Patent Application
20250019718
This patent application claims the benefit and priority of Chinese Patent Application No. 2023108404214 filed with the China National Intellectual Property Administration on Jul. 10, 2023, the disclosure of which is incorporated by reference herein in its entirety as part of the present application.
A computer readable XML file entitled “HLP20230705716_seqlist”, which was created on Sep. 12, 2023, with a file size of about 18,581 bytes, contains the sequence listing for this application, has been filed with this application, and is hereby incorporated by reference in its entirety.
The present disclosure belongs to the field of genetic engineering and specifically relates to a broad-spectrum disease resistance-related protein derived from wheat and a related biomaterial and use thereof.
Wheat stripe rust is a major disease of wheat caused by the wheat stripe rust fungus (Puccinia stiiformis f. sp. tritici), which is a serious hazard worldwide. China is the largest epidemic area of stripe rust in the world, with an average annual incidence of about 4 million hectares. After the stripe rust fungus infests into the host plant, the host plant develops symptoms such as yellow wilt and necrosis. Or when the fungus produces a large number of uredinia on the host surface, green leaves and tissues are reduced, resulting in a great reduction of photosynthesis in the plant.
In nature, plants have evolved sophisticated defense mechanisms against pathogenic microbes. These defense mechanisms include PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). ETI is usually accompanied by the onset of hypersensitive response (HR). HR is the most common form of disease resistance in plants, as evidenced by the formation of localized dieback at the point of infestation and limitation of pathogen growth. Because MAMPs, PAMPs or DAMPs are much conserved, PTIs are readily recognized by most pathogens, whereas effector proteins are highly specific, resulting in differences in ETIs between species or physiological races. Although the activation of ETI and PTI differ in their modes of activation, both induce a range of plant immune responses, including ion mobility at lipid membranes, an increase in intracellular calcium ion concentration, formation of reactive oxygen species (ROS), and activation of the mitogen-activated protein kinase (MAPK) signaling pathway. Subsequent reactions include secretion of antimicrobial proteins, lignification of the cell wall, and so on.
Therefore, it has become an important tool for the sustainable control of wheat stripe rust to improve the disease resistance of wheat through mining wheat disease resistance genes, and understanding the response and signaling mechanisms of wheat under stripe rust stress.
The technical problem to be solved by the present disclosure is to provide a wheat disease resistance protein. The technical problem to be solved is not limited to the described technical subject matter, and other technical subject matters not mentioned herein will be clearly understood by those skilled in the art according to the following description.
To solve the above technical problem, the present disclosure provides the following technical solution.
The present disclosure provides use of a protein, a substance regulating expression of a gene encoding the protein, or a substance regulating activity and/or content of the protein in the following aspects of D1)-D6).
The protein is named as TaCNGC5.1 and is selected from the group consisting of
D1) increasing disease resistance in wheat; D2) preparation of a products to improve disease resistance in wheat;
The protein provided by the present disclosure is derived from the wheat variety Suwon 11 (Triticum aestivum).
In order to facilitate the purification or assay of the protein in A1), protein tags may be attached to the amino terminus or carboxy terminus of the protein with the amino acid sequence of SEQ ID NO: 1 in the sequence listing. The protein tags include, but are not limited to, those listed in Table 1.
The protein TaCNGC5.1 in A2) may be synthesized artificially, or the gene encoding the protein may be synthesized first and then biologically expressed to obtain the protein. The gene encoding the protein TaCNGC5.1 in A2) may be obtained by deletion and/or missense mutation of the DNA sequence set forth in SEQ ID NO: 1, and/or by ligation of the coding sequence with the tag shown in Table 1 at its 5′ end and/or 3′ end.
The nucleotide sequence encoding TaCNGC5.1 of the present disclosure can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides that have been artificially modified to have 75% or greater identity to the nucleotide sequence of TaCNGC5.1 isolated and obtained from the present disclosure are derived from and equivalent to the nucleotide sequence of the present disclosure as long as they encode TaCNGC5.1 and have the same function.
The term “identity” as used herein refers to sequence similarity relative to a natural nucleic acid sequence. Nucleotide sequences having an “identity” includes those having at least 75%, at least 85%, at least 90%, or at least 95% identity to the nucleotide sequence of a protein with the amino acid sequence of SEQ ID NO: 1 of the present disclosure. The identity can be evaluated by naked eyes or by computer software. In computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.
The at least 75% identity may be at least 80%, 85%, 90%, or 95% identity.
Regulating the expression of the gene coding for protein TaCNGC5.1 may be enhancing, up-regulating or increasing the expression of the gene coding for protein TaCNGC5.1. Regulating protein activity and/or content of protein TaCNGC5.1 may be enhancing, up-regulating or increasing the activity and/or content of protein TaCNGC5.1.
The disease resistance in wheat refers to resistance to stripe rust. The stripe rust can be caused by infection with at least one of the pathogens selected from stripe rust physiological race group consisting of CYR31, CYR32, CYR33 and CYR34.
The substances described above are biomaterials selected from the group consisting of
In the biomaterials, the expression cassette containing the nucleic acid molecule encoding TaCNGC5.1 (TaCNGC5.1 gene expression cassette) refers to a DNA capable of expressing TaCNGC5.1 in a host cell, and may include not only a promoter for initiating the transcription of TaCNGC5.1, but also a terminator for terminating the transcription of TaCNGC5.1. Further, the expression cassette may also include an enhancer sequence. Promoters that may be used in the present disclosure include, but are not limited to: constitutive promoters: tissue-, organ- and development-specific promoters and inducible promoters. Suitable transcriptional terminators include, but are not limited to the nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus (CaMV) 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the mannopine synthase (Mas) or octopine synthase (Ocs) terminators.
Recombinant vectors containing the TaCNGC5.1 gene expression cassette can be constructed with expression vectors in the art. The plant expression vectors include, for example, binary Agrobacterium vectors and vectors that can be used for plant particle bombardment, such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1305, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb (CAMBIA Corp.). The expression vectors for plant may also include a 3″-end non-translated region of an exogenous gene, i.e., including a polyadenylate signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylate signal directs the incorporation of polyadenylate into the 3″ end of the mRNA precursor, for example, both Agrobacterium crown gall tumor-inducing (Ti) plasmid genes (e.g., the nopaline synthase gene. Nos), and the non-translated region of transcription at the 3′ end of plant genes (e.g., the embryonic storage protein genes) have such similar functions. When plant expression vectors are constructed using the genes of the present disclosure, enhancers, including translational enhancers and transcriptional enhancers, may also be used, and these enhancer regions may include ATG start codons or neighboring region start codons, etc., provided that it has the same reading frame as that of the coding sequence in order to ensure the correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging and can be natural or synthetic. Translation initiation regions can be derived from transcription initiation regions or structural genes. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used may be processed, e.g., by incorporating genes encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), marker genes for antibiotics (such as the nptII gene conferring resistance to kanamycin and related antibiotics, the bar gene conferring resistance to the herbicide phosphinothricin, the hph gene conferring resistance to the antibiotic hygromycin, the dhfr gene conferring resistance to methotrexate, and the EPSPS gene conferring resistance to glyphosate), or chemical marker genes such as those conferring resistance to phytocide, or the mannose-6-phosphate isomerase gene having the ability to metabolize mannose. In view of the safety of transgenic plants, transformed plants can be directly screened under stress without adding any selective marker gene.
In the biological biomaterials, the vector may be a plasmid, a cosmid, a phage or a viral vector.
In the biomaterials, the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium.
In the biomaterials, none of the transgenic plant cell lines is for propagation.
The present disclosure also provides a method for breeding transgenic wheat with improved disease resistance, including up-regulating or enhancing or increasing the expression of a gene encoding the foregoing protein or the content of the protein in target wheat to obtain disease-resistant wheat having higher disease resistance than the target wheat.
Disease resistance of the wheat in the method is resistance to stripe rust. The stripe rust can be caused by infection with at least one of the pathogens selected from stripe rust physiological race group consisting of CYR31, CYR32, CYR33 and CYR34. The resistance to stripe rust is specified in any one of the following (1)-(3): (1) the spore production of the stripe rust bacteria infesting the transgenic wheat is lower than that of the recipient plant under the stripe rust stress conditions: (2) the expression level of the disease course-related genes of the transgenic plant is higher than that of the recipient plant under the stripe rust stress conditions; and (3) the area of the stripe rust mycelium infestation of the stripe rust fungus infesting the transgenic wheat is lower than that of the recipient plant under the stripe rust stress conditions. The stripe rust stress conditions are compatible or incompatible treatments, with CYR31 strain for compatible treatment and CYR23 strain for incompatible treatment.
The wheat is specifically the wild type wheat Fielder.
Up-regulating, enhancing or increasing the expression of the gene encoding the protein or the content of the protein in the target wheat in the method is to introduce the gene encoding the protein into the target wheat.
In an embodiment of the present disclosure, the gene encoding the TaCNGC5.1 protein (i.e., the nucleotide of SEQ ID NO: 2) is introduced into Agrobacterium tumefaciens EHA105 by means of a recombinant vector CUB-TaCNGC5.1 containing an expression cassette for the gene encoding the TaCNGC5.1 protein. The recombinant vector CUB-TaCNGC5.1 is introduced into Agrobacterium tumefaciens EHA105 by homologous recombination to insert a DNA fragment of TaCNGC5.1 into the CUB vector while keeping the other sequences of the CUB vector intact. The vector cleavage site is BamH1.
In the above method, the transgenic plant is understood to include not only the first generation of transgenic plants obtained by transforming the recipient plant with the TaCNGC5.1 gene, but also its progeny. In the case of transgenic plants, the gene can be propagated in the species or can be transferred into other varieties of the same species, including in particular commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, complete plants and cells.
The aforementioned proteins, the biomaterials in B1) to B4) also fall within the scope of the present disclosure.
The experiments of the present disclosure have demonstrated that the TaCNGC5.1 gene discovered in the present disclosure is induced to be expressed by Puccinia striiformis f, sp. tritici. and that the transgenic wheat obtained by introducing the TaCNGC5.1 gene into wheat is more resistant to multiple physiological races of Puccinia striiformis f, sp. tritici than the wild-type wheat. The proteins and genes provided by the present disclosure provide a basis for artificially controlling the expression of disease resistance-related genes, and will play an important role in breeding plants with broad-spectrum disease resistance.
The present disclosure is described in further detail below in conjunction with specific examples, which are given for the sole purpose of elucidating the present disclosure and not for the purpose of limiting the scope of the present disclosure. The examples provided below may serve as a guide for further improvements by a person of ordinary skill in the art, and do not in any way limit the present disclosure.
The experimental methods in the following examples, if not otherwise specified, are conventional methods, and are performed in accordance with the techniques or conditions described in the literature in the field or in accordance with the instructions for the product. The materials, reagents, and the like used in the following examples are commercially available, if not otherwise specified.
The GFP16318 (green fluorescent protein) vector in the following example is documented in the literature “Molecular characteristics and functional identification of foxtail millet transcription factor WRKY36 [J]. Scientia Agricultura Sinica, 2015, 48(5): 851-860.”, which is available to the public from the Northwest A&F University.
The wheat stripe rust physiological race CYR23 in the following example is described in the literature “Liu P, Guo J, Zhang R, et al. TaCIPK10 interacts with and phosphorylates TaNH2 to activate wheat defense responses to stripe rust [J]. Plant biotechnology journal, 2019, 17(5): 956-968.”. It is available to the public from the Northwest A&F University.
The wheat variety Fielder, the wheat stripe rust physiological races CYR32, CYR33 and CYR34 in the following example are described in the literature “Bai X, Zhan G, Zhang R, Tian S, et al. Transcription factor BZR2 activates chitinase Cht20.2 transcription to confer resistance to wheat stripe rust [J]. Transcription factor BZR2 activates chitinase Cht20.2 transcription to confer resistance to wheat stripe rust [J]. Plant physiology, 2021, 187:2749-2762.”. They are available to the public from the Northwest A&F University.
The wheat stripe rust fungus CYR31 in the following example is described in the literature “Fengle Wang, Liren Wu, Shichang Xu, Shelin Jin, Qiuzhen Jia, Wenhuan Yuan, Jiaxiu Yang. The discovery and studies on new races CYR30 and CYR31 of wheat stripe rust in china [J]. Acta Phytophylacica Sinica, 1996(01): 39-44.”. It is available to the public from Northwest A&F University.
The wheat variety suwon 11 in the following example is described in the literature “Cao Zhangjun, Jing Jinxue, Wang Meinan, et al. Relation analysis of stripe rust resistance gene in wheat important cultivar suwon 11, suwon 92 and hybrid 46 [J]. Acta Botanica Boreali-Occidentalia Sinica, 2003, 23(1): 64-68.” It is available to the public from Northwest A&F University.
Cellulase R10 (YaKult Honsha) cellulase (Yakult, C6270-1g), Mecerozyme R10 (YaKult Honsha) pectinase (Rongxing Biotechology, RX-L0042-100 mg), Mannitol (Beijing Mengyimei Trade Center, M0122-500g), KOH (Beijing Xiyanghuizhi Technology Co., Ltd, XYHZ-2017-05185), KCl (Beijing Biorj Science and Technology Co., Ltd, 7447-40-7), MES (Beijing BioDee Biotechnology Co., Ltd, DE-E169-100g), CaCl2) (Beijing BioDee Biotechnology Co., Ltd, 031-00435), NaCl (Beijing BioDee Biotechnology Co., Ltd., 7647-14-5), MgCl2 (Beijing BioDee Biotechnology Co., Ltd., DE-0288-500g), Glucose (Beijing BioDee Biotechnology Co., Ltd., 049-31165), PEG4000 (Beijing BioDee Biotechnology Co., Ltd., BR-0084), Bovine Serum Albumin (BSA, Beijing ZEPING Bioscience & Technology Co., Ltd, 0219989980), β-Mercaptoethanol (Beijing Readerbio technology Co., Ltd., 0482-100ML).
The formulations of the reagents used in the following examples are as follows.
I. Isolation of mRNA and Amplification of TaCNGC5.1
Seedlings of normally grown 7-day-old wheat suwon 11 were taken, snap-frozen in liquid nitrogen and stored at −80° C., for later use.
Total RNA from wheat leaves was extracted using a Quick RNA isolation kit (Huayueyang Biotechnology (Beijing) Co., Ltd.), and the first-strand cDNA was synthesized with reverse transcriptase XL (AMV). The cDNA was synthesized by the Switching Mechanism at 5′ End of RNA Template (SMART) method. PCR was carried out with the cDNA as a template, and TaCNGC5.1-F and TaCNGC5.1-R as primers. The PCR product was detected by 1.0% agarose gel electrophoresis, and a PCR product of 2070 bp was obtained. The amplification primers were:
The PCR product was shown to have the nucleotide of SEQ ID NO: 2 in the sequence listing according to the sequencing results, which was named as TaCNGC5.1 gene. The gene encodes a protein with the amino acid sequence of SEQ ID NO: 1 in the sequence listing, and was named as TaCNGC5.1 protein.
II. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Detection of TaCNGC5.1 Expression Induced by Puccinia striiformis f, Sp. Tritici.
P. striformis f. sp. tritici inoculation was performed according to the method of Zhensheng Kang et al. (1984, Journal of Northwest A & F University). The leaves of suwon 11 were inoculated with CYR23, a incompatible race of stripe rust fungus, or CYR31, an compatible race of stripe rust fungus, to form a combination of incompatible and compatible interactions, and leaves inoculated with sterile water were used as a control.
Samples in the treatment groups were taken at 0 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h after inoculation, respectively, and the same for that in the control. For sampling, fresh leaves were cut, wrapped in tinfoil paper, snap-frozen in liquid nitrogen, and then stored at −80° C. Total RNA of wheat leaves was extracted by Trizol method (TianGen), and the first strand cDNA was synthesized by reverse transcriptase XL (AMV). The cDNA was synthesized using the SMART method.
Specific quantitative PCR primers were designed based on the sequences of wheat TaCNGC5.1 and the elongation factor TaEF-1α (GenBank accession number: U76744).
RT-PCR primer sequences were:
Quantitative PCR primers were tested for the specificity of their amplification products and amplification efficiency (>90%) before use, and TaEF-la was used as an internal reference gene in Real-time PCR analysis. Real-time quantitative PCR amplification was performed using AceQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) and Bio-Rad CFX Manager quantitative PCR instrument (Bio-rad, Hercules, California), following the instructions with the cDNAs of the sampling points of each treatment as templates, respectively. At least 3 replicates were set for each reaction, and the Ct values for each replicate, as well as their mean and standard deviation, were generated by the quantitative PCR instrument by manually adjusting the baseline. Three replicates were set for each reaction, the Ct values were averaged, and the experimental data were analyzed by Delta Delta Ct method to determine the relative expression of the genes.
The results of quantitative RT-PCR (qRT-PCR) of TaCNGC5.1 in suwon 11 after inoculation with stripe rust incompatible race CYR23 and stripe rust compatible race CYR31, respectively, are shown in
The PCR product TaCNGC5.1 fragment amplified in “I. Isolation of mRNA and amplification of TaCNGC5.1” was ligated into GFP16318 (green fluorescent protein) vector after BamH I digestion, to obtain the recombinant vector TaCNGC5.1-GFP, which expresses the fusion protein TaCNGC5.1-GFP.
The primer sequences for TaCNGC5.1-GFP used for subcellular localization of TaCNGC5.1 were as follows (underline indicates the cleavage site of BamH I):
Methods for preparing wheat protoplasts for transformation:
The following operations were performed at room temperature of 23° C.
Protoplasts transfected with GFP16318 vector was used as a control.
Afterwards, GFP tag expression was observed under a laser confocal microscope.
Protoplasts after 18 h of dark culture were pressed into slices, and then GFP (green fluorescent protein) fluorescence was observed in a laser scanning confocal microscope (LSMC, Biorad MicroRadiance) and scanned and photographed. The working parameters of the LSCM were Ex=488 nm, Em=525+15 nm, Power=10%, Zoom 7, Medium speed scanning, Frame512×512. The software was TIME-COURSE and PHOTOSHOP5.0.
The results are shown in
I. Acquisition of Wheat Transformed with TaCNGC5.1 Gene
The CUB vector is described in “Shuzhang Xie, Kairong Lei, Xiaoyan Yang et al. Study on insect-resistant gene of GmCry1F transformation in maize by Agrobacterium-mediated [J]. Southwest China Journal of Agricultural Sciences, 2015, 28(3):962-966” and is available to the public from Northwest A&F University.
TaCNGC5.1-CUB vector was specifically constructed as follows:
PCR amplification was carried out with TaCNGC5.1-CUB-F and TaCNGC5.1-CUB-R as primers, and TaCNGC5.1 full-length CDS amplified in the first part of Example I as a template, and the amplified fragment (nucleotides 1-2067 of SEQ ID NO: 2 in the Sequence listing) was constructed into a BamH I digested CUB vector by homologous recombination using ClonExpress II One Step Cloning Kit (vazyme) to obtain the recombinant vector TaCNGC5.1-CUB, which is a recombinant vector obtained by inserting the sequence of nucleotides 1-2067 of SEQ ID NO:2 in the sequence listing into the CUB vector, with the rest of the bases remained unchanged.
Note: the underline indicates the enzyme cleavage site of BamH I.
2. Acquisition of Wheat Transformed with TaCNGC5.1 Gene
The recombinant vector TaCNGC5.1-CUB was used to infect the callus of wild-type wheat Fielder using Agrobacterium to obtain TO generation of TaCNGC5.1 transgenic wheat. The TO generation TaCNGC5.1 transgenic wheat was cultivated until two lines of T3 generation TaCNGC5.1 transgenic wheat were obtained.
Genomic DNA from leaves of two lines of T3 generation TaCNGC5.1 transgenic wheat for and wild-type control (Fielder) were extracted separately using CTAB, and TaCNGC5.1 overexpression assay primers (F/R) were utilized to verify whether the aforementioned plants were transgene-positive plants.
Molecular assays were performed on T3 generation transgenic plants (upstream primer F is located on the TaCNGC5.1 gene and downstream primer R is located on the NOS terminator). Water was used as a blank control. Ten plants were randomly taken from each line.
The results are shown in
Wild-type wheat Fielder, TaCNGC5.1 overexpression line 17 and TaCNGC5.1 overexpression line 29 were planted in an incubator at 25/23° C., with day/night temperature and 16 h light/8 h dark photoperiod. After the second leaf unfolded (
The phenotypic results are shown in
Stripe rust fungi biomass was measured in wheat 14 days after inoculation with Stripe rust fungi. The biomass assay was performed according to “Tuo Qi, Jia Guo, Peng Liu, Fuxin He, Cuiping Wan, Md Ashraful Islam, Brett M Tyler, Zhensheng Kang, Jun Guo, Stripe Rust Effector PstGSREI Disrupts Nuclear Localization of ROS-Promoting Transcription Factor TaLOL2. Effector PstGSREI Disrupts Nuclear Localization of ROS-Promoting Transcription Factor TaLOL2 to Defeat ROS-Induced Defense in Wheat. Mol. Plant. 2019 Dec. 2: 12(12): 1624-1638.”
The results are shown in
The above results of disease resistance characterization indicated that under the treatments of four physiological races, CYR31, CYR32, CYR33 and CYR34, TaCNGC5.1 transgenic wheat showed strong resistance. This is a verified advantage in production.
It is shown that TaCNGC5.1 is an important gene involved in the response process of stripe rust resistance in wheat.
The present disclosure is described in detail as above. For those skilled in the art, the present disclosure may be practiced in a wide range of equivalent parameters, concentrations and conditions without departing from the purpose and scope of the present disclosure and without undue experimentation. Although particular embodiments of the present disclosure are given, it should be understood that further improvements can be made to the present disclosure. In summary, in accordance with the principles of the present disclosure, the present application is intended to include any modifications, uses or changes to the present disclosure, including changes that are out of the scope of what has been disclosed in the present application using conventional techniques known in the art.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2023108404214 | Jul 2023 | CN | national |
