A method for alfalfa branch-related gene mining

Abstract

The present application belongs to the field of forage grass molecular biology and genetic engineering, and particularly relates to a method for mining alfalfa branching-related genes. The method comprises the following steps: mowing alfalfa seedlings; on the 0th day to the 7th day after mowing, taking tissue samples from the mowing part; extracting total RNA, reverse transcribing cDNA, and performing qPCR detection with branching negative regulatory genes as targets; selecting samples with the expression of the branching negative regulatory genes showing a first decrease and then a rebound for mining branching-related genes. The present application clearly specifies key parameters such as the growth days of seedlings, mowing positions (cotyledon nodes or first leaf base), and sampling time points, and consistent results can be obtained by different experimenters and different batches of experiments, thereby solving the problem of non-uniform parameters in existing research.

Description

Technical Field

[0001] This invention belongs to the field of forage molecular biology and genetic engineering, specifically relating to a method for mining branching-related genes in alfalfa. Background Technology

[0002] alfalfa ( Medicago sativa Alfalfa is the most widely cultivated legume forage crop globally. Known as the "King of Forages" due to its high protein content and palatability, it is an indispensable high-quality forage for ruminants such as dairy cows. Therefore, developing high-yielding and stable-yielding new varieties of alfalfa is an important goal in forage breeding.

[0003] Biomass is a core indicator determining forage yield, but as a complex quantitative trait, direct genetic improvement is relatively inefficient. Studies have shown that the number of branches per plant is a core structural factor constituting canopy structure and determining the number of harvestable individuals, exhibiting a significant positive correlation with biomass (correlation coefficient exceeding 0.7). Therefore, the number of branches has become a key lever trait for improving alfalfa yield.

[0004] The fate of axillary buds is the initiation step in branching, precisely regulated by a network of hormones including strigolactone, auxin, and cytokinin, ultimately leading to... TB1 / BRC1 This pathway involves the execution of key transcription factors. Although it has been studied extensively in model plants such as Arabidopsis and rice, significant gaps remain in the research on its conservation and regulatory mechanisms in allotetraploid and perennial leguminous crop alfalfa. Currently, there is a lack of standardized operating parameters and methods for mining alfalfa branching-related genes associated with the branching regulatory pathway, making it difficult to support high-throughput screening of branching-related genes. Summary of the Invention

[0005] To develop standardized operating parameters and a method for mining alfalfa branching-related genes associated with branching regulatory pathways, thereby supporting high-throughput screening of these genes, this invention provides a method for mining alfalfa branching-related genes. This method establishes a standardized parameter system for mowing treatment and uses the dynamic changes in the expression of negatively regulating genes in branching as a molecular validation indicator to achieve quantitative detection of the expression response of branching-related genes. To achieve the above objectives, this invention employs the following technical solution.

[0006] This invention provides a method for mining branching-related genes in alfalfa, comprising the following steps: The alfalfa seedlings are cut 4-6 mm above or below the cotyledon node or 4-6 mm above or below the growing point at the base of the first leaf to remove the above-ground parts; wherein the alfalfa seedlings are characterized by a plant height of 5-6 cm, cotyledons that are spread out and form an angle of 160°-180° with the hypocotyl, and the length of the first true leaf being 0.3-0.8 cm.

[0007] Cutting treatment: Cut the seedlings 4 mm to 6 mm above or below the cotyledon node or 4 mm to 6 mm above or below the growing point at the base of the first leaf to remove the above-ground parts.

[0008] Dynamic sampling: Tissue samples were taken from the cut site on days 0 to 7 after the cutting treatment.

[0009] Total RNA was extracted from tissue samples from each cut site and reverse transcribed into cDNA. qPCR was then performed targeting negatively regulatory genes in the branched regions.

[0010] Samples showing a decrease followed by a rebound in the expression levels of negatively regulated genes in branching were selected for use in the mining of branching-related genes.

[0011] This invention aims to provide a method for mining branching-related genes in alfalfa. This method provides molecular validation evidence and clarifies its positioning as a research tool. It differs from agronomic yield-increasing methods by introducing branching-regulating genes (such as...) OsBRC1 The qPCR detection of homologous genes serves as molecular evidence and aims to become an effective research foundation for screening and functional verification of branched genes.

[0012] This invention addresses the lack of standardized parameters and molecular validation in existing technologies by establishing a standardized system integrating "seedling growth standards, cutting site parameters, sampling time windows, and molecular validation indicators." The standard for alfalfa seedlings is defined as a height of 5-6 cm, fully expanded cotyledons, and the emergence of the first true leaf, ensuring consistency of the starting material. During the cutting stage, the cutting site (4-6 mm above and below the cotyledon node or the base of the first leaf) and the method of removing the aboveground parts are precisely defined to ensure the reproducibility of the physical treatment. In the sample collection stage, a daily sampling time window is set from day 0 to day 7 after cutting to cover the critical cycle of gene expression dynamics. In the molecular validation stage, qPCR detection is performed targeting negatively regulating genes in branching, and the "expression level first decreases then rebounds" criterion is used to correlate the physical treatment of cutting with the molecular response of the branching regulatory pathway. This provides a reproducible and quantifiable standardized research method for high-throughput screening and functional validation of branching-related genes.

[0013] Furthermore, the alfalfa seedlings are obtained by sowing alfalfa seeds in a culture medium and cultivating them under a photoperiod of 15.5 to 16.5 hours of light / 7.5 to 8.5 hours of darkness for 9 to 11 days to obtain the alfalfa seedlings (seedlings with uniform growth).

[0014] The selection criteria for culturing seedlings for 9 to 11 days were as follows: the seedlings showed a high degree of consistency in growth status among individual seedlings, the branching-related genes were sensitive to the mowing signal and exhibited strong regularity in expression changes, and the cotyledonary nodes and the base of the first leaf showed clear tissue differentiation, making them easy to sample accurately.

[0015] Furthermore, the seedlings with uniform growth are characterized by a plant height of approximately 5-6 cm, fully expanded cotyledons, and the first true leaf emerging.

[0016] Furthermore, the selection of the eight time points from 0 to 7 days is based on the following criteria: fully covering the entire gene expression cycle from "cutting stress response" to "axillary bud germination initiation", with days 1-3 being the rapid gene expression response period and days 5-7 being the expression recovery period.

[0017] Furthermore, the trend of the branched negative regulatory gene expression level first decreasing and then increasing means that the expression level of the branched negative regulatory gene drops to its lowest point on day 1 to day 3 after the mowing treatment, and then gradually increases on day 5 to day 7.

[0018] Furthermore, tissue samples from the cut site are immediately placed in liquid nitrogen for quick freezing after sampling and stored at -78℃ to -82℃ for later use.

[0019] Furthermore, the culture medium (nutrient soil) is a mixture of peat soil, vermiculite and perlite in a mass ratio of 2.5~3.5:0.5~1.5:1.

[0020] Furthermore, the cultivation conditions for the alfalfa seeds also include a temperature of 23℃~27℃ and a relative humidity of 60%~70%.

[0021] Furthermore, the branching negative regulatory genes include MsBRC1 The gene, whose nucleotide sequence is shown in SEQ ID NO.1.

[0022] Furthermore, the qPCR detection uses 2 -ΔΔCt The relative expression levels of the branched negative regulatory genes were calculated using the method, with internal reference genes as the reference. MsActin or MsUBQ Standardize it.

[0023] The MsActin The nucleotide sequence is shown in SEQ ID NO.2.

[0024] Furthermore, the reaction system for the qPCR detection is as follows: 1.8 μL to 2.2 μL cDNA template, 0.36 μL to 0.44 μL 10 μM upstream primer, 0.36 μL to 0.44 μL 10 μM downstream primer, 9 μL to 11 μL 2×SYBR Green Master Mix, and 6.5 μL to 7.9 μL double-distilled water, for a total volume of 20 μL.

[0025] Furthermore, the reaction procedure for the qPCR detection is as follows: pre-denaturation at 94℃~96℃ for 28~32 seconds; then 38~42 cycles are performed, each cycle including denaturation at 94℃~96℃ for 5~10 seconds, annealing and extension at 55℃~60℃ for 30~34 seconds; finally, melting curve analysis is performed: 94℃~96℃ for 14~16 seconds, 58℃~61℃ for 58~62 seconds, and 94℃~96℃ for 14~16 seconds.

[0026] Furthermore, the method is used for screening candidate genes related to alfalfa branching, including: performing transcriptome sequencing or qPCR detection on samples after mowing and control group samples, and screening for genes whose expression is significantly downregulated on day 1 to day 3 after mowing or whose expression is significantly upregulated on day 5 to day 7 after mowing.

[0027] Furthermore, the method is used for functional verification of alfalfa branching-related genes, including: silencing or overexpressing candidate genes obtained through screening, and verifying the function of the gene in branching regulation in combination with the standardized mowing method.

[0028] Furthermore, the cutting system I is suitable for screening genes involved in the regulation of axillary bud initiation, and the cutting system II is suitable for screening genes involved in the regulation of axillary bud elongation.

[0029] This invention also provides a standardized experimental system for mining alfalfa branching-related genes, comprising the following components: (1) Standardized seedling materials: alfalfa seedlings that have grown for 10 days and are in the stage where the cotyledons are fully expanded and the first true leaf is emerging.

[0030] (2) Standardized cutting treatment: Cutting treatment is carried out 5 mm above or below the cotyledon node or 5 mm above or below the growth point at the base of the first leaf.

[0031] (3) Standardized sampling plan: Dynamic sampling at 8 time points on day 0, day 1, day 2, day 3, day 4, day 5, day 6 and day 7 after the cutting treatment.

[0032] (4) Criteria for judging the effectiveness of standardization: based on MsBRC1 Gene expression levels show a trend of first decreasing and then increasing, serving as a molecular marker of system effectiveness.

[0033] The standardized experimental system provided by this invention is used to establish a reproducible and comparable platform for mining branch-related genes, serving basic research and molecular breeding of alfalfa.

[0034] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides a method for mining branching-related genes in alfalfa. The method first employs purely physical mowing without introducing any exogenous hormones or transgenic elements, avoiding interference from exogenous substances on endogenous gene expression, making it suitable for studying the molecular mechanisms of branching. Second, a standardized parameter system is established, including a 10-day seedling culture cycle, a seedling standard of 5-6 cm in height with fully expanded cotyledons and the first true leaf emerging, a mowing location 4-6 mm above or below the cotyledon node or the base of the first leaf, and a sampling time window for daily sampling from day 0 to day 7 after mowing, ensuring consistent results for different experimenters and batches. Third, negative regulatory genes for branching (such as...) are introduced. MsBRC1 As a molecular marker, its expression level, detected by qPCR, showed a dynamic trend of "first decreasing and then increasing," providing molecular-level evidence for the effectiveness of mowing treatment and thus linking physical treatment with branching regulatory pathways. Finally, a standardized research method that is reproducible, quantifiable, easy to operate, and low-cost was established to achieve high-throughput screening and functional verification of branching-related genes. This addresses the problem in existing technologies that lack standardized operating parameters and molecular verification systems associated with branching regulatory pathways, making it impossible to provide a reproducible method for high-throughput screening of branching-related genes for alfalfa branching-related gene mining.

[0035] 2. The method provided by this invention also has the following advantages: 1) Easy to operate and low cost: This invention requires only conventional seedling cultivation and harvesting operations, without the need for complex equipment, expensive reagents, or exogenous chemicals. Compared to genetic transformation methods (which require 6-8 months and cost thousands of yuan per sample), this invention can complete the process within one week, with negligible cost per sample.

[0036] 2) No external interference, suitable for molecular mechanism research: This invention employs a purely physical cutting process without introducing any exogenous hormones, pesticides, or transgenic elements, thus avoiding interference from exogenous substances on the expression of endogenous genes and making it more suitable for elucidating the molecular mechanisms of branching.

[0037] 3) Standardized parameters ensure high repeatability: This invention clearly specifies key parameters such as the number of days of seedling growth (10 days), the cutting position (cotyledon node or base of the first leaf), and the sampling time point (dynamic sampling from 0 to 7 days). Different experimenters and different batches of experiments can obtain consistent results, which solves the problem of inconsistent parameters in existing research.

[0038] 4) Molecular-level verification confirms system reliability: This invention introduces branching negative regulatory genes OsBRC1 Homologous genes were used as validation markers, and their expression changes were detected by qPCR, providing direct molecular-level evidence for the effectiveness of the system, rather than relying solely on phenotypic statistics. Attached Figure Description

[0039] Figure 1 This invention relates to alfalfa seedling mowing system I; wherein: A is a schematic diagram showing the alfalfa seedlings harvested 0 days prior; B represents the sampling site for System I of alfalfa seedling harvesting; C represents the growth process of alfalfa seedlings in system I, from 0 to 7 days.

[0040] Figure 2 The results of real-time quantitative PCR for 14 branch homologous genes in alfalfa seedling mowing system I of this invention are shown; among which: A is OsBRC1 (Msa1330940) Real-time quantitative PCR results; B is OsTAD1(M5sa1366660) Real-time quantitative PCR results; C is AtFHY3 (Msa0347950) Real-time quantitative PCR results; D is MsG0580027071.01 (Msa1393360) Real-time quantitative PCR results; E is OsBRZ1 (Msa0430380) Real-time quantitative PCR results; F is OsSLR1 (Msa0410780) Real-time quantitative PCR results; G is OsSLP15 (Msa1075170) Real-time quantitative PCR results; H is OsMOC1 (Msa1094440) Real-time quantitative PCR results; I am OsFAR1 (Msa0179120) Real-time quantitative PCR results; J is MsG0580027633.01 (Msa0809890) Real-time quantitative PCR results; K is MsG0580027072.01 (Msa13779150) Real-time quantitative PCR results; L is MsG80027632.01 (Msa0485060) Real-time quantitative PCR results; M is OsCCA1 (Msa1448870) Real-time quantitative PCR results; N is OsIPA1 (Msa1162500) Results of real-time quantitative PCR.

[0041] Figure 3 This is the alfalfa seedling mowing system II of the present invention; wherein: A is a schematic diagram showing the alfalfa seedlings harvested 0 days prior; B represents the sampling site of System II for alfalfa seedling harvesting; C represents the growth process of alfalfa seedlings in the II harvesting system from 0 to 7 days.

[0042] Figure 4 The results of real-time quantitative PCR for 14 branch homologous genes in the alfalfa seedling mowing system II of this invention are shown; among which: A is OsIPA1 (Msa1162500) Real-time quantitative PCR results; B is OsSLP15 (Msa1075170) Real-time quantitative PCR results; C is OsFAR1 (Msa0179120) Real-time quantitative PCR results; D is AtFHY3 (Msa0347950) Real-time quantitative PCR results; E is MsG0580027071.01 (Msa1393360) Real-time quantitative PCR results; F is OsBRC1 (Msa1330940) Real-time quantitative PCR results; G is OsBRZ1 (Msa0430380) Real-time quantitative PCR results; H is OsSLR1 (Msa0410780) Real-time quantitative PCR results; I am OsTAD1 (M5sa1366660) Real-time quantitative PCR results; J is OsMOC1 (Msa1094440) Real-time quantitative PCR results; K is MsG0580027633.01 (Msa0809890) Real-time quantitative PCR results; L is MsG0580027072.01 (Msa13779150) Real-time quantitative PCR results; M is MsG80027632.01 (Msa0485060) Real-time quantitative PCR results; N is OsCCA1 (Msa1448870) Results of real-time quantitative PCR.

[0043] Figure 5 In this invention OsBRC1 (Msa1330940) Real-time quantitative PCR results in alfalfa seedling mowing system I and alfalfa seedling mowing system II; among which: A is OsBRC1 (Msa1330940) Real-time quantitative PCR results in alfalfa seedling harvesting system I; B is OsBRC1 (Msa1330940) Real-time quantitative PCR results in alfalfa seedling harvesting system II.

[0044] Figure 6 In this invention AtFHY3 (Msa0347950) and MsG0580027071.01 (Msa1393360) Both genes were upregulated under both harvesting systems and can be considered as candidate genes for further research; among them: A is AtFHY3 (Msa0347950) Real-time quantitative PCR results in alfalfa seedling harvesting system I; B is AtFHY3(Msa0347950) Real-time quantitative PCR results in alfalfa seedling harvesting system II; C is MsG0580027071.01(Msa1393360) Real-time quantitative PCR results in alfalfa seedling harvesting system I; D is MsG0580027071.01(Msa1393360) Results of real-time quantitative PCR in alfalfa seedling harvesting system II. Detailed Implementation

[0045] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0046] Example 1: Establishment and Validation of Alfalfa Seedling Harvesting System I 1.1 Experimental Materials Seeds of the alfalfa variety “Zhongmu No. 4” were preserved and provided by the laboratory of the College of Grassland Science, Qingdao Agricultural University (Address: 700 Changcheng Road, Chengyang District, Qingdao, Shandong Province). For related information on this variety, please refer to: “Zhongmu No. 4” Homologous Chromosome-Level Genome Assembly Study (Long et al., Genomics Proteomics & Bioinformatics, 2022, DOI: 10.1016 / j.gpb.2022.01.002), completed by the Forage Breeding and Cultivation Innovation Team of the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences.

[0047] 1.2 Seedling Cultivation Seeds of 'Zhongmu No. 4' were sown in seedling trays filled with nutrient soil, 30 seeds per tray, and placed in a light-controlled culture room. Culture conditions: 16 hours light / 8 hours dark, temperature 25℃, light intensity 200 μmol·m⁻¹ -2 ·s -1 The relative humidity was 65%. After 10 days of cultivation, seedlings with uniform growth were obtained (plant height approximately 5.5cm ± 0.5cm, cotyledons fully expanded, and the first true leaf emerging).

[0048] The nutrient soil is Tuopu 70L seedling substrate (implementation standard NY / T2118-2012), which was purchased from Shouguang Yixiandu Agricultural Technology Co., Ltd. The product number is Tuopu 70L seedling substrate (NY / T2118-2012), which is a mixture of peat soil, vermiculite and perlite in a mass ratio of 3:1:1.

[0049] 1.3 Harvesting Process Use a sterile scalpel blade to cut 5 mm above and below the cotyledon node of the seedling, removing the above-ground parts (such as...). Figure 1 A and Figure 1 (As shown in B in the figure). A total of 60 seedlings were treated and divided into 8 groups (8 seedlings in each group), which were used for sampling at different time points.

[0050] Among them, 8 groups correspond to day 0, day 1, day 2, day 3, day 4, day 5, day 6, and day 7 after mowing, with one group for each time point, used for dynamic sampling analysis (e.g., Figure 1 (as shown in C).

[0051] 1.4 Dynamic Sampling Tissue samples (including the cotyledonary node and approximately 5 mm of surrounding tissue) from the cut sites were collected at 0, 1, 2, 3, 4, 5, 6, and 7 days post-cutting. Samples from eight seedlings at each time point were mixed and immediately flash-frozen in liquid nitrogen at -80°C for later storage. Simultaneously, corresponding parts from uncut seedlings of the same age served as a control group.

[0052] The tissue samples of the control group were 5 mm above and below the cotyledon nodes of seedlings of the same age that had not undergone mowing treatment.

[0053] 1.5 RNA extraction and qPCR detection (1) Total RNA was extracted from tissue samples at different time points in each group using the Trizol method, and the integrity and concentration of RNA were detected by 1% agarose gel electrophoresis and Nanodrop.

[0054] (2) Total RNA was reverse transcribed into cDNA using the PrimeScript™ RT kit.

[0055] (3) with alfalfa MsBRC1 Gene-specific qPCR primers were designed. The primer sequences were of standard design in this field and obtained from the publicly available alfalfa genome sequence.

[0056] The primers are as follows: The nucleotide sequence of the upstream primer is shown in SEQ ID NO.3: 5'-TCCCTTTCAAGACAGCCACC-3'.

[0057] The nucleotide sequence of the downstream primer is shown in SEQ ID NO.4: 5'-AGTTTGAAGGCCGCTGATGA-3'.

[0058] MsBRC1 The nucleotide sequence of the gene is shown in SEQ ID NO.1:

[0059] (4) with MsActin The internal reference gene was used for qPCR detection.

[0060] The reaction system for qPCR detection is as follows: SYBR Green Master Mix 10 μL, upstream primer / downstream primer 0.4 μL each, cDNA template 2 μL, ddH2O to bring the total to 20 μL.

[0061] The reaction procedure for qPCR detection is as follows: Pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 15 s, annealing at 60℃ for 30 s, extension at 72℃ for 30 s, for a total of 40 cycles.

[0062] MsActin The nucleotide sequence is shown in SEQ ID NO.2: .

[0063] (5) Using 2 ⁻ΔΔCt The relative expression level is calculated using this method.

[0064] 1.6 Experimental Results qPCR test results showed: MsBRC1 Gene expression trends: Expression begins to decline 1 day after mowing, reaches its lowest point at 3 days, and gradually rises again from 5 to 7 days (e.g., ...). Figure 5As shown in A). The expression trends of the remaining 14 branch homologous genes are as follows: 4 genes showed an expression pattern of first decreasing and then increasing (as shown in A). Figure 2 A~ Figure 2 In the D group, two genes showed a continuous downward trend. Figure 2 E~ Figure 2 In the F group, the expression changes of other genes showed no obvious pattern and exhibited unstable fluctuations. Figure 2 G~ Figure 2 (N in the text).

[0065] MsBRC1 The gene was expressed at a higher level in the control group.

[0066] One day after the cutting treatment, MsBRC1 Expression levels began to decrease significantly (to 40% of the control group).

[0067] Three days after the cutting treatment, MsBRC1 The expression level dropped to the lowest level (15% of the control group).

[0068] 5-7 days after the cutting treatment, MsBRC1 The amount of expression gradually recovered.

[0069] The trend of this expression change was consistent in all three independent replicate experiments.

[0070] From the above, we can see that MsBRC1 It is a known negative regulator of branching; its decreased expression signifies the relief of branching inhibition and enhanced axillary bud germination potential. In this invention... MsBRC1 The expression level decreased rapidly after mowing, proving that the mowing system effectively inhibited the negative regulatory pathway of branching and could induce branching, which can be used for the research on the discovery of branching-related genes.

[0071] Example 2: Establishment and Validation of Alfalfa Seedling Harvesting System II 2.1 Experimental Materials Same as Example 1.

[0072] 2.2 Seedling Cultivation Same as Example 1.

[0073] 2.3 Harvesting Process Using a sterile scalpel blade, make a cut 5 mm above and below the growing point at the base of the first leaf of the seedling to remove the above-ground part (e.g., Figure 3 A and Figure 3 (As shown in B in the diagram).

[0074] 2.4 Dynamic Sampling Same as Example 1 (e.g.) Figure 3 (as shown in C).

[0075] 2.5 RNA Extraction and qPCR Detection Same as Example 1.

[0076] The above qPCR test results show that MsBRC1 The expression trend of the gene in the mowing system II was consistent with that in system I: it began to decline 1 day after mowing, reached its lowest point on 3 days, and gradually rebounded from 5 to 7 days. Figure 5 (B in the original text). The expression trends of the remaining 14 branch homologous genes are as follows: 2 genes showed a continuous upward trend (…). Figure 4 A and Figure 4 In the B group, the three genes showed a trend of first decreasing and then increasing. Figure 4 C~ Figure 4 In E), one gene showed a continuous downward trend ( Figure 4 The expression changes of the remaining genes (F in the original text) showed no obvious pattern and exhibited unstable fluctuations. Figure 4 G~ Figure 4 (N in the text).

[0077] The above results verify the reliability and repeatability of the cutting system of the present invention.

[0078] 2.6 Screening of candidate genes with consistent expression trends in the two mowing systems Based on the qPCR detection results of 14 branch homologous genes in Examples 1 and 2 under two harvesting systems, genes with consistent expression trends in both systems were selected as candidate target genes for subsequent research.

[0079] The selection criteria are as follows: In both the cutting system I and the cutting system II, the gene expression change trends were the same; the expression trends had clear biological significance (e.g., first decreasing and then increasing, or continuously decreasing); the trends were consistent in the three independent replicate experiments.

[0080] The above screening results are as follows Figure 6 As shown, two genes with consistent expression trends were identified in both harvesting systems. AtFHY3 (Msa0347950) (like Figure 6 A and Figure 6 (as shown in B in the image) MsG0580027071.01 (Msa1393360) (like Figure 6 C and Figure 6 (D in the figure) is used as a candidate target gene for subsequent research on branching regulation mechanisms.

[0081] As can be seen from the above, this invention differs substantially from existing technologies in terms of technical objectives, degree of parameter standardization, and verification methods: existing technologies either introduce exogenous chemical substances to interfere with endogenous expression or lack standardized parameters and molecular verification; while this invention, for the first time, explicitly positions the mowing method as a gene mining tool, establishing a purely physical, highly reproducible induction system through standardized parameters (10-day seedlings, mowing at the cotyledonary node / base of the first leaf, dynamic sampling from 0-7 days) and MsBRC1 molecular marker verification. These differences are not obvious and solve the long-standing technical problem in existing technologies of "treatment without standards, effects without verification," demonstrating significant inventiveness.

[0082] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0083] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. A method for mining branching-related genes in alfalfa, characterized in that, Includes the following steps: The alfalfa seedlings are cut 4-6 mm above or below the cotyledon node or 4-6 mm above or below the growing point at the base of the first leaf to remove the above-ground parts; wherein, the alfalfa seedlings are characterized by a plant height of 5-6 cm, cotyledons that are spread out and form an angle of 160°-180° with the hypocotyl, and the length of the first true leaf being 0.3-0.8 cm; Tissue samples were taken from the cut site on days 0 to 7 after the cutting treatment. Total RNA was extracted from tissue samples from each cut site and reverse transcribed into cDNA. qPCR was then performed targeting negatively regulatory genes in the branched regions. Samples showing a decrease followed by a rebound in the expression levels of negatively regulated genes in branching were selected for use in the mining of branching-related genes.

2. The method according to claim 1, characterized in that, The alfalfa seedlings were obtained by sowing alfalfa seeds in a culture medium and cultivating them under a photoperiod of 15.5 to 16.5 hours of light / 7.5 to 8.5 hours of darkness for 9 to 11 days.

3. The method according to claim 2, characterized in that, The culture medium is composed of peat moss, vermiculite and perlite in a mass ratio of 2.5~3.5:0.5~1.5:

1.

4. The method according to claim 2, characterized in that, The alfalfa seeds were cultured under the following conditions: temperature 23℃~27℃ and relative humidity 60%~70%.

5. The method according to claim 1, characterized in that, The branching negative regulatory genes include MsBRC1 The gene, whose nucleotide sequence is shown in SEQ ID NO.

1.

6. The method according to claim 1, characterized in that, The qPCR detection used 2 -ΔΔCt The relative expression levels of the branched negative regulatory genes were calculated using the method, with internal reference genes as the reference. MsActin Standardize; The MsActin The nucleotide sequence is shown in SEQ ID NO.

2.

7. The method according to claim 6, characterized in that, The reaction system for the qPCR detection was as follows: 1.8 μL to 2.2 μL cDNA template, 0.36 μL to 0.44 μL 10 μM upstream primer, 0.36 μL to 0.44 μL 10 μM downstream primer, 9 μL to 11 μL 2×SYBR GreenMaster Mix, and 6.5 μL to 7.9 μL double-distilled water, for a total volume of 20 μL.

8. The method according to claim 6, characterized in that, The reaction procedure for the qPCR detection is as follows: pre-denaturation at 94℃~96℃ for 28 to 32 seconds; then 38 to 42 cycles are performed, each cycle including denaturation at 94℃~96℃ for 5 to 10 seconds, annealing and extension at 55℃~60℃ for 30 to 34 seconds; Melting curve analysis: 94℃~96℃ 14~16 seconds, 58℃~61℃ 58 seconds~62 seconds, 94℃~96℃ 14 seconds~16 seconds.

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