Use of lpGID1 gene in regulating plant salt tolerance and / or growth and development traits

Abstract

This invention belongs to the field of plant molecular biology and relates to the application of the LpGID1 gene in regulating salt tolerance and / or growth and development traits in plants. The LpGID1 gene encodes a ryegrass transcription factor, LpGID1. This invention demonstrates that this gene can negatively regulate salt tolerance in plants, and that overexpression of this gene can also promote plant growth and development. This invention has significant practical implications for the breeding of new ryegrass varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology technology and relates to the application of the LpGID1 gene in regulating plant salt tolerance and / or growth and development traits. Background Technology

[0002] Ryegrass (Lolium perenne L.) is an excellent cool-season grass with advantages such as rapid growth, rich nutrition, and high economic benefits. Compared with other grass species, it can maintain good growth even under high salinity conditions. Therefore, it is widely used in pasture construction, ecological restoration, and soil protection, playing an important role in improving grassland productivity, conserving water and soil, and improving the ecological environment, and has high economic and ecological value.

[0003] However, soil salinization is one of the major abiotic stresses affecting plant growth and development and limiting agricultural production. Salt stress leads to ion imbalance, osmotic stress, and oxidative damage in plants, thereby inhibiting seed germination, slowing growth rate, reducing biomass, and in severe cases, even causing plant death. In the case of ryegrass, salt stress significantly inhibits its growth, manifested as reduced plant height, decreased biomass accumulation, decreased growth rate, and reduced tiller number. Furthermore, salt stress also leads to a decline in the appearance quality of ryegrass, an increase in leaf wilting coefficient, and an increase in the content of free proline, lipid peroxides, and soluble sugars. Therefore, improving the growth and development capacity of ryegrass has become an urgent goal of breeding and biotechnology research. Summary of the Invention

[0004] In view of this, the present invention provides the application of the LpGID1 gene in regulating plant salt tolerance and / or growth and development traits. The LpGID1 gene can negatively regulate plant salt tolerance, and overexpression of this gene can also promote plant growth and development. The purpose of the present invention is to solve or at least partially solve the problems in the prior art.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides the application of the LpGID1 gene in regulating plant salt tolerance and / or growth and development traits.

[0007] Preferably, the nucleotide sequence of the LpGID1 gene is shown in SEQ ID NO.1; and the amino acid sequence of the transcription factor LpGID1 is shown in SEQ ID NO.2.

[0008] Preferably, the growth and development traits include at least one of leaf length, leaf width, number of rosette leaves, and plant height.

[0009] Preferably, the regulation includes positive regulation or negative regulation.

[0010] More preferably, the positive regulation includes: positively regulating plant growth and development traits by promoting the expression of the LpGID1 gene; the negative regulation includes: negatively regulating plant salt tolerance by promoting the expression of the LpGID1 gene.

[0011] More preferably, promoting the expression of the LpGID1 gene includes the following steps:

[0012] The pCAMBIA1305-LpGID1-OE overexpression vector was constructed, and plant plants were transformed to obtain transgenic plants with increased LpGID1 gene expression.

[0013] Alternatively, construct the pCAMBIA1301U-LpGID1-OE overexpression vector, transform plant plants, and culture transgenic plants with increased LpGID1 gene expression.

[0014] More preferably, constructing the pCAMBIA1305-LpGID1-OE overexpression vector includes the following steps:

[0015] Using the LpGID1 gene as a template, PCR amplification was performed using primers pCAMBIA1305-LpGID1-F and pCAMBIA1305-LpGID1-R to obtain amplified fragment I. Amplified fragment I was then ligated into the pCAMBIA1305 vector via homologous recombination to obtain the pCAMBIA1305-LpGID1-OE overexpression vector.

[0016] More preferably, the nucleotide sequences of the primers pCAMBIA1305-LpGID1-F and pCAMBIA1305-LpGID1-R are shown in SEQ ID NO.5-6, respectively.

[0017] More preferably, constructing the pCAMBIA1301U-LpGID1-OE overexpression vector includes the following steps:

[0018] Using the LpGID1 gene as a template, PCR amplification was performed using primers pCAMBIA1301U-LpGID1-F and pCAMBIA1301U-LpGID1-R to obtain amplified fragment II. Amplified fragment II was then ligated into the pCAMBIA1301U vector via homologous recombination to obtain the pCAMBIA1301U-LpGID1-OE overexpression vector.

[0019] More preferably, the nucleotide sequences of primers pCAMBIA1301U-LpGID1-F and pCAMBIA1301U-LpGID1-R are shown in SEQ ID NO.7-8, respectively.

[0020] More preferably, the transformed plant includes the following steps:

[0021] The pCAMBIA1305-LpGID1-OE overexpression vector was transferred into plant plants using Agrobacterium-mediated transformation.

[0022] Alternatively, the pCAMBIA1301U-LpGID1-OE overexpression vector can be transferred into plant plants via Agrobacterium-mediated transformation.

[0023] Preferably, the plant is Arabidopsis thaliana or ryegrass.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0025] This invention cloned and identified the LpGID1 gene from the ryegrass genome. Overexpression of this gene significantly reduces the plant's tolerance to salt stress and promotes plant growth and development. This invention has important practical significance for breeding new varieties of ryegrass. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The figure shows the analysis results of the amino acid sequence of transcription factor LpGID1;

[0028] Figure 2 This is a phylogenetic tree analysis result of transcription factor LpGID1;

[0029] Figure 3 The images show the phenotypic results of three transgenic Arabidopsis thaliana lines after planting; Figure A shows the leaf results; Figure B shows the plant width results; and Figure C shows the plant height results.

[0030] Figure 4The following are statistical analysis charts of phenotypic data of three transgenic Arabidopsis thaliana lines after planting: Chart A shows the statistical analysis of leaf length; Chart B shows the statistical analysis of leaf width; Chart C shows the statistical analysis of the number of rosette leaves; Chart D shows the statistical analysis of plant height at 14 days; and Chart E shows the statistical analysis of plant height at 21 days.

[0031] Figure 5 The figure shows the phenotypic results of Arabidopsis thaliana plants under salt stress.

[0032] Figure 6 Figure 1 shows the results of physiological indicators in the salt stress experiment of Arabidopsis thaliana plants; Figure 2 shows the statistical analysis of relative conductivity; Figure 3 shows the statistical analysis of MDA content; Figure 4 shows the statistical analysis of soluble sugar content; and Figure 5 shows the statistical analysis of 20-day survival rate.

[0033] Figure 7 Figure A shows the phenotypic results of salt stress experiments on ryegrass plants; Figure B shows the phenotypic results of plant height in ryegrass plants.

[0034] Figure 8 Figure 1 shows the results of physiological indicators of salt stress experiments on ryegrass plants; Figure A is a statistical analysis of relative conductivity; Figure B is a statistical analysis of soluble sugar content; and Figure C is a statistical analysis of MDA content. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0036] It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of embodiments. Furthermore, in the description of this invention, the term "comprising" means "including but not limited to". Various embodiments of the invention may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single digits within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Additionally, whenever a numerical range is indicated herein, it means including any referenced number (fraction or integer) within the indicated range.

[0037] Gibberellins are key hormones regulating plant growth and development, widely involved in processes such as seed germination, stem and leaf elongation, and flowering. DELLA family proteins, as repressors of the gibberellin signaling pathway, can improve plant survival rates under salt stress by restricting growth. The core of the GA signaling pathway is the inhibitory effect of DELLA proteins on downstream growth processes. When GA binds to its receptor GID1, it promotes the degradation of DELLA proteins, thereby relieving their inhibitory effect and activating downstream gene expression. However, research on the specific function of the GID1 homolog (LpGID1) is even more lacking. Whether it participates in the salt stress response and how to use this gene to improve the salt tolerance of ryegrass through genetic engineering are all urgent scientific problems and technological gaps that need to be addressed. Therefore, in-depth analysis of the function of the ryegrass LpGID1 gene is of significant theoretical and applied value for overcoming the bottleneck in ryegrass stress resistance breeding and cultivating new salt-tolerant varieties.

[0038] The LpGID1 gene is a homolog of the GID1 gene, but its specific function is currently unclear. Based on the potential application of the LpGID1 gene in plant trait regulation, this invention explores in depth the application of the LpGID1 gene in regulating plant salt tolerance and growth and development traits.

[0039] The following specific embodiments further illustrate the application of the LpGID1 gene in regulating plant salt tolerance and / or growth and development traits. This section further illustrates the content of the invention with reference to specific embodiments, but should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, such as the conditions described in *Molecular Cloning: A Laboratory Manual (Fourth Edition)* published by Cold Spring Harbor Laboratory or the conditions recommended by the manufacturer. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in the art.

[0040] In the following embodiments, the ryegrass is the commercially available variety "Perennial Ryegrass L261", the seeds of which were purchased from Beijing Zhengdao Seed Industry Co., Ltd., and planted in the growth room of the North Building of the Gardening Building of Huazhong Agricultural University.

[0041] In the following embodiments, the pCAMBIA1301U vector was obtained by the method described in the reference ([1] Sun Tianxiao. Functional study of heat shock transcription factors HSFAs and HSFCs subfamily genes in perennial ryegrass [D]. Huazhong Agricultural University, 2021. DOI:10.27158 / d.cnki.ghznu.2021.000047.).

[0042] In the following embodiments, the Arabidopsis thaliana is Columbia-0, which was originally obtained by our research group through experimental techniques such as genetic cultivation. The public can obtain relevant germplasm resources from the applicant, which can only be used to repeat the experiments of this invention and may not be used for other purposes.

[0043] In the following embodiments, the inflorescence infection method is based on the method described in the published patent (CN120665887A, A tulip transcription factor TgTCP2 that regulates plant sugar transport and growth and development and its application).

[0044] In the following embodiments, the callus infection method is described in reference ([1] Sun Tianxiao. Functional study of heat shock transcription factors HSFAs and HSFCs subfamily genes in perennial ryegrass [D]. Huazhong Agricultural University, 2021. DOI:10.27158 / d.cnki.ghznu.2021.000047.). In the following embodiments, the genes involved, encoded proteins and related primer sequences are shown in Table 1.

[0045] Table 1: Sequences of genes, encoded proteins, and related primers in the embodiments of the present invention

[0046]

[0047] In the following examples, all data are expressed as mean ± standard deviation (Mean ± SD); statistical analysis was performed using IBM SPSS statistics software (IBM Corporation, USA); one-way ANOVA was used for comparisons among multiple groups, and Tukey's posthoc test was used for pairwise comparisons if significant differences were found; P < 0.05 indicated that the difference was statistically significant. "Indicates a significant difference (P < 0.05)," "" indicates a highly significant difference (P < 0.01).

[0048] Example 1 Cloning of the LpGID1 gene

[0049] GID1 protein, an important binding protein in the gibberellin signaling pathway, plays a crucial role in plant stress resistance. It is known that the binding of GID1 protein to active GA facilitates the transmission of GA signals to the DELLA protein, thereby regulating the expression of GA synthesis genes. Furthermore, the degradation of DELLA protein can improve plant survival under stress conditions by reducing the accumulation of reactive oxygen species (ROS). The LpGID1 gene is a homolog of the GID1 gene, and its relative expression levels have the potential to influence plant growth, development, and salt tolerance.

[0050] 1. Total RNA extraction and cDNA synthesis

[0051] Total RNA was extracted from ryegrass using a standard RNA extraction kit (Beijing TransGen Biotech Co., Ltd., catalog number: ET101-01-V2), and cDNA was obtained by reverse transcription using a reverse transcription kit (Beijing TransGen Biotech Co., Ltd., catalog number: AT311-02).

[0052] 2. PCR amplification

[0053] Using the cDNA obtained in Section 1.1 of this embodiment as a template, the LpGID1 gene (as shown in SEQ ID NO.3) was amplified using primers LpGID1-F (as shown in SEQ ID NO.3) and LpGID1-R (as shown in SEQ ID NO.4).

[0054] Figure 1 The diagram shows the analysis results of the amino acid sequence of transcription factor LpGID1. Figure 2 The phylogenetic tree analysis results for transcription factor LpGID1 are shown. The results show that transcription factor LpGID1 has 224 amino acids (as shown in SEQ ID NO.2).

[0055] Example 2: Construction and genetic transformation of overexpression vectors

[0056] 1. Constructing overexpression vectors

[0057] (1) Using the LpGID1 gene as a template, PCR amplification was performed using primers pCAMBIA1305-LpGID1-F (as shown in SEQ ID NO.5) and pCAMBIA1305-LpGID1-R (as shown in SEQ ID NO.6) to obtain amplified fragment I. Amplified fragment I was ligated into the pCAMBIA1305 vector (Biovector NTCC Inc., catalog number: 3700276) via homologous recombination. The upstream restriction enzyme site was XbaI, and the downstream restriction enzyme site was KpnI. Amplified fragment I was located downstream of the CaMV 35S promoter to obtain recombinant plasmid 1. Recombinant plasmid 1 was transformed into Agrobacterium strain GV3101 (Beijing Coollab Technology Co., Ltd., catalog number: CC405). Single-clone colony PCR identification was performed. The positive result obtained was the pCAMBIA1305-LpGID1-OE overexpression vector, which was used to transform Arabidopsis thaliana plants.

[0058] (2) Using the LpGID1 gene as a template, PCR amplification was performed using primers pCAMBIA1301U-LpGID1-F (as shown in SEQ ID NO.7) and pCAMBIA1301U-LpGID1-R (as shown in SEQ ID NO.8) to obtain amplified fragment II. Amplified fragment II was ligated into the pCAMBIA1301U vector via homologous recombination, with the upstream restriction enzyme site being KpnⅠ and the downstream restriction enzyme site being SpeI. Amplified fragment II was located downstream of the CaMV 35S promoter, resulting in recombinant plasmid 2. Recombinant plasmid 2 was transformed into Agrobacterium strain EHA105 (Beijing Cooler Master Technology Co., Ltd., catalog number: CC403). Single-clone colony PCR identification was performed, and the positive result obtained was the pCAMBIA1301U-LpGID1-OE overexpression vector, which was used to transform ryegrass plants.

[0059] 2. Genetic transformation

[0060] Agrobacterium GV3101 containing the pCAMBIA1305-LpGID1-OE overexpression vector was introduced into Arabidopsis plants via inflorescence infection. Agrobacterium EHA105 containing the pCAMBIA1301U-LpGID1-OE overexpression vector was introduced into ryegrass callus tissue via callus infection for overexpression. After determining the transgenic lines, Arabidopsis were propagated to the T2 generation, and positive ryegrass plants were selected for uniformization. Conditional propagation culture (the plants were dug up with roots, divided into small plants containing 1-2 tillers, and the upper 1 / 3 of the leaves were cut off to reduce transpiration) was used to obtain 3 transgenic Arabidopsis thaliana lines (OE-14#, OE-15# and OE-16#) and 3 transgenic Arabidopsis thaliana lines (OE-4#, OE-5# and OE-6#) with LpGID1 gene overexpression, which were used to verify the reproducibility of this genetic transformation operation.

[0061] Example 3: Application of the LpGID1 gene in promoting plant growth and development

[0062] This embodiment further verifies the role of the LpGID1 gene in plant growth and development by comparing and analyzing the phenotypic data of three transgenic Arabidopsis thaliana lines and wild-type Arabidopsis thaliana plants during the vegetative and reproductive growth stages. Specifically, wild-type Arabidopsis thaliana without transgenic treatment was used as the control group (WT). After 14 days of germination, plants with consistent growth were selected from each group and planted. Phenotypic characteristics of each group were recorded by photographing after planting began (leaf size, plant width, and plant height; dynamic recording of leaf changes; plant width recorded at week 2 of planting; plant height recorded at week 3 of planting). Detailed phenotypic data of each group (leaf length, leaf width, number of rosette leaves, plant height at 14 days, and plant height at 21 days) were recorded at week 3 of planting.

[0063] Figure 3 The following figures show the phenotypic results of three transgenic Arabidopsis thaliana lines after planting: Figure A shows the leaf results; Figure B shows the plant width results; and Figure C shows the plant height results. Figure 4The following are statistical analysis figures of phenotypic data of three Arabidopsis thaliana transgenic lines after transplanting: Figure A shows the statistical analysis of leaf length; Figure B shows the statistical analysis of leaf width; Figure C shows the statistical analysis of rosette leaf number; Figure D shows the statistical analysis of plant height at 14 days; and Figure E shows the statistical analysis of plant height at 21 days. The results showed that 14 days after transplanting, the leaf length, leaf width, and rosette leaf number of the Arabidopsis thaliana transgenic group were significantly higher than those of the wild-type control group; 21 days after transplanting, the plant height of the Arabidopsis thaliana transgenic group was slightly higher than that of the wild-type control group. These results indicate that overexpression of the LpGID1 gene significantly promotes the vegetative growth of the aboveground parts of Arabidopsis thaliana and the development of rosette leaves, resulting in larger leaf area and faster tillering.

[0064] Example 4: Application of LpGID1 gene in negative regulation of salt tolerance in Arabidopsis thaliana

[0065] All experiments in this embodiment were grouped according to the procedures described in Example 3, divided into a wild-type control group (WT) and Arabidopsis transgenic plants (plants OE-14# and OE-16#). The control and transgenic plants were disinfected with a 1% sodium hypochlorite solution and sown on 1 / 2 MS medium (Beijing Cooler Master Technology Co., Ltd., catalog number: PM1062-307). After 3 days of vernalization at low temperature, they were placed vertically and grown for 4 days before being transferred to a mixed substrate of peat moss and vermiculite in a 4:1 ratio. The control and transgenic plants were subjected to salt stress treatment by irrigation with 1 / 2 Hoagland nutrient solution (Beijing Cooler Master Technology Co., Ltd., catalog number: NSP1021) containing a 200 mM NaCl solution for 20 consecutive days, with 3 biological replicates per group. An equal volume of 1 / 2 Hoagland nutrient solution was used as a mock control group to perform the above operations. Photos were taken at the beginning and 10 days after the salt stress treatment. Phenotypic characteristics of seedlings in each group at d and 20 days; relative conductivity (%), MDA content (malondialdehyde, μg / g), soluble sugar content (μg / g), and survival rate (%) of each group of plants 20 days after salt stress treatment were measured respectively; relative conductivity was determined according to the method described in the published patent (CN121182858A, Application of VDAC1 / 3a alleles in regulating cold tolerance of tomato); MDA content was determined using a plant malondialdehyde MDA detection kit (Shanghai Enzyme-Linked Biotechnology Co., Ltd., catalog number: ml094965); and soluble sugar content was determined using a plant soluble sugar content test kit (Shanghai Enzyme-Linked Biotechnology Co., Ltd., catalog number: ml076787).

[0066] Figure 5 The results of the salt stress experiment on Arabidopsis thaliana plants are shown in the figure. Figure 6 The results of physiological indicators in the Arabidopsis thaliana plant salt stress experiment are shown in the figure; Figure A is a statistical analysis of relative conductivity; Figure B is a statistical analysis of MDA content; Figure C is a statistical analysis of soluble sugar content; and Figure D is a statistical analysis of 20-day survival rate. The results show that the relative conductivity of the Arabidopsis thaliana transgenic group (…) Figure 6 Figure A in the diagram shows that the MDA content was higher than that in the wild-type control group. Figure 6 Figure B in the diagram) and soluble sugar content ( Figure 6 The C-axis in the figure clearly showed an increase, further indicating that plants overexpressing the LpGID1 gene suffered more severe physiological damage under salt stress; 20 days after treatment, the leaves of the Arabidopsis transgenic plants showed large-scale whitening, wilting, and even death. Figure 5 The survival rate of the virgin plants is only about 60%, while the survival rate of wild-type plants remains above 95%. Figure 6 (See Figure D in the diagram). The results indicate that overexpression of the LpGID1 gene can negatively regulate the salt tolerance of Arabidopsis thaliana.

[0067] Example 5: Application of the LpGID1 gene in negatively regulating salt tolerance in ryegrass

[0068] In this embodiment, all experiments were divided into wild-type ryegrass control group (WT) and ryegrass transgenic plants (plants OE-4#, OE-5#, and OE-6#). Both control and transgenic ryegrass plants were divided and cultured, with four biological replicates per plant line and nine or ten tillers per pot. After the plants recovered their growth, they were irrigated with 1 / 2 Hoagland nutrient solution every 5 days for one month, then uniformly pruned to a height of 12 cm. Salt stress treatment was then applied using a 600 mM NaCl solution. A mock control group was also irrigated with an equal amount of water. The growth and height of each group of plants were photographed and recorded at the start of planting after salt stress treatment, on day 7, day 14, and day 17. Relative conductivity, soluble sugar content, and MDA content were measured on day 14 after salt stress treatment, using the same methods as described in Example 4.

[0069] Figure 7 The results of the salt stress experiment on ryegrass plants are shown in the figure; Figure A shows the phenotypic results of the plant height of ryegrass plants; Figure B shows the statistical analysis of the plant height of ryegrass plants. Figure 8The results of the physiological indicators of ryegrass plants under salt stress are shown in the figure. Figure A shows the statistical analysis of relative conductivity; Figure B shows the statistical analysis of soluble sugar content; and Figure C shows the statistical analysis of MDA content. The results showed that after 7 days of treatment, the growth of both the transgenic and control groups was inhibited by salt stress, with differences in plant height. The height of the control group plants was 14.61 cm, while the heights of the transgenic plants were 16.36 cm, 17.66 cm, and 17.16 cm, respectively. Figure 7 The MDA content in the transgenic group did not change significantly after stress, but the MDA content in the control group increased significantly, approximately twice that of the transgenic group. Figure 8 (See Figure C in the diagram). The results showed that the transgenic plants exhibited less lipid peroxidation in their cell membranes and greater membrane system stability. Furthermore, the soluble sugar content of all plants increased after salt stress, with the control group showing a particularly significant increase, reaching 2.38 times the pre-treatment level. Its total sugar content was also significantly higher than that of the transgenic lines. Figure 8 (See Figure B in the table). The results indicate that overexpression of the LpGID1 gene can negatively regulate the salt tolerance of ryegrass.

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of LpGID1 gene in regulating plant salt tolerance and / or growth and development traits.

2. The application as described in claim 1, characterized in that, The nucleotide sequence of the LpGID1 gene is shown in SEQ ID NO. 1; Alternatively, the amino acid sequence of the transcription factor LpGID1 is shown in SEQ ID NO.

2.

3. The application as described in claim 1, characterized in that, The growth and development traits include at least one of leaf length, leaf width, number of rosette leaves, and plant height.

4. The application as described in claim 1, characterized in that, The regulation includes positive regulation or negative regulation; specifically, positive regulation of plant growth and development traits is achieved by promoting the expression of the LpGID1 gene; negative regulation of plant salt tolerance is achieved by promoting the expression of the LpGID1 gene.

5. The application as described in claim 4, characterized in that, Promoting LpGID1 gene expression involves the following steps: The pCAMBIA1305-LpGID1-OE overexpression vector was constructed, and plant plants were transformed to obtain transgenic plants with increased LpGID1 gene expression. Alternatively, construct the pCAMBIA1301U-LpGID1-OE overexpression vector, transform plant plants, and culture transgenic plants with increased LpGID1 gene expression.

6. The application as described in claim 5, characterized in that, The construction of the pCAMBIA1305-LpGID1-OE overexpression vector includes the following steps: Using the LpGID1 gene as a template, PCR amplification was performed using primers pCAMBIA1305-LpGID1-F and pCAMBIA1305-LpGID1-R to obtain amplified fragment I. Amplified fragment I was then ligated into the pCAMBIA1305 vector via homologous recombination to obtain the pCAMBIA1305-LpGID1-OE overexpression vector. The nucleotide sequences of primers pCAMBIA1305-LpGID1-F and pCAMBIA1305-LpGID1-R are shown in SEQ ID NO.5-6, respectively.

7. The application as described in claim 5, characterized in that, The construction of the pCAMBIA1301U-LpGID1-OE overexpression vector includes the following steps: Using the LpGID1 gene as a template, PCR amplification was performed using primers pCAMBIA1301U-LpGID1-F and pCAMBIA1301U-LpGID1-R to obtain amplified fragment II. Amplified fragment II was then ligated into the pCAMBIA1301U vector via homologous recombination to obtain the pCAMBIA1301U-LpGID1-OE overexpression vector. The nucleotide sequences of primers pCAMBIA1301U-LpGID1-F and pCAMBIA1301U-LpGID1-R are shown in SEQ ID NO.7-8, respectively.

8. The application as described in claim 5, characterized in that, The transformed plantlets include the following steps: The pCAMBIA1305-LpGID1-OE overexpression vector was transferred into plant plants using Agrobacterium-mediated transformation. Alternatively, the pCAMBIA1301U-LpGID1-OE overexpression vector can be transferred into plant plants via Agrobacterium-mediated transformation.

9. The application as described in any one of claims 1-8, characterized in that, The plant in question is either Arabidopsis thaliana or ryegrass.

Images