Gene PtoTAR2 for improving drought tolerance and ABA sensitivity of poplar and application of gene PtoTAR2

Abstract

The invention discloses a gene PtoTAR2 for improving drought tolerance and ABA sensitivity of poplars and application of the gene PtoTAR2. The nucleotide sequence of the gene is shown as SEQ ID NO.1, and the amino acid sequence of encoded protein is shown as SEQ ID NO.3. The gene can negatively regulate the drought tolerance and ABA sensitivity of the poplars, and has the advantages that the gene PtoTAR2 can be used for negatively regulating the drought tolerance and ABA sensitivity of the poplars; a candidate gene is provided for cultivating new populus germplasm which is improved in stress resistance and does not affect growth, and the breeding period is effectively shortened. The invention further discloses a method for improving drought tolerance and ABA sensitivity of the poplar and related application, the drought tolerance and ABA sensitivity of the poplar are specifically enhanced by silently expressing the gene PtoTAR2 in a poplar body, and an effective way is provided for cultivating new poplar germplasm with improved stress resistance and no influence on growth; the method is of great significance in promoting sustainable development of forestry.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and particularly relates to a gene PtoTAR2 for improving drought tolerance and sensitivity to ABA of poplar and application thereof. BACKGROUND

[0002] The growth process of plants is affected by various environmental stress factors. In addition to biological stress such as disease and pest, overgrazing, human activities and the like, drought is the most serious non-biological stress that causes damage to plants. In the long-term evolution process, some plants have evolved mechanisms to adapt to drought stress. However, more plants on earth are still threatened by drought stress. With the continuous growth of the world population and the increasing shortage of freshwater resources, the problem of drought is becoming increasingly serious, and the research on drought resistance of plants has become a research hotspot.

[0003] Abscisic acid (ABA) is a key hormone in plants, which widely regulates physiological processes such as seed dormancy, organ abscission, and seed and fruit development. ABA can induce plant organ abscission, inhibit growth, promote dormancy, and regulate stomatal closure to affect transpiration and water use. With further research, the role of ABA in plant stress response has been revealed: when plants encounter drought, salt stress, low-temperature freezing damage and other stresses, ABA synthesis and signaling pathways are activated, which enhances plant stress tolerance by regulating the expression of stress-related genes, accumulation of osmotic adjustment substances, and other processes, and becomes an important regulatory hub for responding to environmental stress.

[0004] Tryptophan Aminotransferase of Arabidopsis 1 / Tryptophan Aminotransferase Related protein (TAA1 / TAR) was first named in dicotyledonous model plant Arabidopsis thaliana. TAA1 is also called WEI8 and SAV3, and TAR is a homologous protein of TAA1. TAA1 / TAR gene belongs to the Pyridoxal-5'-phosphate (PLP) dependent amino acid transaminase superfamily, and its function is mainly involved in the biosynthesis of IAA, which plays an important role in plant growth and development and metabolic processes. Arabidopsis thaliana contains 5 TAA1 / TAR family members, and these proteins play a key role in embryogenesis, flower organ development, seedling growth, vascular tissue formation, lateral root formation, tropic response, shade avoidance and temperature-dependent hypocotyl elongation. In monocot crops such as wheat, weak expression of TaTAR2 can inhibit the growth of aboveground biomass of wheat, and also inhibit the number of root tips and the length of roots.

[0005] At present, the research on TAR2 gene in woody plants is still relatively scarce, especially the functional analysis of its response to stress. Therefore, it is necessary to analyze the role of TAR2 gene in poplar stress resistance, to improve the growth and survival ability of poplar in drought areas, to cultivate new poplar germplasm with improved stress resistance without affecting growth, and to promote genetic improvement of forest tree stress resistance. SUMMARY

[0006] In order to overcome the above problems, the present inventors have screened a drought negative regulatory factor PtoTAR2 related to the sensitivity of plant hormone abscisic acid (ABA), and the transgenic poplar plants with silenced PtoTAR2 gene have significantly enhanced sensitivity to abscisic acid (ABA) and significantly better drought resistance, and their growth conditions are not significantly different from those of the control plants under drought stress, providing an effective way for cultivating new poplar germplasm with improved stress resistance without affecting growth, which is of great significance for improving the growth and survival ability of poplar in drought areas and promoting the sustainable development of forestry, thereby completing the present application.

[0007] Specifically, the present application aims to provide the following aspects:

[0008] In a first aspect, a gene PtoTAR2 for regulating drought tolerance and sensitivity to ABA of poplar is provided, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

[0009] In a second aspect, a protein for regulating drought tolerance and sensitivity to ABA of poplar is provided, and the protein is the encoded protein of the gene PtoTAR2, and the sequence is shown as SEQ ID NO. 3.

[0010] In a third aspect, the gene PtoTAR2 of the first aspect or the protein of the second aspect is applied to regulate drought tolerance and sensitivity to ABA of poplar.

[0011] In a fourth aspect, a recombinant vector is provided, and the recombinant vector is a silenced recombinant vector of the gene PtoTAR2 of the first aspect, which is obtained by constructing an interference fragment of the gene PtoTAR2 on a basic vector.

[0012] In a fifth aspect, a method for improving drought tolerance and sensitivity to ABA of poplar is provided, and the method comprises the step of silencing the expression of the gene PtoTAR2 of the first aspect in poplar.

[0013] In a sixth aspect, the gene PtoTAR2 of the first aspect, the protein of the second aspect, the recombinant vector of the fourth aspect, and the method of the fifth aspect are applied to poplar breeding.

[0014] The present application has the following beneficial effects:

[0015] (1) The application successfully clones the gene PtoTAR2 in poplar, constructs a PtoTAR2 overexpression recombinant vector and a silencing recombinant vector, and transforms the two vectors into Populus tomentosa respectively to perform function verification, proves that silencing expression of the gene PtoTAR2 can improve the drought resistance and sensitivity to ABA of poplar, provides a candidate gene for cultivating new germplasm of poplar with improved stress resistance without affecting growth, and effectively shortens the breeding cycle;

[0016] (2) The method for improving the drought resistance and sensitivity to ABA of poplar provided by the application can specifically enhance the drought resistance and sensitivity to ABA of poplar by silencing expression of the gene PtoTAR2, and the growth condition of the poplar is not significantly different from that of the control plant, thereby providing an effective way for cultivating new germplasm of poplar with improved stress resistance without affecting growth;

[0017] (3) The gene PtoTAR2, the protein, the recombinant vector and the method for improving the drought resistance and sensitivity to ABA of poplar provided by the application have application in poplar breeding, provide a new idea for further research on tree stress resistance mechanism, and have important significance for improving the growth and survival ability of poplar in drought areas and promoting the sustainable development of forestry. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 A comparison chart showing the expression amount of the PtoTAR2 gene in different tissues of Populus tomentosa detected by RT-qPCR in Example 1 is shown; Figure 2 A comparison chart showing the expression amount of the PtoTAR2 gene in Populus tomentosa under 20% PEG6000 treatment for different times (0-24h) detected by RT-qPCR in Example 1 is shown; Figure 3 A comparison chart showing the expression amount of the PtoTAR2 gene in Populus tomentosa under 50 μmol / L ABA treatment for different times (0-24h) detected by RT-qPCR in Example 1 is shown; Figure 4 A comparison chart showing the transcription level of PtoTAR2 in overexpression plants and silencing plants detected by RT-qPCR in Example 3 is shown, and the error bars represent standard deviations; Figure 5 A comparison chart showing the morphological phenotype of WT, PtoTAR2 overexpression transgenic plants (OE-1, OE-5) and silencing transgenic plants (RNAi-3, RNAi-6) under normal conditions in Example 4 is shown; Figure 6 A comparison chart showing the morphological phenotype of WT, PtoTAR2 overexpression transgenic plants (OE-1, OE-5) and silencing transgenic plants (RNAi-3, RNAi-6) after drought treatment for 10 days in Example 4 is shown; Figure 7The diagram shows a comparison of stomatal conductance, Fv / Fm, net photosynthetic rate, and transpiration rate of WT and PtoTAR2 overexpressing transgenic plants (OE-1, OE-5, OE-6) and silent transgenic plants (RNAi-1, RNAi-3, RNAi-6) before and after drought treatment in Example 4. Figure 8 The root morphology phenotypes of WT and PtoTAR2 overexpressing transgenic plants (OE-5) and silent transgenic plants (RNAi-3) in Example 5 after treatment with 5 μmol / L ABA are shown. Figure 9 The diagram shows the DAB and NBT staining of leaves from transgenic plants with WT and PtoTAR2 overexpression (OE-1, OE-5) and silence (RNAi-3, RNAi-6) in Example 5 after treatment with 5 μmol / L ABA. Detailed Implementation

[0019] The present invention will be further described in detail below through preferred embodiments and examples. Through these descriptions, the features and advantages of the present invention will become clearer and more apparent.

[0020] The term “exemplary” as used herein means “serving as an example, embodiment, or illustration.” Any embodiment illustrated herein as “exemplary” is not necessarily to be construed as superior to or better than other embodiments.

[0021] In a first aspect, the present invention provides a gene PtoTAR2 that regulates the drought resistance and ABA sensitivity of poplar trees, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0022] Preferably, the poplar is a white poplar, and more preferably a hairy white poplar.

[0023] In a preferred embodiment, the gene PtoTAR2, which regulates the drought resistance and ABA sensitivity of poplar, is located on chromosome 15 of the Populus tomentosa genome (http: / / db.cngb.org / cnsa / project / CNP0004290_40760cfc / reviewlink / ), with a start position of 9163816 and an end position of 9173548.

[0024] Preferably, the nucleotide sequence of the gene PtoTAR2 can be found in the following file: National Gene Bank: Genome sequencing of Populus tomentosa; Project No.: CNP0004290, Sample No.: CNS0752799, Assembly No.: CNA0069009.

[0025] In a preferred embodiment, the coding region nucleotide sequence of the gene PtoTAR2, which regulates the drought resistance and ABA sensitivity of poplar trees, is shown in SEQ ID NO.2.

[0026] The nucleotide sequence of the coding region of the gene PtoTAR2 can be found in the following file: National Gene Bank: Genome sequencing of Populus tomentosa; Project No.: CNP0004290, Sample No.: CNS0752799, Assembly No.: CNA0069009; PtoTAR2 location: Chromosome 15, start position 9164612, end position 9164883; start position 9166423, end position 9166726; start position 9172097, end position 9172377; start position 9172461, end position 9172759; start position 9172870, end position 9173087. For details, please refer to http: / / db.cngb.org / cnsa / project / CNP0004290_40760cfc / reviewlink / .

[0027] In a second aspect, the present invention provides a protein encoded by the gene PtoTAR2 described in the first aspect, the sequence of which is shown in SEQ ID NO.3.

[0028] A third aspect of the present invention provides the application of the gene PtoTAR2 described in the first aspect or the protein described in the second aspect in regulating the drought resistance and ABA sensitivity of poplar trees.

[0029] Preferably, the poplar is a white poplar, and more preferably a hairy white poplar.

[0030] In a preferred embodiment, the regulation is achieved by increasing or decreasing the expression level of the PtoTAR2 gene or the content of the protein encoded by the PtoTAR2 gene in poplar trees.

[0031] Preferably, the regulation is achieved by overexpressing or silencing the gene PtoTAR2 in poplar trees.

[0032] In a preferred embodiment, the overexpression is achieved by introducing an overexpression recombinant vector of the PtoTAR2 gene into poplar trees, and the silencing is achieved by introducing a silencing recombinant vector of the PtoTAR2 gene into poplar trees.

[0033] Preferably, the overexpression recombinant vector is obtained by constructing the CDS sequence of the PtoTAR2 gene into a base vector;

[0034] The silencing recombination vector was obtained by constructing an interference fragment of the PtoTAR2 gene onto a base vector.

[0035] More preferably, the CDS sequence of the gene PtoTAR2 is shown in SEQ ID NO.2, and the interference fragment sequence of the gene PtoTAR2 is shown in SEQ ID NO.4.

[0036] In a preferred embodiment, when the gene PtoTAR2 is overexpressed in poplar, the drought resistance and ABA sensitivity of poplar decrease; when the gene PtoTAR2 is silenced in poplar, the drought resistance and ABA sensitivity of poplar increase.

[0037] In this invention, the gene PtoTAR2 is a drought negative regulator associated with ABA sensitivity.

[0038] Preferably, the drought resistance and ABA sensitivity of poplar trees are reduced by overexpressing the gene PtoTAR2 in poplar trees, and the drought resistance and ABA sensitivity of poplar trees are improved by silencing the gene PtoTAR2 in poplar trees.

[0039] This invention discloses for the first time the silent expression gene PtoTAR2, which can improve the drought resistance and ABA sensitivity of poplar. This gene can serve as a candidate gene for cultivating new poplar germplasm with enhanced stress resistance without affecting growth, which is of great significance for improving the growth and survival ability of poplar in arid areas and promoting the sustainable development of forestry.

[0040] In a fourth aspect, the present invention provides a recombinant vector, which is a silencing recombinant vector for the gene PtoTAR2, obtained by constructing an interference fragment of the gene PtoTAR2 described in the first aspect onto a base vector.

[0041] Preferably, the interfering fragment of the PtoTAR2 gene is PtoTAR2-RNAi, the sequence of which is shown in SEQ ID NO.4;

[0042] The base vector is the pBI121 vector.

[0043] The pBI121 vector, totaling 13629 bp, contains a strong 35S promoter (CaMV35S), is resistant to kanamycin, and can be digested with XbaI and BamHI.

[0044] A fifth aspect of the present invention provides a method for improving the drought resistance and ABA sensitivity of poplar trees, the method comprising the step of silencing the expression of the gene PtoTAR2 in poplar trees.

[0045] Preferably, the method includes the following steps:

[0046] Step 1: Construct a silencing recombinant vector for the PtoTAR2 gene.

[0047] Preferably, the silencing recombinant vector for the PtoTAR2 gene is obtained by constructing an interference fragment of the PtoTAR2 gene into the pBI121 vector.

[0048] In a preferred embodiment, the interfering fragment of the PtoTAR2 gene is PtoTAR2-RNAi, the sequence of which is shown in SEQ ID NO.4.

[0049] In this invention, the target region of the interference fragment of the PtoTAR2 gene was obtained by constructing a gene family phylogenetic tree and performing phylogenetic analysis on the sequence characteristics of the poplar PtoTAR2 gene and its homologous genes in closely related species. This interference fragment precisely avoids highly conserved redundant sequences in the gene family, ensuring not only efficient silencing of the target gene PtoTAR2, but also blocking cross-interference with other functional genes in the same family at the sequence-specific level, thus solving the off-target problem caused by cross-binding of conserved sequences in conventional designs.

[0050] Preferably, the interference fragment of the PtoTAR2 gene is amplified using primers PtoTAR2-RNAi-F and PtoTAR2-RNAi-R, and their sequences are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0051] Step 2: The silent recombination vector was introduced into poplar trees to obtain transgenic plants with the PtoTAR2 gene.

[0052] Preferably, the poplar is a white poplar, and more preferably a hairy white poplar.

[0053] In a preferred embodiment, the plasmid of the silencing recombinant vector is transformed into Agrobacterium, and then the callus tissue of poplar is infected with Agrobacterium tumefaciens to obtain complete plants.

[0054] Step 3: Identify and perform phenotypic analysis on the transgenic plants.

[0055] Preferably, using gDNA from the transgenic plant as a template, PCR is used for preliminary identification at the DNA level. The identification procedure is as follows:

[0056] The PCR reaction program was as follows: 95℃ for 2 min; (94℃ for 30 s; 55℃ for 30 s; 72℃ for 60 s) for 35 cycles; 72℃ for 5 min; 4℃ for ∞.

[0057] More preferably, the PCR-positive plants were identified by transcriptional RT-qPCR using the detection primers PtoTAR2-qPCR-F and PtoTAR2-qPCR-R, whose sequences are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively.

[0058] The preferred testing procedure is: 95℃ for 30s; (95℃ for 5s; 60℃ for 35s) 40 cycles; 95℃ for 15s; 60℃ for 1min; 95℃ for 15s.

[0059] This invention discovers that by using specific detection primers and detection procedures at the DNA and transcriptional levels, transgenic plants can be accurately identified, reducing the false positive detection rate and effectively improving experimental efficiency.

[0060] In a preferred embodiment, the phenotypic analysis of the silenced transgenic plants includes drought resistance analysis and ABA sensitivity analysis.

[0061] The drought resistance analysis includes growth status analysis, maximum photochemical efficiency (Fv / Fm) analysis, stomatal conductance analysis, photosynthetic rate analysis, and transpiration rate analysis; the sensitivity analysis to ABA includes root morphology analysis and reactive oxygen species level analysis.

[0062] According to an embodiment of the present invention, after 10 days of drought stress treatment, the leaves of wild-type plants and overexpressing transgenic plants showed more severe wilting and yellowing than those of silent transgenic plants.

[0063] Ten days after drought stress treatment, the photosynthetic capacity of the plants showed a clear trend: silent transgenic plants > wild-type plants > overexpressing transgenic plants. Specifically, the vapor-stomach conductance (GSW) of all treated plants decreased significantly, with the gene-silenced lines showing relatively higher GSW. The stomata remained partially open to meet the basal transpiration requirements, and the trend of transpiration rate (E) was consistent with that of GSW. Analysis of the maximum photochemical efficiency (Fv / Fm) of photosynthetic system II showed that the Fv / Fm values ​​of the gene-silenced lines were significantly higher than those of the WT and overexpressing lines. Net photosynthetic rate data further confirmed that drought stress had a smaller impact on the gene-silenced lines, while the photosynthetic capacity of the WT and overexpressing lines was significantly reduced. The overexpressing lines had lost their photosynthetic capacity, and their leaves simultaneously initiated an irreversible death and abscission program.

[0064] After culturing in 1 / 2 MS medium containing 5 μmol / L ABA for 10 days, the rooting inhibition of the silent transgenic plants was significantly higher than that of the wild-type and PtoTAR2 overexpressing transgenic plants.

[0065] Under 5 μmol / L ABA treatment, the leaves of wild-type and PtoTAR2 overexpressing transgenic poplars showed a deep blue (NBT staining) or brownish-red (DAB staining) color after NBT and DAB staining, with a relatively deep coloring degree, indicating a high level of ROS accumulation in their bodies. In contrast, the leaves of PtoTAR2 silenced transgenic plants showed a significantly lighter coloring degree after NBT and DAB staining, suggesting a lower level of ROS accumulation in their bodies.

[0066] The method for improving the drought resistance and ABA sensitivity of poplar trees provided by this invention, by silencing the expression of the PtoTAR2 gene, can specifically enhance the drought resistance and ABA sensitivity of poplar trees, providing a new approach for further research on tree stress resistance mechanisms.

[0067] The sixth aspect of the present invention is the application of the gene PtoTAR2 described in the first aspect, the protein described in the second aspect, the recombinant vector described in the fourth aspect, or the method described in the fifth aspect in poplar breeding.

[0068] Preferably, the breeding is: compared with the wild type, the PtoTAR2 gene is silenced to breed new poplar varieties with high drought resistance and / or high sensitivity to ABA.

[0069] More preferably, the poplar is a white poplar, and more preferably a hairy white poplar.

[0070] The application of the gene PtoTAR2, protein, recombinant vector, and methods for improving the drought resistance and ABA sensitivity of poplar provided by this invention in poplar breeding shows that, compared with wild-type and PtoTAR2-overexpressing poplar plants, poplar plants with silenced PtoTAR2 gene have significantly enhanced sensitivity to abscisic acid (ABA) and significantly better drought resistance. Moreover, under drought stress conditions, their growth status is not significantly different from that of control plants. This provides an effective way to cultivate new poplar germplasm with "enhanced stress resistance without affecting growth", which is of great significance for improving the growth and survival ability of poplar in arid areas and promoting the sustainable development of forestry.

[0071] Example

[0072] The present invention is further described below through specific examples; however, these examples are merely exemplary and do not constitute any limitation on the scope of protection of the present invention.

[0073] Unless otherwise specified, the reagents involved in the following examples are all commercially available conventional reagents, and the methods used are all methods commonly used in this technical field.

[0074] Example 1 Fluorescent quantitative detection of PtoTAR2 gene of poplar

[0075] (1) To further clarify the tissue-specific expression pattern of the PtoTAR2 gene during the growth and development of Populus tomentosa, two-month-old hydroponic Populus tomentosa with consistent growth status were selected as experimental materials, and tissues (including roots, stems, root tips, young leaves, mature leaves, and terminal buds) at different functional and developmental stages were systematically sampled. RNA was extracted from the collected plant tissues according to the instructions of the MolPure Plant RNA Kit from Yisheng Biotechnology Co., Ltd.

[0076] Using the RNA obtained above as a template, cDNA was obtained by reverse transcription using HiScript IV All-in-One Ultra RT SuperMix for qPCR-R433 (Nanjing Novizan Biotechnology Co., Ltd.). The reaction system (20 μL) is shown in Table 1.

[0077] Table 1

[0078]

[0079] The reaction procedure was: 50℃ for 5 min; 85℃ for 5 sec.

[0080] Primers PtoTAR2-qPCR-F and PtoTAR2-qPCR-R were designed based on the CDS sequence of the PtoTAR2 gene from Populus tomentosa (as shown in SEQ ID NO.2). The expression level of the PtoTAR2 gene in different tissues was determined using quantitative real-time PCR. The nucleotide sequences of the primers are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively. The internal control primers were Actin-F and Actin-R, with nucleotide sequences shown in SEQ ID NO.9 and SEQ ID NO.10, respectively.

[0081] RT-qPCR experiments were performed using 2×ChamQ SYBR Color qPCR Master Mix. The reaction volume (20 μL) is shown in Table 2.

[0082] Table 2

[0083]

[0084] The reaction program was as follows: 95℃ for 30s; (95℃ for 5s; 60℃ for 35s) for 40 cycles; 95℃ for 15s; 60℃ for 1min; 95℃ for 15s.

[0085] The results of the real-time PCR reaction are as follows Figure 1As shown, the expression of the PtoTAR2 gene varies significantly among different tissues of Populus tomentosa, and its relative expression level in the root tip is significantly higher than that in other tissues such as roots, stems, young leaves, mature leaves and terminal buds. This indicates that the gene is most highly expressed in the root tip of Populus tomentosa.

[0086] (2) To investigate the response pattern of the PtoTAR2 gene to abscisic acid (ABA) signaling and drought stress, two-month-old hydroponic Populus tomentosa were treated with ABA (50 μmol / L) and simulated drought stress (20% PGE 6000). Populus root tissues were collected at 0, 0.5, 1, 2, 4, 6, 8, 16 and 24 h after ABA and 20% PEG 6000 treatment, and RNA was extracted from the collected root tissues. RNA extraction was performed according to the instructions of the MolPure Plant RNA Kit from Yisheng Biotechnology Co., Ltd.

[0087] The relative expression levels of the PtoTAR2 gene in the roots of Populus tomentosa at different time points under two stress treatments were determined using real-time quantitative PCR. The concentrations of components in the qRT-PCR reaction system (such as primers, template cDNA, and fluorescent dyes) and the amplification program (including pre-denaturation temperature and time, annealing temperature, extension time, and cycle number) were all set according to the method described in step (1) above to ensure consistency of experimental procedures and reliability of data.

[0088] The results of the real-time PCR reaction are as follows Figure 2 and Figure 3 As shown, the expression level of the PtoTAR2 gene significantly decreased with the extension of ABA and drought treatment duration. Therefore, it is speculated that the PtoTAR2 gene plays an important regulatory role in the response of Populus to ABA and drought stress in Populus tomentosa.

[0089] Example 2 Obtaining of PtoTAR2 overexpression transgenic poplar and silencing transgenic poplar

[0090] (1) Construction of PtoTAR2 gene overexpression vector and silencing recombinant vector (RNAi recombinant vector)

[0091] (1.1) Obtaining the target fragment of the PtoTAR2 gene

[0092] RNA was extracted from LM50 Populus tomentosa using the Plant RNA Extraction Kit from Beijing TransGen Biotechnology Co., Ltd.; the RNA was reverse transcribed into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (Nanjing Novizan Biotechnology Co., Ltd.).

[0093] Using cDNA as a template and referring to the Populus tomentosa genome file (http: / / db.cngb.org / cnsa / project / CNP0004290_40760cfc / reviewlink / ), primers for amplifying the target fragments were designed using the Primer BLAST tool (NCBI, https: / / blast.ncbi.nlm.nih.gov) after comprehensively considering various primer design principles. The target fragments included the target fragment for constructing the overexpression vector (the CDS sequence of the PtoTAR2 gene) and the target fragment for constructing the RNAi recombinant vector (a partial reverse sequence of the PtoTAR2 CDS gene).

[0094] The amplification primers for the CDS sequence of PtoTAR2 are PtoTAR2-OE-F and PtoTAR2-OE-R, and their sequences are shown in SEQ ID NO.11 (agaacacgggggactATGGCGAGGCTTTTCAGTGTTTT) and SEQ ID NO.12 (acccccggggatcctCTCCGCCGTCTCATCTAC), respectively.

[0095] The PtoTAR2 interference fragment PtoTAR2-RNAi was amplified using primers PtoTAR2-RNAi-F and PtoTAR2-RNAi-R, and its sequences are shown in SEQ ID NO.5 (acccccggggatcctATAGTATGCCTAGTGAGGCACAT) and SEQ ID NO.6 (agaacacgggggactCGGATCGCCATGATCAAGATTG), respectively.

[0096] The gene PtoTAR2 is located on chromosome 15 of the Populus tomentosa genome (http: / / db.cngb.org / cnsa / project / CNP0004290_40760cfc / reviewlink / ), with a start position of 9163816 and an end position of 9173548.

[0097] PCR amplification was performed using 2x Phanta Max Master Mix (Dye Plus) from Nanjing Vazyme Company. The PCR reaction system (50 μL) is shown in Table 3.

[0098] Table 3

[0099]

[0100] The reaction program was as follows: 95℃ for 3 min; (95℃ for 25 s; 55℃ for 30 s; 72℃ for 60 s (amplification efficiency 45 s / kb)) 36 cycles; 72℃ for 5 min; 4℃ to infinity.

[0101] The final CDS sequence of the PtoTAR2 gene was obtained, as shown in SEQ ID NO.2. The amino acid sequence of the protein it encodes is shown in SEQ ID NO.2, and the nucleotide sequence of the interfering fragment PtoTAR2-RNAi is shown in SEQ ID NO.4.

[0102] (1.2) Vector digestion

[0103] The pBI121 vector enzyme digestion reaction system is shown in Table 4:

[0104] Table 4

[0105]

[0106]

[0107] The enzyme digestion reaction conditions are: 37℃ water bath for 1-2 hours.

[0108] The enzyme digestion products were purified by gel extraction using the Kangwei Century GelExtraction Kit and purified using the Kangwei Century DNA Clean-up Kit. The purified products were then used as vector backbones and stored at -20°C.

[0109] (1.3) Connection transformation

[0110] Using the Uniclone One Step Seamless Cloning Kit from Beijing Jinsha Biotechnology Co., Ltd., the CDS and CDS partial reverse sequences of PtoTAR2 were constructed into the pBI121 vector, resulting in overexpression recombinant vector and RNAi recombinant vector, respectively. Ligation was performed at 50℃ for 10 min.

[0111] Take 50 μl of DH5α competent cells, add 5 μl of ligation product, gently tap to mix, and incubate on ice for 30 minutes. Heat shock in a 42°C water bath for 45 seconds, then quickly transfer to ice to cool for 2 minutes. Add 700 μl of antibiotic-free sterile LB liquid medium to a clean bench, mix well, and incubate at 37°C and 220 rpm for 45 minutes to allow the cells to recover. After recovery, centrifuge at 6000 rpm for 1 minute, collect the cells, resuspend the cells in 100 μl of supernatant, and spread on LB agar plates containing kanamycin (100 mg / ml). Incubate at 37°C upside down for 14-16 hours.

[0112] (1.4) Identification of positive clones by bacterial culture PCR

[0113] Single colonies from the plate were picked sequentially using sterilized pipette tips and added to 250 μl of LB broth containing kanamycin. The culture was incubated at 37°C with shaking at 200 rpm for approximately 3 hours to serve as amplification templates. PCR amplification was performed using Taq Plus Master Mix from Nanjing Vazyme. The PCR reaction system is shown in Table 5.

[0114] Table 5

[0115]

[0116] The sequence of primer pBI121-R is shown in SEQ ID NO.13.

[0117] PCR products were detected by 1% agarose gel electrophoresis.

[0118] (1.5) Extraction of positive clone plasmids

[0119] PCR-positive clones were aspirated into 6 ml of LB liquid medium containing kanamycin and cultured overnight at 37°C with shaking at 200 rpm. Plasmids were extracted using a plasmid miniprep kit from Jiangsu Kangwei Reagent Co., Ltd., and sequenced by Beijing Ruiboxingke Biotechnology Co., Ltd. After confirming that the sequences were correct, the overexpression recombinant vector (PtoTAR2-OE) and the RNAi recombinant vector (PtoTAR2-RNAi) were constructed.

[0120] (2) Genetic transformation of the PtoTAR2 gene

[0121] (2.1) Transformation of Agrobacterium with recombinant plasmids

[0122] Take about 1 μg of PtoTAR2-OE recombinant vector plasmid and PtoTAR2-RNAi recombinant vector plasmid and add them to 100 μL of Agrobacterium GV3101 competent cells, respectively, and mix gently.

[0123] The culture was incubated sequentially on ice for 5 minutes, then flash-frozen in liquid nitrogen for 5 minutes, then in a 37°C water bath for 5 minutes, and finally in an ice bath for 5 minutes. 700 μL of antibiotic-free YEP liquid medium was added to the bacterial culture, and the mixture was thoroughly mixed. The culture was then incubated at 28°C and 200 rpm with shaking for 2–3 hours. After the incubation period, the culture was centrifuged at 6000 rpm for 1 minute, and a portion of the supernatant was discarded. 100 μL of the supernatant was mixed with the bacterial culture, and the mixture was evenly spread onto the surface of YEP solid medium containing 50 mg / L rifampicin and 50 mg / L kanamycin. The culture was then incubated upside down in a 28°C incubator for 72–90 hours.

[0124] After single colonies have grown, several single-clone plaques are picked up with a sterile pipette tip and placed in a 2 mL centrifuge tube containing 250 μL of YEP liquid medium (1:1000 with added Rif and Kana). The tubes are then incubated on a shaker at 30°C and 200 rpm for 2 hours. After incubation, the bacterial culture is aspirated for PCR identification. Positive bacterial cultures that have been correctly identified are added to 50% glycerol, flash-frozen in liquid nitrogen, and stored at -80°C for subsequent genetic transformation experiments.

[0125] (2.2) Activation of Agrobacterium

[0126] Take Agrobacterium tumefaciens containing PtoTAR2 overexpression recombinant vector plasmid and RNAi recombinant vector plasmid from the -80℃ freezer, streak them onto YEP solid medium (1:1000 with added Rif and Kana), seal the plates, and incubate upside down in a 28℃ dark incubator for 2-3 days.

[0127] Using a sterile pipette tip, pick a single colony from the plate and inoculate it into 3 mL of YEP liquid medium (1:1000 with added Rif and Kana). Incubate overnight at 30°C and 200 rpm with shaking. Transfer 1 mL of the bacterial culture to a 250 mL sterile Erlenmeyer flask containing 100 mL of YEP liquid medium (1:1000 with added Rif and Kana). Incubate at 30°C and 200 rpm with shaking for 4-5 hours, until the OD600 reaches 0.3-0.5.

[0128] Take 100 mL of bacterial suspension, centrifuge at 2560 g for 20 min at 4 °C, discard the supernatant, and resuspend the bacterial cells in 100 mL of WPMB resuspension solution in a sterile wide-mouth bottle. This bacterial suspension will be used for subsequent infection experiments.

[0129] (2.3) Healing method of infection

[0130] Select leaves from healthy, sterile poplar seedlings (dark green leaves with thicker texture). Using a sterile scalpel, make 2-3 horizontal incisions along the midrib of the leaf. Lay the leaves face down on callus medium (CIM) and incubate in the dark at 25°C. After 20-30 days of leaf growth, loose white callus will form at the incision sites. Peel the callus off the leaf, divide it into soybean-sized pieces, and place them in a new callus medium. Transfer all the prepared callus to a sterile wide-mouth bottle containing bacterial solution and incubate on a shaker at 28°C and 160 rpm for 15-20 minutes.

[0131] In a clean bench, use filter paper to absorb excess bacterial solution from the callus and spread it evenly on a co-culture plate (WPMC). Incubate in the dark at 25°C for 2 days.

[0132] After dark culture, the callus was transferred to differentiation medium (WPMD). The medium was changed approximately every 20 days for the first time, and then every 10 days thereafter. During this period, the callus will turn green, harden, and then partially turn red. This stage takes about two months, and the entire process is carried out in a 25°C light incubator.

[0133] After the adventitious buds grow to about half a centimeter, use sterile tweezers or a scalpel to cut them off and place them in a bud elongation medium to grow. When the adventitious buds grow to 1-2 centimeters, cut them off individually and place them on a rooting medium for rooting culture. The adventitious buds will grow roots in about 10 days and grow into complete plants.

[0134] The formulation of the culture medium is shown in Table 6:

[0135] Table 6

[0136]

[0137] After preparation, sterilize at 121℃ for 20 minutes under high temperature and pressure.

[0138] Example 3 Identification of PtoTAR2 overexpression transgenic poplar and silencing transgenic poplar

[0139] (1) Crude extraction of DNA from wild-type Populus tomentosa, PtoTAR2 gene-overexpressing transgenic Populus tomentosa, and silent transgenic Populus tomentosa:

[0140] (i) Take a leaf sample to be extracted, place it in a 2mL centrifuge tube and add a grinding bead. After quick freezing with liquid nitrogen, use a tissue homogenizer to homogenize for 5 minutes.

[0141] (ii) After the mixture is broken into powder, add 500 μL of TBS buffer to the centrifuge tube, mix well, and then heat in a 65°C water bath for 10 min.

[0142] (iii) Centrifuge at 12000 rpm for 15 min, transfer the supernatant to a new centrifuge tube, add an equal volume of isopropanol, and allow to settle at room temperature for 30 min. A white flocculent precipitate will be visible.

[0143] (iv) Centrifuge at 12000 rpm for 15 min, slowly discard the supernatant, and add 1 ml of 75% ethanol to wash the precipitate.

[0144] (v) Centrifuge at 7500 rpm for 10 min, slowly pour off the supernatant and remove the excess liquid with a pipette.

[0145] (vi) Place in a 40℃ oven for 2 hours.

[0146] (vii) Add 50 μL of ddH2O to the centrifuge tube to dissolve the precipitate and store it at -20°C.

[0147] (2) Identification of transgenic poplar trees with PtoTAR2 gene overexpression and those with silenced transgenic poplar trees

[0148] (2.1) Identification at the DNA level

[0149] Using crudely extracted gDNA from transgenic plants as a template, PCR identification was performed using Taq Plus Master Mix. The reaction system is shown in Table 5.

[0150] The PCR reaction program was as follows: 95℃ for 2 min; (94℃ for 30 s; 55℃ for 30 s; 72℃ for 60 s) for 35 cycles; 72℃ for 5 min; 4℃ for ∞.

[0151] The PCR products were detected by agarose gel electrophoresis to check if the bands were correct. If correct, the plant was either a transgenic plant overexpressing the PtoTAR2 gene or a transgenic plant with silenced genes.

[0152] PCR testing revealed that plants numbered OE-1, OE-5, and OE-6 were PtoTAR2 overexpressing transgenic plants, while plants numbered RNAi-1, RNAi-3, and RNAi-6 were PtoTAR2 silent transgenic plants.

[0153] (2.2) Identification of transcription level

[0154] The transcriptional level of PtoTAR2 in overexpressing and silent transgenic plants was detected by RT-qPCR.

[0155] (i) RNA was extracted from the leaves of the plant to be tested and reverse transcribed to obtain template cDNA. RNA extraction and reverse transcription of cDNA were performed according to the method described in Example 1.

[0156] (ii) RT-qPCR reaction was performed using 2×ChamQ SYBR Color qPCR Master Mix, and the reaction system was as described in Table 2 of Example 1.

[0157] The reaction program was as follows: 95℃ for 30s; (95℃ for 5s; 60℃ for 35s) for 40 cycles; 95℃ for 15s; 60℃ for 1min; 95℃ for 15s.

[0158] Test results as follows Figure 4 As shown, the expression levels of OE-1 and OE-5 in PtoTAR2 overexpressing transgenic plants were higher, at 134.25 times and 152 times that of wild-type plants (WT), respectively; while the expression levels of RNAi-3 and RNAi-6 in PtoTAR2 silent transgenic plants were lower, at 0.047 times and 0.073 times that of wild-type plants (WT), respectively.

[0159] Example 4 Drought resistance analysis of PtoTAR2 overexpression transgenic plants and silencing transgenic plants

[0160] Poplar plants that have grown uniformly for 25 days under tissue culture conditions were washed with warm water to remove the agar from their roots and transplanted into pre-prepared soil (a 1:1.5 mixture of substrate and vermiculite). After transplanting, the plants were placed in a 25°C constant temperature culture room for 4 weeks, with a photoperiod of 16 hours of light and 8 hours of darkness.

[0161] The plant's growth status is as follows Figure 5 As shown, it can be seen that overexpression of the PtoTAR2 gene can promote the growth of poplar trees, making them taller.

[0162] After 8 weeks of soil cultivation, select healthy and robust seedlings for drought treatment. Stop watering and photograph their growth. After 10 days of drought stress treatment, the plant growth is as follows: Figure 6 As shown, after drought stress treatment, there was no significant difference in plant height among the different lines. However, the leaves of wild-type (WT) and PtoTAR2 overexpressing transgenic plants showed more severe wilting and yellowing than those of PtoTAR2 silenced plants. This indicates that silencing the PtoTAR2 gene can significantly enhance the drought resistance of plants.

[0163] Furthermore, the LI-COR LI-6800 portable photosynthetic fluorescence detection system was used to measure the photosynthetic parameters of the plants before and after drought stress. The specific operating procedure is as follows:

[0164] (1) Dark adaptation treatment and determination of initial fluorescence (Fo) and maximum fluorescence (Fm)

[0165] Before determining the maximum photochemical efficiency (Fv / Fm) of photosystem II (PSII), the leaves need to be fully dark adapted: wrap the leaves to be tested with tin foil for 30 minutes to ensure that all PSII reaction centers in the photosynthetic apparatus of the leaves are fully open and that the photosynthetic energy dissipation processes such as non-photochemical quenching (NPQ) are fully reversed, so as to eliminate the interference of light stress on fluorescence parameters.

[0166] During the test, the instrument parameters were first set: the inlet CO2 concentration was controlled at 400 μmol·mol⁻¹. -1 The water vapor concentration was maintained at 20 mmol·mol⁻¹ -1 The sample flow rate stabilized at 500 μmol·s. -1When measuring the minimum fluorescence (Fo) under dark adaptation, the modulation frequency was set to 2 kHz. The dark-adapted leaf was then carefully placed into the leaf chamber, ensuring the leaf completely covered the measurement area and the leaf chamber was well-sealed to avoid interference from external gases. After starting the fluorescence measurement program, the instrument automatically applied a saturation flash with an intensity sufficient to completely shut down all PSII reaction centers, simultaneously recording the Fo (minimum fluorescence under dark adaptation) and Fm (maximum fluorescence under dark adaptation) values. Finally, the Fv / Fm value was calculated using the formula Fv / Fm = (Fm - Fo) / Fm (Fv is variable fluorescence).

[0167] (2) Measurement of light adaptation treatment and other photosynthetic parameters

[0168] After the Fo and Fm measurements were completed, the leaf chamber light source was turned on and the light intensity was set to 1800 μmol·m. -2 ·s -1 Leaves were allowed to adapt to light intensity for 20 minutes. Once the fluctuation range of parameters such as net photosynthetic rate and transpiration rate was less than 5% (i.e., steady state was reached), other photosynthetic parameters such as stomatal conductance, photosynthetic rate, and transpiration rate were measured and recorded simultaneously.

[0169] Results of photosynthesis-related index measurements as follows Figure 7 As shown, under normal growth conditions, the photosynthetic capacity of PtoTAR2 overexpressing transgenic plants is not significantly different from that of wild-type plants.

[0170] After 10 days of drought stress treatment, the photosynthetic capacity of the plants showed a clear trend: silent transgenic plants > wild-type plants > overexpressing transgenic plants. Specifically, the vapor-stomach conductance (GSW) of all treated plants decreased significantly, with the gene-silenced lines showing relatively higher GSW, indicating that stomata remained partially open to meet basal transpiration requirements. The trend of transpiration rate (E) also remained consistent with that of GSW. Analysis of the maximum photochemical efficiency (Fv / Fm) of photosynthetic system II showed that the Fv / Fm values ​​of gene-silenced lines were significantly higher than those of WT and overexpressing lines, indicating a lower degree of damage to their photosynthetic system. In contrast, the Fv / Fm value of overexpressing lines was below 0.75, indicating irreversible damage to their photosynthetic enzyme system. Net photosynthetic rate data further confirmed that drought stress had a smaller impact on gene-silenced lines, while the photosynthetic capacity of WT and overexpressing lines was significantly reduced. The overexpressing lines had lost their photosynthetic capacity, and their leaves simultaneously initiated irreversible death and abscission programs.

[0171] Example 5 ABA sensitivity analysis of PtoTAR2 overexpression transgenic plants and silencing transgenic plants

[0172] To investigate the sensitivity of PtoTAR2 transgenic poplar to abscisic acid (ABA), this study conducted an ABA sensitivity analysis experiment based on 1 / 2 MS medium. The specific procedures were as follows: PtoTAR2 transgenic poplar tissue culture seedlings and wild-type poplar tissue culture seedlings with consistent growth were selected and inserted into 1 / 2 MS medium containing different ABA concentrations (0 μmol / L and 5 μmol / L), respectively. Three biological replicates were set for each group to ensure the reliability of the results. The medium was then cultured under suitable tissue culture conditions for 10 days, and the phenotypic differences between the groups were observed and recorded.

[0173] The results are as follows Figure 8 As shown, the rooting process of all lines was significantly inhibited in 1 / 2 MS medium containing 5 μmol / L ABA. Further comparison revealed that the rooting inhibition of the silenced transgenic plants was significantly higher than that of the wild-type and PtoTAR2 overexpressing transgenic plants, indicating that PtoTAR2 gene silencing can significantly enhance the sensitivity of poplar to ABA.

[0174] To analyze the accumulation levels of reactive oxygen species (ROS) in plants under different ABA treatments, the NBT staining kit and DAB staining kit from Solarbio were used to stain the leaves of plants treated with 0 μmol / L and 5 μmol / L ABA, respectively, so as to visually reflect the differences in ROS accumulation through the staining depth.

[0175] The results are as follows Figure 9 As shown, under 5 μmol / L ABA treatment, the leaves of wild-type and PtoTAR2-overexpressing transgenic poplars showed deep blue (NBT staining) or brownish-red (DAB staining) colors after NBT and DAB staining, with a relatively deep coloring degree, indicating a high level of ROS accumulation in their bodies. Conversely, the leaves of PtoTAR2-silenced transgenic plants showed significantly lighter NBT and DAB staining degrees, suggesting a lower level of ROS accumulation in their bodies. This result further demonstrates that silencing PtoTAR2 can increase the sensitivity of poplars to ABA.

[0176] The present invention has been described in detail above with reference to specific embodiments and exemplary examples. However, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the present invention, and all such modifications and improvements fall within the scope of the present invention.

Claims

1. A gene, PtoTAR2, that regulates drought resistance and ABA sensitivity in poplar trees, characterized in that... The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A protein that regulates the drought resistance and ABA sensitivity of poplar trees, characterized in that, The protein is encoded by the gene PtoTAR2, and its amino acid sequence is shown in SEQ ID NO.

3.

3. The application of the gene PtoTAR2 as described in claim 1 or the protein as described in claim 2 in regulating the drought resistance and ABA sensitivity of poplar trees.

4. The application according to claim 3, characterized in that, The regulation is achieved by overexpressing or silencing the gene PtoTAR2 in poplar trees.

5. The application according to claim 3, characterized in that, When the gene PtoTAR2 is overexpressed in poplar, the drought resistance and ABA sensitivity of poplar decrease; when the gene PtoTAR2 is silenced in poplar, the drought resistance and ABA sensitivity of poplar increase.

6. A recombinant vector, characterized in that, The recombinant vector is a silencing recombinant vector of the gene PtoTAR2 as described in claim 1, which is obtained by constructing an interference fragment of the gene PtoTAR2 onto a base vector.

7. A method for improving the drought resistance and ABA sensitivity of poplar trees, characterized in that, The method includes the step of silencing the expression of the gene PtoTAR2 of claim 1 in poplar trees.

8. The method according to claim 7, characterized in that, The method includes the following steps: Step 1: Construct a silencing recombination vector for the PtoTAR2 gene; Step 2: The silent recombination vector was introduced into poplar trees to obtain transgenic plants with the PtoTAR2 gene. Step 3: Identify and perform phenotypic analysis on the transgenic plants.

9. The application of the gene PtoTAR2 of claim 1, the protein of claim 2, the recombinant vector of claim 6, and the method of claim 7 or 8 in poplar breeding.

10. The application according to claim 9, characterized in that, The breeding program aims to cultivate new poplar varieties with high drought resistance and / or high sensitivity to ABA by silencing the PtoTAR2 gene expression compared to the wild type.

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