A gene pto tar2 for improving drought resistance and sensitivity to abscisic acid of poplar and application thereof

Abstract

The application discloses a gene PtoTAR2 for improving drought resistance and sensitivity to ABA of poplar and application thereof, a nucleotide sequence of the gene is shown in SEQ ID NO. 1, an amino acid sequence of a coded protein is shown in SEQ ID NO. 3, the gene can negatively regulate drought resistance and sensitivity to ABA of the poplar, provides a candidate gene for cultivating new germplasm of the poplar with improved stress resistance and without affecting growth, and effectively shortens a breeding cycle. The application also discloses a method for improving drought resistance and sensitivity to ABA of the poplar and related application, and the drought resistance and the sensitivity to ABA of the poplar are specifically enhanced by silencing expression of the gene PtoTAR2 in the poplar, which provides an effective way for cultivating new germplasm of the poplar with improved stress resistance and without affecting growth, and has important significance for 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 wheat 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 7Figure 6 shows the stomatal conductance, Fv / Fm, net photosynthetic rate, transpiration rate of WT, PtoTAR2 overexpression transgenic plants (OE-1, OE-5, OE-6) and silencing (RNAi-1, RNAi-3, RNAi-6) transgenic plants in Example 4 before and after drought treatment; Figure 8 Figure 7 shows the root morphological phenotype of WT, PtoTAR2 overexpression transgenic plants (OE-5) and silencing transgenic plants (RNAi-3) after 5 μmol / L ABA treatment in Example 5; Figure 9 Figure 8 shows the leaf DAB, NBT staining of WT, PtoTAR2 overexpression (OE-1, OE-5) and silencing (RNAi-3, RNAi-6) transgenic plants after 5 μmol / L ABA treatment in Example 5. DETAILED DESCRIPTION

[0019] The application will be further described in details by preferred embodiments and examples. The features and advantages of the present application will become more apparent through these descriptions.

[0020] The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any implementation described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other implementations.

[0021] In a first aspect, the present application provides a gene PtoTAR2 for regulating drought tolerance and sensitivity to ABA of Populus, the nucleotide sequence of which is shown in SEQ ID NO. 1.

[0022] Preferably, the Populus is Populus tomentosa, preferably Populus tomentosa Carr.

[0023] In a preferred embodiment, the gene PtoTAR2 for regulating drought tolerance and sensitivity to ABA of Populus is located on chromosome 15 of 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 number: CNP0004290, sample number: CNS0752799, assembly number: CNA0069009.

[0025] In a preferred embodiment, the nucleotide sequence of the coding region of the gene PtoTAR2 for regulating drought tolerance and sensitivity to ABA of poplar is shown as 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 number: CNP0004290, sample number: CNS0752799, assembly number: CNA0069009; PtoTAR2 position: 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, see http: / / db.cngb.org / cnsa / project / CNP0004290_40760cfc / reviewlink / for details.

[0027] In a second aspect of the present application, a protein is provided, which is the coding protein of the gene PtoTAR2 in the first aspect, and the sequence of the coding protein is shown as SEQ ID NO. 3.

[0028] In a third aspect of the present application, the gene PtoTAR2 in the first aspect or the protein in the second aspect is applied to regulate drought tolerance and sensitivity to ABA of poplar.

[0029] Preferably, the poplar is white poplar, preferably P. tomentosa.

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

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

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

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

[0034] The silencing recombination vector is obtained by constructing the interference fragment of the gene PtoTAR2 to a basic vector.

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

[0036] In a preferred embodiment, when the gene PtoTAR2 is overexpressed in a poplar, the drought tolerance and sensitivity to ABA of the poplar are reduced; when the gene PtoTAR2 is silenced in a poplar, the drought tolerance and sensitivity to ABA of the poplar are improved.

[0037] In the present application, the gene PtoTAR2 is a drought negative regulatory factor related to sensitivity to ABA.

[0038] Preferably, the drought tolerance and sensitivity to ABA of a poplar are reduced by overexpressing the gene PtoTAR2 in the poplar, and the drought tolerance and sensitivity to ABA of a poplar are improved by silencing the expression of the gene PtoTAR2 in the poplar.

[0039] The silencing expression of the gene PtoTAR2 is disclosed for the first time in the present application, which can improve the drought tolerance and sensitivity to ABA of a poplar, and the gene can be used as a candidate gene for breeding a new poplar germplasm with improved stress resistance and without affecting growth, which has important significance for improving the growth and survival ability of a poplar in a drought area and promoting the sustainable development of forestry.

[0040] In a fourth aspect of the present application, a recombination vector is provided, which is a silencing recombination vector of the gene PtoTAR2, and is obtained by constructing the interference fragment of the gene PtoTAR2 of the first aspect to a basic vector.

[0041] Preferably, the interference fragment of the gene PtoTAR2 is PtoTAR2-RNAi, and its sequence is as shown in SEQ ID NO. 4.

[0042] The basic vector is a pBI121 vector.

[0043] The pBI121 vector has a total length of 13629 bp, contains a 35S strong promoter (CaMV35S), has a resistance of kanamycin (Kana), and can be subjected to enzyme digestion with XbaI and BamHI.

[0044] In a fifth aspect of the present application, a method for improving the drought tolerance and sensitivity to ABA of a poplar is provided, which comprises the step of silencing the expression of the gene PtoTAR2 in the poplar.

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

[0046] Step 1, constructing a silencing recombination vector of gene PtoTAR2.

[0047] Preferably, the silencing recombination vector of gene PtoTAR2 is obtained by constructing an interference fragment of gene PtoTAR2 on a pBI121 vector.

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

[0049] In the present application, the target region of the interference fragment of gene PtoTAR2 is screened by constructing a gene family phylogenetic tree and performing systematic evolution analysis on the sequence characteristics of Populus PtoTAR2 gene and its homologous genes in related species. This interference fragment precisely avoids the redundant sequences highly conserved in the gene family, not only ensuring efficient silencing of the target gene PtoTAR2, but also blocking cross interference on other functional genes in the same family from the sequence specificity level, solving the off-target problem caused by cross combination of conserved sequences in conventional design.

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

[0051] Step 2, introducing the silencing recombination vector into Populus to obtain transgenic plants of gene PtoTAR2.

[0052] Preferably, the Populus is Populus tomentosa, preferably Populus tomentosa.

[0053] In a preferred embodiment, the plasmid of the silencing recombination vector is transformed into Agrobacterium, and then the Agrobacterium liquid is used to infect Populus callus to obtain a complete plant.

[0054] Step 3, identifying and performing phenotype analysis on the transgenic plants.

[0055] Preferably, gDNA of the transgenic plants is used as a template to perform preliminary identification at the DNA level by PCR, and the identification procedure is as follows:

[0056] The PCR reaction procedure is 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 plants identified positive by PCR are subjected to RT-qPCR identification at transcription level, and the detection primers used are PtoTAR2-qPCR-F and PtoTAR2-qPCR-R, the sequences of which are shown in SEQ ID NO. 7 and SEQ ID NO. 8, respectively.

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

[0059] The present application finds that, by using the specific detection primers and detection procedure described above at DNA level and transcription level, the transgenic plants can be accurately identified, the false positive detection rate is reduced, and the test efficiency is effectively improved.

[0060] In a preferred embodiment, the phenotype analysis of the silenced transgenic plants comprises drought tolerance analysis and ABA sensitivity analysis,

[0061] The drought tolerance analysis comprises growth state analysis, maximum photochemical efficiency (Fv / Fm) analysis, stomatal conductance analysis, photosynthetic rate analysis and transpiration rate analysis; and the ABA sensitivity analysis comprises root morphology analysis and active oxygen level analysis.

[0062] According to the embodiments of the present application, after 10 days of drought stress treatment, the leaves of wild type plants and overexpression transgenic plants exhibit more severe wilting and yellowing than the silenced transgenic plants.

[0063] After 10 days of drought stress treatment, the photosynthetic capacity of the plants shows a clear trend of: silenced transgenic plants > wild type plants > overexpression transgenic plants, specifically: the water vapor stomatal conductance (GSW) of all treated plants decreases significantly, among which the GSW of the gene-silenced lines is relatively high, and the stomata still maintain a partially open state to meet the basic transpiration demand, and the change trend of the transpiration rate (E) is consistent with that of gsw; the maximum photochemical efficiency of photosystem II (Fv / Fm) analysis shows that the Fv / Fm value of the gene-silenced lines is significantly higher than that of the WT and overexpression lines; the net photosynthetic rate data further confirms that the drought stress has less effect on the gene-silenced lines, and the photosynthetic capacity of the WT and overexpression lines is significantly reduced, among which the overexpression lines have lost photosynthetic capacity, and their leaves simultaneously initiate irreversible death and abscission programs.

[0064] After 10 days of culture in 1 / 2MS medium containing 5 μmol / L ABA, the rooting inhibition degree of the silenced transgenic plants is significantly higher than that of the wild type and PtoTAR2 overexpression transgenic plants;

[0065] Under the treatment of 5 μmol / L ABA, the leaves of wild type and PtoTAR2 overexpression transgenic poplar plants were all presented dark blue (NBT staining) or brown (DAB staining) after NBT and DAB staining, and the coloring degree was deeper, indicating that the accumulation of ROS in vivo was higher; on the contrary, the coloring degree of NBT and DAB staining of the leaves of PtoTAR2 silenced transgenic plants was significantly lighter, indicating that the accumulation of ROS in vivo was lower.

[0066] The method for improving drought tolerance and ABA sensitivity of poplar provided by the application can specifically enhance the drought tolerance and ABA sensitivity of poplar by silencing the expression of the gene PtoTAR2, and provides a new idea for further studying the stress resistance mechanism of trees.

[0067] In a sixth aspect, the gene PtoTAR2 of the first aspect, the protein of the second aspect, the recombinant vector of the fourth aspect or the method of the fifth aspect are applied in poplar breeding.

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

[0069] More preferably, the poplar is white poplar, preferably Populus tomentosa.

[0070] The gene PtoTAR2, the protein, the recombinant vector and the method for improving drought tolerance and ABA sensitivity of poplar provided by the application are applied in poplar breeding, and compared with wild type and PtoTAR2 overexpression poplar plants, the sensitivity of PtoTAR2 gene silenced poplar plants to abscisic acid (ABA) is significantly enhanced, the drought resistance is obviously better, and under drought stress conditions, the growth condition is not significantly different from that of the control plants, which provides an effective way for breeding new varieties of poplar with improved stress resistance without affecting growth, and has important significance for improving the growth and survival ability of poplar in drought areas and promoting the sustainable development of forestry.

[0071] Examples

[0072] The application will be further described below by specific examples, but these examples are only exemplary and do not constitute any limitation on the scope of protection of the application.

[0073] Unless otherwise specified, the reagents involved in the following examples are commercially available conventional reagents, and the methods used are commonly used methods in the 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 gene PtoTAR2 in the growth and development process of Populus tomentosa, two-month-old water-cultured Populus tomentosa with consistent growth states were selected as experimental materials, and different functional and developmental stage tissues (including roots, stems, root tips, young leaves, mature leaves, and terminal buds) were systematically sampled. The RNA of the collected plant tissues was extracted according to the MolPure Plant RNA Kit kit instructions of Yixing Bioengineering Company.

[0076] The RNA obtained above was used as a template, and HiScript IV All-in-One Ultra RT SuperMix for qPCR-R433 (Nanjing Novogene Bioinformatics Technology Co., Ltd.) was used for reverse transcription to obtain cDNA. The reaction system (20 μL) is shown in Table 1:

[0077] Table 1

[0078]

[0079] The reaction program was 50 °C for 5 min and 85 °C for 5 sec.

[0080] According to the CDS sequence of the gene PtoTAR2 of Populus tomentosa (as shown in SEQ ID NO. 2), the primers PtoTAR2-qPCR-F and PtoTAR2-qPCR-R for the fluorescence quantitative PCR reaction were designed, and the expression amount of PtoTAR2 gene in different tissues was determined by fluorescence quantitative PCR. The nucleotide sequences of the primers are shown in SEQ ID NO. 7 and SEQ ID NO. 8, respectively. The internal reference primers are Actin-F and Actin-R, and the nucleotide sequences are shown in SEQ ID NO. 9 and SEQ ID NO. 10, respectively.

[0081] 2xChamQ SYBR Color qPCR Master Mix was used for RT-qPCR experiment, and the reaction system (20 μL) is shown in Table 2:

[0082] Table 2

[0083]

[0084] The reaction program was 95 °C for 30 s, 40 cycles of (95 °C for 5 s, 60 °C for 35 s), 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s.

[0085] The results of the fluorescence quantitative PCR reaction are shown in Figure 1As shown in

[0086] (2) To explore the response mode of PtoTAR2 gene to abscisic acid (ABA) signal and drought stress, two-month-old water-cultured P. tomentosa were treated with ABA (50 μmol / L) and simulated drought stress (20% PEG 6000), respectively. The root tissues of P. tomentosa were collected at 0, 0.5, 1, 2, 4, 6, 8, 16, and 24 h after ABA and 20% PEG 6000 treatment, and the RNA of the collected root tissues was extracted. The RNA extraction was performed according to the MolPure Plant RNA Kit kit manual of Norgen Biotek Corporation.

[0087] The relative expression amount of PtoTAR2 gene in the roots of P. tomentosa at each time point under the two kinds of stress treatments was determined by real-time fluorescent quantitative PCR technology. The component concentrations (such as primers, template cDNA, fluorescent dyes, etc.) and amplification procedures (including pre-denaturation temperature and time, annealing temperature, extension time, and cycle number) of the qRT-PCR reaction system were set according to the method described in the above step (1) to ensure the consistency of experimental operation and the reliability of data.

[0088] The results of the fluorescent quantitative PCR reaction are shown in Figure 2 and Figure 3 As can be seen, with the extension of the ABA treatment and drought treatment time, the expression amount of PtoTAR2 gene was significantly reduced. Therefore, it is speculated that the gene PtoTAR2 plays an important regulatory role in the process of corresponding ABA and drought stress of P. 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 of PtoTAR2 gene target fragment

[0092] The RNA of LM50 P. tomentosa was extracted by the plant RNA extraction kit of Beijing Zison Biological Technology Co., Ltd., and the RNA was reverse transcribed into cDNA by HiScript III 1st Strand cDNA Synthesis Kit (Nanjing Novogene Bioinformatics Technology Co., Ltd.).

[0093] The primers for amplifying the target fragments were designed by referring to the genomic file of P. tomentosa (http: / / db.cngb.org / cnsa / project / CNP0004290_40760cfc / reviewlink / ), comprehensively considering various principles of primer design, and using the primer blast tool (NCBI, https: / / blast.ncbi.nlm.nih.gov). The target fragments include the target fragment for constructing the overexpression vector (CDS sequence of gene PtoTAR2) and the target fragment for constructing the RNAi recombinant vector (part of the reverse sequence of CDS of gene PtoTAR2).

[0094] The amplification primers of 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 amplification primers of the interference fragment PtoTAR2-RNAi of PtoTAR2 are PtoTAR2-RNAi-F and PtoTAR2-RNAi-R, and their 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 P. tomentosa genome (http: / / db.cngb.org / cnsa / project / CNP0004290_40760cfc / reviewlink / ), and the start position is 9163816 and the end position is 9173548.

[0097] The 2x Phanta Max Master Mix (Dye Plus) of Nanjing Vazyme Company was used for PCR amplification, and the PCR experimental reaction system (50 μL) is shown in Table 3:

[0098] Table 3

[0099]

[0100] Reaction procedure: 95℃ 3min; (95℃ 25s; 55℃ 30s; 72℃ 60s (45s / kb for amplification efficiency) 36 cycles; 72℃ 5min; 4℃ ∞.

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

[0102] (1.2) Vector enzyme 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 1-2h.

[0108] The enzyme-digested products were recovered and purified by gel recovery, using Kangweishiji Gel Extraction Kit reagent kit, and purified using Kangweishiji DNA Clean-up Kit reagent kit. After purification, it was used as a vector skeleton and stored at -20℃.

[0109] (1.3) Ligation transformation

[0110] The CDS of PtoTAR2 and the CDS partial reverse sequence were constructed into the pBI121 vector using the seamless cloning kit Uniclone One Step Seamless Cloning Kit of Beijing Jinsha Biotechnology Co., Ltd., respectively, to obtain overexpression recombinant vectors and RNAi recombinant vectors. 50℃ ligation for 10min.

[0111] Take 50μl DH5α competent cells, add 5μl ligation product, mix gently and stand on ice for 30min, 42℃ water bath heat shock for 45s, quickly transfer to ice for cooling 2min. Add 700μl of sterile LB liquid medium without antibiotics in the super-clean bench, mix and beat evenly, then place in a 37℃, 220rpm shaking incubator for 45min to recover the bacterial cells. After recovery, centrifuge at 6000rpm for 1min, collect the bacterial cells, resuspend the bacterial cells with 100μl supernatant, and spread on LB solid plate medium containing kanamycin (100mg / ml), and incubate at 37℃ for 14-16h.

[0112] (1.4) Bacterial liquid PCR identification of positive clones

[0113] Single colonies on the plate were picked with a sterilized gun head into 250 μl of LB liquid medium containing kanamycin, and cultured at 37°C and 200 rpm / min for about 3 h as an amplification template. Taq Plus Master Mix from Nanjing Vazyme was used for PCR amplification, and 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] The PCR product was detected by 1% agarose gel electrophoresis.

[0118] (1.5) Positive clone plasmid extraction

[0119] The positive clone detected by PCR was taken into 6 ml of LB liquid medium containing kanamycin and cultured at 37°C and 200 rpm overnight. Plasmid extraction was performed using a plasmid extraction kit from Jiangsu Kangwei Reagent Co., Ltd., and sequencing was performed by Beijing Ruibo Xingke Biotechnology Co., Ltd. After the sequence was determined and compared without error, the construction of the overexpression recombinant vector (PtoTAR2-OE) and the RNAi recombinant vector (PtoTAR2-RNAi) was completed.

[0120] (2) Genetic transformation of PtoTAR2 gene

[0121] (2.1) Transformation of recombinant plasmid into Agrobacterium

[0122] About 1 μg of PtoTAR2-OE recombinant vector plasmid and PtoTAR2-RNAi recombinant vector plasmid was added to 100 μL of Agrobacterium GV3101 competent cells, and mixed gently.

[0123] In order, stand on ice for 5 min, quick-freeze in liquid nitrogen for 5 min, 37°C water bath for 5 min, ice bath for 5 min. Add 700 μL of YEP liquid medium without antibiotics to the bacterial solution, mix thoroughly, and incubate at 28°C and 200 rpm for 2-3 hours. After recovery culture, centrifuge at 6000 rpm for 1 min, discard part of the supernatant, reserve 100 μL of supernatant and mix with the bacterial solution, evenly spread the bacterial solution on the surface of YEP solid medium containing 50 mg / L rifampicin and 50 mg / L kanamycin, and then place in a 28°C incubator for inverted culture for 72-90 hours.

[0124] After the single colony was grown, several single colony spots were picked with a sterile gun tip and placed in a 2 mL centrifuge tube containing 250 μL YEP liquid medium (1:1000 Rif, Kana) and cultured in a shaker at 30°C and 200 rpm for 2 h. After the culture was completed, the bacterial liquid was taken for PCR identification. The correct positive bacterial liquid was added with 50% glycerol, quickly frozen in liquid nitrogen, and stored in a -80°C refrigerator for subsequent genetic transformation experiments.

[0125] (2.2) Activation of Agrobacterium

[0126] The Agrobacterium containing the PtoTAR2 overexpression recombinant vector plasmid and the RNAi recombinant vector plasmid was taken out from the -80°C refrigerator, streaked on YEP solid medium (1:1000 Rif, Kana), and cultured in a 28°C dark incubator for 2-3 days.

[0127] A single colony was picked from the plate with a sterile gun tip and inoculated into 3 mL YEP liquid medium (1:1000 Rif, Kana), and cultured in a shaker at 30°C and 200 rpm overnight. 1 mL of the bacterial liquid was transferred to a 250 mL sterile conical flask containing 100 mL YEP liquid medium (1:1000 Rif, Kana), and cultured in a shaker at 30°C and 200 rpm for 4-5 h until the OD600 was 0.3-0.5.

[0128] 100 mL of the bacterial liquid was taken, centrifuged at 2560 g and 4°C for 20 min, and the supernatant was discarded. The bacterial pellet was resuspended in a sterile jar with 100 mL of resuspension solution (WPMB), and the bacterial liquid was used for subsequent infection experiments.

[0129] (2.3) Wound method infection

[0130] The leaves of healthy and sterile P. tomentosa seedlings (dark green leaves and thick texture) were selected, and 2-3 horizontal wounds were made on the main veins of the leaves using a sterile scalpel. The leaves were placed on the wound medium (CIM) with the front down, and cultured in a 25°C dark environment. After the leaves grew for 20-30 days, white and loose calluses grew at the wound sites. The calluses were peeled off from the leaves, divided into soybean-sized pieces, and transferred to a new wound medium. The prepared calluses were transferred to a sterile jar containing the bacterial liquid, and cultured in a shaker at 28°C and 160 rpm for 15-20 min.

[0131] In a clean bench, the excess bacterial liquid on the calluses was absorbed with filter paper, and the calluses were placed on the co-culture plate (WPMC) and cultured in a 25°C dark environment for 2 days.

[0132] After the end of dark culture, the callus is transferred to differentiation medium (WPMD). The medium is changed once in 20 days at the first time, and then once in 10 days. During the period, the callus will turn green, harden, and then partially turn red. This stage is about two months, and the whole process is cultured in a 25℃ light incubator.

[0133] After the adventitious buds grow to about half a centimeter, sterile tweezers or a surgical knife is used to cut off the adventitious buds and place them in bud elongation medium for growth. When the adventitious buds grow to 1-2 centimeters, they are cut off individually and placed in rooting medium for rooting culture. After 10 days or so, the adventitious buds will grow roots, and the rooting culture will become a complete plant.

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

[0135] Table 6

[0136]

[0137] After configuration, 121℃, 20min high temperature and high pressure sterilization is performed.

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

[0139] (1) Crude extraction of wild-type poplar, PtoTAR2 gene overexpression transgenic poplar and silencing transgenic poplar DNA:

[0140] (i) Take a piece of the sample leaf to be extracted and place it in a 2mL centrifuge tube and add 1 grinding bead. After quick freezing in liquid nitrogen, use a tissue crusher to crush for 5min.

[0141] (ii) After being crushed into powder, 500μL TBS buffer is added to the centrifuge tube. After mixing, it is placed in a 65℃ water bath for heating for 10min.

[0142] (iii) 12000rpm centrifugation for 15min, the supernatant is transferred to a new centrifuge tube, an equal volume of isopropanol is added, and room temperature sedimentation is performed for 30min. A white flocculent precipitate can be seen.

[0143] (iv) 12000rpm centrifugation for 15min, the supernatant is slowly poured off, and 1ml 75% ethanol is added to wash the precipitate.

[0144] (v) 7500rpm centrifugation for 10min, the supernatant is slowly poured off and the remaining liquid is cleaned with a pipette.

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

[0146] (vii) Add 50μL ddH2O to the centrifuge tube to dissolve the precipitate, and store in a -20℃ refrigerator.

[0147] (2) Identification of PtoTAR2 gene overexpression transgenic poplar and silencing transgenic poplar

[0148] (2.1) Identification at DNA level

[0149] PCR identification was performed using Taq Plus Master Mix with crude extracted gDNA of transgenic plants as template, and the reaction system was referred to Table 5.

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

[0151] The PCR products were detected by agarose gel electrophoresis to check whether the bands were correct. If correct, the plants were PtoTAR2 gene overexpression transgenic plants or silencing transgenic plants.

[0152] The PCR detection showed that the plants numbered OE-1, OE-5 and OE-6 were PtoTAR2 overexpression transgenic plants, and the plants numbered RNAi-1, RNAi-3 and RNAi-6 were PtoTAR2 silencing transgenic plants.

[0153] (2.2) Identification at transcription level

[0154] RT-qPCR was used to detect the transcription level of PtoTAR2 in overexpression transgenic plants and silencing transgenic plants:

[0155] (i) RNA of leaves of plants to be detected was extracted, and template cDNA was obtained by reverse transcription. 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 referred to Table 2 in Example 1.

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

[0158] The detection results are shown in Table 3. Figure 4 As can be seen, the expression levels of OE-1 and OE-5 in PtoTAR2 overexpression transgenic plants were higher, which were 134.25 times and 152 times of wild type plants (WT), respectively; the expression levels of RNAi-3 and RNAi-6 in PtoTAR2 silencing transgenic plants were lower, which were 0.047 times and 0.073 times of wild type plants (WT), respectively.

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

[0160] The poplar plants grown under tissue culture conditions for 25 days with consistent growth were washed with warm water to remove the root agar, transplanted into the prepared soil (substrate mixed with vermiculite 1:1.5), and placed in a 25°C constant temperature culture room for 4 weeks after transplantation. During the culture period, a light cycle condition of 16h light / 8h dark was set.

[0161] The growth state of the plants is shown in Figure 5 It can be seen that overexpression of PtoTAR2 gene can promote the growth of poplar and make the plant height higher.

[0162] After 8 weeks of soil culture, healthy and vigorous soil culture seedlings were selected for drought treatment, watering was stopped and the growth state was recorded by taking pictures. After 10 days of drought stress treatment, the growth state of the plants is shown in Figure 6 It can be seen that after drought stress treatment, there is no significant difference in plant height among the different lines, but the leaves of wild type (WT) and PtoTAR2 overexpression transgenic plants show more severe wilting and yellowing than PtoTAR2 silenced plants, which indicates that silencing PtoTAR2 gene can significantly enhance the drought resistance of the plants.

[0163] Further, the photosynthesis-related parameters of the plants before and after drought stress were determined by LI-COR LI-6800 portable photosynthesis and fluorescence detection system, and the specific operation process 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 test leaves with tin paper for 30 min to ensure that all PSII reaction centers in the photosynthetic apparatus of the leaves are fully open, and the non-photochemical quenching (NPQ) and other photosynthetic energy dissipation processes are fully reversed, to exclude the interference of light stress on fluorescence parameters.

[0166] During detection, first set the instrument parameters: the CO2 concentration is controlled at 400 μmol·mol -1 , the water vapor concentration is maintained at 20 mmol·mol -1 , and the sample flow rate is stabilized at 500 μmol·s -1; when measuring minimum fluorescence (Fo) under dark adaptation, the modulation frequency was set to 2 kHz. Subsequently, the dark-adapted leaf was carefully clamped into the leaf chamber, ensuring that the leaf completely covered the measurement area and the leaf chamber was sealed well to avoid interference from external environmental gases. After starting the fluorescence measurement program, the instrument automatically applied saturated flashes of light intensity sufficient to completely close all PSII reaction centers, and simultaneously recorded the Fo (minimum fluorescence under dark adaptation) and Fm (maximum fluorescence under dark adaptation) values. Finally, the Fv / Fm value (Fv is the variable fluorescence) was calculated according to the formula Fv / Fm = (Fm-Fo) / Fm.

[0167] (2) Light adaptation treatment and determination of other photosynthetic parameters

[0168] After the determination of Fo and Fm was completed, the light source of the leaf chamber was turned on and the light intensity was set to 1800 μmol·m -2 ·s -1 The leaves were subjected to 20 min light adaptation at this light intensity. After the instrument monitored that the fluctuation amplitude of the net photosynthetic rate, transpiration rate and other parameters of the plants was less than 5% (i.e., reached a steady state), other photosynthesis-related parameters such as stomatal conductance, photosynthetic rate and transpiration rate were simultaneously determined and recorded.

[0169] The determination results of the photosynthesis-related indicators are shown in Table 1. Figure 7 As can be seen, under normal growth conditions, the photosynthetic capacity of the PtoTAR2 overexpression transgenic plants had no significant difference from that of the wild type.

[0170] After 10 days of drought stress treatment, the photosynthetic capacity of the plants showed a clear trend of silencing transgenic plants > wild type plants > overexpression transgenic plants. Specifically, the water vapor stomatal conductance (GSW) of all treated plants decreased significantly, among which the GSW of the gene silencing lines was relatively high, and the stomata still maintained a partially open state to meet the basic transpiration demand. The change trend of the transpiration rate (E) was consistent with that of gsw; the analysis of the maximum photochemical efficiency of photosystem II (Fv / Fm) showed that the Fv / Fm value of the gene silencing lines was significantly higher than that of the WT and overexpression lines, indicating that the damage degree of their photosynthetic systems was lower. The Fv / Fm value of the overexpression lines was lower than 0.75, indicating that their photosynthetic enzyme systems had suffered irreversible damage; the data of the net photosynthetic rate further confirmed that the drought stress had less effect on the gene silencing lines, while the photosynthetic capacity of the WT and overexpression lines decreased significantly, among which the overexpression lines had lost the photosynthetic capacity, and their leaves simultaneously initiated the irreversible death and abscission program.

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

[0172] To explore the sensitivity of PtoTAR2 transgenic poplar to abscisic acid (ABA), the present example carried out ABA sensitivity analysis experiment based on 1 / 2MS medium. The specific operation is as follows: select the PtoTAR2 transgenic poplar and wild type poplar with the same growth state, and cut them into 1 / 2MS medium containing different ABA concentrations (0 μmol / L, 5 μmol / L), set 3 biological replicates for each group to ensure the reliability of the results; then place the medium in the appropriate tissue culture conditions for 10 days, observe and record the phenotype differences of each group of plants.

[0173] The results are shown in Figure 8 It can be seen that: in the 1 / 2MS medium containing 5 μmol / L ABA, the rooting process of each strain is significantly inhibited. Further comparison found that: the rooting inhibition degree of the silenced transgenic plants is significantly higher than that of the wild type and PtoTAR2 overexpression transgenic plants, indicating that PtoTAR2 gene silencing can significantly enhance the sensitivity of poplar to ABA.

[0174] To analyze the active oxygen (ROS) accumulation level in plants under different ABA treatment, NBT staining kit and DAB staining kit of Solabio Company were used to stain the leaves of plants treated with 0 μmol / L, 5 μmol / L ABA, respectively, to intuitively reflect the accumulation difference of ROS through the color depth.

[0175] The results are shown in Figure 9 It can be seen that: under the treatment of 5 μmol / L ABA, the leaves of wild type and PtoTAR2 overexpression transgenic poplar after NBT and DAB staining are dark blue (NBT staining) or brown (DAB staining), and the coloring degree is deeper, indicating that the ROS accumulation in vivo is higher; On the contrary, the NBT and DAB staining coloring degree of PtoTAR2 silenced transgenic plant leaves is significantly lighter, indicating that the ROS accumulation in vivo is lower. This result further proves that silencing PtoTAR2 can improve the sensitivity of poplar to ABA.

[0176] The above detailed description of the present application is combined with the specific embodiments and exemplary examples, but these descriptions cannot be understood as limitations of the present application. Those skilled in the art understand that the technical solutions and embodiments of the present application can be variously replaced, modified or improved without deviating from the spirit and scope of the present application, and these all fall within the scope of the present application.

Claims

1. Silencing a gene PtoTAR2 or reducing the amount of a protein encoded by a gene PtoTAR2 for use in increasing drought tolerance and increasing sensitivity to ABA in poplar, characterized in that The nucleotide sequence of the gene PtoTAR2 is shown as SEQ ID NO. 1; the amino acid sequence of the encoded protein of the gene PtoTAR2 is shown as SEQ ID NO.

3.

2. A method for improving drought tolerance and ABA sensitivity in poplar, characterized in that, The method involves silencing gene expression in poplar trees. PtoTAR2 The steps, the gene PtoTAR2 The nucleotide sequence is shown in SEQ ID NO.

1.

3. The method of claim 2, wherein, The method comprises the following steps: Step 1, construction of the gene PtoTAR2 silencing recombination vector; Step 2, introducing the silent recombination vector into poplar to obtain transgenic plants of the gene PtoTAR2 ​ Step 3, identification and phenotype analysis of transgenic plants.

4. gene PtoTAR2 , the gene PtoTAR2 encoding protein or recombinant vector in poplar breeding, characterized in that, The nucleotide sequence of the gene PtoTAR2 The amino acid sequence of the encoded protein of the gene PtoTAR2 is shown as SEQ ID NO. 3; The recombinant vector is a silencing recombinant vector of a gene PtoTAR2 obtained by constructing an interfering fragment of a gene PtoTAR2 on a base vector; The breeding is: compared with wild type, silencing expression of genes PtoTAR2 , cultivating new varieties of poplar with high drought tolerance and / or high sensitivity to ABA.

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