Herbicide-resistant gene and application thereof

Abstract

The invention provides a herbicide-resistant gene and application thereof, and belongs to the technical field of molecular biology. The amino acid sequence coded by the herbicide-resistant gene disclosed by the invention is SEQ ID No. 1. After the herbicide-resistant gene is knocked out, the herbicide resistance of plants can be remarkably improved, the growth of the plants is remarkably promoted, and the net photosynthetic rate and the lignin content are increased. Therefore, the herbicide-resistant gene disclosed by the invention can be used for cultivating a new variety of plants with high yield, high quality and herbicide resistance, has a great application value for genetic improvement of comprehensive stress resistance of forest trees, and provides a new molecular tool for genetic improvement of herbicide resistance of forest trees.

Description

Technical Field

[0001] The invention belongs to the field of molecular biotechnology, and specifically relates to a herbicide resistance gene and application thereof. Background Art

[0002] With the large-scale application of plant direct seeding cultivation technology in agricultural production, the problem of farmland weeds in plant direct seeding production has become an important factor affecting agricultural and forestry production. Since the traditional breeding and domestication process does not involve herbicide tolerance traits, resistance resources in natural genetic resources are extremely rare. Using herbicides to inhibit target genes and screen and identify endogenous resistance mutants are currently feasible ways to develop plant herbicide-resistant germplasm resources. At the same time, with the repeated application of a single type of herbicide, the acquired resistance of field weeds increases rapidly. Therefore, it is very necessary to discover new herbicide resistance genes in plants and obtain herbicide resistance traits through transgenic means to cultivate herbicide-resistant plants.

[0003] Glufosinate ammonium is one of the most widely used non-selective herbicides. It binds to glutamine synthetase (GS) and inhibits the synthesis of glutamine, thereby hindering nitrogen assimilation and leading to excessive accumulation of ammonium, ultimately leading to plant death. It is known that increasing the activity of endogenous GS and reducing the sensitivity of GS to glufosinate can confer glufosinate resistance to plants. Point mutations in the endogenous GS gene can increase the resistance of plants to glufosinate, and this change in GS produces typical target resistance, thereby conferring insensitivity to herbicide inhibition. The GS gene has become the main target for the development of glufosinate-resistant crops through gene editing technology. However, whether there are other mechanisms in plants besides GS target resistance remains unclear.

[0004] The SPL gene family is one of the plant-specific transcription factor families, which is widely involved in regulating multiple processes of plant growth and development, including embryonic development, leaf primordium development, transition from juvenile to adult stage, floral organ development, fruit ripening, etc. In addition, SPL genes are also related to plant hormone and light signal transduction, anthocyanin synthesis and response to adverse stress. However, there are few studies on the effects of the SPL family on forest herbicide resistance. Summary of the invention

[0005] The invention provides a herbicide resistance gene and application thereof. The herbicide resistance gene can be used to effectively regulate the herbicide resistance property of plants.

[0006] The present invention provides a herbicide resistance gene, wherein the herbicide resistance gene encodes an amino acid sequence shown in any one of the following:

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

[0008] (2) a sequence having 85% or more homology with the amino acid sequence shown in SEQ ID No. 1;

[0009] (3) A sequence having similar effects while replacing, adding or reducing one or more amino acids in the sequence shown in (1) or (2).

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

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

[0012] The present invention also provides the use of the above-mentioned herbicide-resistant gene as a screening marker in plant transgenic culture.

[0013] The present invention also provides a herbicide resistance molecular marker system and / or a molecular marker method developed using the herbicide resistance gene.

[0014] In a specific embodiment of the present invention, the herbicide includes glufosinate-ammonium.

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

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

[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 the herbicide resistance of plants, comprising knocking out or inhibiting the expression of the herbicide resistance gene.

[0019] In one embodiment of the present invention, the plants include trees.

[0020] The present invention also provides the use of the above-mentioned herbicide-resistant gene or the above-mentioned gene editing vector in creating herbicide-resistant plant germplasm.

[0021] Beneficial effects: The present invention screens and isolates an endogenous herbicide-resistant gene from poplar, and the poplar herbicide-resistant gene is the PtSPL5 gene. The nucleotide sequence shown in SEQ ID No. 4 is shown in the embodiment. The present invention uses a gene editing method to knock out the PtSPL5 gene in poplar. Compared with wild-type poplar plants, the PtSPL5 knockout plants significantly improve the herbicide resistance under glufosinate treatment. At the same time, the growth traits are also tested, and it is found that the PtSPL5 knockout plants significantly promote the growth of poplars, increase the net photosynthetic rate and increase the lignin content compared with wild-type poplar plants. The present invention confirms that knocking out the PtSPL5 gene in poplar plants can significantly improve the herbicide characteristics of plants under glufosinate treatment conditions, and will not inhibit the growth characteristics of plants. Therefore, the PtSPL5 gene can be used to cultivate high-yield, high-quality, herbicide-resistant new varieties, which has great application value for the genetic improvement of comprehensive stress resistance of forest trees, and provides a new molecular tool for the genetic improvement of forest herbicide resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[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 This is a diagram showing the gene editing site and mutation information detection results of the PtSPL5 knockout mutant material;

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

[0025] Figure 4 Plant height, ground diameter, water use efficiency, net photosynthetic rate, intercellular CO2 concentration and lignin content of wild-type poplar plants and PtSPL5 knockout positive plants;

[0026] Figure 5 These are the leaf development traits of wild-type poplar plants and PtSPL5-positive knockout plants under glufosinate-ammonium treatment. DETAILED DESCRIPTION

[0027] The present invention provides a herbicide resistance gene, wherein the herbicide resistance gene encodes an amino acid sequence shown in any one of the following:

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

[0029] (2) a sequence having 85% or more homology with the amino acid sequence shown in SEQ ID No. 1;

[0030] (3) A sequence having similar effects while replacing, adding or reducing one or more amino acids in the sequence shown in (1) or (2).

[0031] The herbicide resistance gene of the present invention is an endogenous gene of poplar, and the amino acid sequence encoded by the gene is shown in SEQ ID No. 1: MLLVVLPTFFTFYFTLSPTDLHFSTSKATPHLASYHTIPQLCDILPKIMAARTLEGKHSSLKEKMINKDDFLIEDELDDDMEEYESGGGAGLADDEKKKGAGVMHGKRGTGSGGASPPSCQVEKCGANLTDAKRYHRRHKVCEVHAKSPAVVVAGLRQRFCQQCSRFHELAEFDETKRSCRRRLAGHNERRRKSTAESYGEASNRKGVNAPLKESPCRQADERGRFQINIPPQGSSSYKRSQIR*. Of course, mutations can be made to the amino acid sequence shown in SEQ ID No. 1, as long as the homology is more than 85% and the same or similar effects are met, the herbicide resistance gene of the present invention can be used.

[0032]

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

[0034] It has been verified by the examples that after the CDS sequence of the PtSPL5 gene is knocked out in poplar, the herbicide resistance of the plant can be significantly improved, and photosynthesis can be improved. The herbicide of the present invention can be glufosinate-ammonium, and after the gene is knocked out, the resistance of the plant to the herbicide is enhanced, which is mainly manifested in alleviating the symptoms of excessive accumulation of ammonium in plant leaves. The plant of the present invention can be a forest tree, and in one embodiment, poplar is used as an example for illustration.

[0035] The present invention also provides the use of the above-mentioned herbicide-resistant gene as a screening marker in plant transgenic culture.

[0036] The herbicide-resistant gene of the present invention has the function of regulating the plant's resistance to herbicides, and the gene can be used as a screening marker. If the herbicide-resistant gene exists in the transgenic cultured plant or the herbicide-resistant gene is overexpressed, it indicates that the transgenic plant has weak resistance to herbicides, and the target transgenic cultured plant can be screened by adjusting the herbicide concentration; if the herbicide-resistant gene is not contained in the transgenic cultured plant or the expression of the herbicide-resistant gene is inhibited, it indicates that the transgenic plant has strong resistance to herbicides, and the target transgenic cultured plant can be screened by using a high concentration of herbicide.

[0037] The present invention also provides a herbicide resistance molecular marker system and / or a molecular marker method developed using the herbicide resistance gene.

[0038] The herbicide-resistant gene of the present invention, especially 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 use the herbicide-resistant gene as a target gene.

[0039] In a specific embodiment of the present invention, a pair of primers for amplifying the PtSPL5 gene is also provided, and the primer sequences thereof 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-ammonium is 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 a specific embodiment of the present invention, the CRISPR-Cas9 method is used; 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, and the nucleotide sequence of the gRNA is as follows:

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

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

[0048] The gRNA provided by the present invention can knock out the PtSPL5 gene in the plant, thereby improving the herbicide resistance of the plant.

[0049] The present invention also provides a method for enhancing the herbicide resistance of plants, comprising knocking out or inhibiting the expression of the herbicide resistance gene.

[0050] In a specific embodiment of the present invention, the PtSPL5 gene is knocked out in a plant by a gene knockout method, and the plant may be a forest tree, such as the poplar tree used in the embodiment. The knockout of the present invention comprises the following steps: transforming the above-mentioned gene editing vector into the cells of the target plant to knock out the PtSPL5 gene. In one embodiment of the present invention, the transformation is completed by Agrobacterium transformation, and the Agrobacterium is GV3101. The present invention can effectively improve the net photosynthetic rate and water use efficiency of the plant by knocking out the PtSPL5 gene in the plant and regulating the expression amount of the PtSPL5 gene in the plant body, promoting plant height increase, and improving the herbicide resistance of the plant, which has great application value for the genetic improvement of the comprehensive stress resistance of forest trees.

[0051] The present invention also provides the use of the above-mentioned herbicide-resistant gene or the above-mentioned gene editing vector in creating herbicide-resistant plant germplasm.

[0052] In order to further illustrate the present invention, a herbicide-resistant gene and its application provided by the present invention are described in detail below in conjunction with examples, but they should not be construed as limiting the protection scope of the present invention.

[0053] Example 1

[0054] 1. Construction of PtSPL5 gene editing vector

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

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

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

[0058] (2) Vector construction: Select the CRISPR / Cas9 vector pSpCas9 suitable for plant cells ( Figure 1 ), connect gRNA into the vector to obtain the knockout vector.

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

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

[0061] Thaw the competent Agrobacterium GV3101 on ice, add 0.01-1μg of plasmid DNA containing the knockout vector to 100μL of GV3101 competent, mix well, and place on ice for 5min; cool quickly in liquid nitrogen for 5min, then quickly transfer to a 37℃ water bath for 5min, and place on ice for 5min; add 700μL of LB liquid culture medium without antibiotics and culture at 28℃, 230r / min vibration for 2-3h. After centrifugation at 6000rpm for 1 minute, take out 100μL of supernatant, resuspend the bacteria and spread on the LB plate containing antibiotics, and culture at 28℃ inverted for 2-3h to obtain Agrobacterium containing the knockout vector.

[0062] (2) Pre-cultivation of Poplar Leaves

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

[0064] (3) Infection and differentiation culture

[0065] The Agrobacterium containing the knockout vector was cultured in LB medium until the logarithmic growth phase. The culture conditions were 28°C and shaken at 180 rpm until the Agrobacterium grew to an OD of 600 The value is 0.6. The bacterial cells are centrifuged, the supernatant is discarded, and the cells are resuspended in a suspension (1 / 2MS + 30g / L sucrose + 100μmol / LAS). Gently shake the bacterial cells to fully resuspend them in the suspension, and then place them on ice for use. The cross-sectioned leaves are placed in the resuspended bacterial solution for 12 minutes, shake slowly, and after absorbing the excess bacterial solution with sterilized filter paper, the leaves are placed on the co-culture medium and cultured in the dark at 25°C for 2 to 3 days. The transformed leaf discs are obtained and transferred to the differentiation medium (MS + 6-BA 0.5mg / L + NAA 0.05 mg / L + sucrose 30g / L + 200mg / L timentin + 1mg / L hygromycin + agar 7g / L), and the light cycle is set to 16h light / 8h dark, and the ambient temperature is maintained at 25°C to promote stable transformation and expression of DNA.

[0066] (4) Rooting culture

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

[0068] 3. PtSPL5 knockout poplar plant growth characteristics improved

[0069] When the PtSPL5 knockout poplar transgenic plants and wild-type poplar plants (plants without foreign genes) reached 4 weeks old, their tissue culture seedlings were removed from the culture medium, transplanted into pre-prepared soil, and placed in a poplar culture room for 12 weeks of soil culture. Figure 3 As shown in Figure 2, compared with wild-type poplar plants, PtSPL5 knockout plants significantly promoted poplar growth, increased net photosynthetic rate, and increased lignin content ( Figure 4). Compared with wild-type poplar plants, PtSPL5 knockout poplar transgenic plants showed significant improvements in multiple growth indicators. Specifically, the plant height of the transgenic plants increased by 10.8% and the ground diameter increased by 14.6%. These changes indicate that PtSPL5 knockout plants promoted plant growth and development. In terms of photosynthesis and physiological adaptability, PtSPL5 knockout poplar plants also showed significant enhancements. Compared with the wild type, the net photosynthetic rate of PtSPL5 knockout plants increased by 0.26 μmol·m - 2 s - 1, indicating that it can more effectively utilize light energy for photosynthesis, improving the efficiency of energy conversion and organic matter synthesis. At the same time, the increase in intercellular CO2 concentration is 6μmol·mol - 1. It increases the raw material supply for photosynthesis and helps improve photosynthetic efficiency. In addition, the stomatal conductance increases by 2.8mmol·m - 2s - 1, indicating that the plant's ability to regulate gas exchange has been enhanced, and it can photosynthesize more efficiently while maintaining water balance. Water use efficiency increased by 0.07mmol·mo l- 1 shows that the plants are more economical in water utilization and can produce more photosynthetic products while consuming less water. The increase in maximum photochemical efficiency of 14.5% means that the plants are more efficient in converting light energy into chemical energy, which helps to improve the overall efficiency of photosynthesis. It is worth mentioning that the increase in lignin content by 20.4% not only enhances the mechanical strength of the plants, but also improves their disease resistance, making the plants stronger overall. These improvements not only enhance the growth potential of the plants, but also significantly improve their ability to adapt to environmental changes, which is of great significance for improving the productivity and ability of poplars to tolerate adversity.

[0070] 4. PtSPL5 knockout poplar plants have improved herbicide resistance

[0071] The PtSPL5 knockout plants were subcultured, and wild-type and knockout poplar tissue culture seedlings that had grown healthily for 30 to 40 days were selected. Fresh and disease-free leaves were taken as materials for glufosinate stress treatment. After the surface of the leaves was cleaned and disinfected, 2 to 3 wounds were made along the main veins of the leaves with a sterile scalpel, and they were temporarily placed on MS solid culture medium (MS + sucrose 20 g / L + agar 6 g / L) 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 in the figure, the PtSPL5 knockout plants significantly improved their herbicide resistance under glufosinate treatment. After 7 days of glufosinate treatment, the leaves of wild-type poplars and PtSPL5 knockout poplars showed obvious chlorosis and yellowing. Compared with wild-type poplar plants, the degree of leaf damage in PtSPL5 knockout poplars was significantly reduced, mainly manifested as local chlorosis. At the same time, with the increase of glufosinate concentration, the degree of damage to the leaves of wild-type poplars increased. After 14 days of glufosinate treatment, the leaves of wild-type poplars began to die irreversibly. The application of glufosinate triggered the generation of a large amount of reactive oxygen in the leaves, followed by the accumulation of ammonia, which led to the rapid disintegration of chloroplasts and the collapse and death of cells. However, the leaves of PtSPL5 knockout poplars were less damaged. Although the leaves showed slight yellowing in some parts, they remained green as a whole. Especially under the treatment of 6g / L glufosinate concentration, the leaves of wild-type poplars were most severely damaged, showing an overall yellowing and wilting scene, while the leaves of PtSPL5 knockout poplars showed a completely opposite situation. It can be seen that after being damaged by glufosinate, the PtSPL5 knockout poplars were still able to survive after a period of recovery, showing strong resistance. In contrast, wild-type poplars lacked the ability to recover from glufosinate damage and ultimately could not resist the impact of the damage.

[0072] It can be seen from the above examples that knocking out the PtSPL5 gene in poplar plants can significantly improve the herbicide properties of the plants under glufosinate-ammonium treatment conditions without inhibiting the growth characteristics of the plants.

[0073] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A herbicide resistance gene, characterized in that: The herbicide-resistant gene encodes an amino acid sequence as shown in any of the following: (1) the amino acid sequence shown in SEQ ID No. 1; (2) a sequence having 85% or more homology with the amino acid sequence shown in SEQ ID No. 1; (3) A sequence having similar effects while replacing, adding or reducing one or more amino acids in the sequence shown in (1) or (2).

2. Use of the herbicide-resistant gene according to claim 1 in regulating plant resistance to herbicides.

3. The application according to claim 2, characterized in that: The regulation includes inhibiting the expression of the herbicide resistance gene and enhancing the plant's resistance to herbicides.

4. Use of the herbicide-resistant gene according to claim 1 as a screening marker in plant transgenic culture.

5. A herbicide-resistant molecular marker system and / or molecular marker method developed using the herbicide-resistant gene according to claim 1.

6. The herbicide resistance molecular marker system and / or molecular marker method according to claim 5, characterized in that: The herbicide includes glufosinate-ammonium.

7. A gene editing vector for knocking out or inhibiting the herbicide-resistant gene according to claim 1.

8. The gene editing vector according to claim 7, characterized in that: The knockout or inhibition method includes using CRISPR-Cas9 method; The nucleotide sequences of the sgRNA used in the CRISPR-Cas9 method include SEQ ID No.2 and SEQ ID No.

3.

9. A method for enhancing plant resistance to herbicides, characterized in that: The method comprises knocking out or inhibiting the expression of the herbicide-resistant gene according to claim 1.

10. Use of the herbicide-resistant gene according to claim 1 or the gene editing vector according to claim 7 or 8 in creating herbicide-resistant plant germplasm.

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