Application of PagWOX1c gene in improvement of callus formation and bud induction of poplar
By overexpressing the PagWOX1c gene in poplar, the problem of low callus and bud induction efficiency in poplar was solved, and the callus induction rate and bud induction rate were significantly improved, providing a new optimization method for plant genetic engineering technology.
Patent Information
- Application Number
- CN202511921759.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-18
- Publication Date
- 2026-01-23
AI Technical Summary
Different species and genotypes of poplar face difficulties and low efficiency in callus and bud induction, which affects their biotechnology breeding progress.
By overexpressing the PagWOX1c gene in poplar or using an expression vector containing the PagWOX1c gene, the callus and bud induction abilities of poplar can be improved. The specific methods include ligating the PagWOX1c gene into a plant expression vector and introducing it into poplar through Agrobacterium-mediated transformation, followed by co-culture, screening, and rooting culture, ultimately obtaining poplar lines with enhanced callus and bud induction abilities.
It significantly improved the callus induction rate and bud induction rate of poplar, provided new gene resources and technical means for optimizing plant tissue culture systems, and promoted the development of plant genetic engineering technology.
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Figure CN121380179A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of plant genetic engineering and biotechnology, and particularly relates to application of a PagWOX1c gene in improving callus formation and bud induction of poplar. BACKGROUND
[0002] Plant cells have totipotency, that is, a single cell can develop into a complete plant under suitable conditions. Plant callus refers to a mass of undifferentiated parenchyma cells formed on the surface of a wound after a plant is wounded, and it is not only an important structure for plant wound repair, but also a key link in biological technology research and application such as plant tissue culture, genetic transformation and gene editing. In plant breeding, variety improvement, secondary metabolite production and plant gene function research, an efficient callus induction system is the basis for ensuring the smooth development of related work. However, the callus induction capacity of different species and different genotypes is significantly different, and many important tree species and crop varieties have problems such as difficult callus induction and low efficiency, which seriously restricts the process of biological technology breeding.
[0003] 84K poplar (Populus alba x P. glandulosa) is a model tree species widely used in forestry production and biotechnology research, and has the advantages of fast growth, strong adaptability and high genetic transformation efficiency. However, in the actual tissue culture process, the callus induction rate of 84K poplar is affected by many factors such as genotype, explant type and culture conditions, and in some cases the induction efficiency is low, which restricts the efficiency of genetic improvement and biotechnology application.
[0004] The WOX (WUSCHEL-related homeobox) gene family is a plant-specific transcription factor family, and the function of the genes in this family is very wide, involving plant growth and development processes such as embryonic development, embryonic polarity establishment, maintenance of meristem stem cells, development of lateral organs, seed formation and induction of in vitro tissues and organs. The functions of different WOX gene members are specific, and some members have been confirmed to be involved in the regulation of plant development and stress tolerance.
[0005] Therefore, it is of great significance to mine key genes regulating plant callus and bud induction, clarify their functions and apply them to improve plant callus induction rate and bud induction rate, in order to optimize plant tissue culture system and promote the development of plant genetic engineering technology. SUMMARY
[0006] The technical problem to be solved by the present application is to provide a method for improving callus induction rate and bud induction rate of poplar.
[0007] The technical scheme of the present application is: application of a PagWOX1c gene or an expression vector containing the PagWOX1c gene in improving the callus induction ability and bud induction ability of a poplar, wherein the PagWOX1c gene is a DNA fragment encoding the amino acid sequence shown in SEQ ID No. 2.
[0008] Further, the improving method is overexpression of the PagWOX1c gene in the poplar.
[0009] Further, the nucleotide sequence of the PagWOX1c gene is shown in SEQ ID No. 1.
[0010] A method for creating a poplar strain with enhanced callus induction ability and bud induction ability, comprising the following steps:
[0011] (1) connecting the PagWOX1c gene to a plant expression vector to obtain a PagWOX1c gene overexpression vector;
[0012] (2) introducing the overexpression vector constructed in step (1) into a wild-type poplar through an agrobacterium-mediated leaf disc method;
[0013] (3) obtaining a positive plant through co-cultivation, screening culture, rooting culture and identification, which is a poplar strain with enhanced callus induction ability and bud induction ability.
[0014] Compared with the prior art, the present application has the following beneficial effects:
[0015] In this study, by observing the callus induction of PagWOX1c transgenic poplar strains and wild-type 84K poplar and calculating the induction rate, it was found that overexpression of PagWOX1c increased the callus induction rate and bud induction rate of the transgenic poplar, while knock-out of PagWOX1c reduced the callus induction rate and bud induction rate of the transgenic poplar. This finding reveals that the PagWOX1c gene improves the callus and bud induction ability of plants, and provides new gene resources and technical means for optimizing plant tissue culture systems. BRIEF DESCRIPTION OF DRAWINGS
[0016] Figure 1 The photos of wild-type and transgenic poplar plants in Example 2.
[0017] Figure 2 The front and back view observation results of leaf blades in callus formation in Example 3.
[0018] Figure 3 The DAB staining diagram of callus in Example 3.
[0019] Figure 4 The callus fresh weight statistics diagram in Example 3.
[0020] Figure 5 Figure 4 is a graph of the statistical results of the induction rate of callus in Example 3.
[0021] Figure 6 Figure 5 is a graph of the observation results of the induction of buds from leaves in Example 4.
[0022] Figure 7 Figure 6 is a graph of the statistical results of the number of buds from leaves in Example 4.
[0023] Figure 8 Figure 7 is a graph of the statistical results of the induction rate of buds in Example 4. DETAILED DESCRIPTION
[0024] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from commercial channels unless otherwise specified.
[0025] Example 1 Cloning and vector construction of Populus PagWOX1c gene
[0026] 1.1 Obtaining of the sequence of the target gene
[0027] According to the sequence number in the Phytozome database of Populus trichocarpa, cDNA sequences homologous to the 84K Populus PagWOX1c gene were screened, and primers were designed using primer 5 software according to the sequences, and the full-length gene was amplified by PCR. The primers are shown in Table 1.
[0028] Table 1
[0029]
[0030] The total RNA extracted from different tissues of 84K Populus was reverse transcribed into cDNA using the FastKing cDNA first-strand synthesis kit. According to the high-fidelity enzyme PCR reaction system, gene cloning was performed, and the reaction system (50 μL) was as follows: PrimeSTAR 25 μl, forward primer 2 μl; reverse primer 2 μl; 84K Populus cDNA 4 μl; sterile ddH2O to 50 μl. The reaction program was as follows: 95 ℃, 5 min; (95 ℃, 10 s; 57 ℃, 30 s; 72 ℃, 50 s) 40 cycles; 72 ℃, 2 min. The product was recovered by Novizen gel recovery kit, and the recovered product was connected to T vector, transformed into Escherichia coli DH5α, and sequenced. The nucleotide sequence of the PagWOX1c gene is shown as SEQ ID No. 1, and the encoded amino acid sequence is shown as SEQ ID No. 2.
[0031] 1.2 Vector construction
[0032] The WOX1C CDS was inserted into the CaMV 35S promoter of the plant expression vector pCAMBIA1302 to construct a 35S:PagWOX1c overexpression vector; a gRNA was designed for a specific target of the PagWOX1c gene and cloned into the CRISPR / Cas9 vector pBUE411 to construct a pU6-gRNA / Cas9-PagWOX1c editing vector. The ligated vector was transformed into E. coli, and single colony liquid was picked for liquid PCR verification, and positive colonies were sequenced. The successfully constructed PagWOX1c overexpression and gene editing vectors.
[0033] Example 2 Obtaining of PagWOX1c transgenic 84K poplar
[0034] 2.1 Preparation of explants: select healthy 84K poplar seedlings, take the top fully expanded leaves, cut the leaves into 0.5 cm x 0.5 cm leaf discs with a sterile scalpel, and use them as explants.
[0035] 2.2 Agrobacterium activation and infection: the overexpression and gene editing vectors obtained in Example 1 were transformed into Agrobacterium GV3101 by heat shock method, then inoculated into LB liquid medium containing kanamycin and rifampicin, and cultured at 28°C, 200 rpm for 2-3 days until the OD value was 0.6-0.8. The bacterial solution was centrifuged at 4°C, 5000 rpm for 5 min, the supernatant was discarded, and the bacterial solution was resuspended in MS liquid medium to adjust the OD value to 0.4-0.5 to obtain the infection solution. The prepared leaf discs were placed in the infection solution and soaked for 10-15 min, with constant gentle shaking. 600
[0036] 2.3 Co-culture and selection culture: after infection, place the leaf discs upside down in the co-culture medium (WPM medium Phytotech L449 2.4 g + MES 0.5 g / L + sugar 20 g + gel 3.5 g + pH 5.9 + AS 100 µM) in the dark for 3 days, then transfer to the differentiation selection medium (WPM449 2.4 g + MES 0.5 g / L + NAA 0.05 mg / L + 6-BA 0.5 mg / L + sugar 20 g + gel 3.5 g + PH 5.9 + Timentin 1 ml (200 mg / ml) + Hyg 2-3 mg / L) and cultured under light. The buds sprouted after 15-20 days. The sprouted buds were transferred to the differentiation selection medium to promote their growth.
[0037] 4.4 Rooting culture and identification: The large seedlings obtained by screening were directly inoculated into rooting screening medium (MS medium Phytotech M519 2.2 g + MES 0.5 g / L + NAA 0.02 mg / L + IBA 0.05 mg / L + sugar 20 g + gel 3.5 g + temin 1 mL (200 mg / mL)) for rooting screening. After the emergence of healthy root system, transgenic seedlings were obtained. Through PCR, RT-qPCR and sequencing analysis, transgenic plants of PagWOX1c overexpression (71C-7 and 71C-11) and gene editing (UR-4a-2 and UR-4a-5) were identified respectively. Figure 1 .
[0038] Example 3 Callus induction rate analysis
[0039] 3.1 Callus induction culture
[0040] Take transgenic and wild type poplar leaves, place the leaves on sterile filter paper, use a scalpel to draw 2-3 lines vertically along the middle vein of the leaves, then use tweezers to inoculate the callus induction medium (4.4g MS medium Phytotech M519 + MES 0.5g / L + 2,4-D 2mg / L + NAA 0.1mg / L + KT 0.1mg / L + sugar 20g + gel 3.5g + pH 5.9) on the leaves. Inoculate 4-6 leaves per dish (90mm). Place the inoculated dishes in the dark for 20d to allow the callus to grow. As shown in Figure 2 , the overexpression PagWOX1c transgenic poplar grows a large amount of callus at the wound, while the wild type 84K has a small amount of callus growth, and the callus growth of the PagWOX1c gene edited poplar is even less.
[0041] 3.2 DAB staining
[0042] Prepare 1.97g of Tris-HCl to 250mL, adjust the pH value to 5.5, add 0.25g of DAB powder and shake well to prepare 1mg / mL DAB staining solution. Soak the callus induced for 20d from the leaves of WT and each transgenic strain in 10mL DAB staining solution, and stain at 37°C for 12-16h, then decolorize with 95% alcohol. Observe the color, and the results are shown in Figure 3 , compared with the wild type 84K, the overexpression PagWOX1c transgenic poplar callus stains darker, indicating that it induces more callus, while the callus of the PagWOX1c gene edited poplar is lighter, and the callus induced is less than the wild type 84K.
[0043] 3.3 Analysis of callus fresh weight and growth rate
[0044] The fresh weight of callus was statistically analyzed using a weighing method. The fresh weight of the leaves 20 days after callus induction was subtracted from the initial fresh weight of the leaves; the difference was the callus fresh weight. Results are as follows: Figure 4 As shown, the fresh weight of callus from the overexpressing PagWOX1c transgenic poplar was significantly greater than that of the wild-type 84K, while the fresh weight of callus from the PagWOX1c gene-edited poplar was significantly lower than that of the wild-type 84K.
[0045] To avoid the influence of differences in initial leaf size on callus fresh weight, the ratio of pure callus fresh weight to leaf disc area was calculated and considered as the callus induction rate. Results are as follows: Figure 5 As shown, the callus induction rate of transgenic poplar overexpressing PagWOX1c was significantly higher than that of wild-type 84K, while the callus induction rate of PagWOX1c-edited poplar was significantly lower than that of wild-type 84K.
[0046] Example 4: Bud Induction Rate Analysis
[0047] 4.1 Bud Induction Culture
[0048] Leaf bud formation was induced using a one-step budding method. WT and transgenic poplar tissue culture seedlings that had been subcultured for 30 days were selected. The third fully expanded leaf was taken and cut into 0.5 × 0.5 mm leaf discs. These discs were placed on bud induction medium (2.4 g WPM medium Phytotech L449 + MES 0.5 g / L + NAA 0.05 mg / L + 6-BA 0.5 mg / L + sugar 20 g + gel 3.5 g + pH 5.9), with 20 leaf discs per dish. The seedlings were then cultured under light for 30 days, and bud formation was observed. The results are as follows: Figure 6 As shown, almost all leaf discs of the PagWOX1c-overexpressing transgenic poplar developed buds, while only some wild-type 84K trees developed buds, and the PagWOX1c-edited poplar trees hardly developed any buds. This indicates that overexpression of PagWOX1c promoted bud induction in the leaves of the transgenic poplar.
[0049] 4.2 Statistics on the number of buds and bud induction rate
[0050] After 30 days of light-induced culture, the number of leaf discs that sprouted in each petri dish was counted, and the shoot induction rate was calculated. Shoot induction rate = number of leaf discs sprouting / total number of leaf discs. Results are as follows: Figure 7 and Figure 8 As shown, the number of buds and the bud induction rate of transgenic poplar trees overexpressing PagWOX1c were significantly higher than those of wild-type 84K, while the number of buds and the bud induction rate of PagWOX1c-edited poplar trees were significantly lower than those of wild-type 84K.
Claims
1. Use of a PagWOX1c gene or an expression vector containing the PagWOX1c gene in improving the callus induction ability and bud induction ability of a poplar, wherein the PagWOX1c gene is a DNA fragment encoding the amino acid sequence shown in SEQ ID No.
2.
2. Use according to claim 1, characterized in that, The improving method is overexpression of the PagWOX1c gene in the poplar.
3. Use according to claim 1 or claim 2, characterised in that, The nucleotide sequence of the PagWOX1c gene is shown in SEQ ID No.
1.
4. A method for creating a Populus line having enhanced capacity for callus induction and shoot induction, the method comprising, The method comprises the following steps: (1) connecting the PagWOX1c gene of claim 1 to a plant expression vector to obtain a PagWOX1c gene overexpression vector; (2) introducing the overexpression vector constructed in step (1) into a wild-type poplar by means of an agrobacterium-mediated leaf disc method; (3) obtaining a positive plant by co-culturing, screening culture, rooting culture and identification, which is a poplar strain with improved callus induction ability and bud induction ability.