A herbicide-resistant gene and its application

Abstract

This invention provides a herbicide-resistant gene and its application, belonging to the field of molecular biotechnology. The amino acid sequence encoded by the herbicide-resistant gene of this invention includes SEQ ID No. 1. Knocking out the herbicide-resistant gene significantly improves the herbicide resistance of plants and significantly promotes plant growth, increasing net photosynthetic rate and lignin content. Therefore, the herbicide-resistant gene of this invention can be used to breed high-yielding, high-quality, herbicide-resistant new plant varieties, and has great application value for the genetic improvement of comprehensive stress resistance in forest trees, providing a new molecular tool for the genetic improvement of herbicide resistance in forest trees.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a herbicide-resistant gene and its application. Background Technology

[0002] With the widespread application of direct seeding technology in agricultural production, weed problems in farmland have become a significant factor affecting agricultural and forestry production. Since traditional breeding and domestication processes do not involve herbicide tolerance traits, resistant resources in natural genetic resources are extremely rare. Utilizing herbicide-inhibiting target genes to screen and identify endogenous resistance mutants is currently a feasible approach to developing herbicide-resistant plant germplasm resources. Simultaneously, with repeated application of single-type herbicides, acquired resistance in field weeds is rapidly increasing. Therefore, discovering novel herbicide resistance genes in plants and obtaining herbicide resistance traits through transgenic methods is essential for cultivating herbicide-resistant plants.

[0003] Glufosinate ammonium is one of the most widely used non-selective herbicides. It binds to glutamine synthase (GS), inhibiting glutamine synthesis, thereby hindering nitrogen assimilation and leading to excessive ammonium accumulation, ultimately causing plant death. It is known that increasing endogenous GS activity and decreasing GS sensitivity to glufosinate can confer glufosinate resistance to plants. Point mutations in the endogenous GS gene can enhance plant resistance to glufosinate; this change in GS produces typical target-site resistance, thus conferring insensitivity to herbicide inhibition. The GS gene has become a primary target for developing glufosinate-resistant crops through gene editing technology. However, whether other mechanisms besides GS target-site resistance exist in plants remains unclear.

[0004] The SPL gene family is one of the plant-specific transcription factor families, widely involved in regulating multiple processes of plant growth and development, including embryonic development, leaf primordium development, the transition from juvenile to adulthood, floral organ development, and fruit ripening. In addition, SPL genes are also associated with plant hormones and light signal transduction, anthocyanin synthesis, and responses to abiotic stress. However, there is currently little research on the effects of the SPL family on herbicide resistance in forest trees. Summary of the Invention

[0005] This invention provides a herbicide-resistant gene and its application, which can effectively regulate the herbicide resistance characteristics of plants.

[0006] This invention provides a herbicide resistance gene, which encodes an amino acid sequence as shown in any of the following:

[0007] (1) The amino acid sequence shown in SEQ ID No. 1;

[0008] (2) A sequence that has more than 85% homology with the amino acid sequence shown in SEQ ID No. 1;

[0009] (3) Replace, add or remove one or more amino acids from the sequence shown in (1) or (2) while maintaining a similar sequence effect.

[0010] The present invention also provides the application of the above-mentioned herbicide resistance gene in regulating plant resistance to herbicides.

[0011] In one specific embodiment of the present invention, the regulation includes inhibiting the expression of the herbicide-resistant gene to enhance the plant's resistance to herbicides.

[0012] This invention also provides the application of the above-mentioned herbicide resistance gene as a screening marker in transgenic plant culture.

[0013] This invention also provides a herbicide resistance molecular marker system and / or molecular marker method developed using the above-mentioned herbicide resistance genes.

[0014] In one specific embodiment of the present invention, the herbicide includes glufosinate.

[0015] The present invention also provides a gene editing vector for knocking out or inhibiting the above-mentioned herbicide resistance gene.

[0016] In one specific embodiment of the present invention, the CRISPR-Cas9 method is employed;

[0017] The nucleotide sequences of the sgRNA used in the CRISPR-Cas9 method include SEQ ID No. 2 and SEQ ID No. 3.

[0018] The present invention also provides a method for enhancing plant resistance to herbicides, including knocking out or inhibiting the expression of the aforementioned herbicide-resistant genes.

[0019] In one specific embodiment of the present invention, the plant includes forest trees.

[0020] This invention also provides the application of the above-mentioned herbicide-resistant gene or the above-mentioned gene-editing vector in the creation of herbicide-resistant plant germplasm.

[0021] Beneficial Effects: This invention isolates an endogenous herbicide resistance gene from poplar trees, namely the PtSPL5 gene, as shown in the nucleotide sequence of SEQ ID No. 4 in the examples. Using gene editing, this invention knocks out the PtSPL5 gene in poplar trees. Compared with wild-type poplar plants, PtSPL5 knockout plants showed significantly enhanced herbicide resistance under glufosinate treatment. Furthermore, growth traits were examined, revealing that PtSPL5 knockout plants significantly promoted poplar growth, increased net photosynthetic rate, and increased lignin content compared to wild-type poplar plants. This invention demonstrates that knocking out the PtSPL5 gene in poplar plants under glufosinate treatment significantly enhances the herbicide resistance of the plants without inhibiting their growth characteristics. Therefore, the PtSPL5 gene can be used to cultivate high-yielding, high-quality, herbicide-resistant varieties, possessing significant application value for the genetic improvement of comprehensive stress resistance in forest trees and providing a new molecular tool for the genetic improvement of herbicide resistance in forest trees. Attached Figure Description

[0022] Figure 1 This is a map of eukaryotic gene editing vectors, where LB represents the left boundary and RB represents the right boundary.

[0023] Figure 2 Image showing the results of gene editing sites and mutation information detection for PtSPL5 knockout mutant materials;

[0024] Figure 3 Growth traits of wild-type poplar plants and PtSPL5-positive plants with knockout;

[0025] Figure 4 The plant height, diameter at ground level, water use efficiency, net photosynthetic rate, intercellular CO2 concentration, and lignin content of wild-type poplar plants and PtSPL5-positive knockout plants were measured.

[0026] Figure 5 Leaf development traits of wild-type poplar plants and PtSPL5-positive plants after glufosinate treatment. Detailed Implementation

[0027] This invention provides a herbicide resistance gene, which encodes an amino acid sequence as shown in any of the following:

[0028] (1) The amino acid sequence shown in SEQ ID No. 1;

[0029] (2) A sequence that has more than 85% homology with the amino acid sequence shown in SEQ ID No. 1;

[0030] (3) Replace, add or remove one or more amino acids from the sequence shown in (1) or (2) while maintaining a similar sequence effect.

[0031] The herbicide-resistant gene described in this invention is an endogenous gene of poplar, and its encoded amino acid sequence is shown in SEQ ID No. 1: MLLVVLPTFFTFYFTLSPTDLHFSTSKATPHLASYHTIPQLCDILPKIMAARTLEGKHSSLKEKMINKDDFLIEDELDDDMEEYESGGGAGLADDEKKKGAGVMHGKRGTGSGGASPPSCQVEKCGANLTDAKRYHRRHKVCEVHAKSPAVVVAGLRQRFCQQCSRFHELAEFDETKRSCRRRLAGHNERRRKSTAESYGEASNRKGVNAPLKESPCRQADERGRFQINIPPQGSSSYKRSQIR*. Of course, mutations based on the amino acid sequence shown in SEQ ID No. 1, as long as they satisfy more than 85% homology and have the same or similar effects, can all be used as the herbicide-resistant gene described in this invention.

[0032]

[0033] The present invention also provides the application of the above-mentioned herbicide resistance gene in regulating plant resistance to herbicides.

[0034] Verification through examples shows that knocking out the CDS sequence of the PtSPL5 gene in poplar significantly enhances the plant's herbicide resistance and improves photosynthesis. The herbicide used in this invention can be glufosinate; knocking out the gene enhances the plant's herbicide resistance, primarily by alleviating symptoms of excessive ammonium accumulation in plant leaves. The plant used in this invention can be a forest tree; poplar is used as an example in one embodiment.

[0035] This invention also provides the application of the above-mentioned herbicide resistance gene as a screening marker in transgenic plant culture.

[0036] The herbicide resistance gene described in this invention plays a role in regulating plant resistance to herbicides. This gene can be used as a screening marker. If the herbicide resistance gene is present or overexpressed in transgenic plants, it indicates that the transgenic plants have weak herbicide resistance, and target transgenic plants can be screened by adjusting the herbicide concentration. If the herbicide resistance gene is not present or its expression is suppressed in transgenic plants, it indicates that the transgenic plants have strong herbicide resistance, and target transgenic plants can be screened using high concentrations of herbicide.

[0037] This invention also provides a herbicide resistance molecular marker system and / or molecular marker method developed using the above-mentioned herbicide resistance genes.

[0038] The herbicide resistance gene described in this invention, particularly the PtSPL5 gene verified in one embodiment, has a significant regulatory effect on herbicide resistance. Molecular markers can be developed based on the gene, and the molecular markers target the herbicide resistance gene.

[0039] In one specific embodiment of the present invention, a pair of primers for amplifying the PtSPL5 gene is also provided, the primer sequences of which include the following:

[0040] PtSPL5-F (SEQ ID No. 6): 5'-ATGTTGCTTGTGGTCCTCCCTA-3';

[0041] PtSPL5-R (SEQ ID No. 7): 5'-TACAAGCGATCCCAGATCAGATAA-3'.

[0042] In one specific embodiment of the present invention, glufosinate was used as a herbicide for experimental verification.

[0043] The present invention also provides a gene editing vector for knocking out or inhibiting the above-mentioned herbicide resistance gene.

[0044] In one specific embodiment of the present invention, the method of CRISPR-Cas9 is employed; the nucleotide sequence of the sgRNA used in the CRISPR-Cas9 method includes SEQ ID No. 2 and SEQ ID No. 3.

[0045] The present invention also provides a gRNA of the PtSPL5 gene described in the above technical solution, the nucleotide sequence of which is shown below:

[0046] SEQ ID No.2: 5'-GAAGAATATGAGAGTGGCGGCGG-3';

[0047] SEQ ID No. 3: 5'-CAGGAGACACAAAGTTTGCGAGG-3'.

[0048] The gRNA provided by this invention can knock out the PtSPL5 gene in plants, thereby enhancing the herbicide resistance of plants.

[0049] The present invention also provides a method for enhancing plant resistance to herbicides, including knocking out or inhibiting the expression of the aforementioned herbicide-resistant genes.

[0050] In one specific embodiment of this invention, the PtSPL5 gene is knocked out in a plant using a gene knockout method. The plant can be a forest tree, such as the poplar used in the example. The knockout process includes the following steps: transforming the gene-editing vector into the cells of the target plant to knock out the PtSPL5 gene. In one embodiment of this invention, the transformation is completed using Agrobacterium-mediated transformation, with Agrobacterium being GV3101. This invention, by knocking out the PtSPL5 gene in plants and regulating its expression level, can effectively improve the net photosynthetic rate and water use efficiency of the plant, promote plant height growth, and enhance the plant's herbicide resistance. It has significant application value for the genetic improvement of comprehensive stress resistance in forest trees.

[0051] This invention also provides the application of the above-mentioned herbicide-resistant gene or the above-mentioned gene-editing vector in the creation of herbicide-resistant plant germplasm.

[0052] To further illustrate the present invention, the following detailed description of a herbicide-resistant gene and its application provided by the present invention is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0053] Example 1

[0054] 1. Construction of PtSPL5 gene editing vector

[0055] (1) gRNA design: Using the online tool CRISPR-P2.0, the sequence of the PtSPL5 gene (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR) was input to design a gRNA targeting this gene. The first exon was selected as the target site to improve the knockout efficiency. The final designed gRNA sequence is as follows:

[0056] gRNA-1 (SEQ ID No. 2): 5'-GAAGAATATGAGAGTGGCGGCGG-3';

[0057] gRNA-2 (SEQ ID No. 3): 5'-CAGGAGACACAAAGTTTGCGAGG-3'.

[0058] (2) Vector construction: The CRISPR / Cas9 vector pSpCas9, suitable for plant cells, was selected. Figure 1 The gRNA was then ligated into the vector, resulting in the knockout vector.

[0059] 2. Genetic transformation using PtSPL5 gene editing vector

[0060] (1) Transformation of Agrobacterium tumefaciens with recombinant plasmid

[0061] Thaw competent Agrobacterium GV3101 cells on ice. Add 0.01–1 μg of plasmid DNA containing the knockout vector to 100 μL of competent GV3101 cells, mix thoroughly, and incubate on ice for 5 min. Rapidly cool in liquid nitrogen for 5 min, then quickly transfer to a 37°C water bath for 5 min, and incubate on ice for 5 min. Add 700 μL of antibiotic-free LB broth and incubate at 28°C with shaking at 230 rpm for 2–3 h. After centrifugation at 6000 rpm for 1 min, resuspend the cells in 100 μL of the supernatant and plate onto LB agar plates containing antibiotics. Incubate upside down at 28°C for 2–3 h to obtain Agrobacterium containing the knockout vector.

[0062] (2) Pre-culture of poplar leaves

[0063] Select healthy poplar seedlings that have grown for 30-40 days and take fresh, disease-free leaves as leaf disc transformation material. After cleaning and disinfecting the surface of the leaves, make 2-3 incisions along the main vein of the leaf using a sterile scalpel, and temporarily place them on a co-culture medium (MS + 30g / L sucrose + 6.5g / L agar + 0.05mg / L NAA + 0.5mg / L 6-BA) for pre-culture for 2-3 days.

[0064] (3) Infection and Differentiation Culture

[0065] Agrobacterium containing the knockout vector was cultured on LB medium to the logarithmic growth phase. The culture conditions were 28°C with shaking at 180 rpm until the Agrobacterium reached the OD value. 600 The value was 0.6. The bacterial cells were centrifuged, the supernatant was discarded, and the cells were resuspended in a suspension (1 / 2 MS + 30 g / L sucrose + 100 μmol / L LAS). The cells were gently shaken to ensure complete resuspending, and then placed on ice until use. Cross-sections of leaves were immersed in the resuspended bacterial solution for 12 min, gently shaken, and excess solution was absorbed with sterile filter paper. The leaves were then placed on a co-culture medium and incubated in the dark at 25°C for 2–3 days. Transformed leaf discs were obtained and transferred to differentiation medium (MS + 6-BA 0.5 mg / L + NAA 0.05 mg / L + sucrose 30 g / L + 200 mg / L termethin + 1 mg / L hygromycin + 7 g / L agar). A photoperiod of 16 h light / 8 h dark was set, and the ambient temperature was maintained at 25°C to promote stable DNA transformation and expression.

[0066] (4) Rooting culture

[0067] After culturing on differentiation medium at 25℃ under 16h light / 8h dark conditions for 3-4 weeks, when adventitious shoots have grown to 1-2cm, individual adventitious shoots are cut off and placed on rooting medium (1 / 2 MS + IBA 0.05mg / L + NAA 0.05mg / L + sucrose 20g / L + agar 7g / L) containing selection pressure (1mg / L hygromycin) and sterilization agent (200mg / L termethin) for rooting culture to obtain selected plants. RNA is extracted from the selected plants, the PtSPL5 gene is cloned, and the target site editing type is detected by sequencing to verify the successful introduction of the gene editing vector. Plants containing the gene editing vector are PtSPL5 knockout plants. Figure 2 ).

[0068] 3. PtSPL5 knockout enhances the growth characteristics of poplar plants.

[0069] When PtSPL5 knockout poplar transgenic plants and wild-type poplar plants (plants without the introduced foreign gene) reached 4 weeks of age, their tissue culture seedlings were removed from the culture medium and transplanted into pre-prepared soil. They were then placed in a poplar cultivation room for 12 weeks of soil culture. Figure 3 As shown, compared with wild-type poplar plants, PtSPL5 knockout plants significantly promoted poplar growth, increased net photosynthetic rate, and increased lignin content. Figure 4Compared to wild-type poplar plants, PtSPL5 knockout poplar transgenic plants showed significant improvements in multiple growth indicators. Specifically, the plant height increased by 10.8%, and the ground diameter increased by 14.6%. These changes indicate that PtSPL5 knockout promoted plant growth and development. PtSPL5 knockout poplar plants also showed significant enhancements in photosynthesis and physiological adaptability. Compared to the wild type, the net photosynthetic rate of PtSPL5 knockout plants increased by 0.26 μmol·m⁻¹. - 2·s - 1. This indicates that light energy can be utilized more effectively for photosynthesis, improving the efficiency of energy conversion and organic matter synthesis. Simultaneously, the intercellular CO2 concentration increased by 6 μmol·mol⁻¹. - 1. It enhances the supply of raw materials for photosynthesis, thus helping to improve photosynthetic efficiency. Furthermore, the stomatal conductance increases by 2.8 mmol·m⁻². - 2s - 1. This indicates that the plant's ability to regulate gas exchange has been enhanced, enabling it to perform photosynthesis more efficiently while maintaining water balance. The water use efficiency increased by 0.07 mmol / mol. l- The figure 1 indicates that the plants are more economical in water use, producing more photosynthetic products with less water consumption. The 14.5% increase in maximum photochemical efficiency means that the plants are more efficient in converting light energy into chemical energy, contributing to improved overall photosynthetic efficiency. Notably, the 20.4% increase in lignin content not only enhances the mechanical strength of the plants but also improves their disease resistance, making them more robust overall. These improvements not only enhance the plants' growth potential but also significantly improve their adaptability to environmental changes, which is of paramount importance for improving poplar productivity and resilience to adversity.

[0070] 4. PtSPL5 knockout enhances herbicide resistance in poplar plants.

[0071] PtSPL5 plants were subcultured, and healthy wild-type and knockout poplar seedlings grown for 30-40 days were selected. Fresh, disease-free leaves were used as materials for glufosinate stress treatment. After surface cleaning and disinfection, 2-3 incisions were made along the midrib using a sterile scalpel. The leaves were temporarily placed on MS solid medium (MS + 20 g / L sucrose + 6 g / L agar) containing different concentrations of glufosinate (0 g / L, 1 g / L, 2 g / L, 4 g / L, and 6 g / L). The photoperiod was set to 16 h light / 8 h dark, the ambient temperature was maintained at 25 °C, and the treatment time was 14 days. The results are as follows: Figure 5As shown, PtSPL5 knockout plants exhibited significantly enhanced herbicide resistance under glufosinate treatment. Seven days after glufosinate treatment, both wild-type and PtSPL5 knockout poplar leaves showed marked chlorosis and yellowing. Compared to wild-type poplar, the PtSPL5 knockout poplar leaves showed significantly less damage, primarily manifesting as localized chlorosis. Simultaneously, with increasing glufosinate concentration, the damage to wild-type poplar leaves intensified. After 14 days of glufosinate treatment, irreversible leaf death began to occur in wild-type poplar leaves. Glufosinate application triggered the generation of large amounts of reactive oxygen species within the leaves, followed by ammonia accumulation, leading to rapid chloroplast disintegration and cell death. However, the PtSPL5 knockout poplar leaves showed less damage; although some leaves exhibited slight yellowing, they remained green overall. Especially at a glufosinate concentration of 6 g / L, wild-type poplar leaves suffered the most severe damage, exhibiting an overall appearance of yellowing and wilting, while the leaves of poplars knocked out by PtSPL5 showed the opposite condition. This indicates that PtSPL5-knocked poplars, despite suffering some damage from glufosinate, can survive after a period of recovery, demonstrating strong resilience. In contrast, wild-type poplars lack the ability to recover from glufosinate damage and ultimately succumb to its effects.

[0072] As can be seen from the above examples, knocking out the PtSPL5 gene in poplar plants can significantly enhance the herbicide properties of the plants under glufosinate treatment without inhibiting the growth characteristics of the plants.

[0073] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. PtSPL5 The application of genes in regulating plant resistance to herbicides is characterized by, The regulation is the knockout of the symbol shown in SEQ ID No.

1. PtSPL5 Genes enhance poplar's resistance to glufosinate.

2. A knockout method PtSPL5 Gene editing vectors, characterized in that, The CRISPR-Cas9 method was used to knock out the variant shown in SEQ ID No.

1. PtSPL5 gene The nucleotide sequence of the sgRNA used is selected from SEQ ID No. 2 and SEQ ID No. 3; the sgRNA is ligated into a vector.

3. A method for enhancing the resistance of poplar trees to glufosinate, characterized in that, To knock out the one shown in SEQ ID No. 1 PtSPL5 Gene.

4. The application of the gene editing vector according to claim 2 in the creation of glufosinate-resistant poplar germplasm.

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