Alfalfa seed specific promoter and application thereof

Abstract

The invention discloses an alfalfa seed specific expression promoter and application thereof in rapid screening and identification of transgenic plants. The invention belongs to the technical field of biology, and particularly relates to an alfalfa seed specific expression promoter and application thereof in rapid screening and identification of transgenic plants. The promoter is a DNA molecule of a), b) or c) as follows: a) a DNA molecule at the 25th-2896th site of which the nucleotide sequence is SEQ ID No: 1; b) a DNA fragment which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of identity with the nucleotide sequence limited by the a), is derived from alfalfa and has a promoter function; c) a DNA fragment which is hybridized with the nucleotide sequence limited by a) or b) under strict conditions and has the promoter function. The promoter can be used for improving the specific expression of genes in seeds and is applied to specific marking of plant seeds or improvement of seed traits.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an alfalfa seed-specific expression promoter and its application in the rapid screening and identification of transgenic plants. Background Technology

[0002] Alfalfa is an important leguminous forage crop, possessing excellent characteristics such as high protein content and good palatability, making it an indispensable feed resource for animal husbandry. my country's supply of high-quality alfalfa hay and seeds is highly dependent on imports, and existing alfalfa varieties are far from meeting production needs. Currently, alfalfa breeding work still mainly relies on recurrent selection, resulting in long breeding cycles and a limited number of bred varieties. With the development of biotechnology, new technologies and methods such as male-sterile line hybridization breeding, cell engineering breeding, and space breeding are gradually being applied to the alfalfa breeding process.

[0003] In the field of biotechnology breeding, commonly used transgenic markers in alfalfa include hygromycin resistance and glufosinate resistance, but there is a lack of seed-specific, rapid, and non-destructive phenotypic markers for transgenic screening. During the breeding process, seed-specific expression promoters driving the expression of fluorescently labeled genes can effectively achieve transgenic seed screening. Seed-specific expression promoters are an important tool for improving plant seed genetic engineering.

[0004] Because alfalfa is a homotetraploid with high heterozygosity, its gene function analysis faces significant challenges. The closely related alfalfa has advantages such as a smaller diploid genome, self-pollination, short growth period, and ease of genetic transformation, and is often used as the model species of leguminous plants, providing convenience for the exploration of gene function in alfalfa species.

[0005] In conclusion, developing a seed-specific promoter for alfalfa is of great significance for basic research and bio-breeding applications of alfalfa. Summary of the Invention

[0006] The main problem this invention aims to solve is how to make alfalfa seeds specifically express their characteristics, thus facilitating alfalfa breeding.

[0007] To address the above problems, the present invention provides DNA molecules as follows: a), b), or c): a) The nucleotide sequence is the DNA molecule with positions 25-2896 of SEQ ID No:1; b) A DNA fragment that has 99%, 95%, 90%, 85%, or 80% or more identity with the nucleotide sequence defined in a), and is derived from alfalfa and has promoter function; c) A DNA fragment that hybridizes to the nucleotide sequence defined in a) or b) under strict conditions and has promoter function.

[0008] The aforementioned 75% or higher consistency can be expressed as 80%, 85%, 90%, or 95% or higher consistency.

[0009] The present invention also provides expression cassettes or recombinant vectors containing the DNA molecules described above.

[0010] The expression cassette containing the PHApro promoter refers to DNA capable of expressing a target gene in a host cell. This DNA includes not only the PHApro promoter to initiate the target gene but may also include a terminator to terminate the transcription of the target gene. Furthermore, the expression cassette may also include an enhancer sequence. The transcription terminators mentioned include, but are not limited to: Agrobacterium carmine synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and carmine and octopine synthase terminators (see, for example: Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627).

[0011] The recombinant vector can be a recombinant expression vector or a recombinant cloning vector.

[0012] In one specific embodiment, the recombinant vector may be pEarleyGate100- PHA pro :RFP Carrier. The pEarleyGate100- PHA pro :RFP The structure of the vector is described as follows: It is a recombinant vector obtained by inserting a DNA fragment (sequence 19-3582 of SEQ ID No:1) between the 5'-GCAGCAGCTGACGCGTAC-3' and 5'-TCTAGAGAGTTAATTAAG-3' fragments of the starting vector pEarleyGate100, while keeping the other sequences of the pEarleyGate100 vector unchanged. pEarleyGate100-PHA pro :RFP The vector can express RFP fluorescent protein, whose amino acid sequence is SEQ ID No:2.

[0013] The present invention also provides recombinant microorganisms containing the DNA molecules described above.

[0014] The recombinant microorganisms may specifically be bacteria, yeast, algae, and fungi. Among them, bacteria may be derived from the genus *Escherichia* (…). Escherichia Erwinia () Erwinia ), Agrobacterium tumefaciens ( Agrobacterium Flavobacterium ( Flavobacterium Alcaligenes ( ) Alcaligenes ), Pseudomonas spp. Pseudomonas ), Bacillus spp. ( Bacillus (etc.) The transgenic cell lines mentioned do not include plant propagation material.

[0015] The present invention also provides a transgenic cell line containing the DNA molecules described above.

[0016] This invention also provides the application of the DNA molecule described above as a promoter.

[0017] The application of DNA molecules in the construction of alfalfa smart maintainer lines, as described above, also falls within the scope of protection claimed in this invention.

[0018] This invention also provides the application of the DNA molecules described above in initiating the expression of target genes in plants.

[0019] Furthermore, the activation of the target gene expression refers to the specific expression of the target gene in plant seeds.

[0020] This invention also provides the application of the aforementioned DNA molecules or recombinant vectors or expression cassettes, recombinant microorganisms or transgenic cell lines in the cultivation of transgenic plants or plant breeding.

[0021] The term "transgenic plant" is understood to include not only the first-generation transgenic plants obtained by transforming a target gene into a target plant, but also its progeny. For transgenic plants, the gene can be propagated within the species, or it can be transferred into other varieties of the same species using conventional breeding techniques, particularly commercial varieties. The transgenic plant includes seeds, callus tissue, complete plants, and cells.

[0022] Furthermore, the plant may be as follows: N1) Dicotyledons; N2) Leguminosae; N3) Leguminosae (family legumes); N4) Alfalfa species; N5) Tribulus terrestris or alfalfa.

[0023] This invention provides an alfalfa seed-specific promoter, PHApro, and its application method. This promoter can be used to enhance gene expression in a seed-specific manner, and can be applied to seed-specific labeling or seed trait improvement. For example, this promoter drives the formation of a molecular module of fluorescently labeled proteins, enabling efficient and non-destructive visual sorting of transgenic seeds. Linking this seed phenotypic marker module with other functional modules and transferring it into recipient plants enables efficient and non-destructive sorting of transgenic seeds. This method provides an efficient phenotypic marker for transgenic seeds, facilitating rapid and non-destructive identification of transgenic seeds, and also provides a powerful tool for seed-specific trait improvement, which is of great significance for biobreeding. Attached Figure Description

[0024] Figure 1 pEarleyGate100- PHA pro :RFP A structural diagram.

[0025] Figure 2 To obtain Agrobacterium tumefaciens-mediated genetic transformation of alfalfa PHA pro :RFP The process of regenerating genetically modified plants.

[0026] Figure 3 Genetically transformed and regenerated plants of Alfalfa truncatum PHA pro :RFP -1、 PHA pro :RFP -2、 PHA pro :RFP -3 Transgenic identification results. In the figure, the marker represents the standard molecular weight of DNA; "-" indicates a negative control, using wild-type R108 DNA as a template; "+" indicates a positive control, using pEarleyGate100- PHA pro :RFP Plasmid DNA is used as a template.

[0027] Figure 4 Genetically modified plants PHA pro :RFP -1、 PHA pro :RFP -2、 PHA pro :RFP The results of the bar test strip test at -3°C were obtained. R108 was used as a negative control.

[0028] Figure 5 Wild-type R108 and transgenic positive plants PHA pro :RFP -1 Seed fluorescence detection phenotype. Detailed Implementation

[0029] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0031] The wild-type alfalfa seeds used in the following examples are wild-type Alfalfa R108 seeds, as described in: Ye, QY, Meng, XZ, Chen, H., Wu, JL, Zheng, LH, Shen, C., Guo, D., Zhao, YF, Liu, JL, Xue, QX, Dong, JL*, and Wang, T.* (2022). Construction of genic male sterility system by CRISPR / Cas9 editing from model legume to alfalfa. [[ID=3⑧]]Plant Biotechnology Journal 20, 613-615. The biological material may be obtained from the applicant by the public and may only be used for the purpose of repeating the experiments of the present invention and may not be used for any other purpose.

[0032] The pE3025-cRFP in the following examples is described in: Guo, D., Liu, P., Liu, QW, Zheng, LH, Liu, SK, Shen, C., Liu, L., Fan, SS, Li, N., Dong, JL, and Wang, T. (2023). Legume-specific SnRK1 promotes malate supply to bacteroids for symbiotic nitrogen fixation.Molecular Plant 16, 1396-1412. The biological material is available to the public from the applicant and is intended solely for the purpose of repeating experiments of this invention and may not be used for any other purpose.

[0033] Culture medium: vermiculite:perlite (volume ratio) is 5:2.

[0034] The culture medium formulations used in the examples are as follows: YEP medium (1 L): 10 g yeast extract, 5 g sodium chloride, 10 g peptone, and 15 g agar added to the solid medium.

[0035] 10×N6 large-volume mother liquor (1 L): MgSO4·7H2O 1.85 g, KNO3 28.3 g, (NH4)2SO4 4.63 g, CaCl2·2 H2O 1.66 g, KH2PO4 4 g.

[0036] 1000×SH micro-volume stock solution (100 mL): MnSO4·H2O 1 g, H3BO3 500 mg, ZnSO4·7H2O 100 mg, KI 100 mg, Na2MoO4·2H2O 10 mg, CuSO4·5H2O 20 mg, CoCl2·6H2O 10 mg.

[0037] 1000×SH Organic Mother Liquor (100 mL): Nicotinic acid 500 mg, pyridoxine hydrochloride 500 mg, thiamine hydrochloride 500 mg.

[0038] 50×EDFS iron salt mother liquor (500 mL): NaFe·EDTA 3.487 g.

[0039] The formulation of SH3a medium (1 L) is as follows: 100 mL of 10×N6 large volume stock solution, 1 mL of 1000×SH micro volume stock solution, 1 mL of 1000×SH organic stock solution, 20 mL of 50×EDFS iron salt stock solution, 0.4 mL of 2,4-D stock solution (10 mg / mL), 0.5 mL of 6-BAP stock solution (1 mg / mL), 100 mg of inositol, 30 g of sucrose, and pH adjusted to 5.85. 3.2 g of plant gel was added to the solid medium.

[0040] The formulation of SH9 medium (1 L) is as follows: 100 mL of 10×N6 large volume stock solution, 1 mL of 1000×SH micro volume stock solution, 1 mL of 1000×SH organic stock solution, 20 mL of 50×EDFS iron salt stock solution, 100 mg of inositol, 20 g of sucrose, and pH adjusted to 5.85. 8 g of agar is added to the solid medium.

[0041] The formulation of 1 / 2 MS liquid culture medium (1L) is: MURASHIGE & SKOOG (MS) BASAL MEDIUM (M519) (Phyto Technology Laboratories) TM 2.22g of sucrose, 12g of sugar, and pH adjusted to 5.85. Add 8g of agar to the solid culture medium.

[0042] Example 1: Transgenic Alfalfa PHA pro :RFP Obtaining the plant I. Carrier pEarleyGate100- PHA pro :RFP Build Primers synthesized by Beijing Liuhe BGI Genomics Co., Ltd.: Table 1, pEarleyGate100- PHA pro :RFP Vector construction primers

[0043] Note: Underlined areas indicate enzyme cleavage sites.

[0044] First, amplification was performed using *Alfalfa* R108 DNA as a template, employing primers ProPHA-F and ProPHA-R. The amplification product (nucleotide sequence SEQ ID No:3) was recovered from a DNA gel and then... Eco RI and Bam Double digestion with HI was performed. Simultaneously, the vector pE3025-cRFP was utilized... Eco RI and Bam The sample was double-digested with HI. The digested fragment was then recovered and ligated using T4 ligase to obtain pE3025-. PHA pro :RFP Carrier.

[0045] pE3025- PHA pro :RFP The structure of the carrier is described as follows: it is based on the starting carrier pE3025-cRFP. Eco RI and Bam A recombinant vector was obtained by inserting a DNA fragment from positions 25-2896 of SEQ ID No:1 between the two HI restriction sites, while keeping the other sequences of the vector pE3025-cRFP unchanged. pE3025- PHA pro :RFPThe vector can express RFP fluorescent protein, whose amino acid sequence is SEQ ID No:2.

[0046] pE3025- PHA pro :RFP Using plasmid DNA as a template, amplification was performed using primers ProPHA:RFP-F and ProPHA:RFP-R, and the reaction products were recovered. Simultaneously, the vector pEarleyGate100 was... Bst BI and Xba I was subjected to double enzyme digestion, and the digestion products were recovered; the above amplification products and enzyme digestion products were subjected to a recombination reaction to obtain pEarleyGate100- [[ID=6②]]PHA pro :RFP carrier ( Figure 1 ).

[0047] pEarleyGate100- PHA pro :RFP The structure of the vector is described as follows: It is a recombinant vector obtained by inserting a DNA fragment (serial number 19-3582) between the 5'-GCAGCAGCTGACGCGTAC-3' and 5'-TCTAGAGAGTTAATTAAG-3' restriction enzyme sites of the starting vector pEarleyGate100, while keeping the other sequences of the pEarleyGate100 vector unchanged. PHA pro :RFP The vector can express RFP fluorescent protein, whose amino acid sequence is SEQ ID No:2.

[0048] II. Genetically Modified Alfalfa PHA pro :RFP Obtaining the plant Using Agrobacterium tumefaciens-mediated leaf disc transformation, pEarleyGate100- PHA pro :RFP The vector was transformed into an alfalfa R108 receptor background, and the desired results were obtained. PHA pro :RFP Transgenic materials, including the co-culture, dedifferentiation, redifferentiation, and emergence stages, such as... Figure 2 The specific steps are as follows: 1. Electroporation of Agrobacterium tumefaciens The plant transformation vector pEarleyGate100- PHA pro :RFP50 ng of plasmid was transferred into competent Agrobacterium EHA105 cells (purchased from Beijing Coollab Technology Co., Ltd., catalog number: EC403-10×50 μL). After incubating on ice for 5 min, the mixture was added to a pre-chilled electroporation cuvette and electroporated at 2100 V. Then, 500 μL of antibiotic-free YEP liquid medium was added to the cuvette, and the mixture was thoroughly mixed. The bacterial culture was transferred to a 2 ml centrifuge tube and cultured at 28℃ and 200 rpm for 1 h with shaking. 30 μL of the bacterial culture was spread onto YEP solid medium containing Rifampicin (75 mg / L) and Kanamycin (50 mg / L) and cultured in the dark at 28℃ for 24-48 h with inverted incubation. Clones were picked and cultured in 700 μL of YEP liquid medium containing Rifampicin (75 mg / L) and Kanamycin (50 mg / L) for 24-48 h with shaking. h, plasmids were extracted and identified by PCR. The identification primers ProPHA:RFP-JP-F (5'- CTACAAAACTAGCACCAA-3') and ProPHA:RFP-JP-R (5'- ACTCGTGGCCGTTCACGG-3') were used for amplification. Positive clones showed a specific band of 2145 bp. Positive clones were selected for the next step of infection.

[0049] 2. Explant preparation First, prepare sterile seedlings of wild-type Alfalfa R108. Aliquot 50 Alfalfa R108 seeds into 2mL centrifuge tubes, treat with 1mL of 98% H2SO4 for 8 min, rinse 5 times with pre-cooled deionized water; then sterilize with 1mL of 30% Libai disinfectant for 12 min, rinse 5 times with sterile water in a clean bench; spread the seeds evenly on 0.8% water agar plates and incubate upside down at 4℃ in the dark for 3 days. Subsequently, incubate upside down at room temperature in the dark for 12 h, and then sow the germinated seeds on 1 / 2 MS medium in a clean bench. After 3-4 weeks of growth at 22℃ for 16 h light and 8 h dark, the seeds can be used for genetic transformation.

[0050] Collect fully expanded leaves of R108 sterile seedlings in a clean bench, and cut each single leaf into 2-4 pieces with a sterile knife for use as explants.

[0051] 3. Preparation of Agrobacterium infection solution Will be transferred to pEarleyGate100- PHA pro :RFPEHA105 Agrobacterium-positive colonies were transferred to 200 mL of YEP liquid medium containing Rifampicin (75 mg / L) and Kanamycin (50 mg / L) and cultured at 28°C with shaking at 230 rpm until OD200 was reached. 600 The concentration was 0.6-0.8. The bacterial culture was transferred to a sterile centrifuge bottle and centrifuged at 5000 rpm for 10 min at room temperature. The supernatant was removed in a clean bench, and the culture was resuspended in an equal volume of SH3a liquid medium containing 0.1 mM acetylsuccinone to prepare pEarleyGate100- PHA pro :RFP The staining solution for converting materials.

[0052] 4. Agrobacterium-mediated genetic transformation and co-culture The prepared explants were placed in the prepared infection solution under sterile conditions, and a vacuum was applied to -0.09 MPa for 30 min, followed by slow degassing. The explants were then cultured on a horizontal shaker at 50 rpm in the dark for 1.5 h. Subsequently, the infection solution was removed in a clean bench, and excess liquid on the surface of the explants was blotted dry with sterile filter paper. The explants were then spread evenly on SH3a solid medium (containing 0.1 mM acetylsyleugenol) covered with a single layer of filter paper and cultured at 22°C in the dark for 3 days.

[0053] 5. Callus induction and differentiation Explants were transferred to SH3a solid medium containing glufosinate (10 mg / L) and termethin (200 mg / L) to induce resistant callus. The culture was carried out at 22°C in the dark, with subculture every 2 weeks for a total of three subcultures over 6 weeks. Subsequently, the callus was transferred to SH9 solid medium containing glufosinate (5 mg / L) and termethin (200 mg / L) to induce shoot differentiation. The culture was carried out at 22°C, under 16 h light and 8 h dark conditions, with subculture every 3 weeks until regenerated seedlings were produced. The regenerated seedlings were then transferred to 1 / 2 MS solid medium without antibiotics for rooting, and cultured at 22°C, under 16 h light and 8 h dark conditions, with subculture every 3-4 weeks. After rooting, the seedlings were transferred to nutrient soil and cultured under greenhouse conditions with plastic wrap to maintain humidity. The wrap was removed after 5 days. The greenhouse conditions were 22°C, 16 h light, 8 h dark, and 70%-80% humidity.

[0054] III. Genetically Modified Alfalfa PHA pro :RFP Genotyping of plants 1. DNA level detection PHA pro :RFP Transgenic positive plants.

[0055] extract PHApro :RFP Genomic DNA from transgenic plants was used as a template, and ProPHA:RFP-TF and ProPHA:RFP-TR were used as primers for amplification. Wild-type R108 genomic DNA was used as a negative control, and pEarleyGate100- PHA pro :RFP The plasmid was used as a positive control. The total volume of the reaction system was 20 μL, including 10 μL of 2× Taq mix, 1 μL of primer F, 1 μL of primer R, 1 μL of template DNA, and 7 μL of ddH2O.

[0056] ProPHA:RFP-TF:5'-CAATACACAAACACACGCTAAA-3'; ProPHA:RFP-TR:5'-TAGTCCCGGGTCTTAATTAACTC-3'.

[0057] The amplification reaction program was as follows: Round 1: denaturation at 95℃ for 5 min; Round 2: denaturation at 94℃ for 30 sec, annealing at 55℃ for 30 sec, extension at 72℃ for 1 min, 33 cycles; Round 3: extension at 72℃ for 10 min. After the program was completed, the results were detected by 1.0% agarose gel electrophoresis.

[0058] The results are as follows: positive transgenic plants can amplify a fragment of 773 bp in size. PHA pro :RFP- 1. [[ID=⑨2]]PHA pro :RFP- 2. PHA pro :RFP- All 3 were transgenic positive plants ( Figure 3 ).

[0059] 2. Protein level detection in alfalfa PHA pro :RFP Transgenic positive plants.

[0060] The transgenic positive plant materials identified by PCR were tested using the transgenic PAT / bar rapid test kit (catalog number: AA1032-LS) from Shanghai Youlong Biotechnology Co., Ltd. Plant leaf tissue was placed between the cap and the body of a disposable tissue extraction tube, and the cap was quickly closed. This was repeated twice to obtain two circular leaf tissues. The pestle was inserted into the tube, and the leaf tissue was crushed by rotating the pestle, pressing continuously for 20-30 seconds. 0.2 mL (approximately 8 drops) of extraction buffer EB004 was added. The crushing step was repeated to ensure thorough mixing of the sample and buffer. The pestle was removed. After the rapid test strip and the sample solution were brought to room temperature, the rapid test strip was removed from the test strip container and directly inserted into the sample slot. The time was 8-10 minutes, and the results were read. Positive materials showed two bands, a C line and a T line, on the Bar rapid test strip, while the negative control only showed a C line band. No bands were considered due to improper operation or test strip failure. The results showed that... PHA pro :RFP- 1. PHA pro :RFP- 2. PHA pro :RFP- All three Bar tests were positive. Figure 4 ).

[0061] Four, PHA pro :RFP Phenotypic identification of transgenic positive plants Wild-type R108 and transgenic positive plants PHA pro :RFP- Fluorescence observation of seeds 1: Under an OLYMPUS SZX7 stereomicroscope, using the OLYMPUS SZX7 stereomicroscope light source and the RFP excitation source of the LUYOR-3415 dual-wavelength fluorescent protein excitation source from LUYOR Instruments Co., Ltd., fluorescence was observed in R108, PHA pro :RFP- Mature seeds were irradiated with laser.

[0062] The results are as follows Figure 5 As shown: R108 seeds showed no RFP fluorescence signal under excitation light. PHA pro :RFP- Seeds of variety 1 showed a clear RFP fluorescence signal under excitation light. Therefore, PHA pro :RFP Seed-specific expression promoters that bind to fluorescently labeled genes enable effective screening of transgenic seeds.

[0063] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. DNA molecules as shown in a), b), or c): a) The nucleotide sequence is the DNA molecule with positions 25-2896 of SEQ ID No:1; b) A DNA fragment that has 99%, 95%, 90%, 85%, or 80% or more identity with the nucleotide sequence defined in a), and is derived from alfalfa and has promoter function; c) A DNA fragment that hybridizes to the nucleotide sequence defined in a) or b) under strict conditions and has promoter function.

2. An expression cassette or recombinant vector containing the DNA molecule of claim 1.

3. A recombinant microorganism containing the DNA molecule of claim 1.

4. A transgenic cell line containing the DNA molecule of claim 1.

5. The application of the DNA molecule as a promoter as described in claim 1.

6. The application of the DNA molecule of claim 1 in the construction of alfalfa smart maintainer lines.

7. The application of the DNA molecule of claim 1 in initiating the expression of a target gene in plants.

8. The application according to claim 7, characterized in that: The target gene expression is the specific expression of the target gene in plant seeds.

9. The application of the DNA molecule of claim 1, the recombinant vector or expression cassette of claim 2, the recombinant microorganism of claim 3, or the transgenic cell line of claim 4 in plant breeding.

10. The application according to claim 9, characterized in that, The plants mentioned are as follows: N1) Dicotyledons; N2) Leguminosae; N3) Leguminosae (family legumes); N4) Alfalfa species; N5) Tribulus terrestris or alfalfa.

Images