Knockout m 6 A demethylase PagALKBH10A / B gene in improving poplar drought stress tolerance

Abstract

The application discloses knockout m 6 A demethylase PagALKBH10A / B gene in improving drought stress tolerance of poplar. The application knocks out ALKBH10A and ALKBH10B genes in poplar through gene editing, and the obtained mutant AlkB has stronger drought stress tolerance compared with WT, mainly showing that the root system is more developed, the survival rate is higher, the growth speed is faster, the water conducting capacity is stronger, the antioxidant capacity is stronger, the gas exchange and photosynthetic performance are stronger under drought stress; and further shows that the knockout of PagALKBH10A / B gene is to improve the drought resistance of poplar by reducing the m 6 A methylation level of transcription factor WRKY40 in poplar, thereby improving the drought resistance of poplar. The application has great significance for improving the drought resistance of poplar, and has wide application prospect for preparing drought-resistant poplar varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, specifically relating to the knockout of m 6 Application of the PagALKBH10A / B demethylase gene in improving drought stress tolerance in poplar trees. Background Technology

[0002] Plants live in a dynamically changing natural environment, and numerous adverse environmental conditions affect their growth and development. Currently, over 170 forms of RNA chemical modifications have been discovered, with mRNA being the most abundant source of internal modifications. 6 A's research indicates that RNA m 6 A-methyltransferase (writer), demethylase (eraser), and binding protein (reader) work by controlling m 6 The dynamic regulation of A-modification is involved in plant growth, development, and stress response.

[0003] m 6 The discovery of A demethylase indicates that m 6 A is a reversible modification. In mammals, FTO and ALKBH5 belong to the AlkB family of m... 6 AlKBH is a demethylase, and there are 13 AlKBH homologs in Arabidopsis thaliana, five of which (ALKBH9A / 9B / 9C / 10A / 10B) are homologous to ALKBH5 in mammals. The demethylation activities of ALKBH9B and ALKBH10B have been demonstrated in Arabidopsis thaliana. ALKBH9B was the first to be shown to remove m from single-stranded RNA and alfalfa mosaic virus RNA molecules. 6 The protein A may represent a plant regulatory strategy for controlling RNA viruses replicating in the cytoplasm, but the m of the alkbh9b mutant... 6 The A content did not change significantly. ALKBH10B can remove m from single-stranded RNA and RNA extracted from Arabidopsis thaliana. 6 A, and in most tissues of the alkbh10b mutant m 6 An increase in A levels indicates that ALKBH10B is a true m 6 A demethylase. Studies in tomatoes have shown that SlALKBH2 is an RNA m-type localized in the endoplasmic reticulum. 6 Demethylases are essential for the normal ripening of tomato fruits. It is noteworthy that FTO and ALKBH5 are nuclear-localized proteins, ALKBH9B and SlALKBH2 are cytoplasmic proteins, while ALKBH10B is distributed in both the nucleus and cytoplasm. This suggests that different demethylases may have different functions in plants.

[0004] In recent years, climate change has exacerbated drought stress, severely impacting forest biomass and productivity. Exploring the molecular mechanisms of tree responses to drought and enhancing drought resistance through breeding techniques is crucial for promoting forestry production and maintaining ecological stability. 6 The regulatory mechanism of A modification is not yet fully understood, and the reported regulatory mechanisms vary among different species. This necessitates further research into the regulation of A-modification in forest trees. 6 Functional analysis of genes involved in A modification is of great significance. Summary of the Invention

[0005] To overcome the shortcomings and deficiencies of existing technologies, this invention aims to provide a method for knocking out m 6 Application of the PagALKBH10A / B demethylase gene in improving drought stress tolerance in poplar trees.

[0006] The first objective of this invention is to provide the application of knocking out the poplar ALKBH10A and ALKBH10B genes in improving the drought stress tolerance of poplar trees. The ALKBH10A gene is a gene that encodes a protein with the amino acid sequence shown in SEQ ID NO.6, and the ALKBH10B gene is a gene that encodes a protein with the amino acid sequence shown in SEQ ID NO.8.

[0007] Preferably, the nucleotide sequence of the ALKBH10A gene is shown in SEQ ID NO.5, and the nucleotide sequence of the ALKBH10B gene is shown in SEQ ID NO.7.

[0008] Preferably, the application of knocking out the ALKBH10A and ALKBH10B genes in poplar trees to promote growth, enhance antioxidant capacity, improve water conduction capacity, enhance gas exchange capacity, and / or enhance photosynthetic capacity of poplar trees under drought stress.

[0009] Preferably, the knockout of the poplar ALKBH10A and ALKBH10B genes is achieved by knocking out the poplar ALKBH10A and ALKBH10B genes using CRISPR / Cas9 multi-gene editing technology.

[0010] Preferably, the nucleotide sequence of the knockout vector used in the CRISPR / Cas9 multi-gene editing technology that targets the ALKBH10A gene is shown in SEQ ID NO.12 or SEQ ID NO.13; and the nucleotide sequence of the knockout vector used in the CRISPR / Cas9 multi-gene editing technology that targets the ALKBH10B gene is shown in SEQ ID NO.14 or SEQ ID NO.15.

[0011] Preferably, knocking out the ALKBH10A and ALKBH10B genes in poplar improves the drought stress tolerance of poplar by reducing the m of WRKY40 in poplar. 6 Modification A increases the expression level of the poplar WRKY40 gene, thereby improving the poplar's drought stress tolerance. The nucleotide sequence of the WRKY40 gene is shown in SEQ ID NO.11.

[0012] The second objective of this invention is to provide a method for obtaining poplar trees with strong drought stress tolerance, comprising the steps of knocking out the poplar ALKBH10A gene and the ALKBH10B gene; wherein the ALKBH10A gene is a gene encoding a protein with an amino acid sequence as shown in SEQ ID NO.6, and the ALKBH10B gene is a gene encoding a protein with an amino acid sequence as shown in SEQ ID NO.8.

[0013] Preferably, the nucleotide sequence of the ALKBH10A gene is shown in SEQ ID NO.5, and the nucleotide sequence of the ALKBH10B gene is shown in SEQ ID NO.7.

[0014] Preferably, the knockout of the poplar ALKBH10A and ALKBH10B genes is performed using CRISPR / Cas9 multi-gene editing technology; the nucleotide sequence of the specific target site of the ALKBH10A gene on the vector used in the CRISPR / Cas9 multi-gene editing technology is shown in SEQ ID NO.12 or SEQ ID NO.13; the nucleotide sequence of the specific target site of the ALKBH10B gene on the knockout vector used in the CRISPR / Cas9 multi-gene editing technology is shown in SEQ ID NO.14 or SEQ ID NO.15.

[0015] A third objective of the present invention is to provide poplar trees with strong drought stress tolerance prepared using any one of the methods described herein.

[0016] The beneficial effects of this invention are: This invention utilizes gene editing technology to simultaneously knock out the ALKBH10A and ALKBH10B genes to obtain poplar RNA m 6 Editing AlkB plants with demethylase genes revealed that the absence of ALKBH10A and ALKBH10B in poplar trees reduced WRKY40 m under drought stress. 6The A modification enhanced WRKY40 expression, thereby increasing the plant's drought resistance. Overall, gene editing of poplar AlkB under drought stress resulted in immediate transcription factor regulation that improved the plant's drought tolerance, providing an important theoretical basis for the subsequent breeding of drought-resistant forest tree germplasm. Attached Figure Description

[0017] Figure 1 The effect of drought treatment on RNA m in poplar trees 6 The effect of demethylase-encoding gene A; where A represents the phenotype of poplar trees under normal and drought treatments for different durations under sand culture conditions, scale bar 5 cm; B represents the RNA m in poplar trees under normal and drought treatments for different durations under sand culture conditions. 6 A represents the relative expression level of the demethylase-encoding gene; C represents the RNA m in poplar trees treated with normal (15C) and drought (15T) conditions for 15 days under sand culture conditions. 6 The relative expression levels of the gene encoding demethylase A, * and ** indicate significant differences at p<0.05 and p<0.01, respectively.

[0018] Figure 2 The diagram shows the construction of gene editing vectors for poplar ALKBH10A and ALKBH10B (A) and the mutation status of the mutant AlkB (B).

[0019] Figure 3 This section describes the effects of PEG on simulated drought stress and its removal from tissue culture flasks on the mutant AlkB. A shows the poplar root system after two weeks of simulated osmotic stress induced by different concentrations of PEG 6000 in rooting culture (scale bar 5 cm); B shows a scan of the poplar root system after two weeks of simulated osmotic stress induced by different concentrations of PEG 6000 in rooting culture (scale bar 10 cm); and C shows the survival rate of tissue culture seedlings after three weeks of transplanting into soil (scale bar 20 cm).

[0020] Figure 4 This study establishes a soil drought stress system for poplar trees; where A represents the phenotype of WT poplar trees after different days of drought treatment, and B represents the stem water content of WT poplar trees after different days of drought treatment.

[0021] Figure 5This study presents a phenotypic analysis of poplar trees planted in soil for 3 months under drought stress for 12 days. A represents the phenotypic figures of poplar trees under different treatment times (scale bar: 20 cm). B represents the survival rate after 12 days of drought treatment followed by 3 days of rehydration (* indicates significant difference at p < 0.05). C represents the tree height and diameter at ground level of poplar trees under different treatment times. D0 (D-0day) represents before drought treatment, D12 (D-12day) represents after 12 days of drought treatment, R3 (R-3day) represents after 12 days of drought treatment followed by 3 days of rehydration, and R-3week represents after 12 days of drought treatment followed by 3 weeks of rehydration.

[0022] Figure 6 This is a phenotypic analysis of poplar trees planted in soil for 3 months under drought stress for 8 days. Among them, A is a phenotypic diagram of poplar trees planted in soil for 3 months under drought stress for 8 days, with a scale bar of 20 cm; B is the growth height and plant height statistics of poplar trees planted in soil for 3 months under drought stress for 8 days; D represents drought; and N represents normal water supply.

[0023] Figure 7 This represents the water potential of the leaves and stems of poplar trees planted in soil for 3 months after 8 days of drought stress; D indicates drought and N indicates normal water supply.

[0024] Figure 8 These are staining images of poplar trees planted in soil for 3 months and subjected to drought stress for 8 days. Among them, A is the result of DAB staining of the leaves, with a scale bar of 10 mm; B is the result of safranin-fast green staining of the abscission layer cells at the junction of the petiole and stem, with a scale bar of 500 μm; D represents drought, and N represents normal water supply.

[0025] Figure 9 This study measured drought stress-related indicators in poplar trees planted in soil for 3 months under drought stress for 8 days. A, G, and G are the results of hydrogen peroxide, malondialdehyde, abscisic acid, total phenols, CAT, POD, and SOD, respectively. * and ** indicate that the same indicator in the same location has significant differences at p<0.05 and p<0.01, respectively.

[0026] Figure 10 The results were obtained by measuring the photosynthetic and chlorophyll fluorescence indices of poplar trees planted in soil for 3 months under drought stress for 8 days; A, G, and F were obtained in sequence for net photosynthetic rate, transpiration rate, stomatal conductance, water use efficiency, stomatal limitation value, maximum photochemical efficiency, and potential photochemical efficiency; * and ** indicate that the same index in the same part had significant differences at the levels of p<0.05 and p<0.01, respectively.

[0027] Figure 11 This is the functional verification result of Poplar WRKY40; where A is the m of Poplar WRKY40. 6A is a visualization of modified Peak. B is the relative expression levels of ALKB10C, ALKB9A, and ALKB9B in normally growing WT (WT-N), normally growing AlkB (ALK-N), drought-treated WT (WT-D), and drought-treated AlkB (ALK-D). C is the relative expression level of WRKY40 in normally growing WT (WT-N), normally growing AlkB (ALK-N), drought-treated WT (WT-D), and drought-treated AlkB (ALK-D). D is the phenotype of Arabidopsis thaliana overexpressing poplar WRKY40 (OE-PagWRKY40) and wild-type Arabidopsis thaliana (WT) cultured in drought-simulated soil with 20% PEG 6000 solution for 0-5 days. Detailed Implementation

[0028] The technical solution of the present invention will be clearly described below using embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] The wild-type poplar used in the following examples is 84K poplar (Populus alba x Populus glandulosa), which was propagated and preserved in the laboratory using tissue culture. The wild-type Arabidopsis thaliana is Columbia type (Col-0), and the tobacco is Nicotiana benthamiana.

[0030] The rooting medium described in the following examples has the following composition: 2.2 g / L MS519, 0.5 g / L MES, 20 g / L sucrose, 8 g / L agar, 0.05 mg / L NAA, 0.02 mg / L IBA, 200 mg / L termethin, with the balance being water, pH 5.8.

[0031] Example 1: Poplar RNA m under drought stress 6 Expression pattern of A demethylase Based on phylogenetic analysis and analysis of homologous protein domain types and conserved motifs, it is preliminarily inferred that the genes ALKBH9A (nucleotide sequence shown in SEQ ID NO.1, encoding the protein with the amino acid sequence shown in SEQ ID NO.2), ALKBH9B (nucleotide sequence shown in SEQ ID NO.3, encoding the protein with the amino acid sequence shown in SEQ ID NO.4), ALKBH10A (nucleotide sequence shown in SEQ ID NO.5, encoding the protein with the amino acid sequence shown in SEQ ID NO.6), ALKBH10B (nucleotide sequence shown in SEQ ID NO.7, encoding the protein with the amino acid sequence shown in SEQ ID NO.8), and ALKBH10C (nucleotide sequence shown in SEQ ID NO.9, encoding the protein with the amino acid sequence shown in SEQ ID NO.10) in poplar exert RNA m... 6 The function of A demethylase.

[0032] To clarify the RNA m under drought stress in poplar 6 The response of demethylase A was investigated using sand culture and water cut-off to simulate natural drought, exploring the five RNA m-factors in poplar. 6 The expression changes of the demethylase-encoding gene were investigated. Aseptic seedlings of 84K poplar, reaching a height of approximately 8 cm, were selected. After hardening off, they were removed from the culture medium, cleaned, and planted in pure sand substrate thoroughly soaked in nutrient solution. A light-transmitting tray was placed on top to maintain humidity. One week later, the tray was removed. 84K poplars with uniform growth were selected for sand cultivation. After thoroughly soaking the substrate in nutrient solution, watering was stopped, and a drought treatment was initiated. The sand-cultivated poplars were divided into two experimental groups: normal growth and drought treatment. Five sampling time points were established at 0, 3, 6, 13, and 15 days. Under the drought treatment in sand cultivation, the poplars showed obvious leaf dehydration and wrinkling on day 15. Figure 1 (A) Plant tissue samples were collected at various time points to extract RNA, which was then reverse transcribed. Five RNA molecules were detected by quantitative real-time PCR. 6 Changes in the expression levels of demethylases A. Analysis of the overall trend of expression levels of the five demethylases at five time points revealed that the expression levels of ALKBH9A, ALKBH9B, and ALKBH10C gradually increased with increasing drought stress, while ALKBH10A and ALKBH10B showed a decreasing trend. Figure 1 (B in the text). At the 15-day time point when drought stress was clearly observed, compared with normal growth, the expression levels of ALKBH9A, ALKBH9B, and ALKBH10C were significantly increased under drought stress, while the expression levels of ALKBH10A and ALKBH10B were significantly decreased. Figure 1 (C in the text). Under drought stress, poplar RNA m6 Expression pattern analysis of A demethylases showed that the expression patterns of ALKBH10A and ALKBH10B were opposite to those of ALKBH9A, ALKBH9B and ALKBH10C, decreasing with increasing drought stress. It is speculated that the absence of ALKBH10A and ALKBH10B may be beneficial to improving the drought resistance of poplar.

[0033] Example 2: Obtaining gene-edited plants Based on the above results, the simultaneous knockout of poplar ALKBH10A and ALKBH10B using a CRISPR / Cas9 multi-gene editing vector was employed to investigate the effects of related proteins on poplar transcriptome m. 6 Regulation of A-modification and its effects on plant growth and drought resistance. To improve editing efficiency, two target sites were designed for the CDS front ends of ALKBH10A and ALKBH10B, respectively. Different promoters, target sites (target 1 and target 2 are for ALKBH10A, nucleotide sequences as shown in SEQ ID NO. 12 and SEQ ID NO. 13, respectively; target 3 and target 4 are for ALKBH10B, nucleotide sequences as shown in SEQ ID NO. 14 and SEQ ID NO. 15, respectively) and gRNA were linked together to form expression cassettes. The four expression cassettes were then assembled into a binary vector using a golden gate ligation method to complete the construction of a multi-gene editing vector. Figure 2 (A in the middle).

[0034] The constructed gene-editing vector was transformed into Agrobacterium GV3101. Using Agrobacterium-mediated transformation of poplar, a homozygous double-mutant gene-edited poplar plant with both ALKBH10A and ALKBH10B knocked out was obtained and named AlkB. After comparison with the WT (84K) genome, the mutation status of the AlkB gene-edited plant is as follows: Figure 2 As shown in B, the first target (target 1) designed for ALKBH10A did not work. ALKBH10A underwent insertions of A and T bases on the two DNA strands of the second target (target 2), respectively. Both targets of ALKBH10B worked. On one DNA strand of ALKBH10B, A base insertion (first target) and GG base deletion (second target) occurred at the two target sites, respectively. On the other DNA strand, a 406 bp fragment was cleaved between the two target sites.

[0035] Example 3: PEG-simulated drought stress experiment and tissue culture seedling transplantation experiment To verify the drought resistance of AlkB gene-edited plants, 2-3 cm long terminal buds of tissue culture seedlings were inoculated onto rooting media with different concentrations (0%, 1%, 2%, and 3% by mass) of PEG 6000. The differences in drought resistance between AlkB and wild-type plants were observed by simulating osmotic stress. After two weeks of cultivation on rooting media with different concentrations of PEG 6000, it was observed that the roots of the AlkB line showed better growth than those of the wild type. On rooting media with PEG 6000 concentrations of 2% and 3% by mass, the growth of wild-type roots was severely inhibited, with only a few short roots remaining, while the growth of AlkB roots was less inhibited. Furthermore, in rooting media without PEG 6000, the growth capacity of AlkB roots was also stronger than that of the wild type. Figure 3 (A in the middle).

[0036] To more directly compare the root growth performance of gene-edited plants with wild-type plants, tissue culture seedlings were cleaned of the culture medium and then placed in water for root scanning. Root scanning results showed that AlkB roots exhibited superior growth compared to wild-type roots at all concentration gradients. Besides having more and longer taproots, AlkB roots also had more lateral roots than wild-type roots. On rooting media with PEG 6000 concentrations of 2% and 3% (w / w), root scanning results showed that wild-type plants had no roots. This was because their roots were shorter and more brittle, and all roots detached during removal from the culture medium. The comparison with AlkB plants better reflects the superior osmotic stress resistance of gene-edited plants. Figure 3 (B in the middle).

[0037] Poplar strains were further propagated through plant tissue culture. Wild-type poplar plants with intact root systems, uniform growth, and a height of approximately 8 cm were selected. After 5 days of hardening off, they were transplanted into soil for physiological phenotype analysis. During the soil planting process, it was found that large-scale water loss and death occurred in the poplars three weeks after transplanting from tissue culture bottles to soil. It is speculated that the drastic change in environmental humidity from tissue culture to soil planting led to the gradual water loss and death of the poplars, and the microorganisms in the environment may also have had an adverse effect. However, a comparison of the survival rate of AlkB and WT showed that AlkB was more adaptable to this environmental change. Of the 24 wild-type poplars planted, only 1 survived, while of the 24 AlkB plants planted, 7 survived, demonstrating a significant difference in adaptability to adversity. Figure 3 (C in the text). Based on the treatment results of PEG 6000, it is speculated that the excellent water conductivity produced by the well-developed root system of AlkB provides it with more water, thereby improving its survival rate.

[0038] Example 4: Soil drought stress experiment To further investigate the drought resistance of poplar trees at a more mature stage of growth, based on literature reports, soil-planted poplar trees for three months were selected for drought resistance analysis. First, wild-type poplar trees were planted in the soil until they were three months old, and watering was stopped. The experimental results showed that on the second day after watering was stopped, there were no obvious physiological changes in the poplar trees. From the fourth day, the leaves began to droop; on the sixth day, the leaves showed slight curling; and on the eighth day, significant drought stress phenotypes appeared. The leaves in the middle of the poplar stems severely lost water, wrinkled, and fell off. By the tenth day, the stem tips began to droop and lose water, wrinkling. Finally, on the twelfth day, the entire poplar tree experienced severe water loss (…). Figure 4 (A) The changes in the water content of poplar stems show that the water content of the plant stems decreased rapidly from the fourth to the eighth day after the water was cut off. After twelve days of water cut off, the water content in the stems tended to level off, indicating that the poplars had reached a state of severe drought. Figure 4 (B in the middle).

[0039] Based on the above results, the phenotype of severe water loss in the entire poplar tree on the twelfth day of drought, combined with the flattening of the poplar stem water content curve, indicates that the poplar has reached a state of severe drought. The twelfth day of water cessation was selected as the time point for drought survival rate statistics in subsequent experiments. Wild-type and AlkB poplar plants, propagated from tissue culture to have intact root systems, uniform growth, and a height of approximately 8 cm, were hardened off for 5 days and then transplanted into soil. After 3 months of planting, plants with no significant differences in growth status were selected for treatment. The soil was thoroughly watered the day before treatment, and watering was stopped for 12 days. Normal watering was then resumed, and the number of surviving plants was counted and the drought stress survival rate was calculated three days after re-watering. Experimental results ( Figure 5 As shown in A), after 12 days of drought stress (D12), most of the wild-type plants treated showed severe drought, with wrinkled and dehydrated leaves, while most AlkB plants only exhibited milder drought stress phenotypes with drooping and wrinkled leaves. After 3 days of rehydration (R3), about half of the wild-type plants withered and died, while most AlkB plants survived, with only the lower and middle leaves of the stems drying and falling off. Survival rate statistics showed that the survival rate of wild-type plants was 58% under 12 days of drought stress, while the survival rate of the AlkB gene-edited lines was 92%, significantly higher than that of wild-type plants. Figure 5 (B in the middle).

[0040] The experiment also statistically analyzed the changes in plant height and diameter at ground level of poplar trees throughout the entire drought stress treatment process. Figure 5In section C), due to the control of the growth status of WT and AlkB plants before treatment, there was no significant difference in plant height and diameter at ground level between the two lines before treatment (D-0 day). After 12 days of drought and 3 days of rehydration, there was still no significant difference in plant height and diameter at ground level between WT and AlkB plants. To compare the growth recovery ability of the two lines after drought stress, they were cultured with normal water supply for three weeks after drought. It was found that after 3 days of rehydration, AlkB plant height and diameter at ground level were not significantly different from or slightly lower than the wild type. However, after three weeks of normal planting, both plant height and diameter at ground level were higher than the wild type. This indicates that AlkB has a better growth recovery ability after drought stress than the wild type. Overall, AlkB not only shows improved drought resistance compared to the wild type, but its growth ability under normal water supply may also be superior to that of wild poplar.

[0041] Example 5: Physiological differences between AlkB and WT under drought stress To further elucidate the underlying mechanism of AlkB's enhanced drought resistance, three-month-old WT and AlkB plants, which showed no significant differences in growth status from soil planting to maturity, were selected for drought stress experiments. Based on the drought stress system established earlier, samples were taken on the eighth day of drought when significant drought stress phenotypes began to appear in the poplars, and the following physiological analyses were performed.

[0042] (1) Phenotypic analysis The experimental results showed that on the eighth day of drought stress, the leaves on the middle and lower parts of the stems of wild-type poplar began to wrinkle and fall off, while the leaves of AlkB plants only showed drooping and curling, indicating that the drought resistance of the AlkB line was significantly better than that of the wild type. Figure 6 (A in the text). Statistical results of plant height before and after drought for the two lines showed that, under normal water supply and drought stress, AlkB's growth ability was significantly better than that of the wild type (A in the text). Figure 6 (B) in the text. Further verification. (2) Water potential analysis Plant water potential is an important indicator of a plant's drought resistance; higher water potential is beneficial for maintaining normal water conduction within the plant. Low water potential in the stem can easily lead to the interruption of the siphon effect of water transport, resulting in air pockets and blockages in the xylem vessels, increasing the damage to the plant caused by drought stress. Therefore, stem segments and leaves 20 cm below the terminal bud were collected for water potential measurement. The results showed that under normal water supply, there was no significant difference in water potential in the leaves and stems of the AlkB and WT lines; under drought stress, the water potential in the leaves and stems of both lines decreased significantly compared to normal water supply, but the water potential in the stems and leaves of the AlkB line was still significantly higher than that of the wild type. Figure 7 This indicates that AlkB has a stronger water absorption or conduction capacity than the wild type, maintaining the normal water conduction within the plant under drought stress.

[0043] (3) Oxidative stress analysis Wild-type plants exhibited leaf drop under drought stress, and drought stress leads to the accumulation of reactive oxygen species within the plants. Therefore, DAB staining was performed on the leaves of AlkB and WT plants to compare the hydrogen peroxide content in the leaves. The DAB staining results showed that under normal water supply, there was no significant difference in leaf color development between WT and AlkB plants. However, under drought stress, the wild-type plants showed more dark brown patches on their leaves compared to AlkB plants. Figure 8 The result (A) indicates that more hydrogen peroxide accumulated in the leaves of the wild-type strain under drought stress. Furthermore, safranin-fast green was used to stain the petiole-stem junction to observe the difference in abscission layers at the leaf drop sites between the two strains. The results clearly show that necrotic abscission cells appeared at the petiole-stem junction of the wild-type strain under drought stress, while no necrotic abscission cells were observed in the AlkB strain. Figure 8 (B in the text). The staining observation results showed that AlkB was less affected by drought stress, with less hydrogen peroxide accumulation in its leaves and no leaf drop, indicating stronger drought tolerance.

[0044] To further investigate the effects of drought stress on AlkB and WT, samples were taken from leaves and stems of plants under drought stress, and drought stress-related indicators were measured. Hydrogen peroxide and malondialdehyde (MDA) are effective indicators of cell membrane oxidative damage. The results showed that the contents of hydrogen peroxide and MDA in the leaves and stems of AlkB under drought stress were significantly lower than those in the wild type. Figure 9 (A / B in the text). Peroxidase activity assays showed that in leaves, there were no significant differences in CAT and POD activities between the two lines, except that SOD activity in AlKB leaves was significantly higher than that in WT; while in AlkB stems, CAT, POD, and SOD activities were all significantly lower than those in WT. Figure 9 (EG in the sample). The content of hydrogen peroxide and malondialdehyde in the leaves of the plant was significantly higher than that in the stem, suggesting that the higher peroxide content in the leaves increased the activity of peroxidase in WT and AlkB. However, due to the excellent water conduction properties of the roots and stems, AlkB has a more sufficient water supply in the stem, resulting in a lower peroxide content in the AlkB stem and a significantly lower enzyme activity than the wild type.

[0045] The abscisic acid (ABA) content in the leaves, petioles, and stems of the two strains was determined, and the results were as follows: Figure 9C) shows that ABA content increases with the distance between each tissue and the root system, with higher ABA content in leaves than in petioles and higher in stems. This may be related to the direction of water transport; tissues closer to the roots have a more abundant water supply and a weaker ABA response. The ABA content in leaves and petioles of AlkB plants is significantly lower than that in WT, while the ABA content in stems is not significantly different from that in WT. It is speculated that AlkB has a stronger water transport capacity, making the plant less affected by drought stress overall.

[0046] Plant phenolic compounds have free radical scavenging and antioxidant effects. Therefore, the total phenolic content of WT and AlkB under normal planting (N) and drought stress (D) was further determined. The results ( Figure 9 D) shows that AlkB plants have a higher total phenol content than WT plants, suggesting that AlkB plants may have more secondary metabolites involved in improving their drought resistance.

[0047] (4) Analysis of gas exchange and chlorophyll fluorescence parameters Gas exchange parameters of poplar trees were measured. Under normal water supply (N), there were no significant differences in parameters between WT and AlkB plants. Compared with normal water supply, under drought stress (D), the net photosynthetic rate, transpiration rate, stomatal conductance, water use efficiency, and stomatal limitation value of both AlkB and WT plants decreased significantly, indicating that gas exchange in both lines was significantly affected by drought stress. However, under drought stress, the net photosynthetic rate, transpiration rate, stomatal conductance, water use efficiency, and stomatal limitation value of AlkB were significantly higher than those of WT. Figure 10 The chlorophyll fluorescence parameters (AE) indicate that AlkB plants are less affected by drought stress and have stronger drought resistance than WT plants. Chlorophyll fluorescence parameter analysis showed that under normal water supply, there was no significant difference in maximum and potential photochemical efficiency between WT and AlkB plants. Under drought stress, the chlorophyll fluorescence parameters of both AlkB and WT lines decreased compared to normal water supply, but AlkB's maximum and potential photochemical efficiency were still significantly higher than WT under drought stress. Figure 10 (FG in the text). Overall, AlkB plants exhibit stronger drought resistance, enabling them to better maintain gas exchange and photochemical reactions under drought stress.

[0048] Example 6: Validation of Regulated Transcription Factors To further understand the differences in response genes between the AlkB and WT lines under drought stress, m-type analysis was performed on drought stress samples from AlkB and WT. 6A-seq was performed, and a series of analyses were conducted, revealing that the transcription factor WRKY40 in poplar (the nucleotide sequence of its encoding gene is shown in SEQ ID NO.11) may play a key regulatory role in response to drought stress.

[0049] To verify the above hypothesis, the IGV software was used to analyze the m on poplar WRKY40. 6 Peak visualization was created based on the methylation modification status. The results show that WRKY40 in AlkB is present near the terminator and 3′UTR. 6 Peak A is lower than WT ( Figure 11 (A) It is speculated that the loss of ALKBH10A and ALKBH10B in AlkB leads to the loss of WRKY40 and NAC4 m. 6 The reduction of A modification led to an increase in its expression level and improved the drought resistance of the plant. ALKBH10A and ALKBH10B were previously predicted as RNA m 6 A demethylase, the absence of which generally leads to overall m in the plant 6 The increased level of modification A was inconsistent with the results of omics assays. Therefore, the three additional RNA m cells screened by bioinformatics analysis were also analyzed. 6 Quantitative qPCR analysis of the expression levels of demethylases ALKBH10C, ALKBH9A, and ALKBH9B showed that drought induced an increase in the expression levels of ALKBH10C, ALKBH9A, and ALKBH9B. Furthermore, the expression levels of ALKBH10C, ALKBH9A, and ALKBH9B in AlkB were higher under both drought and normal water supply conditions than under WT conditions. Figure 11 (B in the text) It is speculated that the high expression of ALKBH10C, ALKBH9A, and ALKBH9B reduces the overall m of AlkB plants. 6 A modifies the level.

[0050] Quantitative results showed that WRKY40 expression increased under drought stress, and the response of WRKY40 to drought in AlkB plants was significantly higher than that in WT plants. Figure 11 (C in the text), suggesting that the deletion of ALKBH10A and ALKBH10B affects other RNA m 6 The expression of A demethylase is affected, thereby reducing the m of WRKY40. 6 A modifier enhances the expression level of the plant, thereby increasing its drought resistance.

[0051] Furthermore, a poplar WRKY40 overexpression vector was constructed and genetically transformed in Arabidopsis thaliana. Arabidopsis plants overexpressing poplar WRKY40 and wild-type (WT) Arabidopsis thaliana were planted in soil for 4 weeks. Then, a 20% (w / w) PEG 6000 solution was applied to the soil until thoroughly watered. Leaf changes were recorded daily to analyze the stress levels of the plants. Results showed that the Arabidopsis plants overexpressing poplar WRKY40 had a larger overall plant size. Leaf color changes also indicated that the stress levels of the overexpressing plants were lower than those of the wild type at different treatment times. Overall, overexpression of poplar WRKY40 in Arabidopsis thaliana improved the plant's growth performance and drought resistance. Figure 11 (D in the middle).

[0052] In conclusion, the deletion of ALKBH10A / B in poplar trees leads to an overall loss of m... 6 The decrease in A modification levels, under drought stress, reduces WRKY40 m 6 Modification A increased the expression level of WRKY40, thereby enhancing the tolerance of poplar to drought stress.

Claims

1. The application of knocking out the ALKBH10A and ALKBH10B genes in poplar trees to improve their drought stress tolerance, characterized in that... The ALKBH10A gene is a gene that encodes a protein with the amino acid sequence shown in SEQ ID NO.6, and the ALKBH10B gene is a gene that encodes a protein with the amino acid sequence shown in SEQ ID NO.

8.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the ALKBH10A gene is shown in SEQ ID NO.5, and the nucleotide sequence of the ALKBH10B gene is shown in SEQ ID NO.

7.

3. The application according to claim 1, characterized in that, To investigate the application of knocking out the ALKBH10A and ALKBH10B genes in poplar trees to promote growth, enhance antioxidant capacity, improve water conduction, increase gas exchange capacity, and / or enhance photosynthetic capacity of poplar trees under drought stress.

4. The application according to claim 1, characterized in that, The aforementioned knockout of the poplar ALKBH10A and ALKBH10B genes was achieved by knocking out the poplar ALKBH10A and ALKBH10B genes using CRISPR / Cas9 multi-gene editing technology.

5. The application according to claim 4, characterized in that, The nucleotide sequence of the knockout vector targeting the ALKBH10A gene in the CRISPR / Cas9 multi-gene editing technology is shown in SEQ ID NO.12 or SEQ ID NO.13; the nucleotide sequence of the knockout vector targeting the ALKBH10B gene in the CRISPR / Cas9 multi-gene editing technology is shown in SEQ ID NO.14 or SEQ ID NO.

15.

6. The application according to claim 1, characterized in that, Knocking out the ALKBH10A and ALKBH10B genes in poplar trees improves their drought stress tolerance by reducing the m of WRKY40 in poplar trees. 6 Modification A increases the expression level of the poplar WRKY40 gene, thereby improving the poplar's drought stress tolerance. The nucleotide sequence of the WRKY40 gene is shown in SEQ ID NO.

11.

7. A method for obtaining poplar trees with strong drought stress tolerance, characterized in that, The method includes the steps of knocking out the poplar ALKBH10A and ALKBH10B genes; the ALKBH10A gene is a gene that encodes a protein with an amino acid sequence as shown in SEQ ID NO. 6, and the ALKBH10B gene is a gene that encodes a protein with an amino acid sequence as shown in SEQ ID NO.

8.

8. The method according to claim 7, characterized in that, The nucleotide sequence of the ALKBH10A gene is shown in SEQ ID NO.5, and the nucleotide sequence of the ALKBH10B gene is shown in SEQ ID NO.

7.

9. The method according to claim 7, characterized in that, The knockout of the poplar ALKBH10A and ALKBH10B genes is performed using CRISPR / Cas9 multi-gene editing technology. The nucleotide sequence of the vector used in the CRISPR / Cas9 multi-gene editing technology that specifically targets the ALKBH10A gene is shown in SEQ ID NO.12 or SEQ ID NO.

13. The nucleotide sequence of the knockout vector used in the CRISPR / Cas9 multi-gene editing technology that specifically targets the ALKBH10B gene is shown in SEQ ID NO.14 or SEQ ID NO.

15.

10. Poplar trees with strong drought stress tolerance prepared by the method according to any one of claims 7-9.

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