Use of lsMOC1 gene or biological material containing lsMOC1 gene-related biological material

Abstract

This invention belongs to the field of plant genetic engineering technology, specifically involving LsMOC1 Genes or containing LsMOC1 Application of gene-related biomaterials. LsMOC1 The nucleotide sequence of the gene is shown in SEQ ID NO.3. This invention utilizes the recombinant expression vector pCAMBIA1300-GFP- LsMOC1 This recombinant expression vector was introduced into the model plant Arabidopsis thaliana, and overexpression was obtained. LsMOC1 Transgenic Arabidopsis thaliana plants. Experiments revealed: LsMOC1 Gene overexpression can delay the phenological stage of plants, slow down growth, prolong the growth cycle, and also improve the plant's resistance to drought stress and gibberellin stress.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically involving LsMOC1 Genes or containing LsMOC1 Application of gene-related biomaterials. Background Technology

[0002] Lysimachia ( Leymus secalinus Leymus chinensis (also known as Elephantgrass) is a perennial high-quality forage grass belonging to the genus Erythrina in the Poaceae family. It is widely distributed in temperate grasslands of Eurasia, playing a particularly important role in the ecosystems of arid, semi-arid, and saline-alkali regions of Northeast, North, and Northwest my country. With its well-developed rhizomatous system, it is a pioneer plant and key species for grassland ecological restoration, degraded grassland management, saline-alkali land improvement, and soil and water conservation. Furthermore, Leymus chinensis is of excellent quality, palatability, and high nutritional value, making it an important forage resource and a significant source of crude protein for ruminant livestock during winter and spring supplementation. However, while wild Leymus chinensis germplasm is abundant, it generally has a short growing season and premature yellowing and withering, resulting in insufficient biomass accumulation within its limited growth cycle. Moreover, its slow regeneration after mowing and the resulting year-round supply shortage severely restrict its large-scale application in grassland animal husbandry and desertification control. Simultaneously, with the continuous changes in global climate change and the ecological environment, Leymus chinensis faces challenges from various abiotic stresses such as drought, salinity, and low temperatures.

[0003] Improving the stress resistance of *Leymus chinensis* not only enhances its survival ability in complex environments but also improves its stability within ecosystems, further promoting vegetation restoration and ecological balance. Extending the growth cycle of *Leymus chinensis* can increase its biomass accumulation, thereby enhancing its economic value as forage and its ecological service functions. While traditional breeding methods can improve the traits of *Leymus chinensis*, they suffer from drawbacks such as long cycles and low efficiency. Furthermore, the complex genome of *Leymus chinensis* and its long breeding cycle limit rapid improvement. Molecular breeding is one of the effective means to achieve rapid, precise, and large-scale improvement. Therefore, identifying and utilizing key genes regulating the traits of *Leymus chinensis* is of great significance for optimizing its production and improving its quality. Summary of the Invention

[0004] To address the above problems, the present invention provides LsMOC1 Genes or containing LsMOC1 Application of gene-related biomaterials. LsMOC1 The nucleotide sequence of the gene is shown in SEQ ID NO.3. Experiments in this invention have shown that overexpression... LsMOC1 After gene modification, the phenological period of plants can be delayed, growth can be slowed down, the growth cycle can be extended, and the plant's resistance to drought stress and gibberellin stress can be improved.

[0005] To achieve the above objectives, the specific technical solution of the present invention is as follows:

[0006] The first aspect of the present invention provides LsMOC1 Genes or containing LsMOC1 The application of gene-related biological materials, wherein the application is to prolong the growth cycle of plants or improve the stress resistance of plants; LsMOC1 The nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0007] Furthermore, the method of application is as follows: improving the quality of the plant through transgenic technology or gene editing technology. LsMOC1 The level of gene expression is adjusted to prolong the plant's growth cycle.

[0008] Furthermore, the stress is any one or more of drought stress and gibberellin stress.

[0009] Furthermore, the method of application is as follows: improving the quality of the plant through transgenic technology or gene editing technology. LsMOC1 The expression level of genes can be adjusted to enhance the plant's ability to resist drought or gibberellin stress.

[0010] Furthermore, the biomaterial includes any one of the following:

[0011] a. The above LsMOC1 A gene-encoded protein, the amino acid sequence of which is shown in SEQ ID NO.4;

[0012] b. Includes the above LsMOC1 Recombinant gene expression vectors;

[0013] c. Includes the above LsMOC1 Recombinant microbial strains of genes.

[0014] Furthermore, the plant is *Lysimachia christinae*, *Arabidopsis thaliana*, or tobacco.

[0015] A second aspect of the present invention provides a method for cultivating transgenic plants with long growth cycles, comprising the following steps:

[0016] Will contain the above-mentioned LsMOC1 The recombinant gene expression vector is introduced into the target plant, enabling... LsMOC1 Genes are overexpressed in a target plant to obtain transgenic plants with a longer growth cycle than the target plant.

[0017] Furthermore, the recombinant expression vector is pCAMBIA1300-GFP- LsMOC1 The recombinant expression vector is composed of LsMOC1 The gene was obtained after being introduced into the overexpression vector pCAMBIA1300-GFP.

[0018] Furthermore, the target plant is *Lysimachia christinae*, *Arabidopsis thaliana*, or tobacco.

[0019] A third aspect of the present invention provides a method for cultivating plants with enhanced stress resistance, comprising the following steps:

[0020] Will contain the above-mentioned LsMOC1 The recombinant gene expression vector is introduced into the target plant, enabling... LsMOC1 The gene is overexpressed in the target plant to obtain a plant with enhanced stress resistance, wherein the stress resistance of the enhanced stress resistance plant is stronger than that of the target plant.

[0021] Furthermore, the recombinant expression vector is pCAMBIA1300-GFP- LsMOC1 The recombinant expression vector is composed of LsMOC1 The gene was obtained after being introduced into the overexpression vector pCAMBIA1300-GFP.

[0022] Furthermore, the stress is drought stress or gibberellin stress.

[0023] Furthermore, the target plant is *Lysimachia christinae*, *Arabidopsis thaliana*, or tobacco.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] This invention is disclosed for the first time. LsMOC1 Genes or containing LsMOC1 Application of gene-related biomaterials in plant trait improvement LsMOC1 The nucleotide sequence of the gene is shown in SEQ ID NO.3. LsMOC1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.4. This invention utilizes the construction of the recombinant expression vector pCAMBIA1300-GFP- LsMOC1 This recombinant expression vector was introduced into the model plant Arabidopsis thaliana, and overexpression was obtained. LsMOC1 Transgenic Arabidopsis thaliana plants. Experiments showed that overexpression... LsMOC1 After gene modification, the phenological period of plants can be delayed, growth can be slowed down, the growth cycle can be extended, and the plant's resistance to drought stress and gibberellin stress can be improved. 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 For amplification LsMOC1 The agarose gel electrophoresis image of the target gene shows the DNA marker in the leftmost lane and PCR amplification products in lanes 1-3, each 1281 bp in size.

[0028] Figure 2 This is a map of the pCAMBIA1300-GFP overexpression vector.

[0029] Figure 3 for LsMOC1 Positive colony verification was performed by transforming GV3101 Agrobacterium chemicompetent cells with the overexpression vector pCAMBIA1300-GFP; the leftmost lane is the DNA marker, and lanes 1-6 are for the constructed pCAMBIA1300-GFP- LsMOC1 sample.

[0030] Figure 4 For overexpression LsMOC1 Screening diagram of positive transgenic Arabidopsis thaliana T1 generation seedlings.

[0031] Figure 5 For overexpression LsMOC1 Agarose gel electrophoresis image of transgenic Arabidopsis T1 generation positive seedlings for gene verification. Lane 1 is the control, and lanes 2 to 11 are transgenic Arabidopsis samples.

[0032] Figure 6 For overexpression LsMOC1 Phenotypic and statistical data of the underground parts of transgenic Arabidopsis thaliana. Col-0 It is a wild-type Arabidopsis thaliana. LsMOC1- L1, LsMOC1- L2 and LsMOC1- L3 are all LsMOC1 Transgenic Arabidopsis thaliana overexpressing genetically modified strains; white dashed lines distinguish different Arabidopsis thaliana samples; Figure A shows wild-type Arabidopsis thaliana grown for two weeks and... LsMOC1 Figure B shows the root phenotype of transgenic Arabidopsis overexpressing the gene; Figure C shows the root phenotype data analysis results of two-week-old Arabidopsis; Figure C shows the secondary root quantity data analysis results of two-week-old Arabidopsis. Identical lowercase letters indicate no significant differences between groups. P≥ 0.05.

[0033] Figure 7 For overexpression LsMOC1 Phenotypic and statistical data of aboveground leaf area in transgenic Arabidopsis thaliana Col-0 It is a wild-type Arabidopsis thaliana. LsMOC1- L1, LsMOC1- L2 and LsMOC1- L3 are all LsMOC1Transgenic overexpression of Arabidopsis thaliana, scale bar: 2cm; Figures A and B show the aboveground leaf phenotypes of Arabidopsis thaliana at the bolting stage and the seed-setting stage, respectively; Figures C and D show the statistical graphs of aboveground leaf coverage area of ​​Arabidopsis thaliana at the bolting stage and the seed-setting stage, respectively; different lowercase letters a and b indicate differences between groups. P <0.05.

[0034] Figure 8 For overexpression LsMOC1 Statistical graphs of aboveground phenotypes and data from transgenic Arabidopsis thaliana. Col-0 It is a wild-type Arabidopsis thaliana. LsMOC1- L1, LsMOC1- L2 and LsMOC1- L3 are all LsMOC1 Transgenic overexpression of Arabidopsis thaliana, scale bar: 3cm; Figures A and B show the aboveground main branch phenotype of Arabidopsis thaliana at the seed-setting stage and the aboveground main branch phenotype of Arabidopsis thaliana at maturity, respectively; Figures C, D, and E show the statistical graphs of main branch height of Arabidopsis thaliana at the seed-setting stage, the statistical graph of the number of lateral branches of Arabidopsis thaliana at maturity, and the statistical graph of main branch height of Arabidopsis thaliana at maturity, respectively; different lowercase letters a and b indicate differences between groups. P <0.05.

[0035] Figure 9 Overexpression under salt stress LsMOC1 Phenotypic diagram of transgenic Arabidopsis thaliana. Col-0 It is a wild-type Arabidopsis thaliana. LsMOC1- L1, LsMOC1- L2 and LsMOC1- L3 are all LsMOC1 Transgenic Arabidopsis overexpression, with white dashed lines used to distinguish different Arabidopsis samples; Figure A shows wild-type and overexpressing Arabidopsis samples on MS medium containing 100 mM NaCl. LsMOC1 Root phenotype diagram of transgenic Arabidopsis thaliana; Figure B shows the control group (wild type) and overexpression. LsMOC1 Phenotypic diagram of the aboveground parts of transgenic Arabidopsis thaliana; Figure C shows the pheenotypic diagram of the aboveground parts of Arabidopsis thaliana under 200 mM salt stress treatment.

[0036] Figure 10 Overexpression under salt stress LsMOC1 Phenotypic analysis and enzyme activity data statistics of transgenic Arabidopsis thaliana. Col-0 It is a wild-type Arabidopsis thaliana. LsMOC1- L1, LsMOC1- L2 and LsMOC1- L3 are all LsMOC1Transgenic overexpression of Arabidopsis thaliana; Figures A and B are statistical graphs of taproot length and lateral root number in Arabidopsis thaliana on MS medium containing 100 mM NaCl, respectively; Figures C, D, and E are analyses of POD, MDA, and SOD enzyme activities in Arabidopsis thaliana under 200 mM salt stress treatment, respectively; different lowercase letters a, b, and c indicate differences between groups. P <0.05.

[0037] Figure 11 Overexpression under drought stress LsMOC1 Phenotypic diagram of transgenic Arabidopsis thaliana. Col-0 It is a wild-type Arabidopsis thaliana. LsMOC1- L1, LsMOC1- L2 and LsMOC1- L3 are all LsMOC1 Transgenic Arabidopsis overexpression, with white dashed lines used to distinguish different Arabidopsis samples; Figure A shows wild-type and overexpressing Arabidopsis samples on MS medium containing 50 mM mannitol. LsMOC1 Root phenotype diagram of transgenic Arabidopsis thaliana; Figure B shows the control group (wild type) and overexpression. LsMOC1 Figure C shows the aboveground phenotypic diagram of transgenic Arabidopsis thaliana; Figure C shows the aboveground phenotypic diagram of Arabidopsis thaliana under natural drought treatment.

[0038] Figure 12 Overexpression under drought stress LsMOC1 Phenotypic analysis and enzyme activity data statistics of transgenic Arabidopsis thaliana. Col-0 It is a wild-type Arabidopsis thaliana. LsMOC1- L1, LsMOC1- L2 and LsMOC1- L3 are all LsMOC1 Transgenic overexpression of Arabidopsis thaliana; Figures A and B are statistical graphs of taproot length and lateral root number of Arabidopsis thaliana on MS medium containing 50 mM mannitol, respectively; Figures C, D, and E are analyses of POD enzyme activity, MDA enzyme activity, and SOD enzyme activity of Arabidopsis thaliana under natural drought treatment, respectively; different lowercase letters a, b, and c indicate differences between groups. P <0.05.

[0039] Figure 13 Overexpression under abscisic acid (ABA) stress treatment LsMOC1 Phenotypic diagram of transgenic Arabidopsis thaliana. Col-0 It is a wild-type Arabidopsis thaliana. LsMOC1- L1, LsMOC1- L2 and LsMOC1- L3 are all LsMOC1 Transgenic Arabidopsis overexpression, with white dashed lines used to distinguish different Arabidopsis samples; Figure A shows wild-type and overexpressing Arabidopsis samples on MS medium containing 25 µM ABA. LsMOC1 Root phenotype diagram of transgenic Arabidopsis thaliana; Figure B shows the control group (wild type) and overexpression. LsMOC1 Phenotypic diagram of the aboveground parts of transgenic Arabidopsis thaliana; Figure C shows the pheenotypic diagram of the aboveground parts of Arabidopsis thaliana under 100 µM ABA stress treatment.

[0040] Figure 14 Overexpression under abscission acid stress treatment LsMOC1 Analysis and statistical chart of enzyme activity data of transgenic Arabidopsis thaliana. Col-0 It is a wild-type Arabidopsis thaliana. LsMOC1- L1, LsMOC1- L2 and LsMOC1- L3 are all LsMOC1 Transgenic Arabidopsis overexpressing; Figures A and B are statistical graphs of taproot length and lateral root number in wild-type and transgenic Arabidopsis on MS medium containing 25 µM ABA, respectively; Figures C, D, and E are analyses of POD, MDA, and SOD enzyme activities in Arabidopsis under 100 µM ABA stress treatment, respectively; different lowercase letters a, b, c, d, and e indicate differences between groups. P <0.05.

[0041] Figure 15 Overexpression of gibberellin (GA3) under gibberellin (GA3) stress treatment LsMOC1 Phenotypic diagram of transgenic Arabidopsis thaliana. Col-0 It is a wild-type Arabidopsis thaliana. LsMOC1- L1, LsMOC1- L2 and LsMOC1- L3 are all LsMOC1 Transgenic Arabidopsis overexpression, with white dashed lines used to distinguish different Arabidopsis samples; Figure A shows wild-type and overexpressing Arabidopsis samples on MS medium containing 50 µM GA3. LsMOC1 Root phenotype diagram of transgenic Arabidopsis thaliana; Figure B shows the control group (wild type) and overexpression. LsMOC1 Phenotypic diagram of the aboveground parts of transgenic Arabidopsis thaliana; Figure C shows the pheenotypic diagram of the aboveground parts of Arabidopsis thaliana under 150µM GA3 stress treatment.

[0042] Figure 16 Overexpression under gibberellin stress treatment LsMOC1 Phenotypic analysis and enzyme activity data statistics of transgenic Arabidopsis thaliana; Col-0 It is a wild-type Arabidopsis thaliana. LsMOC1- L1, LsMOC1- L2 and LsMOC1- L3 are all LsMOC1 Transgenic Arabidopsis overexpressing the gene; Figures A and B are statistical graphs of taproot length and lateral root number in wild-type and transgenic Arabidopsis on MS medium containing 50 µM GA3, respectively; Figures C, D, and E are analyses of POD, MDA, and SOD enzyme activities in Arabidopsis under 150 µM GA3 stress treatment, respectively; different lowercase letters a, b, c, d, and e indicate differences between groups. P<0.05. Detailed Implementation

[0043] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0044] Lysimachia ( Leymus secalinus Leymus chinensis is a pioneer plant and key species for grassland ecological restoration, degraded grassland management, saline-alkali land improvement, and soil and water conservation. With the continuous changes in climate and ecological environment, Leymus chinensis faces challenges from various abiotic stresses such as drought, salinity, and low temperature. Improving the stress resistance of Leymus chinensis can not only enhance its survival ability in complex environments but also improve its stability in the ecosystem, further promoting vegetation restoration and ecological balance. Extending the growth cycle of Leymus chinensis can increase its biomass accumulation, enhancing its economic value as forage and its ecological service functions. While traditional breeding can improve the traits of Leymus chinensis, it suffers from drawbacks such as long cycles and low efficiency. Furthermore, the complex genome of Leymus chinensis and its long breeding cycle limit its rapid improvement. Molecular breeding is one of the effective means to achieve rapid, precise, and large-scale improvement. Therefore, identifying and utilizing key genes regulating the traits of Leymus chinensis is of great significance for optimizing Leymus chinensis production and improving its quality.

[0045] This invention provides LsMOC1 Genes or containing LsMOC1 Application of gene-related biomaterials. LsMOC1 The nucleotide sequence of the gene is shown in SEQ ID NO.3. This invention utilizes the recombinant expression vector pCAMBIA1300-GFP- LsMOC1 This recombinant expression vector was introduced into the model plant Arabidopsis thaliana, and overexpression was obtained. LsMOC1 Transgenic Arabidopsis thaliana plants. Experiments revealed: LsMOC1 Gene overexpression can delay the phenological stage of plants, slow down growth, prolong the growth cycle, and also improve the plant's resistance to drought stress and gibberellin stress.

[0046] Example 1: LsMOC1 Cloning of genes

[0047] 1. Total RNA extraction and cDNA synthesis

[0048] Following the instructions of the RNA extraction kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.) and the reverse transcription kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.), RNA was extracted from three tissues of Leymus chinensis: roots, stem base (0.5 cm above ground), and leaves, as well as from the terminal buds of the rhizomes in different growth directions (upward, downward, and horizontal). The RNA was then reverse transcribed into cDNA.

[0049] 2. PCR amplification

[0050] Using the cDNA obtained in step 1 as a template, LsMOC1 -F-SalI-1300 and LsMOC1 -R-KpnI-1300 is the amplification primer. Perform PCR amplification according to the following PCR reaction system and PCR reaction procedure to obtain the PCR primers. LsMOC1 The sequence of -F-SalI-1300 is shown in SEQ ID NO.1. LsMOC1 The sequence of -R-KpnI-1300 is shown in SEQ ID NO.2.

[0051] SEQ ID NO.1: 5'-GTCGACATGATCGGCTCACTCCACTCTTCCTCG-3';

[0052] SEQ ID NO.2: 5'-GGTACCCTGCCACGACGACACGGACAGCAG-3';

[0053] PCR reaction system: 1 μL cDNA, 10 μM LsMOC1 -F-SalI-1300 1μL, 10μM LsMOC1 -R-KpnI-1300 1μL, ApexHF HS DNA Polymerase CL 0.5μL, 2×ApexHF CL Buffer (Mg 2+ Add 10 μL of dNTP plus and RNase-free ddH2O to a final volume of 20 μL.

[0054] PCR reaction program: 94℃ pre-denaturation for 1 min; 98℃ denaturation for 10 s, 60℃ annealing for 15 s, 68℃ extension for 75 s, 35 cycles.

[0055] 3. PCR product recovery

[0056] The PCR products obtained in step 2 were detected by 1.2 w / v% agarose gel electrophoresis. The correct bands were excised and recovered to obtain the target gene. Figure 1 ).

[0057] 4. Connection

[0058] The target gene obtained in step 3 was ligated into the pEASY-Blunt Simpl vector to obtain the recombinant vector pEASY-Blunt Simple- LsMOC1 .

[0059] 5. Escherichia coli competent transformation

[0060] a. Remove DH5α competent cells from the ultra-low temperature storage environment of -80℃ and immediately place them on ice until the cell suspension is in an ice-water mixed state;

[0061] b. Aseptically transfer 10 μL of the recombinant vector pEASY-Blunt Simple- LsMOC1 Add to 50 μL of competent cells, gently mix by tapping the tube wall, and then let stand in an ice bath for 30 min;

[0062] c. Transfer the mixture to a 42°C constant temperature water bath for 90 seconds of heat shock treatment, followed by a rapid 3-minute ice bath, avoiding centrifuge tube vibration throughout the process.

[0063] d. Add 900 μL of LB liquid culture medium and mix thoroughly. Transfer to a constant temperature shaker and set to 37°C and 200 rpm for 1 hour to complete cell recovery.

[0064] e. Centrifuge at 5000 rpm for 3 min at 4℃, remove 900 μL of supernatant using a pipette, resuspend the cells in the remaining culture medium and spread evenly on LB agar plates containing 100 mg / L kanamycin.

[0065] f. After the liquid has been completely absorbed, invert the culture dish and place it in a 37°C constant temperature incubator for 12 hours of inverted culture.

[0066] The monoclonal positive strains obtained through culture were sent to a sequencing company for DNA sequencing. The sequencing results were then compared with the target gene ( LsMOC1 The gene sequences were compared and analyzed. The results showed that the two sequences were completely identical, indicating that cloning was successful. LsMOC1 Gene.

[0067] LsMOC1 The nucleotide sequence of the gene is shown in SEQ ID NO.3. LsMOC1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.4. The protein sequence encoded by the ORF of the LsMOC1 gene contains 426 amino acids, with a molecular weight of 45249.21 Da, an isoelectric point pI value of 6.36, and a GRAVY of -0.015, indicating that it is a hydrophilic protein.

[0068] SEQ ID NO.3:

[0069]

[0070] MIGSLHSSSSSDTDNNNSNADVRNSSGEGEGDAALLAGGGRALAAAPSTRDLVLACADLLQRGDLAAARRAAEILLSAVSPRGDATDRLAYHFARALVLRVDAKAGLPFSLRPPAGTAPAPSGAYLAFNQIAPFLRFAHLTANQAILEAVEGSRRVHILDLDAAHGVQWPPLLQAIAERADPALGPPEVRITGAGADRDTLLRTGNRLRAFAR SIHLPFHFTPLLLSCAASTHHVAGTSTTQSTAVTSLELHPDETLAVNCVLFLHKLGGQDELTAFLKWVKAMAPAVVTVAEREASGGGIDPIDELPRRVGVAMDHYSA VFEALEATVPPGSRERLAVEQEVLGREIEAAVGSTDGRWWRGLERWATAARGTGFAARPLSAFAVSQARLLLRLHYPSEGYLVQESRGACFLGWQTRPLLSVSSWQ.

[0071] Example 2: Overexpression LsMOC1 Obtaining transgenic plants

[0072] 1. Obtaining the pCAMBIA1300-GFP-LsMOC1 recombinant expression vector

[0073] The pEASY-Blunt Simple- enzyme obtained in Example 1 was digested using a double enzyme digestion method. LsMOC1 endonuclease Room I and endonuclease CPN I was subjected to double enzyme digestion to overexpress the vector pCAMBIA1300-GFP ( Figure 2 ) respectively with endonuclease Room I and endonuclease CPN I undergo double enzyme digestion:

[0074] Enzyme digestion reaction system: pEASY-Blunt Simple- LsMOC1 or pCAMBIA1300-GFP 16μL endonuclease Room I 1μL, endonuclease CPN I 1μL, 10 × Buffer 2μL;

[0075] After mixing the above enzyme digestion reaction system, place it in a PCR instrument at 37°C for 30 min.

[0076] The digested gene fragment was ligated with the digested overexpression vector pCAMBIA1300-GFP using T4 DNA ligase according to the following ligation system and procedure to obtain the recombinant expression vector pCAMBIA1300-GFP. LsMOC1 ;

[0077] Ligation system: 0.5 μL T4 DNA ligase, 2 μL T4 buffer, 5.5 μL digested gene fragment, and 2 μL digested overexpression vector pCAMBIA1300-GFP.

[0078] Ligation procedure: After mixing the above ligation system, place it in a PCR instrument at 16°C for 12 hours.

[0079] 2. Obtaining recombinant bacteria

[0080] 2.1. The recombinant expression vector pCAMBIA1300-GFP- obtained in step 1 was... LsMOC1 Transplanted into DH5α Escherichia coli competent cells:

[0081] a. Remove DH5α competent cells from the ultra-low temperature storage environment of -80℃ and immediately place them on ice until the cell suspension is in an ice-water mixed state;

[0082] b. Aseptically transfer 10 μL of the recombinant vector pCAMBIA1300-GFP- LsMOC1 added In 50 μL of competent cells, gentle mixing was achieved by lightly tapping the tube wall, followed by standing in an ice bath for 30 min;

[0083] c. Transfer the mixture obtained in step b to a 42°C constant temperature water bath for 90 seconds of heat shock treatment, followed by a rapid 3-minute ice bath, avoiding centrifuge tube vibration throughout the process.

[0084] d. Add 900 μL of LB liquid culture medium and mix thoroughly. Transfer to a constant temperature shaker and set to 37°C and 200 rpm for 1 hour to complete cell recovery.

[0085] e. Centrifuge at 5000 rpm for 3 min at 4℃, remove 900 μL of supernatant using a pipette, resuspend the remaining bacterial culture and spread it on LB agar plates containing 100 mg / L kanamycin, and incubate upside down in a 37℃ incubator for 12 hours.

[0086] 2.2. Single colonies were picked for testing. Agarose gel electrophoresis showed that the fragment size was consistent with the target band, confirming the successful acquisition of the fragment containing pCAMBIA1300-GFP. LsMOC1 Escherichia coli plasmids.

[0087] 2.3. Following the instructions of the Pure PLasmid Mini Kit (Beijing Kangwei Century Biotechnology Co., Ltd.), extract plasmids from samples containing pCAMBIA1300-GFP- LsMOC1 pCAMBIA1300-GFP- was extracted from the bacterial culture of E. coli containing plasmid. LsMOC1 Plasmid.

[0088] 2.4 Obtaining Recombinant Agrobacterium

[0089] a. Mix 50 μL of GV3101 Agrobacterium competent cells with 8 μL of pCAMBIA1300-GFP- LsMOC1 The plasmids were mixed, followed by sequential ice bath for 5 min, liquid nitrogen cryogenic treatment for 5 min, constant temperature water bath heat shock at 37℃ for 5 min, and ice bath for 5 min.

[0090] b. Add 700 μL of antibiotic-free YEP liquid medium and incubate at 28°C with shaking for 2 hours. Centrifuge at 5000 rpm for 1 min to collect the bacteria. Take 100 μL of supernatant, gently pipette and resuspend the bacteria, and spread it on a YEP plate containing 100 mg / L kanamycin and 50 mg / L rifampin. After the bacterial suspension is completely absorbed, invert the plate and incubate at 28°C for 2 days.

[0091] c. Select single colonies for verification. When the agarose gel electrophoresis image shows that the fragment size matches the target band, it proves that the desired fragment has been successfully obtained. LsMOC1 Recombinant Agrobacterium ( Figure 3 After being cultured at 28℃, it is stored at -80℃ for later use.

[0092] 3. Overexpression LsMOC1 Cultivation of transgenic Arabidopsis thaliana

[0093] 3.1 Infection of Arabidopsis thaliana

[0094] a. The day before infection, cut off the open flower buds and pods of the Arabidopsis thaliana to be infected, and water it thoroughly;

[0095] b. containing LsMOC1 The bacterial culture of recombinant Agrobacterium was inoculated into 6 mL of YEP liquid medium containing 100 mg / L kanamycin and 50 mg / L rifampin and cultured overnight.

[0096] c. Inoculate again into 30 mL of YEP liquid medium containing 100 mg / L kanamycin and 50 mg / L rifampin, and incubate until the culture medium reaches OD. 600 The value is 1.2;

[0097] d. Pre-cool the culture medium in a centrifuge at 4°C, centrifuge the culture medium at 5000 rpm for 10 min, collect the bacterial cells, and resuspend the bacterial cells using MS suspension.

[0098] Centrifuge at 4℃, 5000 rpm for 10 min, collect the bacterial cells, and resuspend the cells in MS suspension; repeat this step once and adjust the OD of the bacterial culture. 600 The value was 0.8. After standing for 2 hours, the inoculum was obtained.

[0099] f. Immerse the flowers of Arabidopsis thaliana in the infection solution, let stand for 1 minute, cover with black cloth to avoid light and keep moist for 1 day, then culture under normal light, and perform a second infection after 7 days.

[0100] g. Cultivate under light until the seeds mature, collect the seeds, dry them in an oven at 28℃ for 2 days, and store them at 4℃ in the dark to obtain T0 generation transgenic Arabidopsis seeds.

[0101] 3.2 Screening for positive transgenic Arabidopsis seedlings

[0102] The T0 generation Arabidopsis thaliana seeds were sterilized and laid on a board in a clean bench, specifically as follows:

[0103] a. Seed disinfection: T0 generation Arabidopsis thaliana seeds were disinfected sequentially with 75 v / v% alcohol for 1 min, 7.5 v / v% hypochlorous acid for 1 min, and rinsed five times with sterile water;

[0104] b. After sterilization, the seeds were evenly sown onto MS solid medium containing 100 μg / mL hygromycin and 50 μg / mL kanamycin. After drying the surface of the medium, the plates were sealed and vernalized for 2 days at 4°C in the dark.

[0105] c. After vernalization, transfer the petri dishes to an environment with a temperature of 23℃, a day length of 16h / d, and a light intensity of 40 μmol·m⁻². -2 ·s -1 After being cultured in an incubator for 7 days, the culture was transferred to a substrate made of a 3:1 volume ratio of nutrient soil and vermiculite for further cultivation. Figure 4 After two weeks of cultivation, Arabidopsis leaves were harvested, and leaf DNA was extracted using the CTAB method to screen for positive seedlings. Figure 5 ), obtained expression LsMOC1 Transgenic Arabidopsis thaliana seeds were collected after the positive seedlings matured; the seeds were then subcultured to obtain stable T3 generation transgenic seeds for preservation.

[0106] Example 3: LsMOC1 Effects on plant phenotype

[0107] In order to explore overexpression LsMOC1 The effects of gene overexpression on Arabidopsis thaliana phenotype were analyzed. LsMOC1 The phenotypes of the underground and aboveground parts of transgenic Arabidopsis thaliana were determined, and the data were analyzed using software. The experimental methods are as follows.

[0108] overexpression LsMOC1 Effects of genes on root development: Wild-type and overexpression genes prepared in Example 2 were compared. LsMOC1 Transgenic Arabidopsis thaliana seeds were sown in MS medium petri dishes and vernalized for 2 days at 4°C in the dark. After vernalization, the petri dishes were transferred to an incubator at 23°C and 16 h / d for 7 days to promote seed germination and seedling formation. Germinated Arabidopsis seedlings were then transplanted into 13cm × 13cm MS square petri dishes and cultured vertically for another 7 days. After cultivation, root length of the seedlings was measured using ImageJ software, and preliminary data statistics and bar charts were generated. One-way ANOVA and significance analysis were performed using IBM SPSS Statistics 25 software (based on...). P <0.05 is considered significant). Three biological replicates were set up for each control and treatment group.

[0109] overexpression LsMOC1 Effects of genes on aboveground development: Wild-type Arabidopsis thaliana and overexpressing genes... LsMOC1 Transgenic Arabidopsis thaliana seeds were sown individually in the center of 9cm diameter circular flowerpots. The substrate was a composite medium of nutrient soil and vermiculite in a 3:1 volume ratio. The flowerpots were placed in a culture room at 23℃ with a light duration of 14h / d. Phenotypic differences among the lines were observed and photographed daily. Plant height and leaf area were measured using ImageJ software. One-way ANOVA and significance analysis were performed using IBM SPSS Statistics 25. P <0.05). Both the control and treatment sets were configured with 3 biological replicates.

[0110] The experimental results are as follows:

[0111] Depend on Figure 6 It can be seen that overexpression LsMOC1 Compared with the wild type, the taproot length and the number of lateral roots in the Arabidopsis thaliana were not significantly different, indicating that... LsMOC1 The gene had no significant effect on root morphogenesis under normal culture conditions.

[0112] like Figure 7 and Figure 8 As shown, when transgenic lines and wild-type plants grown on MS plates were transplanted into nutrient soil and cultured normally, during the bolting period in the 4th week, both transgenic and wild-type Arabidopsis thaliana bolted simultaneously. However, with the same number of culture days, the transgenic lines bolted more slowly than the wild-type. LsMOC1Arabidopsis thaliana is smaller in size; measurements of leaf coverage show that the wild-type has approximately 1.5 times the leaf coverage of the transgenic lines. After about six weeks of normal culture, the leaf coverage of the transgenic lines grew to a size comparable to the wild-type. Compared to the wild-type, the transgenic lines had more leaves and more bolting points. Simultaneously, during the seed-setting stage... LsMOC1 The transgenic lines had shorter main stems than the wild-type lines, which were approximately 1.3 times taller than the transgenic lines. By week eight, the wild-type Arabidopsis had already reached maturity and the plants withered, while the overexpressing transgenic lines… LsMOC1 The transgenic strains are beginning to enter the mature stage, with the whole plant being dark green. Although the leaves are slightly yellow and some pods are beginning to turn brown, their growth phenology is significantly delayed and their growth cycle is extended compared to the wild type.

[0113] The above experimental results show that LsMOC1 Genes cause Arabidopsis thaliana to lag phenological stages, slow growth, prolong the growth cycle, and affect plant height, leaf area, and number of branches at different growth and development stages.

[0114] Example 4: LsMOC1 Effects on plant stress resistance

[0115] This invention utilizes cultured overexpression LsMOC1 Transgenic homozygous Arabidopsis thaliana lines were subjected to salt stress, drought stress, abscisic acid (ABA) stress, and gibberellin (GA3) stress experiments to observe overexpression under different habitat conditions. LsMOC1 Phenotypic analysis of transgenic Arabidopsis thaliana and analysis of overexpression LsMOC1 The effects on plant stress resistance were investigated. The experimental procedures are as follows.

[0116] Overexpression under stress LsMOC1 Transgenic Arabidopsis root phenotypic experiment:

[0117] wild type and overexpression LsMOC1 After sterilization, transgenic Arabidopsis seeds were sown in MS culture dishes. The dishes were vernalized for 2 days in the dark at 4°C, then transferred to an environment with 14 h / d light and 23°C for 7 days until germination. Germinated seedlings were then transplanted into 13cm × 13cm square MS culture dishes containing different stress factors, including: 50mM mannitol (simulating osmotic stress), 25μM ABA (abscisic acid treatment), 100mM NaCl (salt stress), and 50μM GA3 (gibberellin treatment). Blank MS medium served as a control group.

[0118] The culture dishes were placed vertically and cultured for another 7 days under the same light (16 h / d) and temperature (23℃) conditions. The growth status of Arabidopsis roots in each treatment group under different stress conditions was photographed and recorded. Root length and lateral root number were measured using ImageJ software. The differences in response of each line under drought, ABA, salt, and GA3 treatments were analyzed by comparing the results between groups. One-way ANOVA and significance analysis were performed using IBM SPSS Statistics 25 software (based on...). P <0.05 is considered significant). Three biological replicates were set up for each control and treatment group.

[0119] Overexpression under stress LsMOC1 Phenotypic experiments of transgenic Arabidopsis thaliana aboveground parts:

[0120] wild type and overexpression LsMOC1 After sterilization, transgenic Arabidopsis seeds were sown in MS culture dishes. The dishes were vernalized for 2 days in the dark at 4°C, then transferred to an environment with 16 h / d light and 23°C for 7 days until seed germination. Germinated seedlings were transplanted into square pots filled with nutrient soil, with 4 plants per pot, and cultured for another 2 weeks under 14 h / d light and 23°C conditions, with routine watering management. When the plants reached 4 weeks of age, stress treatment was applied. Plants watered with an equal volume of water served as the control group. Both the control and treatment were performed in triplicate, with leaf samples collected and preserved simultaneously. Enzyme activities were determined according to the instructions of the peroxidase (POD) kit, superoxide dismutase (SOD) kit, and malondialdehyde (MDA) content kit purchased from Ise-Hisashi Biosciences Co., Ltd.

[0121] (1) Drought stress treatment: Stop irrigation and treat the plants with drought for 7 days. After the treatment, cut off the above-ground leaves of Arabidopsis thaliana and store them at 4℃ to determine enzyme activity as soon as possible.

[0122] (2) ABA stress treatment: On the first day, the third day, the fifth day and the seventh day, 100 μM ABA solution was used to irrigate the flower pot until it slightly seeped out. After the treatment, the above-ground leaves of Arabidopsis thaliana were cut off and stored at 4℃ to determine the enzyme activity as soon as possible.

[0123] (3) Salt stress treatment: On the first day, the third day, the fifth day and the seventh day, 200mM NaCl solution was applied to the flower pot until it slightly seeped out. The above-ground leaves were cut off the next day and stored at 4℃ to determine the enzyme activity as soon as possible.

[0124] (4) GA3 stress treatment: On the first day, the third day, the fifth day and the seventh day, 150 μM GA3 solution was applied to the flower pot until it slightly seeped out. The above-ground leaves were cut off the next day and stored at 4℃ to determine the enzyme activity as soon as possible.

[0125] The experimental results are as follows:

[0126] (1) LsMOC1 The results of the effect on the salt stress resistance of plants are as follows: Figure 9 and Figure 10 As shown, on salt-stressed medium, the transgenic lines exhibited a significantly reduced taproot length compared to the wild-type, indicating that overexpression of *Lysimachia christinae*... LsMOC1 The gene did not enhance the salt stress resistance of Arabidopsis thaliana. Aboveground stress experiments showed that, compared to the wild type, overexpression of the gene significantly improved salt stress resistance. LsMOC1 Transgenic Arabidopsis thaliana exhibited higher levels of stress, more wrinkled leaves, and higher MDA enzyme activity, while showing lower activities of antioxidant enzymes POD and SOD, further indicating that... LsMOC1 Genes cannot enhance Arabidopsis thaliana's ability to withstand salt stress.

[0127] (2) LsMOC1 The results of the effect on the drought stress resistance of plants are as follows: Figure 11 and Figure 12 As shown, on drought-stressed medium, the transgenic lines exhibited a significantly reduced taproot length compared to the wild-type; however, the overexpression lines showed a significantly increased number of lateral roots, indicating that overexpression of *Lysimachia christinae*... LsMOC1 This gene may enhance the drought stress resistance of Arabidopsis thaliana by promoting lateral root development. Results from aboveground stress experiments showed that overexpression of this gene in wild-type Arabidopsis thaliana under drought stress significantly improved drought resistance. LsMOC1 The transgenic Arabidopsis thaliana experienced less stress. Although some leaves of the transgenic lines also showed purple discoloration, the leaves remained fully expanded without drying out or withering. Furthermore, the activity of MDA enzyme was lower than that of the wild type, while the activity of the antioxidant enzyme POD was significantly increased, indicating that... LsMOC1 Genes have enhanced the drought resistance of Arabidopsis thaliana.

[0128] (3) LsMOC1 The results of the effect on the plant's resistance to abscission acid stress are as follows: Figure 13 and Figure 14 As shown, on ABA stress medium, the transgenic lines exhibited a significantly reduced taproot length compared to the wild-type; simultaneously, the number of lateral roots in the overexpression lines was also significantly reduced, indicating that overexpression... LsMOC1 The gene did not enhance the ability of Arabidopsis thaliana to respond to ABA stress. Results from aboveground stress experiments showed that, compared to the wild type, overexpression under ABA treatment significantly improved the response of Arabidopsis thaliana. LsMOC1Transgenic Arabidopsis thaliana had smaller and darker leaves, and higher MDA enzyme activity, indicating a higher degree of stress. Simultaneously, they exhibited lower activities of antioxidant enzymes POD and SOD, further indicating... LsMOC1 Genes cannot enhance Arabidopsis thaliana's resistance to ABA stress.

[0129] (4) LsMOC1 The results of the effect on the plant's resistance to gibberellin stress are as follows: Figure 15 and Figure 16 As shown, on GA3 medium, the underground phenotype of the transgenic lines showed no significant difference in taproot length compared to the wild type; however, the number of lateral roots in the overexpression lines was significantly increased compared to the wild-type Arabidopsis thaliana in the treatment group, indicating that overexpression... LsMOC1 The gene showed a tendency to enhance the resistance of Arabidopsis thaliana to high concentrations of GA3 inhibition. Aboveground stress experiments revealed that under GA3 stress, the leaves of both transgenic and wild-type Arabidopsis thaliana turned purple. However, there was no significant difference in leaf size between the transgenic and wild-type Arabidopsis thaliana and the control group. Simultaneously, MDA enzyme activity was lower than that of the wild type. Although there was no significant difference in the antioxidant enzyme POD, SOD activity was significantly increased, indicating that... LsMOC1 The gene increases Arabidopsis' resistance to high concentrations of GA3 by enhancing SOD enzyme activity.

[0130] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0131] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. LsMOC1 gene or a gene-related biological material LsMOC1 application of a gene-related biological material, characterized in that, The LsMOC1 The nucleotide sequence of the gene is shown in SEQ ID NO.3; the improvement of the plant's [specific genetic information] through transgenic technology or gene editing technology is described. LsMOC1 The expression level of genes is used to prolong the plant's growth cycle, improve its drought resistance, or enhance its resistance to gibberellin stress; the plant in question is Arabidopsis thaliana.

2. Use according to claim 1, characterized in that, The biomaterial comprises any one of the following: a、 the LsMOC1 gene encodes a protein, the amino acid sequence of which is shown as SEQ ID NO. 4; b. comprising said LsMOC1 a recombinant expression vector of a gene; c. a recombinant microbial strain comprising said LsMOC1 gene.

3. A method of growing a transgenic plant with an extended life cycle, comprising, The method comprises the following steps: The recombinant expression vector containing the gene of claim 1 is introduced into a plant of interest, so that LsMOC1 the gene is overexpressed in the plant of interest, and a transgenic plant with a long growth cycle is obtained, the growth cycle of the transgenic plant being longer than that of the plant of interest; the plant is Arabidopsis thaliana. LsMOC1 the gene is overexpressed in the plant of interest, and a transgenic plant with a long growth cycle is obtained, the growth cycle of the transgenic plant being longer than that of the plant of interest; the plant is Arabidopsis thaliana.

4. The method of claim 3, wherein, The recombinant expression vector is pCAMBIA1300-GFP- LsMOC1 The recombinant expression vector is composed of LsMOC1 The gene was obtained after being introduced into the overexpression vector pCAMBIA1300-GFP.

5. A method of breeding a plant with enhanced stress resistance, characterized in that, The method comprises the following steps: Containing the contents of claim 1 LsMOC1 The recombinant gene expression vector is introduced into the target plant, enabling... LsMOC1 The gene is overexpressed in the target plant to obtain a plant with enhanced stress resistance, wherein the stress resistance of the enhanced stress resistance plant is stronger than that of the target plant; the stress is drought stress or gibberellin stress; the plant is Arabidopsis thaliana.

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