PagTTG1 gene for regulating and controlling pore opening and closing and drought resistance of poplar and application of PagTTG1 gene

Abstract

The invention relates to a PagTTG1 gene for regulating and controlling pore opening and closing and drought resistance of poplar and application of the PagTTG1 gene, and relates to the technical field of stress-resistant biological genetic engineering. A nucleotide sequence and an amino acid sequence of a coding region of the PagTTG1 gene are respectively shown as SEQ ID NO. 3 and SEQ ID NO. 4. The PagTTG1 gene is knocked out through CRISPR / Cas9, and compared with a wild plant, a TTG1 knockout plant has the advantages that the drought resistance is obviously enhanced, and the pore opening degree is obviously reduced; the WD family protein PagTTG1 gene is indicated to be a key regulatory factor for regulating and controlling pore opening and closing and drought resistance of the poplar. The invention provides a new choice for pore opening and closing and drought resistance regulation and control means, and has important application value in the field of forest stress resistance gene engineering.

Description

Technical Field

[0001] This invention relates to a PagTTG1 gene that regulates stomatal opening and closing and drought resistance in poplar trees and its application, particularly to the PagTTG1 gene, a key regulatory factor in the WD40 repeat protein family that regulates stomatal opening and closing in poplar trees and its application in drought resistance, belonging to the field of plant drought resistance genetic engineering technology. Background Technology

[0002] Environmental changes (temperature, light, water, CO2 concentration) affect forest growth. Adverse environments severely restrict the sustainable use of forest resources and the stability of the ecological environment. Therefore, improving the environmental adaptability of trees, reducing the impact of environmental changes on forest growth, and enhancing the stability and ecological function of forest resources are of great significance. Environmental changes lead to morphological changes in plants. Plant leaves are the primary organs for sensing environmental changes, and their development is regulated by multiple regulatory factors. Understanding these regulatory mechanisms can help to specifically improve the environmental adaptability of woody plants.

[0003] Drought is one of the most significant abiotic stresses affecting plant physiological states in natural ecosystems, playing a crucial role in ecosystem management and protection. With global warming and the continuous depletion of available freshwater resources, drought stress severely impacts plant growth and development. Due to the long growth cycle and complex genetic background of trees, cultivating drought-resistant varieties through traditional breeding methods is difficult. Therefore, targeted breeding of new drought-resistant tree varieties through molecular breeding has become an important task for researchers.

[0004] To develop molecular breeding techniques for trees with strong adaptability and excellent drought resistance, it is first necessary to understand the morphological changes in plants before and after drought stress, as well as the genes and regulatory mechanisms that regulate the drought response of trees. Based on this understanding, molecular-directed design methods can be used to purposefully cultivate new varieties of trees with strong drought resistance and good adaptability, thereby improving the drought resistance and growth stability of trees and promoting the sustainable development of forestry. When faced with drought stress, plants typically employ various physiological mechanisms to protect themselves, reducing water loss and improving water use efficiency. Stomata, as the main structures in plants for gas exchange and water evaporation, play a crucial role in this process.

[0005] TTG1 encodes the WD40 repeat protein, belonging to the WD40 repeat protein family, one of the largest protein families, widely distributed in all eukaryotic cells. In Arabidopsis, numerous studies have shown that the WD40 repeat protein TTG1 plays a central role in all aspects of epidermal cell fate by forming the MBW complex with MYB and bHLH transcription factors. TTG1 is involved in the regulation of plant trichome initiation, anthocyanin biosynthesis, seed coat mucus production, seed development, and responses to biotic (pathogens, pests, etc.) and abiotic (drought, salinity, etc.) stresses. However, the molecular mechanism by which the TTG1 gene regulates stomatal activity in woody plant drought stress responses remains unclear.

[0006] Poplar, as a woody model plant, has important ecological and economic value. In Arabidopsis, TTG1 has been reported to play an important role in regulating epidermal cell fate and also participate in regulating plant abiotic stress responses. However, how to study the regulatory role of TTG1, a member of the WD40 repeat protein family, in stomatal opening and closing and its molecular mechanism of drought resistance in poplar, a model tree species, to enrich the drought resistance gene network of poplar and provide a theoretical basis and gene resources for high-quality molecular design breeding of drought-resistant forest trees, has become an urgent technical problem to be solved in this field. Summary of the Invention

[0007] The purpose of this invention is to provide a PagTTG1 gene that can regulate the stomatal opening and closing of poplar leaves and drought resistance, and to use its knockout vector to transform poplar trees, in order to promote the development of forest tree stress resistance breeding, provide technical means for the cultivation or screening of superior and highly resistant tree species, and at the same time significantly improve the drought resistance of poplar trees.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: Based on the analysis of interacting proteins of the JAZs family members of the poplar jasmonic acid signaling pathway, this invention identifies a gene PagTTG1 that can regulate stomatal movement in poplar leaves. The nucleotide and amino acid sequences of its coding region are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The full length of PagTTG1 CDS is 1023 bp, encoding 341 amino acids.

[0009] Furthermore, sgRNA primers were designed using conserved sequences to construct a CRISPR / Cas9 vector for the PagTTG1 gene, which was then transformed into poplar trees to obtain PagTTG1 knockout plants.

[0010] Furthermore, using the above vector, silver poplar 84K (Populus alba × P. glandulosa) was transformed by Agrobacterium-mediated transformation to obtain PagTTG1 gene knockout plants TTG1-1 and TTG1-2, respectively.

[0011] Furthermore, observations of drought resistance and stomatal phenotype of the different transgenic lines obtained above revealed that the PagTTG1 knockout lines exhibited significantly enhanced drought resistance and a markedly reduced stomatal aperture.

[0012] The above research results demonstrate that PagTTG1 plays a key regulatory role in stomatal opening and closing and drought resistance in poplar trees, and has important application value in molecular drought resistance breeding of forest trees.

[0013] Compared with the prior art, the main beneficial technical effects of the present invention are as follows: This invention provides the PagTTG1 gene with the sequence shown in SEQ ID NO.3 and its application in increasing the drought resistance of poplar. Using Populus aurea 84K as material, the PagTTG1 gene was screened and identified. Phenotypic identification of its knockout plants showed that it can regulate the opening and closing of poplar stomata, thus affecting its drought resistance. This indicates that the PagTTG1 gene is a key regulatory factor in regulating the opening and closing of poplar stomata and its drought resistance, providing a new option for regulating stomatal opening and closing and drought resistance, and has important application value in the field of forest drought resistance genetic engineering. Attached Figure Description

[0014] Figure 1 This is a diagram showing the tissue expression characteristics of the PagTTG1 gene in poplar in Example 1 of the present invention; Figure 2 This is a diagram showing the expression characteristics of Populus tomentosa PagTTG1 under drought stress in Example 1 of this invention. Figure 3 This is a schematic diagram of the gene knockout location in Embodiment 2 of the present invention; Figure 4 This is a schematic diagram of the gene knockout vector construction in Example 2 of the present invention; Figure 5 This is a schematic diagram illustrating the detection of gene editing in the knockout transgenic plant in Example 2 of the present invention; Figure 6 This is a comparison diagram of drought resistance phenotypes between wild-type 84K poplar and transgenic poplar with gene knockout TTG1-1 and TTG1-2 in Example 5 of the present invention; Figure 7 This is a comparison diagram of stomatal opening and closing phenotypes between wild-type 84K poplar and transgenic poplar with gene knockout TTG1-1 and TTG1-2 in Example 5 of the present invention. Figure 8 This is a statistical analysis of the water loss rate of wild-type 84K poplar and transgenic poplar with gene knockout TTG1-1 and TTG1-2 in Example 5 of the present invention. Detailed Implementation

[0015] The present invention will be further described below with reference to specific embodiments. Operations not described in detail in the following embodiments can be performed by referring to the instructions for use of molecular cloning related reagent kits.

[0016] 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.

[0017] Total RNA was extracted from *P. alba* X *P. glandulosa* using the RNeasy Plant Mini kit and the RNase-free DNase I kit (Qiagen, Hilden, Germany). 1.0 μg of RNA was taken from each sample, and the first strand of cDNA was synthesized using the SuperScript III first-strand synthesis system (Life Technologies, Carlsbad, CA, USA). Primers (containing start and stop codons) were designed using Primer Premier 5 software based on the published *P. alba* genome sequence to amplify the full-length gene (the primers incorporated the PBI121 vector homologous recombination adapter). The forward primer PagTTG1ORF, PagTTG1-CDS-F, is shown in SEQ ID NO.1 of the sequence listing, and the reverse primer PagTTG1-CDS-R is shown in SEQ ID NO.2. The PCR reaction system was as follows: 12.5 μl of TaKaRa high-fidelity amplification enzyme mixture PrimeSTAR, 0.5 μl of forward primer (10 μM), 0.5 μl of reverse primer (10 μM), 1 μl of template (Silver Adenophora 84K cDNA), and sterile ddH2O to a final volume of 25 μl; the reaction program was: pre-denaturation at 95℃ for 5 min; 95℃ for 30 s; 56℃ for 30 s; 72℃ for 1 min, 35 cycles; 72℃ for 5 min; the final full-length cDNA sequence of the gene was 1023 bp, named PagTTG1 gene, as shown in SEQ ID NO.3 of the sequence listing, and the sequence of the protein it expresses is shown in SEQ ID NO.4;

[0018] Tissue expression analysis was performed on various tissues and organs of wild *Populus alba* 84K (P. alba X P. glandulosa) plants grown in a culture room for 2 months, including the following parts: root (R); young leaves (YL); mature leaves (ML); young stem (YS): stem segments from the 3rd to 5th nodes; developing xylem + cambium (DIX+C): stem segments from the 15th internode to the base of the plant, the mixed tissue of the developing xylem region and the cambium region of the bark scraped off after peeling the bark; bark (BARK): bark from the 15th internode to the base of the plant, the remaining parts after scraping off the cambium. The plant materials for drought stress response analysis were wild-type Populus alba X P. glandulosa plants that had been grown in the culture room for one month. After treatment with 30% PEG6000 for 0, 3, 6, 9, 12 and 24 h, the sixth leaf of 4 plants was collected for pooled sampling. Each of the above samples contained three biological replicates. All plant materials were flash-frozen in liquid nitrogen and stored at -80°C for later use. RNA was extracted from the samples and cDNA was synthesized by reverse transcription. Poplar ACTIN was used as an internal reference gene. Quantitative PCR was used to analyze the tissue expression characteristics of the PagTTG1 gene and its response pattern to drought stress. The amplification primers were PagTTG1-RT-F as shown in SEQ ID NO.5; and PagTTG1-RT-R as shown in SEQ ID NO.6. The real-time quantitative PCR reaction system is shown in Table 1 below (10 μl reaction system): Table 1 SYBR® Premix Ex TaqTM (2×) 5.0μl PagTTG1-RT-F (10μM) 0.5μl PagTTG1-RT-R (10μM) 0.5μl cDNA 1.0μl <![CDATA[ddH2O]]> 3.0μl The reaction program was as follows: pre-denaturation at 95℃ for 5 min; followed by 45 cycles of amplification, including: denaturation at 95℃ for 20 sec, annealing at 58℃ for 20 sec, and extension at 72℃ for 20 sec; and a final extension at 72℃ for 5 min. Simultaneously, the following program was used to obtain the melting curve: during the temperature increase from 60℃ to 95℃, the temperature was increased at a rate of 0.06℃ / sec (5 acquisitions per℃) until the heating was completed, thus completing the entire program and ending the run. 2 -ΔΔCt The results of quantitative fluorescence analysis were constructed using computational methods. like Figure 1 The diagram shown illustrates the tissue expression characteristics of the PagTTG1 gene in poplar trees in Example 2 of this invention. Quantitative real-time PCR analysis revealed differences in PagTTG1 gene expression levels across different tissues, with relatively high expression levels observed in mature leaves. Figure 2As shown, this is the response pattern of the poplar PagTTG1 gene to drought stress in Example 2 of the present invention. The quantitative real-time PCR analysis revealed that the PagTTG1 gene was upregulated after PEG6000 treatment. Drought stress can induce the upregulation of the PagTTG1 gene. Therefore, it is speculated that this gene may play an important role in stomatal regulation and drought resistance.

[0019] (1) Primer design: Through phylogenetic analysis of Populus alba X P. glandulosa, a homologous gene of TTG1 was found in Populus alba X P. glandulosa. The TTG1 gene was named TTG1.1 and its homologous gene was named TTG1.2. In order to avoid the redundancy of gene function, a target site was designed to knock out TTG1.1 and TTG1.2 at the same time. Based on the genomic sequences of PagTTG1.1 (SEQ ID NO.3) and PagTTG1.2 (SEQ ID NO.7) of Populus alba X P. glandulosa, primers were designed. Using the genomic DNA of Populus alba X P. glandulosa as a template, the genomic sequences of PagTTG1.1 and PagTTG1.2 were cloned. A 20 bp random nucleotide sequence (PAM) was selected as the target sequence at a position without SNP (Single Nucleotide Polymorphism) site. The target primer sequence AtU3dT1F (SEQ ID NO.7) was synthesized. ID NO.8), AtU3dT1R (SEQ ID NO.9), AtU3bT2F (SEQ ID NO.10), AtU3bT2R (SEQ ID NO.11), AtU6-1T3F (SEQ ID NO.12), AtU6-1T3R (SEQ ID NO.13), AtU6-29T4F (SEQ ID NO.13) NO.14), AtU6-29T4R (SEQ ID NO.15); (2) Preparation of target sequence adapter: Dilute the target sequence primer with TE buffer solution or double-distilled water to prepare a 1 μM stock solution. Then, take 10 μl of each of the upstream and downstream target sequence primers, mix them well, denature at 95 °C for 5 min, and then move to room temperature for gradual cooling and annealing to form double strands. (3) GRNA vector digestion and ligation: Take 0.5-1 μg of each of pYLgRNA-AtU3d / LacZ plasmids and digest them with 5-10 U Bsa I (NEB) in 20 μl of the reaction; Prepare a 10 μl system of 1×BsaⅠ enzyme digestion and ligation reaction solution, as shown in Table 2 below; Table 2 pYLgRNA-AtU3d (AtU3b, AtU6-1, AtU6-29) plasmid (20 ng / μl) 1.0μl Target sequence adapter 0.5 μl (final concentration 0.05-0.1 μM) Ⅰ (5 U / μl) 1.0μl 10×NEB T4 DNA ligase buffer (containing 1.0 mM ATP) 0.5μl T4 DNA ligase (35 U / μl) 1.0 μl (finally contains 35 U) CutSmart Buffer 1.0μl <![CDATA[ddH2O]]> 5.0μl The PCR reaction was performed in 5 cycles: 37℃, 5 min; 20℃, 5 min. (4) Amplify the gRNA expression cassette by two rounds of PCR to obtain a more stable and specific target product and to avoid amplification of empty vector products; 1) In the first round of PCR amplification, two PCR reactions were performed for the forward target sequence and the reverse complementary target sequence, respectively. 1 μl of the enzyme digestion and ligation product obtained in step (3) was used as a template, and the primers in SEQ ID NO.8-SEQ ID NO.15 were used for PCR reaction. The primer concentration was 10 μM for each primer. Table 3 Reaction 1: Reaction 2: Enzyme digestion ligation products 1.0μl Enzyme digestion ligation products 1.0μl UF (SEQ ID NO.16) 0.5μl gR-R (SEQ ID NO.17) 0.5μl sgRNA-R 0.5μl sgRNA-F 0.5μl <![CDATA[2×ExTaq buffer (Mg 2+ plus)]]> 10μl <![CDATA[2×ExTaq buffer (Mg 2+ plus)]]> 10μl <![CDATA[ddH2O]]> 8.0μl <![CDATA[ddH2O]]> 8.0μl The reaction conditions for both reaction 1 and reaction 2 were: 95℃, 15 sec; 60℃, 15 sec; 72℃, 15 sec; the number of cycles was 25-28. 5 μl of PCR product was taken and detected by 2% agarose gel electrophoresis. 1 μl of PCR product from each of reaction 1 and reaction 2 was diluted 10 times with ddH2O, and 2 μl of the diluted product was used as the template for the second round of PCR reaction. 2) Second round PCR reaction (50μl system): Pre-mix position-specific primer pairs into 10x working solution, 1.5μM each: B1'(SEQ ID NO.18) + B2(SEQ ID NO.19) (T1); B2'(SEQ ID NO.20) + B3(SEQ ID NO.21) (T2); B3'(SEQ ID NO.22) + B4(SEQ ID NO.23) (T3); B4'(SEQ ID NO.24) + BL(SEQ ID NO.25) (T4); Take 1μl of the products from the first round PCR reaction 1 and reaction 2, dilute 10 times with H2O, take 1μl of each and mix them as templates, 20-50μl of each expression cassette for PCR (50μl for 1 target site); Add 30 μl of each of 2-3 target sites (or 20 μl of each of 4 or more target sites) to 1 / 10 of the working solution for each primer combination (final concentration 0.15 μM). Use an appropriate amount of KOD-Plus or other high-fidelity PCR enzyme to amplify for 15-20 cycles (adjust according to actual conditions): 95℃, 10s, 58℃, 15s, 68℃, 20s. Take 2-3 μl of the product and check the product length by electrophoresis to estimate the approximate concentration of the sample. The reaction conditions are the same as the first round of PCR reaction. Take 5 μl of the obtained PCR product for electrophoresis detection. The remaining 45 μl is purified and recovered using a Takara purification kit for the next step of enzyme digestion and ligation reaction. (5) pYLCRISPR / Cas9-DH final vector enzyme digestion and ligation reaction (15 μl system), see Table 4 below; Table 4 pYLCRISPR / Cas9-DH plasmid (concentration 70-80 ng / μl) 1.0μl Second round PCR product (20-70 ng) 1.0μl Restriction endonuclease Bsa I (5 U / μl) 1.0μl 10×NEB T4 DNA buffer (containing 1.0 mM ATP) 1.5μl T4 DNA ligase (35 U / μl) 0.1 μl (finally contains 35 U) CutSmart Buffer 1.5μl <![CDATA[ddH2O]]> 7.9μl The reaction conditions were as follows: enzyme digestion at 37℃ for 2 min; variable temperature cycling enzyme digestion and ligation for approximately 10-15 cycles: 10℃ for 3 min; 20℃ for 5 min; and finally, enzyme digestion at 37℃ for 2 min. (6) Take the enzyme digestion and ligation reaction solution from step (5) and transform E. coli competent cells to obtain the pYLCRISPR / Cas9-DH vector plasmid containing the target sequence.

[0020] 1. Genetic transformation of PagTTG1 gene knockout vector The constructed gene knockout vector (containing a pYLCRISPR / Cas9-DH vector linking the target sequence) was transformed into Agrobacterium GV3101 via electroporation. The transformation was then carried out in poplar trees via Agrobacterium-mediated genetic transformation. The transformation steps were as follows: Poplar 84K (P. alba X P. glandulosa) tissue culture seedlings used for genetic transformation were cultured at 23-25℃, with a light intensity of 50 μM m⁻² s⁻¹ (16 / 8h day / night). Agrobacterium containing the target expression vector infected the leaves when OD₆₀ = 0.6–0.8. The infected leaves were then placed on adventitious bud induction medium (SIM, Murashige-Skoog (MS) basal medium supplemented with 0.5 mg / L 6-benzyl aminopurine (6-BA) and 0.05 mg / L naphthaleneacetic acid (NAA). Leaves were co-cultured on NAA (N-acetic acid) at 22±2℃ in the dark for 3 days. After co-culture, the leaves were transferred to SIM containing 3 mg / L hygromycin B and 200 mg / L Timentin. Under the conditions of 23-25℃, 16h / 8h light (day / night), and 50 μM m-2 s-1 light intensity, resistant adventitious shoots were induced and screened. After 30 days of induction culture, the resistant adventitious shoots were transferred to rooting medium (RIM, 1 / 2 MS basal medium supplemented with 0.05 mg / L IBA and 0.02 mg / L NAA) containing 3 mg / L hygromycin B and 200 mg / L Timentin until rooting was induced. DNA was extracted from the leaves of the rooted plants for PCR verification. 2. Identification of CRISPR-Cas9-mediated PagTTG1 gene-editing mutant plants Primers PagTTG1-C9-F (as shown in SEQ ID NO.26) and PagTTG1-C9-R (as shown in SEQ ID NO.27) were designed to detect mutant plants. The target band contained the PAM positions of the PagTTG1.1 and PagTTG1.2 genes. DNA was extracted from transgenic plants and used as a template for PCR amplification with a high-fidelity enzyme (TAKARA, Prime STARMax DNA polymerase). The PCR product was ligated into a T vector (TAKARA, pMD™19-T Vector Cloning Kit) and transformed into competent E. coli cells. Clones identified as positive by bacterial culture PCR were sent to a sequencing company for sequencing. Sequence alignment analysis was performed on at least 30 single clones. The sequencing results are shown below. Figure 5 As shown, the results of the TTG1 knockout transgenic plant detection in Example 4 of this invention are as follows: Sequencing results show that both alleles of the PagTTG1.1 and PagTTG1.2 genes in mutants TTG1-1 and TTG1-2 have been edited; Figure 6 As shown, this invention's Example 4 uses wild-type *Populus alba* 84K (P. alba X P. glandulosa) and gene knockout TTG1-1. 、 Comparative phenotypes of drought resistance in TTG1-2 transgenic poplar trees; as shown in the figure. Figure 7 As shown, this invention's Example 4 uses wild-type *Populus alba* 84K (P. alba X P. glandulosa) and gene knockout TTG1-1. 、 Statistics on stomatal opening and closing in TTG1-2 transgenic poplar; such as Figure 8 As shown, this invention's Example 4 uses wild-type *Populus alba* 84K (P. alba X P. glandulosa) and gene knockout TTG1-1. 、 Statistics on water loss rate of transgenic poplar trees of TTG1-2: mutant TTG1-1 、 Compared with the wild type, TTG1-2 exhibits enhanced drought resistance and a significantly reduced stomatal aperture. Therefore, based on phenotypic and statistical data, it is preliminarily inferred that the TTG1 mutant has the function of enhancing the drought resistance of poplar and promoting stomatal closure.

[0021] 1. Observation of drought resistance in TTG1 mutant plants The TTG1 mutant plants and the wild-type silver poplar 84K (P. alba X P. glandulosa) as a control were planted in a greenhouse in three batches, and the phenotypic characteristics of each batch were measured and photographed. like Figure 6The figure shows a comparison of drought resistance between wild-type *Populus alba* 84K (P. alba X P. glandulosa) and TTG1 mutant transgenic poplar in Example 5 of this invention. Wild-type *Populus alba* 84K (P. alba X P. glandulosa) and TTG1 mutant transgenic poplar were grown in a culture room for one month. All plants underwent natural drought treatment. After 7 days of drought treatment, the leaves of wild-type *Populus alba* 84K (P. alba X P. glandulosa) showed an initial drought-induced water loss phenotype, while the leaves of the TTG1 mutant transgenic poplar showed a normal growth phenotype. After 9 days of drought treatment, the leaves of wild-type *Populus alba* 84K (P. alba X P. glandulosa) showed a wilting phenotype, while the leaves of the TTG1 mutant transgenic poplar showed an initial drought-induced water loss phenotype. After 2 days of rehydration, the wild-type *Populus alba* 84K (P. alba X P. glandulosa)... The leaves of wild-type Populus alba XP. glandulosa withered, while the leaves of the TTG1 mutant transgenic poplar recovered to normal growth. Through phenotypic analysis of natural drought treatment on wild-type Populus alba XP. glandulosa and TTG1 mutant transgenic poplar, it is preliminarily speculated that the TTG1 mutant plant can improve the drought resistance of poplar. 2. Observation of stomatal opening and closing in TTG1 mutant plants (1) Take leaves of wild-type silver gland poplar 84K (P. alba X P. glandulosa) and TTG1 mutant plants with a size of 0.5cm*0.5cm after one month of soil cultivation, place them in 2.5% glutaraldehyde solution, vacuum them and store them in a refrigerator at 4℃ for later use. (2.) Sample pretreatment for scanning electron microscopy: The leaf samples after vacuuming were taken out from the 4℃ refrigerator and washed 5 times with 0.1M PB buffer. The samples were then rotated in the 4℃ refrigerator for 15 min each time. The leaf samples were then treated with gradient alcohols, with 30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol respectively. The samples were then rotated in the 4℃ refrigerator for 15 min each time. After that, the samples were dried using a critical point desiccator (Leica CPD300). The dried samples were then stored in clean centrifuge tubes. (3.) Scanning electron microscopy observation: The dried sample was coated using a coating machine (Leica ACE600), and then the opening and closing of the pores of the sample were observed using a scanning electron microscope (Hitachi Regulus 8230). like Figure 7The image shown is a scanning electron microscope comparison of stomatal opening and closing of wild-type silver glandulosa 84K (P. alba X P. glandulosa) and gene knockout TTG1-1 and TTG1-2 mutant transgenic poplar in Example 5 of this invention. Compared with wild-type silver glandulosa 84K (P. alba X P. glandulosa), the stomatal opening of the TTG1-1 and TTG1-2 knockout mutant transgenic poplar is reduced, indicating that TTG1 knockout plants can promote stomatal closure.

[0022] 3. Statistics on water loss rate of TTG1 mutant plants The sixth leaf of wild-type Populus alba X P. glandulosa and TTG1 knockout plants, which have been grown in the culture room for one month, was removed to calculate the water loss rate. The removed leaves were weighed, and then weighed again at 0.5h, 1h, 1.5h, 2h, 3h, 4h, 5h, and 7h. The leaves were then dried and weighed again to calculate the leaf water loss rate of Populus alba X P. glandulosa and TTG1 knockout plants. Six leaves were used for weighing each line. like Figure 8 The figure shown is a statistical analysis of the water loss rate of wild-type silver glandulosa 84K (P. alba X P. glandulosa) and transgenic poplar trees with TTG1-1 and TTG1-2 gene knockout mutants in Example 5 of the present invention. Compared with wild-type silver glandulosa 84K (P. alba X P. glandulosa), the water loss rate of transgenic poplar trees with TTG1-1 and TTG1-2 gene knockout mutants is slower, which is consistent with the result that TTG1 knockout plants can promote stomatal closure. Phenotypic and stomatal opening and closing morphological observations of the TTG1-1 and TTG1-2 mutant transgenic lines obtained above revealed that the TTG1-1 and TTG1-2 mutant transgenic lines can improve the drought resistance of poplar, promote stomatal closure, and slow down the water loss rate. This indicates that the PagTTG1 gene plays a key regulatory role in regulating stomatal opening and closing and improving the drought resistance of poplar, and has important application value in molecular breeding of forest trees for stress resistance.

[0023] This invention utilizes CRISPR / Cas9 to knock out the PagTTG1 gene. Compared with wild-type plants, TTG1 knockout plants exhibited significantly enhanced drought resistance and a markedly reduced stomatal aperture. This demonstrates that the WD family protein PagTTG1 gene is a key regulator of stomatal opening and closing and drought resistance in poplar trees. This invention provides a new option for regulating stomatal opening and closing and drought resistance, and has significant application value in the field of forest tree stress resistance genetic engineering.

[0024] Although the foregoing has described the inventive concept and embodiments in detail, those skilled in the art will recognize that various improvements and modifications can be made to the invention without departing from the scope of the claims, and such improvements and modifications should still fall within the protection scope of the invention.

Claims

1. A PagTTG1 gene that regulates stomatal opening and closing and drought resistance in poplar trees, the nucleotide and amino acid sequences of its coding region are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively; the full length of PagTTG1 CDS is 1023 bp, encoding 341 amino acids.

2. The application of the PagTTG1 gene, as described in claim 1, which regulates stomatal opening and closing and drought resistance in poplar trees, in the process of poplar trees responding to drought stress.

3. The genetic transformation of the PagTTG1 gene knockout vector that regulates stomatal opening and closing and drought resistance in poplar trees as described in claim 1 is carried out by the following steps: the pYLCRISPR / Cas9-DH vector containing the target sequence is transferred into Agrobacterium GV3101 by electroporation, and then transferred into poplar trees by Agrobacterium-mediated genetic transformation.

4. The genetic transformation of the PagTTG1 gene knockout vector for regulating stomatal opening and closing and drought resistance in poplar trees as described in claim 3, specifically the following transformation steps: Silver poplar 84K tissue culture seedlings used for genetic transformation are cultured at a temperature of 23-25℃, a light intensity of 16 / 8h, and a light intensity of 50μM m⁻² s⁻¹. Agrobacterium containing the target expression vector infects the leaves when OD₆₀ = 0.6-0.

8. The infected leaves are placed on adventitious bud induction medium and co-cultured for 3 days in the dark at a temperature of 22±2℃. The co-cultured leaves are then transferred to SIM containing 3 mg / L hygromycin and 200 mg / L termethin. Resistant adventitious buds are induced and screened under conditions of 23-25℃, a light intensity of 16h / 8h, and a light intensity of 50μM m⁻² s⁻¹. After 30 days of induction culture, the resistant adventitious buds are transferred to a medium containing 3 mg / L hygromycin and 200 mg / L termethin. The plants were incubated in a rooting medium containing hygromycin and 200 mg / L termethin until rooting was induced. DNA was then extracted from the leaves of the rooted plants for PCR verification.

5. The genetic transformation of the PagTTG1 gene knockout vector for regulating stomatal opening and closing and drought resistance in poplar trees as described in claim 4, characterized in that: The adventitious bud induction medium was SIM:Murashige-Skoog basal medium supplemented with 0.5 mg / L 6-benzylaminopurine and 0.05 mg / L naphthaleneacetic acid.

6. The genetic transformation of the PagTTG1 gene knockout vector for regulating stomatal opening and closing and drought resistance in poplar trees as described in claim 4, characterized in that: The rooting medium was RIM, and 1 / 2 MS basal medium supplemented with 0.05 mg / L IBA and 0.02 mg / L NAA.

7. The identification of the PagTTG1 gene editing mutant plants that regulate stomatal opening and closing and drought resistance of poplar as described in claim 1, comprising the following steps: designing detection primers PagTTG1-C9-F and PagTTG1-C9-R for mutant plants, the target band containing the PAM positions of the PagTTG1.1 and PagTTG1.2 genes; extracting DNA from transgenic plants as templates, performing PCR amplification with a high-fidelity enzyme, ligating the obtained PCR product into a T vector and transforming it into competent E. coli cells, and identifying positive clones by bacterial PCR.

8. Construction of the PagTTG1 gene knockout vector for regulating stomatal opening and closing and drought resistance in poplar as described in claim 1: sgRNA primers were designed using conserved sequences to construct a CRISPR / Cas9 vector for the PagTTG1 gene, which was then transformed into poplar trees to obtain TTG1 knockout plants.

9. Obtaining the gene knockout plants TTG1-1 and TTG1-2 of the PagTTG1 gene that regulates stomatal opening and closing and drought resistance of poplar as described in claim 1: Using the TTG1 knockout vector, the silver poplar 84K was transformed by Agrobacterium-mediated transformation to obtain the gene knockout plants TTG1-1 and TTG1-2, respectively.

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