transgenic receptor Fielder, the CR-TaSWEET11f-7 strain has 3 base deletions in the first target region of chromosome 5A, 1 base insertion in the first and second target regions of 5B, 4 base deletions in the first target region of 5D, and 1 base insertion in the second target region. The CR-TaSWEET11f-10 strain has 4 base deletions in the first target region of 5A, 1 base insertion in the first target region of 5B, 1 base deletion in the second target region, 2 base deletions in the first target region of 5D, and 1 base insertion in the second target region. It can be seen that the TaSWEET11f gene in the CR-TaSWEET11f-7 strain is knocked out, and the corresponding protein synthesis is blocked.

[0049] Example 2 Gene editing TaSWEET11f improves the resistance of wheat to scab

[0050] The gene editing wheat plants CR-TaSWEET11f-7 and CR-TaSWEET11f-10 obtained in the above-mentioned Example 1 were subjected to scab resistance identification. The method of inoculating the pathogen was according to the method disclosed in NY / T 2954-2016, and the specific method was as follows: one spikelet in the middle and upper part of the booting stage was inoculated with 10 μl of Fusarium graminearum spore solution (Fg1312, preserved in the applicant's laboratory) containing 1×10 5 , and the bag was kept moist for 3 days. The number of diseased spikelets was counted 15 days after inoculation. The phenotype of the gene editing strain 15 days after being infected with scab is shown in Figure 3 , and the number of diseased spikelets is shown in Table 1 and Figure 4 .

[0051] Table 1 Number of diseased spikelets

[0052] Fielder (WT) TaSWEET11 f-7 TaSWEET11 f-10 10 4 5 9 4 2 11 7 2 10 7 2 5 4 5 8 6 4 10 4 5 11 6 3

[0053] It can be seen that the scab resistance level of the gene editing TaSWEET11f wheat plant is significantly improved compared with the receptor wheat Fielder.

[0054] Although the above-mentioned examples have made a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments. People can also obtain other embodiments according to the present embodiments without creativity, and these embodiments all belong to the protection scope of the present application.

Claims

1. Application of simultaneous knockout of wheat TaSWEET11f-A, TaSWEET11f-B, TaSWEET11f-D genes in improving the resistance of wheat to scab; the nucleotide sequences of the TaSWEET11f-A, TaSWEET11f-B, TaSWEET11f-D genes are shown in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3, respectively.

2. Use according to claim 1, characterized in that, The application refers to introducing a vector containing the knockout TaSWEET11f-A, TaSWEET11f-B, TaSWEET11f-D genes or an Agrobacterium strain containing the vector into wheat cells, tissues or individuals to improve the resistance of wheat to scab.

3. A method for improving the resistance of wheat to scab, comprising simultaneously knocking out TaSWEET11f-A, TaSWEET11f-B, TaSWEET11f-D genes by using a CRISPR / Cas9 system; the CRISPR / Cas9 system comprises two sgRNAs targeting the TaSWEET11f-A, TaSWEET11f-B, TaSWEET11f-D genes; the nucleotide sequences of the TaSWEET11f-A, TaSWEET11f-B, TaSWEET11f-D genes are shown in SEQ ID NO. 1-SEQ ID NO. 3, respectively.

4. The method of claim 3, wherein, The sgRNAs are sgRNA1 and sgRNA2 with nucleotide sequences shown in SEQ ID NO. 4 and SEQ ID NO. 5, respectively.

5. The method of claim 4, wherein, The specific steps are as follows: 1) The sgRNA1 and sgRNA2 are connected with a CRISPR / Cas9 gene editing vector to obtain a vector CR-TaSWEET11f; 2) The vector CR-TaSWEET11f is transformed into E. coli, and then a plasmid is extracted; 3) The plasmid obtained in step 2) is transformed into Agrobacterium to obtain a transformed bacterium; 4) The transformed bacterium obtained in step 3) is introduced into a wheat plant by genetic transformation to obtain an edited plant T0 with simultaneous knockout of TaSWEET11f-A, TaSWEET11f-B, TaSWEET11f-D genes, and T0 generation is continuously self-crossed to obtain T2 generation; an edited plant with simultaneous knockout of TaSWEET11f-A, TaSWEET11f-B, TaSWEET11f-D genes and homozygous in T2 generation is selected, which is a gene edited plant with improved resistance to scab.

6. The method of claim 5, wherein, In step 1), the gene editing vector is pCBC-MT1T2.

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