Gene mtjub1, interacting gene for regulating plant type of leguminous plant and application thereof

Abstract

The application belongs to the technical field of molecular biology, and particularly relates to a gene MtJUB1 for regulating the plant type of leguminous plants, an interaction gene and application, a nucleotide sequence of the gene MtJUB1 is shown in a sequence table SEQ ID NO.1, and the interaction gene of the gene MtJUB1 is MtDAG1, a nucleotide sequence of which is shown in a sequence table SEQ ID NO.4.The application provides a new gene resource for regulating the plant type of leguminous plants, and in the future, two key genes can be reformed in leguminous plants through gene editing technology to cultivate varieties with excellent plant types.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a gene MtJUB1 that regulates the plant architecture of leguminous plants, its interacting genes, and their applications. Background Technology

[0002] Legumes are the third largest family of plants, widely distributed throughout the world. Legume crops are primarily grown for their seeds, and common species include soybeans, cowpeas, broad beans, mung beans, kidney beans, peas, peanuts, alfalfa, and mimosa. Legumes generally have significant economic importance, serving as a major source of starch, protein, oil, and vegetables for human consumption, as well as a source of industrial raw materials (biological nitrogen fixation) and building materials. Plant height is a crucial agronomic trait; both excessively tall and short plants negatively impact yield. Therefore, appropriate plant height is an important reference indicator for breeding superior varieties. Currently, there are few reports on genes regulating plant height in legumes, and the related molecular mechanisms are even more underdeveloped. Discovering legume plant height-related genes using molecular biology techniques and further elucidating their molecular regulatory mechanisms will help in future gene editing of these genes to improve plant architecture and ultimately achieve high yields. Currently, legumes generally suffer from incomplete genome annotation, difficulties in genetic transformation, and a lack of mutants. Alfalfa is the model plant of the legume family. This species shares genetic similarities with other legumes, and research results obtained from alfalfa can be directly applied to other legume species. Furthermore, alfalfa possesses advantages such as a small genome, complete genome annotation, a mature tissue culture system, and a large-scale mutant library. Therefore, we selected alfalfa as our research material to explore genes related to plant height and elucidate its molecular mechanisms. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides a gene MtJUB1 that regulates the plant architecture of leguminous plants, its interacting gene, and its applications.

[0004] This invention is achieved by providing a gene MtJUB1 that regulates the plant architecture of leguminous plants, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0005] Preferably, the nucleotide sequence of the genome sequence of the above-mentioned gene is shown in SEQ ID NO.2.

[0006] Further preferred, the amino acid sequence of the protein encoded by the above gene is shown in the sequence listing SEQ ID NO.3.

[0007] The present invention also provides an interacting gene of the above-mentioned gene MtJUB1, namely MtDAG1, whose nucleotide sequence is shown in SEQ ID NO.4.

[0008] Preferably, the nucleotide sequence of the genome sequence of the above-mentioned interacting genes is shown in SEQ ID NO.5.

[0009] Further preferred, the amino acid sequence of the protein encoded by the above-mentioned interacting gene is shown in SEQ ID NO.6.

[0010] This invention also provides the application of the gene MtJUB1 in regulating the plant architecture of legumes. Specifically, the gene MtJUB1 negatively regulates the plant height of legumes.

[0011] Compared with the prior art, the advantages of the present invention are as follows:

[0012] The gene MtJUB1 and its interacting gene MtDAG1 of this invention provide a new gene resource for regulating plant architecture in legumes. In the future, gene editing technology can be used to modify these two key genes in legumes to cultivate varieties with superior plant architecture. Attached Figure Description

[0013] Figure 1 The phenotype of plants overexpressing MtJUB1;

[0014] Figure 2 Phylogenetic analysis of MtJUB1 in leguminous orthologous proteins;

[0015] Figure 3 The results of the screening of the MtJUB1 yeast library;

[0016] Figure 4 The results of the interaction between MtJUB1 and MtDAG1 in tobacco. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0018] Example 1: Construction of alfalfa MtJUB1 overexpression vector.

[0019] A mixed sample of roots, young leaves, and stem tips was collected from wild-type Alfalfa R108 plants at approximately 4 weeks of age. Total RNA was extracted using the Tiangen RNA Extraction Kit instructions. RNA concentration was determined using NanoDrop, and 1 μg of total RNA was extracted using PrimeScript. TMReverse transcription was performed using the 1st Strand cDNA Synthesis Kit. Specific amplification primers were designed based on the MtJUB1 (Medtr5g090970) sequence. To facilitate future CoIP validation, a myc sequence was added to the F-terminus (forward primer) of the MtJUB1 amplification primer. First, pCAMBIA3301 was double-digested with Nco1 and Pml1. The MtJUB1 coding sequence was then ligated into the digested pCAMBIA3301 vector via homologous recombination, resulting in p35S:myc-MtJUB1. The constructed plasmid was transformed into Agrobacterium strain GV3101 using the heat shock method and stored for later use.

[0020] The F-terminal amplification primer sequence for the MtJUB1 overexpression vector is cacgggggactcttgaccATGGAGCAGAAACTCATCTCTGAAGAGGATCTGATGGAGGTGGCAAAATTAG. The R-terminal (reverse primer) sequence is gctggtcacctgtaattcaCTAATTAAAATCTTTACAATC.

[0021] Example 2: Arabidopsis thaliana transformation.

[0022] To quickly verify the function of MtJUB1, we introduced the p35S:myc-MtJUB1 plasmid into Arabidopsis thaliana using the flower-splashing method. The specific procedures are as follows:

[0023] (1) The Agrobacterium strain containing the p35S:myc-MtJUB1 plasmid preserved in the previous step was streaked on the corresponding antibiotic plates. Single colonies were picked and inoculated into 5 ml of liquid LB medium containing the corresponding antibiotics. The culture was carried out at 28°C and 220 rpm for 18-24 h with shaking.

[0024] (2) Then expand the culture by increasing the LB culture medium to 100 mL and culturing for another 4-6 hours.

[0025] (3) When the OD reaches about 1.0, centrifuge at 4000 rpm to precipitate and remove the supernatant. Resuspend the bacterial cells with an equal volume of 5% sucrose solution, let stand for 1-2 hours, and add 0.02%-0.04% SilwetL-77 to the bacterial solution and mix well.

[0026] (4) Dip the Arabidopsis thaliana inflorescence into the bacterial solution for 1 minute.

[0027] (5) Then place the infected Arabidopsis seedlings in a dark, humid environment for 48 hours. Infect them again after one week, until all Arabidopsis flowers stop opening.

[0028] (6) After harvesting Arabidopsis seeds, plant them in new seedling trays. When the seedlings have two cotyledons, begin spraying with a 30 mg / ml herbicide to select positive plants. Spray every 3 days until no new seedlings germinate. The surviving transgenic plants are designated as T1 generation plants and harvested. Plant the seeds from T1 plants. Plants that meet the condition of a 3:1 ratio of normal to abnormal growth are designated as T2 plants and harvested individually. Plant the seeds from T2 plants. Plants that are all homozygous are designated as T3 plants.

[0029] Example 3: Phenotypic analysis of MtJUB1 overexpression transgene.

[0030] Wild-type Arabidopsis thaliana Columbia (Col) and transgenic MtJUB1 were planted under a 16-hour / 8-hour day / night cycle (temperature 22℃ / 18℃), with a light intensity of 150 μEm⁻²s⁻¹ and a relative humidity of 35%-40%. The plants were thoroughly watered and fertilized with 0.1% water-soluble fertilizer every week. After 4 weeks, the wild-type and transgenic plants were photographed and their characteristics were statistically analyzed. Figure 1 The results showed that Col plants were significantly taller than MtJUB1 transgenic plants, and the leaves of the transgenic plants were more compact. This result demonstrates that MtJUB1 negatively regulates plant height.

[0031] Example 4: MtJUB1 legume orthologous protein.

[0032] Example 3 has demonstrated that the MtJUB1 gene in alfalfa negatively regulates plant height. Further analysis of JUB1 homologs in other leguminous plants using sequence alignment and phylogenetic tree analysis was conducted. Figure 2 The results suggest the following evidence that MtJUB1 and its corresponding leguminous orthologs are involved in plant height regulation in different species:

[0033] (1) The phylogenetic tree shows that all JUB1 proteins are homologous and clustered with MtJUB1 on the corresponding branches, which indicates the conservation of their functions.

[0034] (2) Sequence alignment analysis proved that all legume JUB1 genes possess a conserved NAC domain. Functionally conserved proteins generally rely on their specific conserved protein domain to perform their functions. Transgenic experiments have demonstrated that JUB1 in alfalfa negatively regulates plant height, while JUB1 in soybean, peanut, common bean, chickpea, red clover, cowpea, and alfalfa all possess a conserved NAC domain. Therefore, JUB1 may also negatively regulate plant height in legume species in multiple legume species, which is the most direct evidence of the functional conservation of MtJUB1 and its homologs.

[0035] Example 5: Screening for JUB1 interacting proteins using a yeast library.

[0036] We used a yeast library constructed from alfalfa shoot tips to screen for interacting proteins with JUB1, further exploring the molecular regulatory mechanism of MtJUB1 in regulating plant height. The specific procedure is as follows:

[0037] (1) Homologous recombination of the coding sequence of MtJUB1 into the pGBKT7 vector. Using the constructed pGBKT7-MtJUB1 as a bait vector, single colonies of pGBKT7-MtJUB1 yeast were picked and shaken in 10 mL of de SD-Trp liquid medium until the OD value reached 0.8.

[0038] (2) Centrifuge at 1000g for 5 minutes, remove the supernatant, and keep the bacterial precipitate.

[0039] (3) Resuspend in 1 mL of SD-Trp liquid medium.

[0040] (4) Add 1-2 mL of yeast cDNA library from alfalfa stem tips and 1-2 mL of pGBKT7-MtJUB1 yeast suspension to a large Erlenmeyer flask, along with 100 mL of YPDA liquid medium containing Kan. Shake at 30℃ and 30 rpm for 24 h.

[0041] (5) Take out the mixed bacterial solution from the incubator, centrifuge at 1000g for 5 minutes, and remove the supernatant.

[0042] (6) After discarding the supernatant, add 100 mL of YPDA medium containing Kan to clear the cells. Repeat twice, then add 20 mL of 0.5% NaCl solution to the precipitate to suspend the bacterial plaque.

[0043] (7) Spread the culture onto SD-Leu-Trp medium with two deficiencies, incubate upside down at 30°C for 3 days, and then inoculate positive colonies onto SD-Ade-His-Leu-Trp medium with four deficiencies for more stringent screening of interacting colonies. Figure 3 ).

[0044] (8) The obtained colonies were shaken and yeast plasmids were extracted. After being transformed into E. coli, plasmid extraction and sequencing were performed. The genome of Alfalfa tribulus was selected for sequence alignment using the Phytozome online website (https: / / phytozome-next.jgi.doe.gov / ), and its target protein was found to be Medtr8g027295.

[0045] (9) The sequence of Medtr8g027295 was compared with that of Arabidopsis thaliana on the Tair online database (https: / / www.arabidopsis.org / ), and the highest similarity was found to DAG1 in Arabidopsis thaliana. Based on the above results, Medtr8g027295 was named MtDAG1, and it was also preliminarily demonstrated that MtJUB1 may interact with MtDAG1 to regulate plant height.

[0046] Example 6: Bimolecular fluorescence complementation (BiFC) experiment to verify the interaction between MtJUB1 and MtDAG1.

[0047] To further verify the interaction between MtJUB1 and MtDAG1, the full-length coding sequences of MtJUB1 and MtDAG1 were recombined into the pFGC5941 vector, constructing pFGC:nYFP-MtJUB1 and pFGC:cYFP-DAG1, respectively. The successfully constructed recombinant plasmids were transformed into Agrobacterium for later use. Agrobacterium with different recombinant plasmids were shaken in LB broth until the OD value reached approximately 1.0. After centrifugation at 7000 rpm for 5 min at room temperature, the supernatant was discarded, and the bacteria were resuspended in tobacco suspension. The OD values ​​were measured to be 0.5-1.0 to ensure that the OD values ​​of the bacterial suspensions used for interaction verification were consistent. After the resuspension was kept at room temperature in the dark for 2 h, it was injected into the abaxial surface of tobacco leaves. After culturing in the dark for 48 hours, the lower epidermis of the tobacco leaves was peeled off, and the interaction signal was detected using a laser confocal microscope. Figure 4 The results showed that MtJUB1 and MtDAG1 interact within tobacco plants, further verifying that MtJUB1 regulates plant height through MtDAG1.

[0048] The primer sequence for the F-terminal amplification of the MtJUB1 recombinant pFGC:nYFP vector is CATTTAAATCTCGAGGGATCCATGGAGGTGGCAAAATTAG, and the R-terminal sequence is GGTGGCGATGGATCTTCTAGACTAATTAAAATCTTTACAATC.

[0049] The F-terminal amplification primer sequence for the MtDAG1 recombinant pFGC:cYFP vector is GAGGAGGACCTGCTTTCTAGAATGGAAGGGTTATCTCCTAATTC, and the R-terminal sequence is CGCCGGACGGGTACCGGATCCTTACCATGATCCTTCACCCA.

[0050] In summary, this invention demonstrates that MtJUB1 interacts with MtDAG1 to affect plant height in alfalfa. In the future, gene editing technology can be used to modify these two key genes in legumes to cultivate varieties with superior plant types.

Claims

1. Genes that regulate plant architecture MtJUB1 The application, characterized in that, The gene MtJUB1 The encoded amino acid sequence is shown in SEQ ID NO. 3 of the sequence listing, gene. MtJUB1 Overexpression negatively regulates plant height in Arabidopsis thaliana and Alfalfa.

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