Poa pratensis drought-resistant gene PpNRT2.4 and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a poa pratensis drought-resistant gene PpNRT2.4 and application thereof. The invention provides a nitrate transporter gene PpNRT2.4 from poa pratensis, provides a complete coding sequence of the nitrate transporter gene PpNRT2.4 and a protein sequence coded by the nitrate transporter gene PpNRT2.4, clarifies biological functions of the PpNRT2.4 gene, and verifies that the drought resistance of transgenic plants can be remarkably improved by overexpression of the PpNRT2.4 gene. A precise, efficient and safe innovative technical path is provided for creating new drought-resistant germplasm of turfgrass and other crops and breaking through the traditional breeding efficiency bottleneck.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a drought-resistant gene PpNRT2.4 of Kentucky bluegrass and application thereof. BACKGROUND

[0002] With the increasingly serious global water shortage problem, developing water-saving turf with high water use efficiency has become the core demand of urban ecological construction. Kentucky bluegrass (Poa pratensis) is a widely cultivated cool-season turfgrass, and its drought resistance and water use efficiency directly affect the maintenance cost and ecological benefits. Therefore, it is of great significance to further tap its inherent drought resistance genetic potential for cultivating new turfgrass varieties with low water consumption and high tolerance and promoting the sustainable development of landscaping.

[0003] Plants have formed a complex drought resistance regulation network in the long-term evolution process. In recent years, the synergistic mechanism of nitrogen metabolism and water stress response has attracted much attention. Nitrate transporters (NRT) not only dominate the absorption and transport of nitrogen, but also have been confirmed to be widely involved in the response of plants to abiotic stress, especially the NRT2 subfamily. However, current research on drought-resistant genes of plants mainly focuses on dehydrins, LEA proteins and some transcription factor families, and the specific function and molecular mechanism of NRT2 family in the drought resistance of turfgrass are still blank. In the prior art, no NRT2 family gene has been successfully cloned and functionally verified for drought resistance from Kentucky bluegrass, which limits the application of this important genetic resource in molecular breeding.

[0004] Lack of specific drought-resistant gene resources: Currently, there is a lack of efficient drought-resistant genes that are mined from the genetic background of Kentucky bluegrass and functionally verified. This leads to a very limited selection of specific genetic elements when using genetic engineering methods to precisely improve the drought resistance of turfgrass.

[0005] Lack of function recognition and utilization of NRT2 family genes: Although previous studies have suggested that NRT2 family may be involved in stress response, the existing technology has not provided any experimental evidence and clear conclusion on whether the NRT2 gene of Kentucky bluegrass (such as PpNRT2.4) is directly involved in drought resistance regulation, the subcellular localization of the encoded protein, and whether overexpression can confer a heritable drought-resistant phenotype to plants.

[0006] Single and inefficient drought breeding technology path: Traditional hybrid breeding has a long cycle and complex process, and it is difficult to break the reproductive isolation between species. Existing transgenic drought resistance strategies often have low expression efficiency and uncertain ecological risks due to the incompatibility of exogenous genes and the genetic background of the recipient plants, and lack of gene resources from closely related or same species with clear function and safety. SUMMARY

[0007] The application clones and identifies a nitrate transporter gene PpNRT2.4 from Poa pratensis, provides the complete coding sequence of the gene and the protein sequence coded by the gene, fills the blank of the gene resource in the existing database, and clarifies the biological function of the PpNRT2.4 gene, verifies the role of the gene in plant drought resistance through in-vivo and in-vitro experimental systems, clarifies the subcellular localization of the gene, and proves that overexpression of the gene can significantly improve the drought resistance of transgenic plants, so as to transform a potential genetic resource into an evidenced and usable drought resistance gene element. The application provides an application scheme of the PpNRT2.4 gene in molecular breeding, especially in breeding of transgenic plants with enhanced drought resistance, and provides a precise, efficient and safe innovative technical path for creating new germplasm of turfgrass and other crops with drought resistance and breaking through the efficiency bottleneck of traditional breeding.

[0008] In view of the deficiencies of the prior art, the application is implemented by the following scheme:

[0009] The application provides a Poa pratensis gene PpNRT2.4 with drought resistance, and the sequence of the gene PpNRT2.4 is shown as SEQ ID NO:1.

[0010]

[0011] The application also provides a drought-resistant Poa pratensis protein PpNRT2.4, wherein the protein PpNRT2.4 is a protein encoded by the gene PpNRT2.4 in claim 1.

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

[0013] MVTMGKKDVQQEQYYCGDWPVDGVDAEGRATELRPLALSHPHTQAFHLAWLSLFACFFAAFAAPPILPALRPALVLAPSDASTAAVGSLVAALVGRLVMGPVCDLLGPRRASGVASLVCALALALAAVYASSPAGFVALRFCAGLSLSNFVANQHWMSRIFAPSAVGLANAVAAGWANVGSAAAQIAMPLAYDCIVLRLGVPITVAWRVAYLIPCAMLITTGLAVLAFPYDLPQGCAASGGGRDKGGDKGTKGFWKAVRGGVCDYRAWVLLLTYGYCYGVELIMENVAADFFRRRFRLPMEAAGAAAACFGVMNTVARPAGGVASDEVGRRFGMRGRLWALWAVQSTGAVLCVLVGRMGASEAPSLAATMAVMVACGAFVQAASGLTFGIVPFVSKRSMGVVSGMTASGGAVGAIITNRLFFSSSRYTVEEAISFTGLTSLLCTLPLALIYFPRSGGMLCGAYGYEFIDQDCHDDDDVNKDDDYMLLK*.

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

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

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

[0017] The application also provides a method for cultivating drought-resistant plants, which is characterized by overexpressing the gene PpNRT2.4 in plants to obtain plants with enhanced drought stress tolerance.

[0018] Compared with the prior art, the present application has the following beneficial effects:

[0019] (1) A drought-resistant gene resource derived from the NRT2 family of Poa pratensis is provided

[0020] The PpNRT2.4 gene (CDS: 1467 bp) of Poa pratensis is successfully cloned, and rice plants overexpressing the gene are created through transgenic technology. The results of RT-qPCR and physiological and biochemical detection prove that the gene can significantly enhance the drought resistance of rice. This directly converts an unknown genetic sequence into a functional and effective gene element with significant effects, providing a new and efficient specific gene resource for the genetic improvement of drought resistance of turfgrass and other crops.

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

[0022] Bioinformatics analysis confirms that the protein PpNRT2.4 is a hydrophobic integral membrane protein and contains a conserved domain PLN00028 related to stress response, predicting its potential function from the sequence. More importantly, through GFP fusion protein experiments, it is first determined that the protein is localized in both the plasma membrane and the nucleus. This finding not only provides key clues for understanding its possible involvement in signal transduction and gene regulation, but also provides direct cell biology evidence for its drought resistance function at the cellular level.

[0023] (3) A new path for efficient and safe drought-resistant breeding based on homologous genes of closely related species

[0024] The PpNRT2.4 gene of Poa pratensis (monocotyledon) is introduced into rice, which is also a monocotyledon. Since the donor (Poa pratensis) and the recipient (rice) are closely related, their genetic background is more compatible, effectively avoiding the problems of low expression efficiency and potential ecological risks of genes from distant species. The T0 generation plants with significantly enhanced drought resistance are successfully obtained, proving the high feasibility of molecular design breeding using this gene.

[0025] (4) The obtained transgenic germplasm can be directly used for breeding practice, with clear application value

[0026] Rice T0 plants overexpressing the PpNRT2.4 gene are created. These plants are breeding intermediate materials with enhanced drought resistance, which can be used as important germplasm resources for subsequent breeding plans, and also as ideal research materials for in-depth analysis of the drought resistance molecular mechanism of PpNRT2.4 gene. This reflects the transformation of the present application from basic research to application, with strong practicality and clear application value. BRIEF DESCRIPTION OF DRAWINGS

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

[0028] Figure A is a hydrophilicity analysis; Figure B is a transmembrane region analysis; and Figure C is a domain analysis;

[0029] Figure 2 Figure is a subcellular localization map of protein PpNRT2.4;

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

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

[0032] Figure 3 Y271-CKL is a transgenic plant leaf control; Y271-CKG is a transgenic plant root control; Y271-DRL is a transgenic plant leaf drought stress; Y271-DRG is a transgenic plant root drought stress; WT-CKL is a wild type leaf control; WT-CKG is a wild type root control; WT-DRL is a wild type leaf drought stress; and WT-DRG is a wild type root drought stress. DETAILED DESCRIPTION

[0033] The present application will be further described below in conjunction with specific examples, which are used to explain the present application and are not intended to limit the scope of the present application. In the following examples, the test methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0034] EXAMPLE

[0035] (1) Gene cloning and bioinformatics analysis

[0036] The RNA of Poa pratensis was extracted using a conventional method, and the integrity of the RNA was confirmed by agarose gel electrophoresis after quality detection. The cDNA was synthesized using a reverse transcription kit with the extracted RNA as a template. The primers were designed according to the NRT2.4 gene sequence obtained from the transcriptome analysis of Poa pratensis, and the cDNA was used as a template for PCR amplification. After purification using magnetic beads, the product was constructed into a PMD19-T vector to obtain a ligation product. The ligation product was transformed into competent cells and plated on a plate containing an antibiotic. After single colony PCR verification, the positive bacterial solution was sequenced.

[0037] Bioinformatics analysis of PpNRT2.4 gene sequence showed that the CDS of PpNRT2.4 was 1467 bp long, the specific sequence was shown as SEQ ID NO: 1, it encoded 488 amino acids, and the amino acid sequence was shown as SEQ ID NO: 2.

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

[0039] (2) Subcellular localization

[0040] The coding region (without stop codon) of the target gene was used as a template to design homologous recombination primers and perform PCR amplification. The fragment was subjected to homologous recombination with the pBWA(V)HS-eGFP vector digested by BsaI / Eco31I, using Biorun 2×EasyClone Mix. The product was transformed into E. coli DH5α, and the correct recombinant plasmid was verified by kanamycin resistance screening, colony PCR and sequencing, and then transformed into Agrobacterium GV3101. The Agrobacterium containing the recombinant plasmid was resuspended in infiltration buffer to OD 600 Between 0.2 and 1.0, and incubated at room temperature for 1 h. The leaves of N. benthamiana were injected with the Agrobacterium, and then incubated in the dark overnight and normally for 48-72 h. The epidermis was taken for sectioning, and the eGFP fluorescence was observed under a confocal microscope.

[0041] The subcellular localization of PpNRT2.4-GFP fusion protein is shown in Figure 2 The fluorescence signal showed that the protein was mainly localized in the plasma membrane, which was consistent with its classic properties as a membrane transporter protein; signals were also detected in the nuclear region, indicating that it had the ability of nuclear-cytoplasmic shuttle and might be involved in nitrate signal perception and transduction.

[0042] The coding sequence (without stop codon) of PpNRT2.4 gene was used as a template to design specific primers with homologous arms for PCR amplification. The purified PCR product was subjected to homologous recombination with the pBWA(V)HS-eGFP vector digested by BsaI / Eco31I, using Biorun 2×EasyClone Mix. The recombinant product was transformed into E. coli DH5α competent cells, and the positive clones were screened by kanamycin resistance plate, and verified by colony PCR and sequencing to obtain the correct recombinant plasmid, which was transformed into Agrobacterium GV3101 competent cells. Single colonies were selected and cultured in LB liquid medium containing the corresponding antibiotics to the logarithmic growth phase. For injection, the bacterial cells were collected by centrifugation and resuspended in infiltration buffer to OD 600The OD600 value of the Agrobacterium solution was adjusted to 0.2-1.0, and the solution was allowed to stand at room temperature for 1 h. The Agrobacterium solution was injected into the 3rd and 4th leaves of Nicotiana benthamiana, and the injection was performed between the two leaf veins. 48-72 h after the injection, the injected leaves were cut, and the epidermis was peeled off to prepare a sample. The fluorescence signal was observed under a confocal microscope.

[0043] (3) Drought resistance identification of the transgenic plants

[0044] To verify the function of PpNRT2.4, the complete coding sequence of PpNRT2.4 was cloned into the vector pBWA(V)HS-eGFP in the same manner as described above. The obtained recombinant plasmid was transformed into the Agrobacterium EHA105 strain, and the Agrobacterium-mediated method was used to transform the rice ‘Huanghuazhan’ recipient material. Through screening and molecular identification, a transgenic line (OE) overexpressing PpNRT2.4 was obtained. The untransformed wild type (WT) ‘Huanghuazhan’ was used as a control, and the drought resistance phenotype of the transgenic plants was identified.

[0045] The OE and WT plants at the 4th leaf stage were subjected to simulated drought stress with 20% PEG-6000 until the WT leaves were obviously wilted (about 4-5 days), and samples were taken synchronously; normal culture was used as a control. RT-qPCR was used to detect the expression of PpNRT2.4 (reference gene Ubq). The root activity, MDA content, and chlorophyll content were determined using a kit. The data were expressed as mean ± standard deviation, and one-way ANOVA and Tukey test (p < 0.05) were performed using SPSS 20.0.

[0046] The expression level of PpNRT2.4 in the OE plants was significantly higher than that in the WT under normal and stress conditions, and was up-regulated by stress induction Figure 3 A). After drought stress, the root activity of the OE plants was significantly enhanced, while that of the WT decreased Figure 3 B); the MDA content of the OE plants was significantly reduced, while that of the WT increased Figure 3 C); the chlorophyll content of the OE plants was significantly increased, and the chlorophyll content of the WT was also significantly increased, but was significantly lower than that of the OE Figure 3 D).

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

[0048] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and do not limit the protection scope of the present application. For those skilled in the art, based on the above description and ideas, other different forms of changes or modifications can also be made, which are not required or impossible to enumerate all the embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principles of the present application shall be included in the protection scope of the claims of the present application.

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 In order to obtain drought-resistant plants.

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