Application of molecular chaperonin TaBiP1 in wheat scab resistance

Abstract

The invention relates to the technical field of biology, in particular to application of wheat molecular chaperonin TaBiP1 in prevention and treatment of wheat scab. It is found for the first time that after overexpression of the wheat molecular chaperonin TaBiP1, the infection degree of wheat scab can be remarkably reduced. Compared with a transgenic receptor parent Fielder, the TaBiP1 overexpression plant can significantly reduce the infection of the wheat scab, and can be used for creating new germplasm resistant to the wheat scab. The TaBiP1 gene in receptor wheat is edited through a CRISPR / Cas9 gene editing technology, a transgenic plant bip1 is obtained, the wheat scab resistance of the transgenic plant bip1 is weakened compared with that of Fielder, and the positive significance of TaBiP1 in enhancing the wheat scab resistance is proved.

Description

Technical Field

[0001] The present invention relates to the field of plant breeding, in particular to wheat TaBiP1 Application of genes in wheat fusarium head blight resistance. Background Art

[0002] wheat( Triticum aestivum L. ) is the world's largest crop with the largest planting area, the highest yield and the widest distribution. In the past few decades, global wheat production has increased significantly. However, the cultivation and production of wheat in the world are still facing major challenges and serious threats. Various diseases have broken out on a large scale, seriously affecting wheat yield and quality. Wheat head blight (Fusarium head blight, FHB) is a devastating and difficult to control disease caused by Fusarium graminearum ( Fusarium graminearum) The fungal disease caused by Fusarium graminearum is one of the three major diseases of wheat and is known as the "cancer" of wheat (Chen Y, Kistler HC, Ma Z. Fusarium graminearum trichothecenemycotoxins: biosynthesis, regulation, and management[J]. Annual Review of Phytopathology, 2019, 57(1): 15-39.). Epidemics of wheat fusarium rust frequently occur in my country, especially in the middle and lower reaches of the Yangtze River. The average annual incidence area of ​​wheat fusarium rust exceeds 5.33×10 6 hm 2 In Jiangsu Province alone, the annual average incidence area reached 1.2×10 6 hm 2 . (Wang JH, Peng XD, Lin SH, et al. First report of Fusariumhead blight of wheat caused by Fusarium sacchari in China[J]. Plant Disease, 2015, 99(1): 160-160.).

[0003] Although the combined use of tolerant wheat varieties, fungicides, and specific management practices (such as tillage and rotation) can reduce some of the losses caused by the disease, so far, no effective strategy can completely control the prevalence of FHB (Bai G, Su Z, Cai J. Wheat resistance to Fusarium head blight[J]. Canadian Journal of Plant Pathology, 2018, 40(3): 336-346.). Discovering disease-resistant genes and breeding varieties resistant to Fusarium head blight are the most effective and environmentally friendly measures for controlling Fusarium head blight in wheat. However, only 9 genetic loci for wheat resistance to Fusarium head blight have been identified so far( Fhb1 ~ Fhb9 ), and so far, only Fhb1 (Su Z, Bernardo A, Tian B, et al. A deletion mutation in TaHRC confers Fhb1 resistance to Fusarium head blight in wheat[J]. Nature genetics, 2019, 51(7): 1099-1105.) and Fhb7 (Wang H, Sun S, Ge W, et al. Horizontal gene transfer of Fhb7 from fungus underlies Fusarium head blight resistance in wheat. Science 368: eaba5435[EB / OL].(2020)) have been cloned. Therefore, exploring and utilizing efficient genes for wheat resistance to Fusarium head blight is of great significance for promoting the process of breeding for resistance to Fusarium head blight in wheat.

[0004] Heat shock proteins (HSPs) play a role in plant development and responses to abiotic and biotic stress conditions, including drought, salt stress, pathogen and insect infestation, etc. Increasing evidence indicates that the defense mechanism mediated by heat shock protein 70 (HSP70) plays an important role in plant disease resistance (Usman M G, Rafii M Y, Martini M Y, et al. Molecular analysis of Hsp70 mechanisms in plants and their function in response to stress[J]. Biotechnology and Genetic Engineering Reviews, 2017, 33(1): 26-39.). Plant HSP70 is related to endoplasmic reticulum (ER)-mediated immunity. Recent studies have pointed out that plant pathogens can use their effectors to regulate host ER stress to promote their infection. The ER-resident HSP70 protein BiP is involved in plant immunity as a key sensor of ER stress. It has been reported that the effector PsAvh262se produced by Phytophthora sojae stabilizes BiP and inhibits cell death induced by ER stress (Jing M, Guo B, Li H, et al. A Phytophthora sojae effector suppresses endoplasmic reticulum stress-mediated immunity by stabilizing plant binding immunoglobulin proteins[J]. Nature Communications, 2016, 7(1): 11685.). The above background indicates that BiP has the potential for exploration in the field of plant disease resistance. Summary of the Invention

[0005] By means of genetic engineering and the biochemical system of the interaction between Gibberella zeae pathogenic factors and wheat, the present invention identified the key target protein TaBIP1 in wheat responding to Gibberella zeae. Therefore, by overexpression TaBiP1 and knockout expression TaBiP1 to determine its application for the resistance of wheat to Fusarium head blight.

[0006] Based on the research of the present inventors, it was first discovered that overexpression of wheat TaBiP1 can reduce the infection degree of wheat Fusarium head blight; it was first discovered that knockout expression of wheat TaBiP1 can increase the infection degree of wheat Fusarium head blight. Thus, the present invention was completed.

[0007] The present invention first provides TaBiP1Gene information, whose amino acid sequence is shown in SEQ ID No. 2, No. 4 or No. 6.

[0008] Furthermore, the present invention provides an expression element, a recombinant vector and a host cell containing the said gene.

[0009] Preferably, the said gene is overexpressed in plants by transgenic methods.

[0010] The present invention also provides the application of the said gene in creating disease-resistant transgenic plants, wherein it is overexpressed in transgenic plants by transgenic methods.

[0011] Preferably, the plant is a monocotyledonous plant, more preferably the plant is wheat. Even more preferably, the disease resistance refers to Fusarium head blight caused by Fusarium graminearum.

[0012] The present invention relates to the application of TaBiP1 in the disease resistance process. TaBiP1 Overexpression of the gene does not affect the growth and yield of wheat plants; however, TaBiP1 Transgenic plants with overexpressed genes have significant resistance to Fusarium head blight and can inhibit the expansion of Fusarium graminearum in host cells. At the same time, it is found that knocking out TaBiP1 mutant transgenic plants will significantly reduce the resistance to Fusarium head blight. The transgenic plants involved in the present invention do not affect normal growth and seed setting.

[0013] Furthermore, the present invention provides a method for enhancing the resistance of plants to Fusarium head blight, which overexpresses the said gene in transgenic plants by transgenic methods; The present invention is beneficial to the cultivation of disease-resistant wheat varieties and provides a basis for screening highly resistant wheat varieties in the later stage. For example, the present invention can provide TaBiP1 overexpressing plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments.

[0015] Figure 1 Overexpression in wheat TaBiP1 can enhance its resistance to Fusarium head blight. Figure A shows TaBiP1 the relative expression level in the overexpressing wheat lines. Among them, the expression levels of the three lines #6, #7, and #8 are relatively high, so they are selected for subsequent experimental analysis. Figure B shows representative images of wheat ears of WT (Fielder), TaBiP1 -OE three lines 15 days after being infected with Fusarium graminearum under greenhouse conditions. Figure C shows TaBiP1 the quantitative analysis of the disease index of the -OE three lines. The results show that overexpression of TaBiP1It can significantly enhance the resistance to Fusarium head blight of wheat after that.

[0016] Figure 2 At 5 days, the representative images (A) of the leaves of WT, TaBiP1 -OE three lines infected with Fusarium graminearum and the lesion area (B). The data show that TaBiP1 the leaves of -OE three lines have stronger resistance to Fusarium infection.

[0017] Figure 3 At TaBiP1 Details of the DNA sequence editing of the target segment of the gene-edited plants are shown; "." indicates base deletion; the red fields indicate base mutations. A total of three different types of knockout expression lines, namely #2, #3, and #5, were obtained.

[0018] Figure 4 At TaBiP1 The phenotype of the resistance to Fusarium head blight of the T2 generation homozygous transgenic plants with gene editing. Figure A shows the representative images of the wheat spikes of WT (Fielder), bip1 three lines infected with Fusarium graminearum for 15 days. Figure B shows bip1 the quantitative analysis of the disease index of three lines. The results show that after knocking out the expression of TaBiP1 the resistance to Fusarium head blight of wheat is significantly reduced.

[0019] Figure 5 At TaBiP1 The growth conditions of the transgenic plants of -OE three lines in the field. The results show that overexpressing TaBiP1 will not affect the normal growth of the plants.

[0020] In summary, TaBiP1 is a protein that has a positive effect on the resistance to Fusarium head blight of wheat. It can significantly improve the resistance to Fusarium head blight of wheat without affecting the normal growth of wheat. Detailed implementation manners

[0021] The present invention will be described below through specific examples for better understanding, but it does not constitute a limitation to the present invention. Specifically as follows: Example 1: Overexpression of TaBiP1 in wheat can enhance its resistance to Fusarium head blight In the present invention, the said application preferably includes the following steps: (1) Using the In-Fusion cloning technology (Clontech, catalog number 638910), insert the TaBiP1 full-length fragment into the pUbiGW vector with the Ubiquitin promoter through the BamH I site; The said TaBiP1 The nucleotide sequence of the CDS full-length fragment is as shown in SEQ ID No. 1; (2) Transfer the ligation vector obtained in step (1) into Escherichia coli DH5α. After screening and sequencing, extract the plasmid ligated with the BiP1 gene; (3) Transform the plasmid obtained in step (2) into Agrobacterium tumefaciens EHA105 to obtain TaBiP1 Agrobacterium tumefaciens carrying the plasmid; (4) Transform the transformed bacteria obtained in step (3) into the wheat Fielder line. After gene identification, obtain wheat overexpressing TaBiP1 the gene; Wheat transformation refers to the Agrobacterium-mediated wheat transformation method (Goetz H., Cornelia M., and Jochen K. (2021). Agrobacterium-Mediated Transformation of Wheat Using Immature Embryos. Rom. Agric. Res. 38 , 99 - 107 (2021)). Use the Enviologix QuickStix Kit (Envirologix, catalog number AS013) to evaluate the genotypes of transgenic plants. Further confirm whether the T1 and T2 generations are overexpressed by qRT-PCR ( Figure 1 in A).

[0022] Subsequently, identify the resistance of the overexpressing TaBiP1 ([[]]END]] TaBiP1 -OE) (SEQ ID NO: 1) transgenic material to Fusarium head blight. Inoculate Fusarium graminearum under greenhouse conditions, TaBiP1 The disease severity of -OE is significantly lower than that of the wild-type Fielder ( Figure 1 in B, C). We also tested the disease symptoms caused by Fusarium graminearum in the leaves of overexpressing materials. Notably, compared with the wild type, TaBiP1 -OE showed a significant reduction in the lesion area on the leaves ( Figure 2 ).

[0023] Among them, the treatment method for detached leaves: Wild-type and transgenic wheat plants were grown in a growth chamber at 22 °C with a photoperiod of 16 h light and 8 h dark. Collect the detached secondary leaves of 2-week-old plants and transfer them to a square petri dish containing 1% water agar. Culture 10 µL of conidial suspension (about 5×10 4(conidia / mL). At 5 - 6 days after infection, the infection symptoms were recorded using ImageJ (https: / / www.computerbild.de / download / ImageJ-422527.html) to evaluate the necrotic lesions.

[0024] Finally, overexpression TaBiP1 did not affect plant growth and yield ( Figure 5 ).

[0025] Example 2: Knockout of TaBiP1 expression in wheat weakens its resistance to Fusarium head blight A wheat knockout expression construct was used to insert the intermediate vector pMETaU6.1 fragment into the gene editing backbone vector pLGYE-3 through the Bsa1 site using In-Fusion cloning technology (Clontech, catalog number 638910). All constructs were transformed into the Agrobacterium tumefaciens strain EHA105. Wheat transformation was carried out as described above. Genotype evaluation of transgenic plants was performed using nucleotide sequencing, and three different types of homozygous lines #2, #3, and #5 were obtained through subculture ( Figure 3 ).

[0026] Subsequently, the transgenic wheat materials with knockout expression TaBiP1 were identified for their resistance to Fusarium head blight. Under greenhouse conditions, Fusarium graminearum was inoculated and the number of diseased spikes was counted. The results showed that bip1 the disease severity of the three lines was significantly higher than that of the wild type Fielder ( Figure 4 ).

Claims

1. Wheat BiP protein, characterized in that, Its amino acid sequence is as shown in SEQ ID No.2, SEQ ID No.4 or SEQ ID No.

6.

2. A gene encoding the wheat BiP protein as claimed in claim 1.

3. An expression element, a recombinant vector, and a host cell containing the gene as claimed in claim 2.

4. Use of the wheat BiP protein as claimed in claim 1, or its encoding gene in enhancing the disease resistance of plants; Preferably, the plant is a monocotyledonous plant, preferably the plant is wheat; more preferably, the disease resistance refers to Fusarium head blight caused by Fusarium.

5. Use of the wheat BiP protein as claimed in claim 1, or its encoding gene, the expression element, the recombinant vector, and the host cell as claimed in claim 3 in the preparation of transgenic plants with enhanced disease resistance.

6. The application according to claim 5, wherein The plant is a monocotyledonous plant, preferably the plant is wheat.

7. The application according to claim 6, characterized in that, The disease resistance refers to Fusarium head blight caused by Fusarium.

8. A method for preparing a transgenic plant with enhanced disease resistance, characterized in that, Including the step of overexpressing or encoding a gene of wheat protein as described in claim 1 in a transgenic plant by a transgenic method, and screening to obtain a transgenic plant with enhanced resistance to Fusarium head blight of wheat. TaBiP1 ​ 9. The method according to claim 8, wherein The plant is a monocotyledonous plant, preferably the plant is wheat.

10. The method according to claim 9, wherein The disease resistance refers to Fusarium head blight caused by Fusarium.

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