Poa pratensis drought-resistant gene ppnrt2.4 and application thereof

Abstract

This invention belongs to the field of genetic engineering technology, specifically relating to a drought-resistant gene in Kentucky bluegrass. PpNRT2.4 And its applications. This invention provides a nitrate transporter gene derived from Kentucky bluegrass. PpNRT2.4 And provide its complete coding sequence and the protein sequence it encodes, clarifying the... PpNRT2.4 The study investigated the biological functions of the gene and confirmed that its overexpression can significantly improve the drought resistance of transgenic plants, providing a precise, efficient, and safe innovative technical path for creating new drought-resistant germplasm for turfgrass and other crops and breaking through the bottleneck of traditional breeding efficiency.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the drought-resistant gene PpNRT2.4 of Kentucky bluegrass and its application. Background Technology

[0002] With the global water shortage becoming increasingly severe, developing water-saving turfgrass with high water use efficiency has become a core requirement for urban ecological construction. Kentucky bluegrass (Poa pratensis), a widely cultivated cool-season turfgrass globally, directly impacts maintenance costs and ecological benefits due to its drought resistance and water use efficiency. Therefore, in-depth exploration of its inherent drought-resistant genetic potential is of significant strategic importance for cultivating new turfgrass varieties with low water consumption and high tolerance, and for promoting the sustainable development of landscaping.

[0003] Plants have developed complex drought resistance regulatory networks over long periods of evolution, among which the synergistic mechanism of nitrogen metabolism and water stress response has attracted much attention in recent years. Nitrate transporters (NRTs) not only dominate nitrogen absorption and transport, but their family members (especially the NRT2 subfamily) have been shown to be widely involved in plant abiotic stress responses. However, current research on plant drought resistance genes mainly focuses on dehydrogenases, LEA proteins, and certain transcription factor families, while research on the specific functions and molecular mechanisms of the NRT2 family in turfgrass drought resistance remains lacking. Currently, no NRT2 family gene has been successfully cloned and functionally validated for drought resistance from Kentucky bluegrass, which limits the application of this important gene resource in molecular breeding.

[0004] The scarcity of specific drought-resistant gene resources: Currently, there is a lack of functionally validated, highly efficient drought-resistant genes extracted from the genetic background of Kentucky bluegrass itself. This results in a very limited selection of specific genetic elements when using genetic engineering to precisely improve the drought resistance of turfgrass.

[0005] Lack of understanding and utilization of NRT2 family gene functions: Although existing studies have suggested that the NRT2 family may be involved in stress response, current technologies have not provided any experimental evidence or clear conclusions regarding key scientific questions such as whether Kentucky bluegrass NRT2 genes (such as PpNRT2.4) directly participate in drought resistance regulation, the subcellular localization of their encoded proteins, and whether overexpression can confer heritable drought resistance phenotypes in plants.

[0006] Drought-resistant breeding technologies are limited and inefficient: traditional hybridization breeding is time-consuming, complex, and struggles to overcome interspecific reproductive barriers. Existing transgenic drought-resistant strategies often suffer from low expression efficiency and uncertain ecological risks due to incompatibility between exogenous genes and the recipient plant's genetic background. Furthermore, there is a lack of safe and functionally defined gene resources derived from closely related or identical species. Summary of the Invention

[0007] This invention clones and identifies a nitrate transporter gene, PpNRT2.4, derived from Kentucky bluegrass, providing its complete coding sequence and the protein sequence it encodes, filling a gap in existing databases for this gene resource. The biological function of the PpNRT2.4 gene is clarified, its role in plant drought resistance is verified through in vitro and in vivo experimental systems, its subcellular localization is elucidated, and its overexpression is demonstrated to significantly enhance the drought resistance of transgenic plants, thus transforming a potential genetic resource into an empirically proven and usable drought-resistant gene element. The invention also provides a scheme for the application of the PpNRT2.4 gene in molecular breeding, particularly in the cultivation of drought-resistant transgenic plants, offering a precise, efficient, and safe innovative technical path for creating new drought-resistant germplasm for turfgrass and other crops, and overcoming the bottlenecks in traditional breeding efficiency.

[0008] To address the shortcomings of existing technologies, this invention is achieved through the following solution:

[0009] This invention provides a drought-resistant Kentucky bluegrass gene PpNRT2.4, the sequence of which is shown in SEQ ID NO:1:

[0010]

[0011] The present invention also provides a drought-resistant Kentucky bluegrass protein PpNRT2.4, wherein the protein PpNRT2.4 is the protein encoded by the gene PpNRT2.4 as described in claim 1;

[0012] The amino acid sequence of the protein PpNRT2.4 is shown in SEQ ID NO:2:

[0013] MVTMGKKDVQQEQYYCGDWPVDGVDAEGRATELRPLALSHPHTQAFHLAWLSLFACFFAAFAAPPILPALRPALVLAPSDASTAAVGSLVAALVGRLVMGPVCDLLGPRRASGVASLVCALA LALAAVYASSPAGFVALRFCAGLSLSNFVANQHWMSRIFAPSAVGLANAVAAGWANVGSAAAQIAMPLAYDCIVLRLGVPITVAWRVAYLIPCAMLITTGLAVLAFPYDLPQGCAASGGGRDK GGDKGTKGFWKAVRGGVCDYRAWVLLLTYGYCYGVELIMENVAADFFRRRFRLPMEAAGAAAACFGVMNTVARPAGGVASDEVGRRFGMRGRLWALWAVQSTGAVLCVLVGRMGASEAPSLA ATMAVMVACGAFVQAASGLTFGIVPFVSKRSMGVVSGMTASGGAVGAIITNRLFFSSSRYTVEEAISFTGLTSLLCTLPLALIYFPRSGGMLCGAYGYEFIDQDCHDDDVNKDDDYMLLK*.

[0014] This invention provides a recombinant vector containing the gene PpNRT2.4, wherein the backbone of the expression vector is pBWA(V)HS.

[0015] The present invention provides a strain containing the PpNRT2.4 gene, wherein the strain is an Agrobacterium containing the PpNRT2.4 gene.

[0016] The present invention also provides the application of the gene PpNRT2.4 in improving the drought resistance of plants, wherein the gene PpNRT2.4 improves the drought resistance of plants through overexpression.

[0017] The present invention also provides a method for cultivating drought-resistant plants, characterized in that the method comprises: overexpressing the gene PpNRT2.4 in plants to obtain plants with enhanced drought stress tolerance.

[0018] Compared with existing technologies, the present invention has the following advantages:

[0019] (1) It provides drought-resistant gene resources from the Kentucky bluegrass NRT2 family.

[0020] The PpNRT2.4 gene (CDS: 1467 bp) of Kentucky bluegrass was successfully cloned, and rice plants overexpressing this gene were created using transgenic technology. RT-qPCR and physiological and biochemical assays demonstrated that this gene significantly enhances the drought resistance of rice. This directly transforms a previously unknown genetic sequence into a functionally defined and effective usable gene element, providing a novel, efficient, and specific gene resource for the genetic improvement of drought resistance in turfgrass and other crops.

[0021] (2) The protein characteristics and subcellular localization of PpNRT2.4 were revealed, providing key evidence for its functional identification.

[0022] Bioinformatics analysis confirmed that protein PpNRT2.4 is a hydrophobic integrated membrane protein containing the conserved domain PLN00028, which is associated with stress response, and its potential function was predicted from the sequence. More importantly, through GFP fusion protein experiments, it was clarified for the first time that this protein is simultaneously located in the plasma membrane and the cell nucleus. This discovery not only provides key clues for understanding its potential involvement in signal transduction and gene regulation, but also provides direct cell biological evidence for its drought resistance function at the cellular level.

[0023] (3) A new, efficient, and safe approach to drought-resistant breeding based on homologous genes from closely related species

[0024] The PpNRT2.4 gene from Kentucky bluegrass (a monocotyledonous plant) was introduced into rice, another monocotyledonous plant. Because the donor (Kentucky bluegrass) and recipient (rice) are closely related, their genetic background compatibility is higher, effectively avoiding problems such as low gene expression efficiency and potential ecological risks in distantly related species. The successful acquisition of T0 generation plants with significantly enhanced drought resistance demonstrates the high feasibility of using this gene for molecular design breeding.

[0025] (4) The obtained transgenic germplasm can be directly used in breeding practice, and its application value is clear.

[0026] Rice T0 plants overexpressing the PpNRT2.4 gene were created. These plants are intermediate breeding materials with enhanced drought resistance, serving as important germplasm resources for subsequent breeding programs and ideal research materials for in-depth analysis of the drought-resistant molecular mechanism of the PpNRT2.4 gene. This demonstrates the transformation of this invention from basic research to application, highlighting its strong practicality and clear application value. Attached Figure Description

[0027] Figure 1 Bioinformatics analysis of the gene PpNRT2.4 and the protein PpNRT2.4;

[0028] Figure A represents hydrophilicity analysis; Figure B represents transmodal region analysis; Figure C represents structural domain analysis.

[0029] Figure 2 Subcellular localization map of protein PpNRT2.4;

[0030] Figure 3 For RT-qPCR and physiological and biochemical assays;

[0031] Figure A shows RT-qPCR detection; Figure B shows root activity; Figure C shows MDA content; Figure D shows chlorophyll content.

[0032] Figure 3 Y271-CKL represents the leaf control of transgenic plants; Y271-CKG represents the root control of transgenic plants; Y271-DRL represents the leaf drought stress of transgenic plants; Y271-DRG represents the root drought stress of transgenic plants; WT-CKL represents the leaf control of wild-type plants; WT-CKG represents the root control of wild-type plants; WT-DRL represents the leaf drought stress of wild-type plants; WT-DRG represents the root drought stress of wild-type plants. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0034] Example

[0035] (1) Gene cloning and bioinformatics analysis

[0036] RNA was extracted from Kentucky bluegrass using standard methods, and its integrity was confirmed by agarose gel electrophoresis after quality testing. cDNA was synthesized using the extracted RNA as a template via reverse transcription. Primers were designed based on the NRT2.4 gene sequence obtained from Kentucky bluegrass transcriptome analysis, and PCR amplification was performed using the cDNA as a template. After purification with magnetic beads, the cDNA was ligated into the PMD19-T vector. The ligation product was transformed into competent cells and plated on antibiotic-containing plates. Single colonies were picked for colony PCR verification, and positive colonies were sequenced.

[0037] Bioinformatics analysis of the PpNRT2.4 gene sequence showed that the CDS length of PpNRT2.4 is 1467 bp, and the specific sequence is shown in SEQ ID NO:1, encoding 488 amino acids, and the amino acid sequence is shown in SEQ ID NO:2.

[0038] Protein PpNRT2.4 is an integral membrane protein with relatively strong overall hydrophobicity. Figure 1 A, Figure 1 B), containing the NRT2 family conservative core structural domain PLN00028 ( Figure 1 C), indicating an association with plant stress response.

[0039] (2) Subcellular localization

[0040] Using the target gene coding region (with the stop codon removed) as a template, homologous recombination primers were designed and PCR amplified. The fragment was then homologously recombinated with the pBWA(V)HS-eGFP vector, which had been double-digested with BsaI / Eco31I, using Biorun 2×EasyClone Mix. The product was transformed into *E. coli* DH5α, and after kanamycin resistance selection, colony PCR, and sequencing verification, the recombinant plasmid was transformed into *Agrobacterium* GV3101. *Agrobacterium* containing the recombinant plasmid was resuspended in permeate to OD200. 600 The concentration was kept between 0.2 and 1.0 at room temperature for 1 h. The leaves of *Nicotiana benthamiana* were injected and incubated in the dark overnight, followed by normal culture. After 48–72 h, epidermal sections were prepared and eGFP fluorescence was observed under a confocal microscope.

[0041] Subcellular localization of PpNRT2.4-GFP fusion protein as follows Figure 2 As shown in the figure, the fluorescence signal indicates that the protein is mainly located in the plasma membrane, consistent with its classic properties as a membrane transport protein; the signal was also detected in the nucleus, indicating that it has nucleoplasmic shuttle capability and may be involved in nitrate signal sensing and transduction.

[0042] Using the coding sequence of the PpNRT2.4 gene (with the stop codon removed) as a template, specific primers with homologous arms were designed for PCR amplification. The purified PCR product was then combined with the pBWA(V)HS-eGFP vector, which had been double-digested with BsaI / Eco31I, and homologous recombination was performed using Biorun 2×EasyClone Mix. The recombinant product was transformed into *E. coli* DH5α competent cells. Positive clones were screened using kanamycin-resistant plates, and the correct recombinant plasmid was obtained through colony PCR and sequencing verification. This plasmid was then transformed into *Agrobacterium* GV3101 competent cells. Single clones were picked and cultured in LB broth containing the appropriate antibiotic until the logarithmic growth phase. For injection, the cells were collected by centrifugation and resuspended in osmotic buffer to OD200. 600The concentration was 0.2–1.0, and the solution was allowed to stand at room temperature for 1 h. Agrobacterium-mediated infection was injected into the third and fourth leaves from the bottom of Nicotiana benthamiana, infecting between the two veins. 48–72 h after injection, the leaves from the injection site were cut, the epidermis was peeled off to prepare slides, and the fluorescence signal was observed under a confocal microscope.

[0043] (3) Identification of drought resistance in transgenic plants

[0044] To verify the function of the PpNRT2.4 gene, the complete coding sequence of the PpNRT2.4 gene was cloned into the vector pBWA(V)HS-eGFP, using the same method as above. The obtained recombinant plasmid was transformed into Agrobacterium EHA105 strain, and Agrobacterium-mediated transformation was performed on rice 'Huang Huazhan' recipient material. Through screening and molecular identification, overexpressing transgenic lines (OE) were obtained. Using untransformed wild-type (WT) 'Huang Huazhan' as a control, the drought resistance phenotype of the transgenic plants was identified.

[0045] Four-leaf stage OE and WT plants were subjected to simulated drought stress with 20% PEG-6000 until WT leaves showed obvious wilting (approximately 4-5 days), and samples were collected simultaneously; normal culture served as a control. PpNRT2.4 expression (internal reference gene Ubq) was detected using RT-qPCR. Root activity, malondialdehyde (MDA) content, and chlorophyll content were measured using kits. Data are expressed as mean ± standard deviation and were analyzed using SPSS 20.0 with one-way ANOVA and Tukey's test (p < 0.05).

[0046] The expression level of PpNRT2.4 in OE plants was significantly higher than that in WT plants under both normal and stress conditions, and was upregulated under stress. Figure 3 A). After drought stress, the root activity of OE plants was significantly enhanced, while WT decreased ( Figure 3 B); OE plants showed a significant decrease in MDA content, while WT increased ( Figure 3 C); The chlorophyll content of OE plants was significantly increased, and the chlorophyll content of WT plants also increased significantly, but was significantly lower than that of OE plants. Figure 3 D).

[0047] Overexpression of PpNRT2.4 can significantly improve root activity, reduce membrane damage and maintain a high chlorophyll level in rice under drought stress, thereby enhancing the plant's drought resistance.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A gene for drought-resistant Kentucky bluegrass PpNRT2.4 Its characteristics are, The gene PpNRT2.4 The sequence is shown in SEQ ID NO:

1.

2. A drought-resistant protein PpNRT2.4 for Kentucky bluegrass, characterized in that, The protein PpNRT2.4 is the gene described in claim 1. PpNRT2.4 The encoded protein; The amino acid sequence of the protein PpNRT2.4 is shown in SEQ ID NO:

2.

3. A kind containing PpNRT2.4 A gene recombination vector, characterized in that, The backbone of the expression vector is pBWA(V)HS.

4. A type of... PpNRT2.4 The strain of the gene is characterized by, The strain contains genes. PpNRT2.4 Agrobacterium.

5. Genes PpNRT2.4 Its application in improving plant drought resistance is characterized by... The gene PpNRT2.4 Improve plant drought resistance through overexpression.

6. A method for cultivating drought-resistant plants, characterized in that, The method involves overexpressing a gene in a plant. PpNRT2.4 To obtain plants that enhance drought stress tolerance.

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