Poplar root growth related gene PcaHLH151 and application thereof

Abstract

The invention relates to a poplar root growth related gene PcaHLH151 and application thereof, and belongs to the technical field of molecular biology. On one hand, the invention provides the populus amurensis root growth related gene PcaabHLH151 and the encoding protein thereof, and on the other hand, the invention provides the application of the populus amurensis root growth related gene PcaabHLH151. The invention provides an important gene resource capable of regulating and controlling root growth and possibly having universality, and an excellent candidate gene is provided for cultivating plant varieties with more root biomass accumulation.

Description

Technical Field

[0001] This invention relates to a gene related to the root growth of Populus tomentosa. PcabHLH151 Its applications belong to the field of molecular biotechnology. Background Technology

[0002] Poplar ( Populus cathayana Populus is a native tree species unique to my country, belonging to the genus Populus. Populus Poplar (Tacamahaca), also known as the Chinese poplar, possesses excellent characteristics such as rapid growth, wide adaptability, strong sprouting ability, and a well-developed root system. Due to its light, dense, and decay-resistant wood, it is currently widely used in industrial construction, plywood manufacturing, pulp production, and ecological afforestation. In production practice, poplars are often propagated asexually through cuttings to maintain their superior characteristics. The ability of adventitious roots to form roots often determines the efficiency of forest propagation and the survival rate of afforestation. Studies have shown that the rooting ability of poplar germplasm varies significantly across different regions, thus affecting the later growth and development of the plant.

[0003] Currently, there are few reports on genes related to poplar root growth, and the regulatory mechanisms of root growth and development remain unclear. Therefore, elucidating the genetic basis of root-related traits is crucial for clarifying the regulatory pathways of poplar root growth and development, and will lay a foundation for breeding individuals with high biomass and strong regeneration capacity, as well as for subsequent genetic improvement of forest trees.

[0004] Based on genome-wide association analysis of natural populations of Populus tomentosa, the inventors found that Populus tomentosa is significantly associated with root-related traits. PcabHLH151 Gene, PcabHLH151 It belongs to the basic helix-loop-basic helix transcription factor (bHLH), and is similar to that in Arabidopsis thaliana. AtbHLH112 The gene shows high homology, suggesting that it may be involved in the growth and development of poplar roots.

[0005] To the poplar PcabHLH151 Genetic research helps to elucidate the growth and development mechanism of poplar roots, laying a good foundation for subsequent genetic improvement and cultivation of poplars with well-developed root systems and strong rooting ability, thereby increasing timber production and promoting the economic prosperity of industries such as poplar timber and ecological protection. Summary of the Invention

[0006] To address the problems existing in the prior art, the purpose of this invention is to provide a gene related to the root growth of Populus tomentosa. PcabHLH151 Its application helps to elucidate the growth and development mechanism of poplar roots, laying a good foundation for subsequent genetic improvement and cultivation of poplars with well-developed root systems and strong rooting ability, thereby increasing timber production and promoting the economic prosperity of industries such as poplar timber and ecological protection.

[0007] This invention is achieved through the following technical solution: A gene related to the root growth of Populus tomentosa PcabHLH151 Its nucleotide sequence is shown in SEQ ID NO.1.

[0008] Preferably, the amino acid sequence of the Populus alba root growth-related gene PcabHLH151 is shown in SEQ ID NO.2.

[0009] The above-mentioned genes related to the root growth of Populus tomentosa PcabHLH151 Application in regulating plant root biomass.

[0010] Preferably, the application includes: incorporating genes related to poplar root growth into the plant. PcabHLH151 Or cause plants to overexpress genes related to poplar root growth PcabHLH151 Or make the plant to bHLH151 Gene editing is performed.

[0011] Preferably, the application involves constructing a system containing genes related to the root growth of Populus tomentosa. PcabHLH151 The plant expression vector was genetically transformed into *Populus simonii* '84K' (… P. alba × P. glandulosa In '84K', positive plants were obtained through screening. Phenotypic analysis of positive plants and wild-type plants was used to identify plants with increased root biomass.

[0012] Preferably, the application specifically includes the following steps: 1) Roots from superior Populus euphratica germplasm lines 1-15 were collected, RNA was extracted, reverse transcribed into cDNA, and cloned. PcabHLH151 The CDS sequence was obtained, then ligated into a sequencing vector and sequenced. After confirmation that it was correct, an overexpression vector was constructed and genetically transformed into *Populus silverae* '84K' using Agrobacterium-mediated transformation. 2) Targeting Populus euphratica and Populus spp. '84K' bHLH151 Genomic sequence characteristics were used to design a gene sequence that could simultaneously and specifically identify Populus tomentosa and Populus '84K' simultaneously and specifically. bHLH151 The sgRNA sequence of the gene was obtained, and then the bHLH151-Cas9 gene editing vector was constructed based on the CRISPR / Cas9 system. After the sequence was confirmed to be correct, it was genetically transformed into *Populus alba* '84K'. 3) Positive plant screening was performed on overexpressing plants and gene-edited plants using hygromycin resistance and PCR methods to obtain overexpressing plants and gene-edited plants. Phenotypic statistics and nitrogen treatment at different concentrations were then performed to obtain gene-edited plants with reduced root biomass and nitrogen use efficiency.

[0013] The present invention has the following beneficial effects: 1. The Populus cathayana is obtained for the first time in the application PcabHLH151 gene and the encoded protein, and the biological function of regulating root biomass of the plant is verified; 2. The Populus cathayana is obtained for the first time in the application PcabHLH151 gene is overexpressed in the Populus alba '84K', and it is proved by PCR method that the gene has been successfully integrated into the genome of the Populus alba '84K', that is, the positive plant; 3. The Populus cathayana is obtained for the first time in the application bHLH151 gene is knocked out in the Populus alba '84K', and it is proved by high-throughput sequencing method that the gene has been successfully mutated in the genome of the Populus alba '84K', that is, the gene edited plant; 4. The overexpression plant, the gene edited plant and the wild type plant are subjected to phenotype observation and nitrogen treatment of different concentrations in the application, and the results show that the root biomass of the gene edited plant is lower than that of the wild type, and the difference in the biomass of the cutting seedlings of the gene edited plant and the wild type plant under the low nitrogen level is extremely significant, which indicates that PcabHLH151 knocking out of the gene reduces the accumulation of root biomass and the nitrogen utilization efficiency of the transgenic plant. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 For the detection of the overexpression Populus alba '84K' strain positive plant in Example 2 of the application PcabHLH151 Figure 2 For the gene expression level of the positive overexpression Populus alba '84K' strain in Example 2 of the application PcabHLH151 Figure 3 For the editing identification of the gene edited Populus alba '84K' strain in Example 2 of the application bHLH151 Figure 4 For the phenotype of the overexpression and gene edited Populus alba '84K' strain on the 30th day after transplanting in Example 2 of the application PcabHLH151 Figure 5 For the root dry weight statistics of the overexpression and gene edited Populus alba '84K' strain in Example 2 of the application PcabHLH151 Figure 6 For the nitrogen treatment phenotype of the overexpression and gene edited Populus alba '84K' strain cutting seedlings in Example 2 of the application PcabHLH151 Figure 7 For the root dry weight statistics of the overexpression and gene edited Populus alba '84K' strain cutting seedlings in Example 2 of the application PcabHLH151 DETAILED DESCRIPTION

[0015] ​​​​​​​The present invention will be further described below with reference to specific embodiments. It should be understood that the following text is only used to describe one or more specific embodiments of the present application and does not strictly limit the scope of protection specifically claimed in the present application. In the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other.

[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] Example 1: Genes related to the root growth of Populus tomentosa PcabHLH151 Cloning 1. Material preparation Populus tomentosa root samples were collected from superior Populus tomentosa lineages 1-15 ( Populus cathayana Collected from Jiangou, Beijing (116.05°N, 40.07°E, altitude 922m), stored at -80℃ for future use; 2. RNA extraction Referring to the Takara RNAiso plus reagent instructions, the specific steps are as follows: (1) After sterilizing the mortar, spoon and tweezers used in the experiment at high temperature, pre-cool them with liquid nitrogen. Take the previously preserved poplar root system into the mortar, add liquid nitrogen and grind it into powder quickly. (2) Take 200 mg of the ground powder into a 2 ml RNA-free centrifuge tube, add 1 ml RNAiso plus reagent, shake well to mix, and let stand for 5 min; then centrifuge at 12000 g for 5 min at 4 °C. (3) Take 600 μl of the supernatant from step 2 into a new 1.5 ml RNA-free centrifuge tube, add 200 μl of chloroform, shake vigorously to mix for 15 s, let stand for 5 min; then, centrifuge at 12000 g for 15 min at 4 °C. (4) Take 400 μl of the supernatant from step (3) into a new 1.5 ml RNA-free centrifuge tube, add 400 μl of isopropanol pre-cooled at -20℃, invert the tube, mix well, and let stand for 10 min; then, centrifuge at 12000g for 10 min at 4℃. (5) Discard the supernatant, add 1 ml of 70% ethanol, wash the precipitate, centrifuge at 7500g for 5 min at 4℃, and repeat twice; (6) Discard the supernatant, centrifuge again at 7500g for 5 min at 4℃, remove the remaining alcohol from the centrifuge tube, air dry for 20 min, add 50 μl of RNA-free water to dissolve the precipitate; 3. RNA reverse transcription Following the instructions of the PrimerScript RT reagent Kit with gDNA Eraser (Takara), all experimental consumables were RNA-free, and all reactions were performed on ice. (1) Genomic DNA removal Prepare the reaction mixture shown in Table 1 on ice, centrifuge and mix well, and react at 42°C for 2 min; Table 1 Reagent System (10 μl) 5×gDNA Eraser Buffer 2 μl gDNA Erase 1 μl Total RNA 500 ng RNase Free ddH2O up to 10 μl (2) cDNA synthesis In step (1), add the reaction mixture from Table 2 below to the PCR tube, mix thoroughly, centrifuge, and then react again at 37°C for 15 min, 85°C for 5 s, and then immediately cool on ice. Table 2 Reagent System (20 μl) Step (1) reaction solution 10 μl PimerScript RT Enzyme Mix I 1 μl RT Enzyme Mix 1 μl 5×PimerScrip Buffer 4 μl RNase Free ddH2O 4 μl Total 20 μl (3) The cDNA obtained after reverse transcription is diluted 10 times with RNA-free water and then used for subsequent gene cloning and quantitative PCR and other related experiments; 4. Gene cloning (1) Design corresponding CDS primers Extracted from the Populus genotype database PcabHLH151 The cDNA sequence of the gene was used to design primers: PcabHLH151-cds-F as shown in SEQ ID NO.3; and PcabHLH151-cds-R as shown in SEQ ID NO.4. (2) PCR amplification Using cDNA from the roots of Populus tomentosa as a template, PCR amplification was performed using the corresponding primers. The reaction program was as follows: 95℃, 5 min; 95℃, 5 s, 60℃, 30 s, 72℃, 60 s, 30 cycles; 72℃, 10 min. The amplification instrument was an Applied Biosystems™ Veriti™ 96-well rapid thermal cycler. The amplification system is shown in Table 3 below. Table 3 Component System (20 μl) Template (Populus cathayana root cDNA) 1 μl Primer PcabHLH151-cds-F 1 μl Primer PcabHLH151-cds-R 1 μl ddH2O 7 μl 2×Green Taq Mix 10 μl (3) Gel recovery and purification 1.5% agarose gel electrophoresis was used to check whether the band size met expectations, and the next step of gel extraction and recovery was performed for verification. The gel recovery and purification were carried out using the SanPrep column DNA gel recovery kit from Shanghai Sangon Biotech Co., Ltd. (4) Connection reaction Referring to the Takara pMDTM19-T Vector Cloning Kit manual, the connection system is shown in Table 4 below; Table 4 Component System (10 μl) Gel recovery PCR product 4 μl pMD19-T 1 μl DNA Ligation Kit 5 μl (5) The ligation product was transformed into competent E. coli DH5α cells. Referring to the E. coli DH5α transformation instructions from Shanghai Weidi Biotechnology Co., Ltd., the specific steps are as follows: ① Place the purchased E. coli competent cells DH5α on ice for freeze-thaw, add 10 μl of the ligation product from step (4), gently tap the bottom of the tube, mix well, and let stand on ice for 30 min; ②42℃, 90s, then let stand on ice for 2min; ③ Add 400 μl of antibiotic-free LB medium, and incubate at 220 rpm and 37°C with shaking for 1 h; ④ Take 100 μl of culture medium and spread it evenly on Luria-Bertani (LB) medium (containing ampicillin), and incubate at 37℃ for 12 h; ⑤ Select single colonies and place them in a centrifuge tube containing 1 ml of liquid culture medium with the corresponding antibiotic. Incubate at 37°C and 220 rpm for 4-5 hours until the bacterial solution becomes turbid. Then perform bacterial PCR identification. ⑥ Send the positive bacterial culture that was correctly identified in step ⑤ to Shanghai Sangon Biotech Co., Ltd. for sequencing. Select more than 8 single clones for the cloned gene sequence to reduce the impact of mispaired gene sequence confirmation during PCR.

[0018] The final cloned genes related to the poplar root system PcabHLH151 The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the Populus tomentosa root growth-related gene PcabHLH151 is shown in SEQ ID NO.2.

[0019] Example 2: PcabHLH151 Application of gene editing to reduce root biomass and nitrogen use efficiency of *Populus silveraefolia* '84K' 1. PcabHLH151 Screening and culture of positive plants overexpressing *Populus silveraefolius* '84K' (1) PcabHLH151 Construction of overexpression vector and obtaining plants overexpressing *Populus silveraefolia* '84K' ① The cloned product obtained in Example 1 PcabHLH151 The gene sequence was constructed into the pDONR207 intermediate vector using the Gateway method via the BP reaction, and then into the pMDC32 expression vector via the LR reaction. ② The genetic transformation experiment of *Populus silveraefolius* '84K' followed the *Agrobacterium silveraefolius*-mediated callus transformation system previously constructed by our research group: the above-constructed callus was transformed using an electroporation method. PcabHLH151The overexpression vector is introduced into Agrobacterium GV3101, and then introduced into Populus alba '84K' by Agrobacterium-mediated method; the specific steps are as follows: the Populus alba '84K' callus for genetic transformation is cultured in dark at a temperature of 23-25℃, and the Agrobacterium containing the target expression vector is used to infect the callus when OD 600 =0.6-0.8, and the callus after the infection is dried and placed on the Lloyd & McCown Woody Plant Basal Medium with Vitamins (L&M) medium, and co-cultured in dark for 3 days. The callus after the co-culture is transferred to the L&M medium added with 0.5mg / l 6-benzyl aminopurine (6-BA), 0.05mg / l naphthaleneacetic acid (NAA), 3mg / L hygromycin B and 200mg / L Timentin, and induced and screened for resistant adventitious buds under the conditions of a culture temperature of 23-25℃, illumination of 16 / 8h (day / night), and illumination intensity of 50μmol m-2s-1. -2 s -1 After 30-45 days of induction culture, the resistant adventitious buds are transferred to the rooting medium of 1 / 2 Murashige and Skoog (MS) medium added with 0.05mg / L indole-3-butyric acid (IBA), 0.02mg / L NAA, 3mg / L hygromycin B and 200mg / L Timentin, and induced for rooting; (2) DNA extraction and PCR detection ①Preheat an appropriate amount of CTAB solution in a 65℃ water bath, and precool an appropriate amount of isopropanol at 20℃; ②Grind the collected un-identified different strain transgenic poplar leaves into powder under liquid nitrogen, and move to a 2ml centrifuge tube, and add 600μl of preheated CTAB solution and shake well; ③Put the centrifuge tube in step ② in a 65℃ water bath for 45min, and invert and mix well every 5min; ④Add 600μl of chloroform:isopropanol (24:1), and shake vigorously and mix well to emulsify; ⑤Centrifuge at 12 000rpm for 12min, and take 600μl of supernatant to a 1.5ml centrifuge tube; ⑥Add 600μl of pre-cooled isopropanol, shake well and mix, and place in a-20℃ refrigerator for 1h; ⑦Centrifuge at 12 000rpm for 15min, discard the supernatant, and add 1ml of anhydrous ethanol to wash the precipitate; ⑧ Centrifuge at 7500 rpm for 5 min, discard the supernatant, and keep the DNA precipitate; ⑨ After the precipitate has dried in the fume hood, add 50 μl ddH2O to dissolve it; ⑩ The DNA from the different strains obtained above was subjected to PCR detection, and a total of 21 positive strains were finally obtained, such as... Figure 1 As shown, name them respectively as OE#1, OE#3, OE#4, OE#5, OE#6, OE#9, OE#13, OE#15, OE#16, OE#17, OE#23, OE#27, OE#28, OE#29, OE#30, OE#31, OE#32, OE#33, OE#34, OE#35, OE#36; (2) RNA extraction and PcabHLH151 Detection of gene expression levels ①The RNA of the transgenic '84K' was extracted according to the RNA extraction method in Example 1; ② The RNA extracted in step ① above will be processed... PcabHLH151 Gene expression level detection, such as Figure 2 As shown, different PcabHLH151 The expression levels of the overexpressing *Populus silveraefolia* '84K' strains varied considerably. Based on the expression level measurement results and preliminary phenotypic analysis, this invention selected two transgenic plants, OE#17 and OE#23, for subsequent experiments. (3) Cultivation of transgenic silver poplar '84K' Transgenic *Populus alba* '84K' tissue culture seedlings were subcultured on 1 / 2 Murashige and Skoog medium (containing hygromycin and termethin) at a temperature of 24±2℃ and a light exposure of 16h. 2. bHLH151 Obtaining and screening gene-edited '84K' positive plants of *Populus simonii* (1) bHLH151 Gene sequence acquisition and vector construction in *Populus silverae* '84K' ① By comparing similar sources, we can understand PcabHLH151 The homologous gene of the gene in the genome of *Populus simonii* '84K' was obtained, and the gene was cloned by PCR following the DNA extraction steps described above. bHLH151 Genome sequences; comparing Populus to Populus to '84K' bHLH151 Gene sequence alignment was performed, and based on the target site design website (http: / / skl.scau.edu.cn / ), a gene capable of simultaneously and specifically recognizing Populus tomentosa and Populus spp. '84K' was designed and obtained. bHLH151The sgRNA sequence of the gene was used to construct an sgRNA expression cassette using overlapping PCR. Subsequently, the sgRNA expression cassette was assembled into the CRISPR / Cas9 vector pCas9-DH using a "clean-and-ligate" method. (2) bHLH151 Obtaining and identifying gene-edited plants ①The genetic transformation experiment of *Populus simonii* '84K' was conducted according to the above-mentioned overexpression lines; ② bHLH151 DNA extraction from gene-edited plants followed the methods described above: First, gene-edited plants were initially screened for hygromycin resistance. Subsequently, PCR was used to identify the insertion of the target vector. Simultaneously, [the process was further refined using...]. bHLH151 Gene-specific primers were used to detect gene editing in the genomic DNA of positive plants. bHLH151-cas9JD-F is shown in SEQ ID NO. 5; bHLH151-cas9JD-R is shown in SEQ ID NO. 6. The PCR products were then subjected to high-throughput sequencing using the Hi-tom platform at the China National Rice Research Institute. Subsequently, based on the sequencing results, [the following were selected]. bHLH151 The strains KO#2 and KO#40, in which both alleles were edited, underwent further functional validation, such as... Figure 3 As shown; 3. PcabHLH151 Phenotypic identification of overexpression and gene editing of *Populus simonii* '84K' strain (1) bHLH151 Gene editing reduces the accumulation of plant root biomass. Each transgenic *Populus simonii* '84K' line was established with at least 15 biological replicates. Simultaneously, a wild-type *Populus simonii* '84K' (WT) was set up as a control. The plants were planted in an artificial climate chamber at the Chinese Academy of Forestry. After one month of growth, the transgenic *Populus simonii* '84K' lines were photographed and their aboveground and underground biomass was measured. The root systems were rinsed with running water before the biomass was measured. The results are as follows: Figure 4 and Figure 5 As shown; PcabHLH151 The phenotype of the overexpressing *Populus simonii* '84K' strain was quite similar to that of the wild-type *Populus simonii* '84K', with no significant differences; however, bHLH151 The biomass dry weight of the aboveground and underground parts of the gene-edited *Populus simonii* '84K' plant was significantly reduced by 15.33% and 18.67% respectively compared with the wild-type *Populus simonii* '84K'; this result indicates that *Populus simonii*... PcabHLH151 Genes play a role in regulating plant roots to a certain extent. Mutations in these genes can affect plant root growth and further reduce root biomass. (2)bHLH151 Gene editing affects nitrogen utilization efficiency of plants 30 biological replicates of the cuttings of the transgenic Populus alba '84K' line are set, and the cuttings of wild-type Populus alba '84K' are set as controls, and the planting site is a research greenhouse of the Chinese Academy of Forestry; mineral nutrients such as nitrogen (N), phosphorus (P), potassium (K) and the like are essential elements for plant growth, wherein the xylem and phloem of the root system of the plant play an important role in the absorption and long-distance transport of these nutrients, in order to explore whether different degrees of root growth affect the nitrogen utilization efficiency of the plant, the present application sets two concentration gradients of nitrogen treatment for the cuttings, which are low nitrogen treatment (0.5mM N) and normal nitrogen treatment (8mM N); the nutrient solution for treatment is Hoggland nutrient solution without nitrogen element, wherein the low nitrogen treatment nutrient solution is additionally added with 0.375mM KNO3 and 0.063mM NH4NO3, and the normal nitrogen treatment nutrient solution is additionally added with 6mM KNO3 and 1mM NH4NO3; after one month under different concentrations of nitrogen treatment, the cuttings of each line are photographed and recorded, and the biomass is determined; the determination results are shown in Figure 6 and Figure 7 Under different concentrations of nitrogen treatment, PcabHLH151 the phenotype of the overexpression Populus alba '84K' line is close to that of the wild-type Populus alba '84K', and there is no significant difference; under normal nitrogen treatment, bHLH151 the above-ground biomass of the gene-edited Populus alba '84K' cutting is not significantly different from that of the wild-type Populus alba '84K' cutting, wherein the difference in underground biomass is reduced by 11.21%; under normal nitrogen treatment bHLH151 the above-ground phenotype of the gene-edited Populus alba '84K' cutting is recovered, which indicates that under normal nitrogen treatment, the cutting of the line can obtain sufficient nitrogen source for the growth and development of the above-ground part; however, under low nitrogen treatment, bHLH151 the above-ground biomass and the underground biomass of the gene-edited Populus alba '84K' cutting are both much lower than those of the wild-type Populus alba '84K', and are significantly reduced by 10.59% and 12.66%; the results show that under low nitrogen conditions, bHLH151 the gene-edited Populus alba '84K' cutting may not be able to obtain sufficient nitrogen source for growth and development, which indicates bHLH151 the gene editing further reduces the nitrogen utilization efficiency of the plant.

[0020] In one aspect, the present application provides Populus canadensis root growth related genes PcabHLH151 and the encoded proteins thereof, and in another aspect, the present application provides uses of Populus canadensis root growth related genes PcabHLH151 The present application provides an important and possibly universal gene resource for regulating root growth, and provides an excellent candidate gene for cultivating plant varieties with more accumulated root biomass.

[0021] While the foregoing has been set forth in terms of particular embodiments and illustrative concepts, those of ordinary skill in the art will recognize that changes can be made to this disclosure without departing from the scope of the claims, and it is therefore intended that the present disclosure cover modifications and variations of this disclosure provided they come within the scope of the appended claims and their equivalents.

Claims

1. A gene related to the root growth of Populus tomentosa PcabHLH151 Its nucleotide sequence is shown in SEQ ID NO.

1.

2. The protein encoded by the Populus alba root growth-related gene PcabHLH151 according to claim 1, the amino acid sequence of which is shown in SEQ ID NO.

2.

3. The poplar root growth-related gene according to claim 1 PcabHLH151 Applications in regulating plant biomass accumulation and nitrogen use efficiency.

4. The application as described in claim 3, characterized in that: To enable plants to contain genes related to poplar root growth PcabHLH151 Or cause plants to overexpress genes related to poplar root growth PcabHLH151 Or make the plant to bHLH151 Gene editing is performed.

5. The application as described in claim 3, characterized in that: Constructing a structure containing genes related to poplar root growth PcabHLH151 The plant overexpression vector and the bHLH151-Cas9 gene editing vector were genetically transformed into *Populus simonii* '84K'. Overexpression and mutant plants were obtained through screening. Phenotypic analysis of overexpression, mutant and wild-type plants revealed gene-edited plants with reduced root biomass and reduced nitrogen use efficiency.

6. The application as described in claim 3, characterized in that: Specifically, the following steps are included: 1) Collect roots from high-quality Populus euphratica germplasm lines 1-15, extract RNA, reverse transcribe it into cDNA, and clone it. PcabHLH151 The CDS sequence was obtained, then ligated into a sequencing vector and sequenced. After confirmation that it was correct, an overexpression vector was constructed and genetically transformed into *Populus silverae* '84K' using Agrobacterium-mediated transformation. 2) Targeting Populus euphratica and Populus spp. '84K' bHLH151 Based on genomic sequence characteristics, and following the start codon, a design was developed to specifically identify *Populus euphratica* and *Populus simonii* '84K'. bHLH151 The sgRNA sequence of the gene was obtained, and then the bHLH151-Cas9 gene editing vector was constructed based on the CRISPR / Cas9 system. After the sequence was confirmed to be correct, it was genetically transformed into *Populus alba* '84K'. 3) Positive plant selection was performed on overexpressing and gene-edited plants using hygromycin resistance and PCR methods. Overexpressing and gene-edited plants were obtained, and their phenotypic characteristics were statistically analyzed and treated with different concentrations of nitrogen to obtain gene-edited plants with reduced root biomass and nitrogen use efficiency.

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