TaJDR1 gene and its application in improving plant resistance to Fusarium head blight and drought.

Abstract

This invention discloses the TaJDR1 gene and its application in improving plant resistance to Fusarium head blight and drought. The nucleotide sequence of the TaJDR1 gene is shown in SEQ ID NO.1, and its encoded amino acid sequence is shown in SEQ ID NO.2. This invention is the first to clone the TaJDR1 gene from the local wheat variety Wangshuibai and achieve overexpression of this gene in wheat using gene gun-mediated genetic transformation technology. The results show that the transgenic wheat exhibits significantly improved resistance to Fusarium head blight and drought resistance. Since the TaJDR1 gene originates from wheat itself, overexpression does not affect food safety; therefore, this invention can be widely applied in the field of disease and drought resistance breeding for various plants.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the TaJDR1 gene and its application in improving plant resistance to Fusarium head blight and drought. Background Technology

[0002] Plants frequently face various biotic and abiotic stresses during their growth and development, such as drought, high salinity, low temperature, and pests and diseases. These stresses significantly impact their growth, development, and productivity. Biotic and abiotic stresses are diverse in nature and often interact, posing a severe challenge to the overall health of plants. To cope with these stresses, plants have developed multiple defense mechanisms: when facing biotic stresses, plants thicken their cell walls to enhance mechanical strength, preventing pathogen invasion or insect feeding, and activate the immune system and the expression of defense genes. Simultaneously, they limit the spread of pathogens through hypersensitivity reactions and produce a series of secondary metabolites to enhance resistance. When facing abiotic stresses, plants improve their resistance through the synthesis of osmotic regulators, activation of antioxidant systems, and regulation of ion balance.

[0003] Fusarium head blight and drought are two key stressors in wheat production, affecting not only plant growth and development but also significantly reducing yield and quality. Fusarium head blight (FHB) is a disease of the ear caused by various Fusarium spp. species. It typically breaks out in warm, humid climates, especially during the wheat flowering stage. Although Fusarium can cause disease at different growth stages of wheat, ear infection is most severe. Its main symptoms include: shriveled and dried grains, significantly reduced thousand-grain weight; dehydration of ear tissues, affecting nutrient transport; decreased grain quality; and the deoxynivalenol (DON) toxin produced in the grains, posing a serious threat to human and livestock health. On the other hand, drought stress restricts wheat root development, reducing water and nutrient absorption; leaves wither and turn yellow, reducing photosynthetic capacity and thus affecting the plant's energy supply; and grain development is hindered, leading to shriveling and a decrease in thousand-grain weight. The combined effect of these stressors significantly reduces wheat yield and quality. Against this backdrop, identifying potential candidate genes that can enhance plant resistance to Fusarium head blight and drought is crucial and holds broad application potential.

[0004] Plant lectins are widely distributed in various organisms and are a class of natural proteins that can bind to oligosaccharide structures in specific glycoproteins or glycolipids on the cell surface, participating in the regulation of many biological processes. Jacalin-related lectins (JRLs) are a class of plant lectins first isolated from jackfruit (Artocarpus integrifolia), possessing one or more Jacalin domains, and are currently one of the most widely studied plant lectin families. Dirigent proteins are proteins identified from Forsythia suspensa that can guide coniferyl alcohol radicals to produce (+)-pineresinol (a type of lignan) (Davin, LB et al., 1997). DIR family genes can respond to abiotic stress and hormone-induced regulation and participate in lignin synthesis in plants. The C-terminus of the DIR domain can also be fitted with a Jacalin domain, called Jacalin DirigentRelated proteins, abbreviated as JDRs. They play an important role in disease and pest resistance. Currently, JRL proteins from several different plants have been reported, which can respond to adverse environmental stresses and participate in plant growth and development. For example, the VER2 gene isolated from winter wheat plays an important role in vernalization signaling and spike development in winter wheat (Yong et al., 2003); the OsJRL gene in rice negatively regulates the cold resistance of rice by regulating phenylalanine metabolism and flavonoid biosynthesis (An et al., 2023); OsJAC1 is a chimeric rice DIR-JRL protein that, when constitutively expressed in rice or barley, participates in broad-spectrum disease resistance to bacteria, oomycetes, and fungi (Weidenbach et al., 2016). Summary of the Invention

[0005] The purpose of this invention is to provide a method for using the TaJDR1 gene to improve plant resistance to Fusarium head blight and drought, providing a new candidate gene for the research and breeding of new wheat varieties resistant to Fusarium head blight and drought, thereby promoting the development and application of more new varieties resistant to Fusarium head blight and drought.

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

[0007] The application of the TaJDR1 gene in plant resistance to Fusarium head blight and drought, the cDNA sequence of the TaJDR1 gene is shown in SEQ ID NO:1, and its length is 1056 bp.

[0008] The amino acid sequence encoded by the TaJDR1 gene is shown in SEQ ID NO:2. This protein is a mannose-binding lectin protein located in the cytoplasm and nucleus.

[0009] A recombinant plasmid, wherein the recombinant plasmid contains the wheat Fusarium head blight resistance gene TaJDR1; the vector for this plasmid is preferably pUCBS, that is, the recombinant plasmid is preferably pUCBS-TaJDR1. Furthermore, any vector capable of introducing a foreign gene into plants for expression can be used in this invention.

[0010] Adding antibiotic marker genes (such as hygromycin, kanamycin, and gentamicin) to the vector and then using the vector with the antibiotic marker genes for transformation allows antibiotics to be added to the culture medium of the transformed plants to inhibit the growth of non-transgenic cell lines and plants, which helps to obtain transgenic plants quickly and effectively.

[0011] To facilitate the screening of transgenic plants or cell lines, selection markers such as GUS and GFP are added.

[0012] Using a gene gun-mediated stable genetic transformation method, nucleic acid molecules capable of expressing TaJDR1 protein are introduced into recipient plants to obtain transgenic plants.

[0013] The recipient plant is a grass (Poaceae).

[0014] Beneficial effects:

[0015] This invention utilizes plant genetic engineering technology to obtain the TaJDR1 gene from the wheat variety Wangshuibai for the first time. Experiments have demonstrated that transferring the wheat lectin-like protein provided by this invention into plants through genetic engineering can improve the plant's resistance to Fusarium head blight and drought. Since this gene is an endogenous gene present in the grain crop wheat, its overexpression will not affect the food safety of the plant and can be widely applied in resistance breeding processes for various plants. Attached Figure Description

[0016] Figure 1 pAT2 vector map

[0017] Figure 2 These are PCR detection results for positive TaJDR1 transgenic wheat plants. #1-#4 represent transgenic lines.

[0018] Fielder is wild-type.

[0019] Figure 3 The expression level of TaJDR1 was significantly increased in transgenic wheat.

[0020] Figure 4 The genetically modified wheat showed improved resistance to Fusarium wilt.

[0021] Figure 5 The drought resistance of genetically modified wheat has been improved. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials and reagents used in the examples are commercially available. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0024] The wheat variety used in this invention, Wangshuibai, is a very important local wheat variety widely used in wheat genetics research. The wheat variety used for transgenic wheat is Fielder, a commonly used model variety for wheat transgenics, characterized by high transformation efficiency.

[0025] Example 1: Cloning of the wheat lectin gene TaJDR1

[0026] 1. Extraction of total RNA from wheat tissues

[0027] (1) Take an appropriate amount of wheat ear tissue infected by Fusarium graminearum, place it in a 2.0 ml centrifuge tube, freeze it with liquid nitrogen, then grind it into powder in a grinder (Germany Lech MM400), add 1 ml Trizol (Sigma), shake vigorously for 15 s to fully lyse the sample, and place it at room temperature for 5 min.

[0028] (2) Add 200 μL of chloroform, shake vigorously for 15 seconds, and let stand at room temperature for 3 minutes. Then centrifuge at 12,000 rpm for 15 minutes at 4°C.

[0029] (3) Take 480 μL of the upper aqueous phase and transfer it to a clean 1.5 ml centrifuge tube. Add an equal volume of chloroform, vortex vigorously to mix, and incubate at room temperature for 3 min. Then centrifuge again at 12000 rpm for 15 min at 4 °C. (4) Take 300 μL of the upper aqueous phase and transfer it to a clean 1.5 ml centrifuge tube. Add 2 / 3 volume of isopropanol, gently invert to mix, and incubate at room temperature for 20 min. Then centrifuge at 12000 rpm for 20 min at 4 °C. (5) Discard the supernatant, add 800 μL of 75% ethanol (prepared with DEPC-treated water) to wash the RNA precipitate, centrifuge again at 8000 rpm for 5 min at 4 °C, discard the supernatant, and repeat the washing twice.

[0030] (6) Finally, in a clean bench, blow the RNA precipitate dry for about 10-15 minutes until the RNA becomes transparent, and then add 35 μL of RNase-free water to fully dissolve the RNA.

[0031] 2. Reverse transcription to obtain first-strand cDNA

[0032] use One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGold, AT311-02) for Reverse Transcription of cDNA

[0033] The reaction system as indicated in the instruction manual

[0034]

[0035] The reaction procedure is as follows: 42℃ for 30 min, 85℃ for 5 s.

[0036] 3. Cloning and sequencing of the TaJDR1 gene

[0037] This study found that TaJDR1 was upregulated in the spike tissue infected by *Fusarium graminearum* through transcriptome analysis. Gene-specific primers A1 were designed using MacVector software at the 5'UTR and 3'UTR. The A1 primer pair sequence is as follows: F-5'-AGCACCTGGTTTTATCAGCTTCC-3';

[0038] R: 5'-GGGTCGGATACTGAATGATTTAG-3'. Primers were synthesized by Nanjing GenScript Biotech Co., Ltd. Using this cDNA as a template, amplification was performed using primer pair A1 and KOD Fx DNA polymerase (catalog number KFX-101). The amplification system is as follows:

[0039]

[0040] The reaction program is as follows: PCR program: 94℃ for 5 min; 94℃ for 30 s, 57℃ for 30 s, 68℃ for 1 min, 36 cycles; 68℃ for 10 min; product stored at 4℃.

[0041] The cDNA of the product obtained by PCR amplification is 1056 bp in length (SEQ ID NO: 1). The gene corresponding to this PCR product is named TaJDR1. The amino acid sequence of the protein encoded by the TaJDR1 gene is shown in SEQ ID NO: 2.

[0042] Example 2: Obtaining the recombinant plasmid pUCBS-TaJDR1

[0043] Primers for the transgenic overexpression vector A2 were designed using MacVector software. The primer sequences are as follows: F-5'-GTGTTACTTCTGCAGATGGCCACACCCACCGATG-3'; R:5'-TGAACGATCCTGCAGTTAGATGGTGTAAACACCAATTGAAGTGAC-3'. The primers were synthesized by GenScript Biotech in Nanjing. The homologous arm represents the sequence of the intermediate vector pAT2.

[0044] Using the cDNA obtained in Example 1 as a template, PCR amplification was performed using KOD FX DNA polymerase. The PCR product containing the TaJDR1 gene with the homologous recombination arm of the pAT2 vector was obtained. The pAT2 vector was modified from the pMD19-T vector by linking it to a Ubi+Nos expression cassette, as shown in the diagram. Figure 1 As shown, the full sequence of the plasmid is shown in SEQ ID NO: 3.

[0045] The PCR product containing homologous arms obtained in the previous section and the pAT2 vector were used to construct the vector via homologous recombination. The homologous recombinase was supplied by TransGen Biotech Ltd. Seamless Cloning and Assembly Kit. The cloning system is as follows, with the optimal molar ratio of linearized vector to insert fragment being 1:3. The cloning procedure is: 50℃ for 15 min.

[0046] Connection system

[0047]

[0048] Primers A3 for the transgenic overexpression vector were designed using MacVector software. The primer sequences are as follows: F-5'-GTCGACTCTAGAGGATCCAAGCTTTGCAGCGTGACC-3'; R:5'-GGTAATCCAAGGGGATCCAAGCTTGATCTAGTAACATAGATGAC-3'. The primers were synthesized by GenScript Biotech in Nanjing. The homologous arms are the sequences of the vector pUC BS.

[0049] Using the correctly sequenced pAT2 vector as a template, PCR amplification was performed using KOD FX DNA polymerase. The resulting PCR product contained the TaJDR1 gene expression cassette (Ubi:TaJDR1:Nos) of the pUCBS vector homologous recombination arm.

[0050] The PCR template containing homologous arms obtained in the previous section and the pUCBS vector were used to construct the vector via homologous recombination. The homologous recombinase was supplied by Beijing TransGen Biotech Co., Ltd. Seamless Cloning and Assembly Kit. The cloning system is as follows, with the optimal molar ratio of linearized vector to insert fragment being 1:3. The cloning procedure is: 50℃ for 15 min.

[0051] Example 3: Screening and Obtaining Positive Transgenic Wheat

[0052] Following the materials and methods described in the 1993 Weeks article published in Plant Physiology, Vol. 102, pp. 1077-1084, callus tissue generated from short-term culture of immature embryos, capable of regenerating fertile plants at a high frequency, was used as the recipient. The TaJDR1 gene, controlled by the Ubiquitin promoter, was introduced into the wheat variety Fielder using a gene gun method. The gene-gun-transformed embryos were cultured in the dark on induction medium for 30 days to induce callus formation. The callus tissue was then transferred to selection medium containing 20 mg / L G418 for selection. Finally, the regenerated seedlings were transferred to MS medium for rooting. After root development, leaves were harvested, DNA was extracted, and PCR detection was performed. Positive plants were identified as transgenic wheat with TaJDR1 overexpression.

[0053] Transgenic lines were tested for positive plants using the following PCR system: including 20-40 ng DNA template, 10× 1.25 μL of 2.5 mM dNTPs (Trans), 1 μL of EasyTaq (Trans), 0.5 U of primers (10 mM), and sterile water to bring the volume to 12.5 μL. The PCR reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 2 min, 36 cycles; 72℃ extension for 5 min; storage at 4℃; the amplified product was subjected to agarose gel electrophoresis. If a specific band of 1936 bp appeared, it proved that it was a positive plant. Figure 2 The expression level of TaJDR1 transgenic wheat was detected by agarose gel electrophoresis of the amplified products. The presence of a specific band of 267 bp indicated increased expression. Figure 3 ).

[0054] Example 4: Identification of the resistance to Fusarium head blight in TaJDR1 transgenic wheat

[0055] Fusarium head blight inoculation: During the flowering stage, 10 μL of a mixed spore suspension (F15, F301, F1312, GFP) was simultaneously dripped into the leaves of T1 generation transgenic wheat and the basal florets of spikelets in the middle of both transgenic and non-transgenic ears. A label indicating the inoculation date was also attached to the inoculated ears. Phenotypic results showed that 14 days after single-floret drip inoculation, the Fusarium head blight phenotype in the wild-type material Fielder had spread to the entire ear or most of the ear, while in the overexpressing transgenic plants, it had spread to 1 / 3 of the ear. Figure 4 A). Statistical analysis showed that the average pathological axis length of overexpressing transgenic plants was significantly reduced ( Figure 4 B)(P<0.01).

[0056] Example 5: Identification of drought resistance function in TaJDR1 transgenic wheat

[0057] T2 generation overexpressing transgenic plants with identified genotypes and negative-negative plants (Fielder) isolated from the transgene were planted in pots. They were placed in an incubator at 16h / 8h (light / dark), 18℃ / 12℃ (light / dark), and 40% relative humidity, with three replicates. Healthy plants at the two-leaf stage were subjected to drought until obvious wilting phenotypes appeared, then rehydrated for 7 days. Photographs were taken and plant survival rates were assessed. Phenotypic results showed that the survival rate of overexpressing transgenic plants was significantly higher than that of negative-negative plants. Figure 5 ).

[0058] In conclusion, transferring the TaJDR1 gene into plants can enhance their resistance to Fusarium head blight and drought stress.

[0059] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. Overexpression of wheat TaJDR1 The application of genes in improving wheat resistance to Fusarium head blight and drought, the aforementioned TaJDR1 The cDNA sequence of the gene is shown in SEQ ID NO:

1.

2. Contains wheat TaJDR1 The application of recombinant gene expression vectors in improving wheat resistance to Fusarium head blight and drought. TaJDR1 The cDNA sequence of the gene is shown in SEQ ID NO:

1.

3. The application according to claim 2, characterized in that, The recombinant expression vector is a vector for overexpression.

4. The application according to claim 3, characterized in that, The recombinant expression vector is the one described in claim 1. TaJDR1 The gene is first inserted into the pAT2 cloning vector. PstI After restriction enzyme sites, the Ubi+TaJDR1+Nos expression cassette was inserted into the pUCBS expression vector. BamH I Obtained from the enzyme cleavage site.

5. The application according to claim 4, characterized in that, The nucleotide sequence of the pAT2 cloning vector is shown in SEQ ID NO:3.

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