TaJDR1 gene and application thereof in improving gibberellic disease resistance and drought resistance of plants

Abstract

The invention discloses a TaJDR1 gene and application of the TaJDR1 gene to improvement of gibberellic disease resistance and drought resistance of plants. The nucleotide sequence of the TaJDR1 gene is as shown in SEQ ID NO. 1, and the amino acid sequence coded by the TaJDR1 gene is as shown in SEQ ID NO. 2. According to the invention, the TaJDR1 gene is cloned from a wheat local variety Zhuishuibai for the first time, and overexpression of the gene in wheat is realized through a genetic transformation technology mediated by a gene gun. Results show that the gibberellic disease resistance and the drought resistance of the transgenic wheat are remarkably improved. The TaJDR1 gene is derived from wheat, and overexpression does not influence food safety, so that the TaJDR1 gene can be widely applied to the field of disease-resistant and drought-resistant breeding of various plants.

Description

Technical Field

[0001] The invention relates to the technical field of gene engineering, in particular to a TaJDR1 gene and an application thereof in improving plant resistance to scab and drought. Background Art

[0002] During their growth and development, plants often face a variety of biotic and abiotic stresses, such as drought, high salt, low temperature, pests and diseases, which can have a significant impact on their growth, development and productivity. Biological and abiotic stresses vary in nature and often interact with each other, posing severe challenges to the overall health of plants. In order to cope with these stresses, plants have developed a variety of defense mechanisms: when facing biotic stresses, plants enhance mechanical strength by thickening cell walls to prevent pathogen invasion or insect feeding, activate the immune system and expression of defense genes, and limit the spread of pathogens through hypersensitive reactions, and produce a series of secondary metabolites to enhance resistance; when dealing with abiotic stresses, plants will improve their resistance by synthesizing osmotic regulating substances, activating antioxidant systems, and regulating ion balance.

[0003] Fusarium head blight and drought are two key stress factors faced in wheat production. They not only affect the growth and development of plants, but also significantly reduce yield and affect quality. Fusarium head blight (FHB) is a spike disease caused by various Fusarium spp., which is usually prone to outbreaks in warm and humid climates, especially during the flowering period of wheat. Although Fusarium can cause diseases at different growth stages of wheat, the infection of the spike is the most serious. Its main manifestations include: grain shrinkage and shriveling, and a significant reduction in 1,000-grain weight; dehydration of the spike tissue affects the transportation of nutrients; grain quality decreases; in addition, the deoxynivalenol (DON) toxin produced in the grain poses a serious threat to human and livestock health. On the other hand, drought stress will limit the development of wheat roots and reduce the absorption of water and nutrients; leaves will shrink and turn yellow, and the photosynthesis capacity will decrease, which will in turn affect the energy supply of the plant; grain development will be hindered, resulting in shriveling and a decrease in 1,000-grain weight. The combined effects of these stress factors greatly reduce the yield and quality of wheat. In this context, it is very important to discover potential candidate genes that can improve plant resistance to scab and drought, which has broad application prospects.

[0004] Plant lectins are widely present in various organisms. They are a class of natural proteins that can bind to oligosaccharide structures in specific glycoproteins or glycolipids on the cell surface and participate in the regulation of many biological processes. Jacalin-related lectin (JRL) is a class of plant lectins first isolated from Artocarpus integrifolia. It has one or more Jacalin domains and is currently a family of plant lectins that are widely studied. Dirigent protein is a protein identified from Forsythia suspensa that can guide coniferyl alcohol radicals to produce (+)-pinoresinol (a type of lignan) (Davin, LB et al., 1997). DIR family genes can respond to abiotic stress and hormone-induced regulation, and can participate in the synthesis of lignin in plants. The DIR domain can also be chimeric with the Jacalin domain at the C-terminus, called Jacalin DirigentRelated protein, abbreviated as JDR. It plays an important role in disease and insect resistance. At present, JRL proteins from many different plants have been reported, which can respond to adverse environmental stress and participate in plant growth and development. For example, the VER2 gene isolated from winter wheat plays an important role in the vernalization signal and panicle development of winter wheat (Yong et al., 2003); the OsJRL gene in rice negatively regulates the cold tolerance 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, is involved in broad-spectrum disease resistance against bacteria, oomycetes and fungi (Weidenbach et al., 2016). Summary of the invention

[0005] The purpose of the present invention is to provide a method for using the TaJDR1 gene to improve plant resistance to ergot and drought, to provide a new candidate gene for the research and cultivation of new wheat varieties resistant to ergot and drought, and thus to promote the development and application of more new varieties resistant to ergot and drought.

[0006] To achieve the above object, the present invention provides the following technical solutions:

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

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

[0009] A recombinant plasmid, wherein the recombinant plasmid contains the wheat scab resistance gene TaJDR1; the plasmid carrier is preferably pUCBS, that is, the recombinant plasmid is preferably pUCBS-TaJDR1. In addition, any vector that can introduce foreign genes into plants for expression can be used in the present invention.

[0010] Antibiotic marker genes (such as hygromycin, kanamycin and gentamicin) are added to the vector, and the vector with the antibiotic marker gene is used for transformation. Antibiotics can be added to the plant culture medium after transformation to inhibit the growth of non-transgenic cell lines and plants, which helps to quickly and effectively obtain transgenic plants.

[0011] In order to facilitate the screening of transgenic plants or cell lines, selection markers such as GUS, GFP, etc. are added.

[0012] The gene gun-mediated stable genetic transformation method is used to introduce nucleic acid molecules capable of expressing TaJDR1 protein into the recipient plant to obtain transgenic plants.

[0013] The recipient plant is a grass plant.

[0014] Beneficial effects:

[0015] The present invention applies plant genetic engineering technology and obtains the gene TaJDR1 from the wheat variety Wangshuibai for the first time. Experiments prove that the wheat lectin protein provided by the present invention is transferred into plants by genetic engineering methods, which can improve the resistance of plants to scab and drought. Since the gene is an endogenous gene existing in the food crop wheat itself, the overexpression of the gene will not affect the food safety of the plant, and can be widely used in the resistance breeding process of various plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 pAT2 vector map

[0017] Figure 2 The PCR test results of transgenic TaJDR1 wheat positive plants. #1-#4 are transgenic lines.

[0018] Fielder is wild type.

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

[0020] Figure 4 The resistance of transgenic wheat to Gibberella has been improved.

[0021] Figure 5 The drought resistance of transgenic wheat has been improved. DETAILED DESCRIPTION

[0022] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials, reagents, etc. used in the examples are all commercially available unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged.

[0024] The wheat variety Wangshuibai used in the present invention is a very important wheat local variety and is widely used in wheat genetics research. The wheat variety used for transgenic is Fielder, which is a commonly used model variety for wheat transgenic and has the characteristics of high transformation efficiency.

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

[0026] 1. Extraction of Total RNA from Wheat Tissues

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

[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, shake vigorously to mix, let it stand at room temperature for 3 minutes, and centrifuge it again at 12000 rpm for 15 minutes 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, let it stand at room temperature for 20 minutes, and then centrifuge it at 12000 rpm for 20 minutes at 4°C. (5) Discard the supernatant, add 800 μL of 75% ethanol (prepared with DEPC-treated water) to wash the RNA precipitate, centrifuge it again at 8000 rpm for 5 minutes at 4°C, discard the supernatant, and repeat the washing twice.

[0030] (6) Finally, in a clean bench, blow dry the RNA precipitate 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 (Full Gold, AT311-02) for reverse transcription of cDNA

[0033] The reaction system according to the instructions

[0034]

[0035] The reaction procedure was: 42°C for 30 min, 85°C for 5 s

[0036] 3. Cloning and sequencing of TaJDR1 gene

[0037] In this study, transcriptome analysis revealed that TaJDR1 was upregulated in panicle tissues infected by Gibberella. Gene-specific primers A1 were designed in 5'UTR and 3'UTR using MacVector software. The sequences of primer pairs A1 were as follows: F-5'-AGCACCTGGTTTTATCAGCTTCC-3';

[0038] R: 5'-GGGTCGGATACTGAATGATTTAG-3'. The primer was synthesized by Nanjing GenScript Co., Ltd. The cDNA was used as a template and amplified with primer pair A1 and KOD Fx DNA polymerase (Cat. No. KFX-101). The amplification system was as follows:

[0039]

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

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

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

[0043] The transgenic overexpression vector primer A2 was designed using MacVector software. The primer sequences are as follows: F-5'-GTGTTACTTCTGCAGATGGCCACACCCACCGATG-3'; R: 5'-TGAACGATCCTGCAGTTAGATGGTGTAAACACCAATTGAAGTGAC-3'. The primers were synthesized by Nanjing GenScript Co., Ltd. The homology arms were the sequences of the intermediate vector pAT2.

[0044] The cDNA obtained in Example 1 was used as a template and PCR amplified using KOD FX DNA polymerase. The PCR product of TaJDR1 gene containing the homologous recombination arm of pAT2 vector was obtained. The pAT2 vector was transformed from pMD19-T vector and connected to the Ubi+Nos expression cassette. The map is shown in Figure 1 The complete sequence of the plasmid is shown in SEQ ID NO: 3.

[0045] The PCR product containing homology arms obtained in the previous part and the pAT2 vector were used to construct the vector by homologous recombination. The homologous recombination enzyme was purchased from Beijing Quanshijin Company. Seamless Cloning and Assembly Kit. The ligation system is as follows. The optimal molar ratio of linearized vector to insert fragment is 1:3. The ligation procedure is: 50℃15min.

[0046] Connection system

[0047]

[0048] The transgenic overexpression vector primer A3 was designed using MacVector software. The primer sequences are as follows: F-5'-GTCGACTCTAGAGGATCCAAGCTTTGCAGCGTGACC-3'; R: 5'-GGTAATCCAAGGGGATCCAAGCTTGATCTAGTAACATAGATGAC-3'. The primers were synthesized by Nanjing GenScript Co., Ltd. The homology arm was the sequence of the vector pUC BS.

[0049] The above-mentioned correctly sequenced pAT2 vector was used as a template and PCR amplification was performed using KOD FX DNA polymerase to obtain a PCR product of the TaJDR1 gene expression cassette (Ubi: TaJDR1: Nos) containing the homologous recombination arms of the pUCBS vector.

[0050] The vector was constructed by homologous recombination using the PCR template containing homology arms obtained in the previous part and the pUCBS vector. The homologous recombination enzyme was purchased from Beijing Quanshijin Company. Seamless Cloning and Assembly Kit. The ligation system is as follows. The optimal molar ratio of linearized vector to insert fragment is 1:3. The ligation procedure is: 50℃15min.

[0051] Example 3: Screening and obtaining of positive transgenic wheat

[0052] Referring to the materials and methods in the article published by Weeks in Plant Physiology, issue 102, pages 1077-1084 in 1993, the callus tissue produced by short-term culture of immature embryos that can regenerate fertile plants at a high frequency was used as the recipient. The TaJDR1 gene under the control of the Ubiquitin promoter was introduced into the wheat variety Fielder by gene gun method, and the embryos transformed by gene gun were placed on an induction medium and cultured in the dark for 30 days to induce callus tissue, and then the callus tissue was transferred to a screening medium containing 20 mg / L G418 for screening, and finally the regenerated seedlings were transferred to MS medium for rooting, and the leaves were cut after the roots grew out, and DNA was extracted for PCR detection. The positive plants were transgenic wheat with overexpression of TaJDR1.

[0053] Transgenic lines were tested for positive plants using the following PCR system: 20-40 ng DNA template, 10× 1.25μL, 2.5mM dNTPs (Trans) 1μL, EasyTaq (Trans) 0.5U, primer (10mM) 0.25μL, sterile water to make up the volume to 12.5ul. PCR reaction program is: 94℃ pre-denaturation 5min; 94℃ denaturation 30s, 60℃ annealing 30s, 72℃ extension 2min, 36 cycles; 72℃ extension 5min; 4℃ storage; agarose gel electrophoresis of the amplified product, if a specific band of 1936bp appears, it proves that it is a positive plant ( Figure 2 The amplified product was tested by agarose gel electrophoresis to detect the expression level of TaJDR1 transgenic wheat. If a specific band of 267 bp was found, it was proved that the expression level was increased ( Figure 3 ).

[0054] Example 4: Identification of the resistance of TaJDR1 transgenic wheat to scab

[0055] Inoculation of Fusarium head blight: At the flowering stage, 10 μL of mixed spore suspension (F15, F301, F1312, GFP) was dripped onto the leaves of T1 transgenic wheat and the florets at the base of the spikelets in the middle of the spikelets of non-transgenic wheat. At the same time, a label with the inoculation date was placed on the inoculated spikelets. The phenotypic identification results showed that 14 days after the single flower drip inoculation, the Fusarium head blight phenotype of the wild-type material Fielder had spread to the entire spikelet or most of the spikelet, while the overexpressing transgenic plants had spread to 1 / 3 of the spikelet ( Figure 4 A). Statistics showed that the average diseased axis length of the overexpressing transgenic plants was significantly reduced ( Figure 4 B)(P<0.01).

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

[0057] The T2 generation overexpression transgenic plants with identified genotypes and the negative plants (Fielder) isolated from the transgene were planted in pots respectively. They were placed in an incubator at 16h / 8h (light / dark), 18℃ / 12℃ (light / dark), and 40% relative humidity. The experiment was repeated 3 times. The healthy 2-leaf plants were deprived of water and drought until an obvious wilting phenotype appeared. Then they were rehydrated for 7 days, photographed, and the survival rate of the plants was investigated. The results of phenotypic identification showed that the survival rate of the overexpression transgenic plants was significantly higher than that of the negative plants ( Figure 5 ).

[0058] In summary, transferring the gene TaJDR1 into plants can make the plants have stronger resistance to ergot and drought stress.

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

Claims

1. Wheat TaJDR1 gene, characterized in that: The cDNA sequence of the TaJDR1 gene is shown in SEQ ID NO:

1.

2. The protein encoded by the wheat TaJDR1 gene according to claim 1, characterized in that The amino acid sequence is shown in SEQ ID NO:

2.

3. A recombinant expression vector containing the gene according to claim 1.

4. The recombinant expression vector according to claim 3, characterized in that: A recombinant vector is any vector that can direct the overexpression of foreign genes in plants.

5. The recombinant expression vector according to claim 4, characterized in that: The recombinant expression vector is obtained by first inserting the TaJDR1 gene described in claim 1 into the PstI restriction site of the pAT2 cloning vector, and then inserting the Ubi+TaJDR1+Nos expression box into the BamH I restriction site of the pUCBS expression vector.

6. The recombinant expression vector according to claim 5, characterized in that: The nucleotide sequence of the pAT2 cloning vector is shown in SEQ ID NO:

3.

7. Use of the gene according to claim 1 in improving wheat resistance to fusarium head blight and drought.

8. Use of the recombinant expression vector according to any one of claims 4 to 6 in improving wheat resistance to fusarium head blight and drought.

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