Use of ls slr1 gene or biological material containing ls slr1 gene in plant trait improvement

Abstract

This invention belongs to the field of plant genetic engineering technology, specifically involving LsSLR1 Genes or containing LsSLR1 Application of gene-related biomaterials in plant trait improvement. This invention discloses... LsSLR1 The nucleotide sequence of the gene is shown in SEQ ID NO.3. This invention utilizes the recombinant expression vector pCAMBIA1300-GFP- LsSLR1 This recombinant expression vector was introduced into the model plant Arabidopsis thaliana, and overexpression was obtained. LsSLR1 Transgenic Arabidopsis thaliana plants. Experiments revealed: LsSLR1 Gene overexpression can promote lateral root development, dwarf plants, shorten the plant growth cycle, and improve plant resistance to salt stress, drought stress, or abscisic acid stress. LsSLR1 Genes can genetically modify plant traits.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically involving LsSLR1 Genes or containing LsSLR1 Application of gene-related biomaterials in plant trait improvement. Background Technology

[0002] Lysimachia ( Leymus secalinus Leymus chinensis, a perennial clonal plant belonging to the genus Leymus in the family Poaceae, is highly adaptable to various ecological conditions and widely distributed in saline-alkali, arid, and desertified marginal lands in Northwest my country, North China, and the Qinghai-Tibet Plateau. These regions have harsh soil conditions and complex, variable climates, yet Leymus chinensis survives and thrives in these environments thanks to its unique biological characteristics. It possesses a well-developed rhizome-tillering network, a structure that endows it with strong reproductive capacity and ecological adaptability. Its rhizomes can penetrate deep into the soil to absorb water and nutrients, while its tillering ability allows it to form dense plant communities in a short period, effectively covering the ground and reducing soil erosion. Therefore, Leymus chinensis possesses multiple values, including excellent forage, soil and water conservation, and ecological restoration, and is hailed as a "pioneer grass species," showing significant application prospects in ecological governance and livestock development.

[0003] The complex traits of *Leymus chinensis* (such as drought resistance, cold tolerance, and regeneration ability) are mostly quantitative traits regulated by multiple genes. This means that the genetic basis of these traits is complex, influenced by multiple genes, and there are complex interactions between these genes. Traditional breeding methods mainly rely on phenotypic selection, artificially selecting individuals with superior traits for hybridization, and then screening out plants that meet the requirements from the offspring. Although this method can improve the traits of *Leymus chinensis* to some extent, it suffers from problems such as long breeding cycles, low efficiency, and difficulty in overcoming the bottleneck of trait improvement due to the lack of precise regulation at the gene level. Modern molecular biology techniques, such as genome sequencing, gene expression analysis, and gene editing, can deeply analyze the genetic basis of the complex traits of *Leymus chinensis*, identify key genes that regulate these traits, and utilize these genes to achieve precise and rapid improvement of *Leymus chinensis* traits. Therefore, discovering and utilizing its key regulatory genes is of great significance for accelerating the precise genetic improvement of *Leymus chinensis* traits and the breeding of new varieties. Summary of the Invention

[0004] To address the above problems, the present invention provides a... LsSLR1 The application of genes in plant trait improvement. This invention, through the application of genes in plant trait improvement... LsSLR1 Investigating gene function revealed overexpression LsSLR1 Genes can promote the development of lateral roots, dwarf plants, shorten the plant growth cycle, and improve the plant's resistance to salt stress, drought stress, or abscisic acid stress.

[0005] To achieve the above object, the specific technical solutions of the present application are as follows:

[0006] The first aspect of the present application provides LsSLR1 The gene or the biological material containing LsSLR1 The application of the gene-related biological material in plant trait improvement, the gene or the biological material containing LsSLR1 The gene or the biological material containing LsSLR1 The gene or the biological material containing LsSLR1 The nucleotide sequence of the gene is shown as SEQ ID NO. 3.

[0007] Further, the regulation of plant growth is any one or several of promoting plant lateral root development, dwarfing plants and shortening the growth cycle of plants.

[0008] Further, the expression amount of the gene in the plant is increased by transgenic technology or gene editing technology, so as to promote the lateral root development of the plant, dwarf the plant or shorten the growth cycle of the plant. LsSLR1 The gene or the biological material containing

[0009] Further, the stress is any one or several of salt stress, drought stress and abscisic acid stress.

[0010] Further, the expression amount of the gene in the plant is increased by transgenic technology or gene editing technology, so as to promote the lateral root development of the plant, dwarf the plant or shorten the growth cycle of the plant. LsSLR1 The gene or the biological material containing

[0011] Further, the biological material includes any one of the following:

[0012] a. The protein encoded by the gene, and the amino acid sequence of the protein is shown as SEQ ID NO. 4; LsSLR1 b. A recombinant expression vector containing the gene;

[0013] LsSLR1 c. A recombinant microbial strain containing the gene.

[0014] c. A recombinant microbial strain containing the gene. LsSLR1 Further, the recombinant microbial strain is Escherichia coli containing the gene or Agrobacterium containing the gene.

[0015] LsSLR1 Further, the plant is Leymus mollis, Arabidopsis thaliana or tobacco. LsSLR1 The second aspect of the present application provides a method for cultivating a plant with enhanced stress resistance, comprising the following steps:

[0016] The biological material containing the gene is obtained by the method.

[0017] The second aspect of the present application provides a method for cultivating a plant with enhanced stress resistance, comprising the following steps:

[0018] The biological material containing the gene is obtained by the method.​​LsSLR1 The recombinant expression vector of the gene is introduced into the target plant, so that LsSLR1 The gene is overexpressed in the target plant, and a plant with enhanced stress resistance is obtained, and the stress resistance of the plant with enhanced stress resistance is stronger than that of the target plant.

[0019] Further, the stress is salt stress, drought stress or abscisic acid stress.

[0020] Further, the recombinant expression vector is pCAMBIA1300-GFP- LsSLR1 .

[0021] Further, the recombinant expression vector is obtained after the gene is introduced into the overexpression vector pCAMBIA1300-GFP. LsSLR1

[0022] Further, the construction method of the recombinant expression vector comprises the following steps: connecting the enzyme-digested LsSLR1 Gene and enzyme-digested overexpression vector pCAMBIA1300-GFP at 16℃ for 12h to obtain recombinant expression vector pCAMBIA1300-GFP- Xba .

[0023] Further, the enzyme cutting site of the enzyme cutting is Kpn I and LsSLR1 I.

[0024] Further, the target plant is Leymus mollis, Arabidopsis thaliana or tobacco.

[0025] Compared with the prior art, the beneficial effects of the present application are:

[0026] The present application first discloses the application of LsSLR1 Gene or biological material containing LsSLR1 Gene related to plant trait improvement. LsSLR1 The nucleotide sequence of the gene is shown as SEQ ID NO. 3, LsSLR1 The amino acid sequence of the protein encoded by the gene is shown as SEQ ID NO. 4. The present application constructs a recombinant expression vector pCAMBIA1300-GFP- LsSLR1 , and introduces the recombinant expression vector into model plant Arabidopsis thaliana, and obtains transgenic Arabidopsis thaliana plants overexpressing LsSLR1 . Experiments show that: LsSLR1 After overexpression of the gene, (1) the number of plant lateral roots is significantly increased, indicating Figure 1 ​The gene is involved in the development of plant root system, has significant influence on plant root morphogenesis and lateral root development; (2) can make plant phenophase advance, advance bolting, dwarf plant, shorten plant growth cycle; (3) can improve the salt stress resistance of plant, the drought stress resistance and the abscisic acid stress resistance. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0028] LsSLR1 To amplify Figure 2 The agarose gel electrophoresis diagram of the target gene, the leftmost lane is DNA Marker, and lanes 1-4 are PCR amplification products with a size of 1842bp.

[0029] Figure 3 The map of the pCAMBIA1300-GFP overexpression vector.

[0030] LsSLR1 To LsSLR1 The positive colonies of the overexpression vector pCAMBIA1300-GFP transformed into GV3101 Agrobacterium chemically competent are verified; the leftmost lane is DNA Marker, and lanes 1-6 are pCAMBIA1300-GFP- Figure 4 Sample.

[0031] LsSLR1 To overexpress Figure 5 The T1 generation positive seedling screening diagram of transgenic Arabidopsis.

[0032] LsSLR1 To overexpress LsSLR1 The agarose gel electrophoresis diagram of the T1 generation positive seedling gene verification of transgenic Arabidopsis, lane 1 is the control, and lanes 2-11 are overexpression Figure 6 Transgenic Arabidopsis sample.

[0033] LsSLR1 To overexpress Col-0 The underground phenotype and data statistical diagram of transgenic Arabidopsis, LsSLR1-L1 The wild type Arabidopsis, LsSLR1-L2 , LsSLR1-L3 and LsSLR1 are LsSLR1 Transgenic overexpression Arabidopsis, the white dotted line is used to distinguish different Arabidopsis samples; A is the wild type Arabidopsis grown for two weeks andFigure 7 Figure B shows the root phenotype of transgenic Arabidopsis overexpressing the gene; Figure C shows the root length phenotype data analysis results of two-week-old Arabidopsis; Figure D shows the secondary root quantity data analysis results of two-week-old Arabidopsis. Different lowercase letters a and b indicate differences between groups. P <0.05.

[0034] LsSLR1 For overexpression Col-0 Phenotypic diagram of the aboveground parts of transgenic Arabidopsis thaliana. LsSLR1- It is a wild-type Arabidopsis thaliana. LsSLR1-L2 L1 , LsSLR1-L3 and LsSLR1 All Figure 8 Transgenic overexpression of Arabidopsis thaliana; Figure A shows the aboveground leaf phenotype of Arabidopsis thaliana at the bolting stage, Figure B shows the leaf phenotype of Arabidopsis thaliana at the seed-setting stage, Figure C shows the aboveground leaf phenotype of Arabidopsis thaliana at the seed-setting stage, and Figure D shows the main branch phenotype of Arabidopsis thaliana at the mature stage.

[0035] LsSLR1 For overexpression Col-0 Statistical graph of aboveground phenotypes in transgenic Arabidopsis thaliana. LsSLR1-L1 It is a wild-type Arabidopsis thaliana. LsSLR1-L2 , LsSLR1-L3 and LsSLR1 All Figure 9 Transgenic overexpression of Arabidopsis thaliana; Figure A shows the statistical analysis of the main branch height of Arabidopsis thaliana at the seed-bearing stage; Figure B shows the statistical analysis of the main branch height of Arabidopsis thaliana at maturity; Figure C shows the quantitative analysis of the number of lateral branches of Arabidopsis thaliana at maturity; different lowercase letters a and b indicate differences between groups. P <0.05.

[0036] LsSLR1 Overexpression under salt stress Col-0 Phenotypic diagram of transgenic Arabidopsis thaliana. LsSLR1-L1 It is a wild-type Arabidopsis thaliana. LsSLR1-L2 , LsSLR1-L3 and LsSLR1 All LsSLR1 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. LsSLR1 Root phenotype diagram of transgenic Arabidopsis thaliana; Figure B shows the control group (wild type) and overexpression. Figure 10 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.

[0037] LsSLR1 Overexpression under salt stress Col-0 Phenotypic analysis and enzyme activity data statistics of transgenic Arabidopsis thaliana. LsSLR1-L1 It is a wild-type Arabidopsis thaliana.LsSLR1-L2 , LsSLR1-L3 and LsSLR1 All Figure 11 Transgenic overexpression of Arabidopsis thaliana; Figures A and B show the taproot length and lateral root number of Arabidopsis thaliana on MS medium containing 100 mM NaCl, respectively; Figures C, D, and E show the POD, MDA, and SOD enzyme activities of Arabidopsis thaliana under 200 mM salt stress treatment, respectively; different lowercase letters a, b, and c indicate differences between groups. P <0.05.

[0038] LsSLR1 Overexpression under drought stress Col-0 Phenotypic diagram of transgenic Arabidopsis thaliana. LsSLR1-L1 It is a wild-type Arabidopsis thaliana. LsSLR1-L2 , LsSLR1-L3 and LsSLR1 All LsSLR1 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. LsSLR1 Root phenotype diagram of transgenic Arabidopsis thaliana; Figure B shows the control group (wild type) and overexpression. Figure 12 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.

[0039] LsSLR1 Overexpression under drought stress Col-0 Phenotypic analysis and enzyme activity data statistics of transgenic Arabidopsis thaliana. LsSLR1-L1 It is a wild-type Arabidopsis thaliana. LsSLR1-L2 , LsSLR1-L3 and LsSLR1 All Figure 13 Transgenic overexpression of Arabidopsis thaliana; Figures A and B show the taproot length and lateral root number of Arabidopsis thaliana on MS medium containing 50 mM mannitol, respectively; Figures C, D, and E show the POD, MDA, and SOD enzyme activities of Arabidopsis thaliana under natural drought treatment, respectively; different lowercase letters a, b, c, and d indicate differences between groups. P <0.05.

[0040] LsSLR1 Overexpression under abscisic acid (ABA) stress treatment Col-0 Phenotypic diagram of transgenic Arabidopsis thaliana. LsSLR1-L1 It is a wild-type Arabidopsis thaliana. LsSLR1-L2 , LsSLR1-L3 and LsSLR1 All LsSLR1 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.LsSLR1 Root phenotype diagram of transgenic Arabidopsis thaliana; Figure B shows the control group (wild type) and overexpression. Figure 14 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.

[0041] LsSLR1 Overexpression under abscission acid stress treatment Col-0 Phenotypic analysis and enzyme activity data statistics of transgenic Arabidopsis thaliana. LsSLR1-L1 It is a wild-type Arabidopsis thaliana. LsSLR1-L2 , LsSLR1-L3 and LsSLR1 All Figure 15 Transgenic Arabidopsis overexpressing the gene; Figures A and B show the taproot length and lateral root number of wild-type and transgenic Arabidopsis on MS medium containing 25 µM ABA, respectively; Figures C, D, and E show the POD, MDA, and SOD enzyme activities of Arabidopsis under 100 µM ABA stress treatment, respectively; different lowercase letters a, b, c, and d indicate differences between groups. P <0.05.

[0042] LsSLR1 Overexpression of gibberellin (GA3) under stress Col-0 Phenotypic diagram of transgenic Arabidopsis thaliana. LsSLR1-L1 It is a wild-type Arabidopsis thaliana. LsSLR1-L2 , LsSLR1-L3 and LsSLR1 All LsSLR1 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. LsSLR1 Root phenotype diagram of transgenic Arabidopsis thaliana; Figure B shows the control group (wild type) and overexpression. Figure 16 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.

[0043] LsSLR1 Overexpression under gibberellin stress treatment Col-0 Phenotypic analysis and enzyme activity data statistics of transgenic Arabidopsis thaliana. LsSLR1-L1 It is a wild-type Arabidopsis thaliana. LsSLR1-L2 , LsSLR1-L3 and LsSLR1 All LsSLR1Figure A and figure B are the statistical diagrams of the main root length and the lateral root number of wild type and transgenic Arabidopsis thaliana on MS medium containing 50 µM GA3, figure C, figure D and figure E are the analysis of POD enzyme activity, MDA enzyme activity and SOD enzyme activity of Arabidopsis thaliana under 150 µM GA3 stress treatment, different lowercase letters a, b, c, d, e represent that there is difference between groups, P <0.05. DETAILED DESCRIPTION

[0044] The specific embodiments of the present application are described in detail below, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present application. The experimental methods described in the embodiments of the present application are conventional methods, and the materials and reagents used in the following examples are commercially available unless otherwise specified.

[0045] Leymus is a perennial clonal plant of Poaceae Leymus, which has a developed rhizome-tillering network, and has multiple values of excellent forage, soil and water conservation and ecological restoration, and is known as "pioneer grass species". However, the complex traits of Leymus (such as drought resistance, cold tolerance, regeneration ability, etc.) are mostly quantitative traits regulated by multiple genes, and it is difficult to precisely improve them by traditional breeding. Therefore, it is of great significance to excavate and utilize key regulatory genes to accelerate the precise genetic improvement and new variety breeding of Leymus.

[0046] The present application provides a kind of LsSLR1 gene or containing LsSLR1 application of gene related biological material in plant trait improvement. LsSLR1 The nucleotide sequence of the gene is shown in SEQ ID NO.3, LsSLR1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.4. The present application constructs a recombinant expression vector pCAMBIA1300-GFP- LsSLR1 The recombinant expression vector is introduced into model plant Arabidopsis thaliana, and transgenic Arabidopsis thaliana plants overexpressing LsSLR1 are obtained. Experiments have found that: LsSLR1 After overexpression of the gene, (1) the number of plant lateral roots is significantly increased, indicating that LsSLR1 the gene is involved in plant root development and has a significant impact on plant root morphogenesis and lateral root development; (2) it can make the plant phenology advance, advance bolting, dwarf plant, shorten the plant growth cycle; (3) it can improve the ability of plant salt stress resistance, drought stress resistance and abscisic acid stress resistance.

[0047] Example 1: LsSLR1 Cloning of the gene

[0048] 1. Extraction of total RNA and synthesis of cDNA

[0049] According to the operation steps in the instructions of the RNA extraction kit (purchased from Nanjing Norgen Biotek Co., Ltd.) and the reverse transcription kit (purchased from Nanjing Norgen Biotek Co., Ltd.), the RNA of the roots, stem bases (0.5 cm of the aboveground parts), and leaves of Leymus chinensis and the RNA of the rhizome apical buds in different growth directions (upward, downward, and transverse) of Leymus chinensis were extracted and reverse transcribed into cDNA.

[0050] 2. PCR amplification

[0051] The cDNA obtained in step 1 was used as a template, and LsSLR1 -F-XbaI-1300 and LsSLR1 -R-KpnI-1300 were used as amplification primers to perform PCR amplification according to the following PCR reaction system and PCR reaction program, and PCR primers were obtained. LsSLR1 The sequence of -F-XbaI-1300 is shown in SEQ ID NO. 1, LsSLR1 and the sequence of -R-KpnI-1300 is shown in SEQ ID NO. 2.

[0052] SEQ ID NO. 1: 5’-TCTAGAATGAAGCGCGAGTACCAAGAAG-3’;

[0053] SEQ ID NO. 2: 5’-GGTACCCGGCGCGGCCAGGCGCCATGCC-3’;

[0054] PCR reaction system: cDNA 1 μL, 10 μM Figure 1 -F-XbaI-1300 1 μL, 10 μM LsSLR1 -R-KpnI-1300 1 μL, ApexHF HS DNA Polymerase CL 0.5 μL, 2×ApexHF CL Buffer (Mg 2+ and dNTPplus) 10 μL, and RNase-free ddH2O was supplemented to 20 μL.

[0055] PCR reaction program: 94°C pre-denaturation for 1 min; 98°C denaturation for 10 s, 60°C annealing for 15 s, 68°C extension for 105 s, 35 cycles.

[0056] 3. Recovery of PCR products

[0057] The PCR product obtained in step 2 was subjected to 1.2 w / v% agarose gel electrophoresis detection, and the correct band was subjected to gel recovery to obtain the target gene LsSLR1 ).

[0058] 4. Connection

[0059] The target gene obtained in step 3 was connected with pEASY-Blunt Simple vector to obtain recombinant vector pEASY-Blunt Simple- LsSLR1 .

[0060] 5. E. coli competent transformation

[0061] a. The DH5a competent cells were taken out from the ultra-low temperature-80℃ storage environment, and immediately ice-bathed to the ice water mixed state of the cell suspension;

[0062] b. 10 μL of recombinant vector pEASY-Blunt Simple- LsSLR1 was added to 50 μL of competent cells, and gentle mixing was achieved by flicking the tube wall, and then it was placed in ice bath for 30 min;

[0063] c. The mixed system was transferred to a 42℃ constant temperature water bath device for 90S heat shock treatment, and then quickly ice-bathed for 3 min, and the whole process avoided centrifugal tube shock;

[0064] d. 900 μL of LB liquid medium was added and mixed thoroughly, and then transferred to a constant temperature shaker set at 37℃, 200 rpm shaking culture for 1 hour to complete cell recovery;

[0065] e. Centrifuged at 5000 rpm for 3 min at 4℃, removed 900 μL of supernatant with a pipette, and evenly coated the remaining bacterial suspension on LB agar plates containing 100 mg / L of kanamycin;

[0066] f. After the liquid was completely absorbed, the culture dish was turned over and placed in a 37℃ constant temperature incubator for 12 hours of inverted culture.

[0067] The monoclonal positive strain obtained by culture was entrusted to a sequencing company for DNA sequence sequencing, and then the obtained sequencing results were compared and analyzed with the sequence of the target gene (GFP) LsSLR1 gene). The results showed that the sequences were completely consistent, indicating that the LsSLR1 gene was successfully cloned.

[0068] LsSLR1 The nucleotide sequence of the gene is shown in SEQ ID NO. 3, LsSLR1The 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 LsSLR1 gene comprises 613 amino acids, the molecular weight is 64478.41 Da, the isoelectric point pI value is 5.02, and the GRAVY is -0.125, which is a hydrophilic protein.

[0069] SEQ ID NO. 3:

[0070]

[0071] SEQ ID NO.4:

[0072] MKREYQEAGGSGGGDMGSSKDKMMVSSSAGEGEDVDELLAALGYKVRASDMADVAQKLEQLEMAMGMGGVGAGAAPDDSFATHLATDTVHYNPTDLSSWVESMLSELNAPPPPLPPAPTLNASTSSTVTGGAGYFDLPPSVDSSSSTYALRPIPSPADLSADSVRDPKRMRTGGSTTSSSSSSSSSLGGGGARSSVVEAAPPATAAASAPALPVVVVDTQEAGIRLVHALLACAEAVQQENLSAAEALVKQIPLLAASQGGAMRKVAAYFGEALARCVFRFRPQPDSSLLDAAFADLLHAHFYESCPYLKFAHFTANQAILEAFAGCRRVHVVDFGIKQGMQWPALLQALALRPGGPPSFRLTGVGPPQPDETDALQQVGWKLAQFAHTIRVDFQYRGLVAATLADLEPFMLQPEGEEGPNEEPEVIAVNSVFEMHRLLAQPGALEKVLGTVRAVRPRIVTVVEQEANHNSGSFLDRFTESLHYYSTMFDSLEGAGSGGPSEVSSGAAAAAPAGTDQVMSEVYLGRQICNVVACEGAERTERHETLGQWRNRLGNAGFETVHLGSNAYKQASTLLALFAGGDGYKVEEKEGCLTLGWHTRPLIATSAWRLAAP.

[0073] Example 2: Obtaining of transgenic plants overexpressing Xba LsSLR1

[0074] 1. Obtaining of pCAMBIA1300-GFP-LsSLR1 recombinant expression vector

[0075] The pEASY-Blunt Simple-TOPO®-LsSLR1 overexpression vector obtained in Example 1 was double-digested with restriction enzyme Kpn I and restriction enzyme Figure 2 I, and the overexpression vector pCAMBIA1300-GFP (Invitrogen) was double-digested with restriction enzyme Xba I and restriction enzyme Kpn I, respectively: LsSLR1 I and restriction enzyme Xba I:

[0076] Enzyme digestion system: pEASY-Blunt Simple- Kpn or pCAMBIA1300-GFP 16 μL endonuclease LsSLR1 I 1 μL, endonuclease LsSLR1 I 1 μL, 10 × Buffer 2 μL;

[0077] After mixing the above enzyme digestion system, it is placed in a PCR instrument at 37°C for 30 min.

[0078] The digested gene fragment and the digested overexpression vector pCAMBIA1300-GFP are connected by T4 DNA ligase according to the following connection system and connection procedure, and the recombinant expression vector pCAMBIA1300-GFP- ​ is obtained.

[0079] Connection system: T4 DNA ligase 0.5 μL, T4 buffer 2 μL, digested gene fragment 5.5 μL, digested overexpression vector pCAMBIA1300-GFP 2 μL.

[0080] Connection procedure: After mixing the above connection system, it is placed in a PCR instrument at 16°C for 12 h.

[0081] 2, Obtaining of recombinant bacteria

[0082] 2.1, The recombinant expression vector pCAMBIA1300-GFP- ​ obtained in step 1 is transformed into DH5ɑ E. coli competent cells:

[0083] a. The DH5ɑ competent cells are taken out from the ultra-low temperature-80℃ storage environment, and immediately ice-bathed to make the cell suspension present as ice-water mixture;

[0084] b. 10 μL of the recombinant vector pCAMBIA1300-GFP- LsSLR1 is added to 50 μL of the competent cells, and gently mixed by flicking the tube wall, and then placed in ice bath for 30 min;

[0085] c. The mixed system obtained in step b is transferred to a 42℃ constant temperature water bath device for 90S heat shock treatment, and then quickly ice-bathed for 3 min, and the whole process avoids centrifugal tube shaking;

[0086] d. 900 μL of LB liquid medium is added and mixed thoroughly, and then transferred to a constant temperature shaker set at 37℃ and 200 rpm for shaking culture for 1 hour to complete cell recovery;

[0087] 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.

[0088] 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. LsSLR1 Escherichia coli plasmids.

[0089] 2.3. Following the instructions of the Pure PLasmid Mini Kit (Beijing Kangwei Century Biotechnology Co., Ltd.), extract plasmids from samples containing pCAMBIA1300-GFP- LsSLR1 pCAMBIA1300-GFP- was extracted from the bacterial culture of E. coli containing plasmid. LsSLR1 Plasmid.

[0090] 2.4 Obtaining Recombinant Agrobacterium

[0091] a. Mix 50 μL of GV3101 Agrobacterium competent cells with 8 μL of pCAMBIA1300-GFP- LsSLR1 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.

[0092] 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.

[0093] 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. LsSLR1 Recombinant Agrobacterium ( Figure 3 After being cultured at 28℃, it is stored at -80℃ for later use.

[0094] 3. Overexpression LsSLR1 Cultivation of transgenic Arabidopsis thaliana

[0095] 3.1 Infection of Arabidopsis thaliana

[0096] a. The day before infection, cut off the open flower buds and pods of the Arabidopsis thaliana to be infected, and water it thoroughly.

[0097] b. containing LsSLR1The 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.

[0098] 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;

[0099] 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.

[0100] 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 infection solution was obtained.

[0101] f. Immerse the Arabidopsis thaliana flowers in the infection solution, let stand for 1 minute, cover with a black cloth to avoid light and keep moist for 1 day, then culture under normal light, and perform a second infection after 7 days.

[0102] 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.

[0103] 3.2 Screening for positive transgenic Arabidopsis seedlings

[0104] The T0 generation Arabidopsis thaliana seeds were sterilized and laid on a board in a clean bench, specifically as follows:

[0105] 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;

[0106] 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.

[0107] 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 LsSLR1Transgenic Arabidopsis thaliana seeds were collected after the positive seedlings matured; the seeds were then subcultured to obtain stable T3 generation transgenic seeds for preservation.

[0108] Example 3: LsSLR1 Effects on plant phenotype

[0109] In order to explore overexpression LsSLR1 The effects of gene overexpression on Arabidopsis thaliana phenotype were analyzed. LsSLR1 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.

[0110] overexpression LsSLR1 Effects of genes on root development: Wild-type and overexpression genes prepared in Example 2 were compared. LsSLR1 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.

[0111] overexpression LsSLR1 Effects of genes on aboveground development: Wild-type Arabidopsis thaliana and overexpressing genes... LsSLR1 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.

[0112] The experimental results are as follows:

[0113] Depend on Figure 6 It can be seen that overexpression LsSLR1 Compared with the wild type, the taproot length of the Arabidopsis thaliana was not significantly different, but the number of lateral roots increased significantly, about twice that of the wild type, indicating that... LsSLR1The gene is involved in the development of root system and has significant influence on root morphological construction and lateral root development.

[0114] As shown in Figure 7 and Figure 8 , the transgenic lines and wild type grown on MS plates are transplanted into normal culture in nutrient soil, and it is found that the transgenic Arabidopsis thaliana is earlier than the wild type in the bolting period at the fourth week, and there is no obvious difference in leaf coverage area. After being cultured for about six weeks, there is no obvious difference in the main branch height between the transgenic Arabidopsis thaliana and the wild type, but the transgenic Arabidopsis thaliana overexpressing LsSLR1 enters the mature period earlier, and the leaves are dry. At the eighth week, it can be seen that the transgenic Arabidopsis thaliana has stopped elongation at the sixth week, and finally has a trend of increasing the number of lateral branches and reducing the height of the main branch compared with the wild type. It is shown that LsSLR1 the gene can promote the development of Arabidopsis thaliana, and the bolting is earlier, and the growth cycle is shortened.

[0115] The above results show that LsSLR1 the gene can promote the development of Arabidopsis thaliana, make the phenophase earlier, and have the effects of promoting lateral root development and plant dwarfing.

[0116] Example 4: LsSLR1 Influence on the stress resistance of plants

[0117] The present application uses the overexpression LsSLR1 transgenic Arabidopsis thaliana homozygous line cultivated to carry out salt stress, drought stress, abscisic acid (ABA) stress, gibberellin (GA3) stress experiment, and observe the phenotype of the overexpression LsSLR1 transgenic Arabidopsis thaliana under different habitats, and analyze the influence of the overexpression LsSLR1 on the stress resistance of plants. The experimental steps are as follows.

[0118] The overexpression LsSLR1 transgenic Arabidopsis thaliana root system phenotype experiment:

[0119] After the wild type and the overexpression LsSLR1 transgenic Arabidopsis thaliana seed is sterilized, it is sowed in a MS culture dish. The culture dish is placed in a 4℃ dark environment for vernalization for 2 days, and then transferred to an environment with a light duration of 14h / d and a temperature of 23℃ for culture for 7 days to germination. The germinated seedlings are 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), 50μM GA3 (gibberellin treatment), and the blank MS medium as a control group.

[0120] The culture dishes were placed vertically and continued to be cultured under the same light (16 h / d) and temperature (23°C) conditions for 7 days. The growth status of the Arabidopsis roots under different stress conditions was recorded by taking pictures. ImageJ software was used to measure the root length and the number of lateral roots. The differences in the responses of each strain under drought stress, ABA, salt stress, and GA3 treatment were analyzed by inter-group comparison. One-way ANOVA and significant difference analysis (P < 0.05) were performed using IBM SPSS Statistics 25 software. Three biological replicates were set for each control and treatment group. P <0.05 as the significant level). Three biological replicates were set for each control and treatment group.

[0121] Overexpression under stress treatment LsSLR1 Transgenic Arabidopsis thaliana overground phenotype experiment:

[0122] The wild type and overexpression LsSLR1 After the seeds of transgenic Arabidopsis thaliana were sterilized, they were sown in MS culture dishes. The culture dishes were placed in a dark environment at 4°C for 2 days for vernalization, and then the culture dishes were transferred to an environment with light for 16 h / d and a temperature of 23°C for 7 d for seed germination. The germinated seedlings were transplanted into square pots filled with nutrient soil, with 4 plants per pot, and placed in an environment with a light duration of 14 h / d and a temperature of 23°C for further cultivation for 2 weeks, with regular watering management during this period. When the plants grew to 4 weeks old, stress treatment was performed. Plants irrigated with an equal amount of water served as the control group, and the control and treatment were both three biological replicates, with simultaneous leaf sampling and preservation. Enzyme activity determination was performed according to the instructions of the Peroxidase (POD) kit, Superoxide Dismutase (SOD) kit, and Malondialdehyde (MDA) content kit purchased from Iseki Biological Technology Co., Ltd.

[0123] (1) Drought stress treatment: stop irrigation, drought treatment for 7 days, and then cut the Arabidopsis thaliana overground leaves, and store them in a 4°C environment for enzyme activity determination as soon as possible;

[0124] (2) ABA stress treatment: on the first day, on the third day, on the fifth day, and on the seventh day, irrigate the pots with 100 μM ABA solution, and then cut the Arabidopsis thaliana overground leaves, and store them in a 4°C environment for enzyme activity determination as soon as possible;

[0125] (3) Salt stress treatment: on the first day, on the third day, on the fifth day, and on the seventh day, irrigate the pots with 200 mM NaCl solution, and then cut the overground leaves on the next day, and store them in a 4°C environment for enzyme activity determination as soon as possible.

[0126] (4) GA3 stress treatment: On the first day, on the third day, on the fifth day, on the seventh day, 150 μM of GA3 solution was irrigated into the flowerpot to slightly seep out, and the next day the aboveground leaves were cut and stored at 4°C to determine the enzyme activity as soon as possible.

[0127] The experimental results are as follows:

[0128] (1) LsSLR1 The results of the influence of the salt stress resistance of the plants are shown in Figure 9 and Figure 10 By comparing and observing the phenotypes of the underground salt stress experiment, it can be seen that the main root length of the overexpression LsSLR1 transgenic Arabidopsis thaliana lines has no obvious difference from that of the wild type on the salt stress medium, but the number of lateral roots of the overexpression lines is significantly increased, indicating that the overexpression of the Leymus mollis LsSLR1 gene promotes the development of lateral roots under salt stress conditions. It is found by observing the aboveground stress experiment results that the growth of the transgenic lines is better than that of the wild type under salt stress, although the leaves of the transgenic lines are smaller and slightly yellow than those of the control group, but the wild type Arabidopsis thaliana not only has an increased range of yellow leaves but also has wrinkled leaves under salt treatment; the MDA enzyme activity of the wild type is more significantly increased than that of the overexpression LsSLR1 transgenic Arabidopsis thaliana lines, indicating that the stress degree of the overexpression LsSLR1 transgenic Arabidopsis thaliana lines is smaller, and the antioxidant enzyme POD, SOD activities are higher, further indicating that the LsSLR1 gene improves the salt stress resistance of Arabidopsis thaliana.

[0129] (2) LsSLR1 The results of the influence of the drought stress resistance of the plants are shown in Figure 11 and Figure 12 It can be concluded from the experimental results that the main root length and the number of lateral roots of the overexpression LsSLR1 transgenic Arabidopsis thaliana lines are significantly increased compared with those of the wild type; the number of lateral roots of the overexpression LsSLR1 transgenic Arabidopsis thaliana lines is significantly reduced compared with that of the control group, but the overall development of the root system is still better than that of the wild type, and the main root length of the overexpression LsSLR1 transgenic Arabidopsis thaliana lines under stress treatment is also significantly increased compared with that of the control group, indicating that the overexpression of the Leymus mollis LsSLR 1gene can improve the drought stress resistance of Arabidopsis thaliana. The aboveground stress experiment results show that the leaves of the transgenic lines are smaller and purplish than those of the control group under drought stress, but the growth of the transgenic lines is better than that of the wild type, and the wild type Arabidopsis thaliana stops growing and even dies under drought stress; the MDA enzyme activity of the wild type is more significantly increased than that of the overexpression LsSLR1The MDA enzyme activity of the transgenic Arabidopsis was increased more significantly, indicating that the transgenic Arabidopsis was under less stress; at the same time, although the POD activity of the transgenic Arabidopsis under drought stress was not significantly different from that of the wild type, the SOD activity was higher, indicating that the LsSLR1 gene can increase the drought stress resistance of Arabidopsis by increasing the SOD enzyme activity.

[0130] (3) LsSLR1 The results of the effect on the plant resistance to abscisic acid stress are shown in Tables 2 and 3. Figure 13 and Figure 14 On the ABA stress medium, the main root length of the transgenic Arabidopsis overexpressing the LsSLR1 gene was not significantly different from that of the wild type, the number of lateral roots of the transgenic Arabidopsis overexpressing the LsSLR1 gene was significantly lower than that of the control group, but the number of lateral roots of the transgenic Arabidopsis overexpressing the LsSLR1 gene was significantly higher than that of the wild type, indicating that the overexpression of the LsSLR1 gene can increase the ABA stress resistance of Arabidopsis. At the same time, the above-ground stress experiment showed that under ABA stress, the transgenic Arabidopsis overexpressing the LsSLRl had more leaves and larger leaf area, and the MDA enzyme activity was smaller, indicating that the stress degree was lower, and the antioxidant enzymes POD and SOD activities were higher, further indicating that the overexpression of the LsSLR1 gene can increase the ABA stress resistance of Arabidopsis.

[0131] (4) LsSLR1 The results of the effect on the plant resistance to abscisic acid stress are shown in Tables 2 and 3. Figure 15 and Figure 16 By comparing and observing the phenotypes of the underground stress experiment, it was found that the main root length and the number of lateral roots of the transgenic Arabidopsis in the stress treatment group were significantly reduced compared with the control group, and on the GA3 medium, the root length and the number of lateral roots of the transgenic Arabidopsis overexpressing the LsSLR1 gene were also reduced compared with the wild type in the treatment group, indicating that the overexpression of the LsSLRl gene reduced the ability to increase the GA3 stress resistance of Arabidopsis. LsSLR1 The results of the above-ground stress experiment showed that the transgenic Arabidopsis overexpressing the LsSLR1 gene grew worse than the wild type Arabidopsis under GA3 stress, and the transgenic and wild type Arabidopsis had smaller leaves and more serious purple leaves compared with the control group, and the MDA enzyme activity was higher than that of the wild type, while the SOD enzyme activity was significantly reduced, indicating that the overexpression of the LsSLR1 gene reduced the ability of Arabidopsis to resist high-concentration GA3.

[0132] It is to be understood that every range of values disclosed herein is to be understood to encompass any and every sub-range of values within the range. Although the preferred embodiments of the invention have been described above, it will be appreciated that those skilled in the art, on consideration of this disclosure, will be able to devise additional embodiments that, although not explicitly described or shown herein, nonetheless fall within the scope of the present invention. Accordingly, the appended claims are intended to include within their scope all such alternatives, modifications and variations as fall within the scope of the present invention. Various features and aspects of the present invention will become apparent from the following examples, which are intended only to exemplify the invention. It should be understood, of course, that in the various examples of the present invention, the specific phrasing of the claims will depend on the exact nature of the claims sought.

[0133] Obviously, numerous modifications and variations of the present application are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims and their equivalents, the present application can be practiced otherwise than as specifically described herein.

Claims

1. LsSLR1 gene or a nucleic acid LsSLR1 Use of a gene-related biological material in plant trait improvement, characterized in that, The LsSLR1 The nucleotide sequence of the gene is shown in SEQ ID NO.3; the biological material includes any one of the following: a、 the LsSLR1 gene encodes a protein, the amino acid sequence of which is shown as SEQ ID NO. 4; b. comprising said LsSLR1 a recombinant expression vector of a gene; c. a recombinant microbial strain comprising said LsSLR1 gene; The specific application involves: enhancing the [specific properties] of plants through transgenic or gene-editing technologies. LsSLR1 The expression level of the gene is used to promote the development of lateral roots, dwarf the plant, or shorten the plant growth cycle; or to improve the plant's ability to resist salt stress, drought stress, or abscisic acid stress; the plant is Arabidopsis thaliana.

2. A method of breeding a plant with enhanced stress resistance, characterized in that, The plant is Arabidopsis thaliana and the method comprises the following steps: Containing the contents of claim 1 LsSLR1 The recombinant gene expression vector is introduced into the target plant, enabling... LsSLR1 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; wherein the stress is salt stress, drought stress or abscisic acid stress.

3. The method of claim 2, wherein, The recombinant expression vector is pCAMBIA1300-GFP- LsSLR1 The recombinant expression vector is composed of LsSLR1 The gene was obtained after being introduced into the overexpression vector pCAMBIA1300-GFP.

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