Bitter gourd low temperature tolerance gene McADF09 and application thereof

Abstract

The application provides a bitter gourd low-temperature tolerance gene McADF09 and application thereof, and belongs to the technical field of genetic engineering. The application studies expression rules in a cold-resistant material RM and a cold-sensitive material SM of the bitter gourd, and finds that the McADF09 gene plays an important role in plant resistance to low-temperature stress. The application overexpresses the gene in a model organism Arabidopsis thaliana, and the low-temperature stress resistance of the Arabidopsis thaliana plant is enhanced. Therefore, the McADF09 gene and the protein coded by the McADF09 gene can regulate the cold resistance of the plant, and provide a new molecular target for preparation of cold-resistant plants.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the bitter gourd low-temperature tolerance gene McADF09 and its applications. Background Technology

[0002] Bitter melon (Momordica charantial L.), also known as balsam pear, bitter gourd, or bitter melon, is an annual climbing herbaceous plant belonging to the genus Momordica in the Cucurbitaceae family. Bitter melon has outstanding advantages in nutritional value, insect and disease resistance, antibacterial properties, and medicinal and health benefits. Its cultivation area and consumption are large, and its planting and application potential is gradually gaining attention. Bitter melon is mainly distributed in subtropical and tropical regions, preferring warm climates and tolerating heat but not cold. With global climate change and the increasing frequency of extreme weather events and low-temperature disasters, bitter melon often encounters low temperatures during early spring sowing and transplanting in southern regions, affecting seed germination and seedling growth, thus significantly restricting the planting season and market supply. Research on low-temperature stress in bitter melon is helpful in cultivating varieties adapted to climate change, maintaining good growth and yield even under low-temperature adversity, ensuring a stable supply of vegetables, and playing an important role in maintaining food security. The application of cold-resistant bitter gourd varieties can advance or delay the planting season, extend the growth cycle and market supply period, expand the planting area, and enable cultivation in colder regions, thereby improving land utilization and agricultural production efficiency. Therefore, breeding cold-resistant bitter gourd varieties has become a focus of bitter gourd breeding, and in-depth basic research to identify and address the cold-resistant genes in bitter gourd has significant practical implications. Summary of the Invention

[0003] In view of this, the present invention provides the bitter gourd cold-resistant gene McADF09 and its application to solve the above problems.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides the bitter gourd cold-resistant gene McADF09, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0006] The present invention also provides a protein encoded by the aforementioned cryogenic gene McADF09, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] The present invention also provides an overexpression vector containing the bitter melon cold-resistance gene McADF09.

[0008] Preferably, the overexpression vector comprises a basic plasmid and the nucleotide fragment shown in SEQ ID NO.1.

[0009] Preferably, the base plasmid is pCAMBIA1300.

[0010] The present invention also provides a method for enhancing the low-temperature resistance of Arabidopsis thaliana, comprising the following steps:

[0011] S1. Design primers to amplify the McADF09 gene sequence with homologous arms;

[0012] S2. The plant overexpression vector pCAMBIA1300 was digested with enzymes and ligated with a specific sequence of the McADF09 gene with a homologous arm to obtain a recombinant plasmid;

[0013] S3. Introduce the recombinant plasmid into Agrobacterium, and then culture the recombinant Agrobacterium for 36-48 h;

[0014] S4. Transform the recombinant Agrobacterium into Arabidopsis thaliana to obtain the final product.

[0015] Preferably, the sequences of the primers are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0016] Preferably, the plant overexpression vector pCAMBIA1300 is digested with BglII and XbaI. The digestion system is: 12 μL Nuclease-free Water, 2 μL 10× Buffer, 1 μL BglII, and 1 μL XbaI, and the reaction is carried out at 37 °C for 24 h.

[0017] Preferably, the ligation system is: 10 μL Biorun 2×EasyClone Mix, 5 μL McADF09 gene fragment, and 5 μL pCAMBIA1300 linearized vector; reacted at 37 °C for 30 min.

[0018] By adopting the above technical solution, this invention has the following beneficial effects: This invention studies the expression patterns of the cold-resistant material RM and the cold-sensitive material SM in bitter gourd, and finds that the McADF09 gene plays an important role in plant resistance to low-temperature stress. Overexpression of this gene in the model organism Arabidopsis thaliana enhances the plant's resistance to low-temperature stress. Therefore, the McADF09 gene and its encoded protein can regulate the cold resistance of plants, providing a new molecular target for the preparation of cold-resistant plants. Attached Figure Description

[0019] Figure 1Phylogenetic trees for bitter melon, Arabidopsis thaliana, rice, maize, and wheat were constructed using the ADF protein neighbor-joining method, with 1000 replicates. Family members were divided into eight sub-branches of different colors. The abbreviations used in the phylogenetic trees are as follows: Mc, bitter melon; At, Arabidopsis thaliana; Os, rice; Zm, maize; Ta, wheat.

[0020] Figure 2 The conserved motifs and gene structures of McADFs genes.

[0021] Figure 3 It is a sympathetic element in the McADF promoter region.

[0022] Figure 4 The relative expression patterns of 11 McADF genes in RM and SM leaves under low temperature stress (0, 6, 12, and 24 h) are shown. Black bars represent RM, and gray bars represent SM. Data were analyzed using a 2... -ΔΔCt The analysis included three independent replicates. The statistical significance of differences between groups was assessed using Student's t-test, where *P < 0.05 indicated statistical significance, **P < 0.01 indicated highly significant significance, and ns indicated no significant difference.

[0023] Figure 5 To alter the phenotype of cold-sensitive material leaves under low-temperature stress by treating with exogenous salicylic acid (SA).

[0024] Figure 6 The expression levels of McADFs in the leaves of cold-sensitive materials under low-temperature stress were altered by exogenous salicylic acid (SA) treatment. SM represents cold-sensitive materials.

[0025] Figure 7 This is a diagram of the PCR amplification products of the McADF09 gene.

[0026] Figure 8 The image shows the PCR electrophoresis results of the McADF09 gene bacterial culture.

[0027] Figure 9 Phenotypes of wild-type Arabidopsis thaliana and McADF09 overexpression lines before and after low-temperature stress. Detailed Implementation

[0028] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] The nucleotide sequence of the bitter melon cold-resistance gene McADF09 in this invention is shown in SEQ ID No. 1:

[0030] ATGGCAAATTCAGCATCAGGAATGGCAGTGCATGATGAGTGCAAGCTCAAGTTTCTGGATCTGAAGGCCAAGAGAAAATACAGGTTCATAGTGTTCAAGATTGAGGAGAATATCCAGCAGGTGACTGTAGATAAGGTCGGTGGCCACAACGAAACCTACGACGATTTCATCGCCTCCATCCCTGCCAACGAGTGTCGTTATGCTGTCTTTGA TTTTGACTTCACAACCGATGAGAATTGCCAGAAGAGCAAGATTTTCTTCATTTCTTGGTCACCCGACACGTCGAAAATAAGAAGTAAAATGTTGTATGCGAGTTCCAAGGACAGATTCAAGAGAGAGCTGGATGGAATTCAAGTTGAATTACAAGCAACAGATCCCAGTGAGATGAGCTTTGACATCATCAAAGGAAGAGCTATCTGA (SEQ ID No.1).

[0031] The amino acid sequence of the protein encoded by the bitter melon cold-resistance gene McADF09 is shown in SEQ ID NO. 2:

[0032] MANSASGMAVHDECKLKFLDLKAKRKYRFIVFKIEENIQQVTVDKVGGHNETYDDFIASIPANECRYAVFDFDFTTDENCQKSKIFFISWSPDTSKIRSKMLYASSKDRFKRELDGIQVELQATDPSEMSFDIIKGRAI (SEQ ID No. 2).

[0033] Example 1. Identification of cold-resistance genes in bitter melon

[0034] Based on the need for improving cold-resistant varieties of bitter gourd, the cold-resistant genes of bitter gourd were identified and analyzed. The steps are as follows:

[0035] 1. Identification of the ADF gene family at the whole genome level in bitter melon

[0036] The protein sequence and gff3 genomic annotation file of bitter melon were obtained from NCBI (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF_001995035.1 / ). A Hidden Markov Model (HMM) (PF00241) was downloaded from the Pfam database (http: / / pfam.xfam.org / ). HMMs were used to identify possible McADF protein domain genes in bitter melon, with a threshold of E < 1e−5. The integrity of the domains was further confirmed using the online website Pfam (https: / / pfam.xfam.org / ) and NCBI-CDD search (https: / / www.ncbi.nlm.nih.gov / cdd / ). Ultimately, 11 members of the bitter melon ADF gene family were identified.

[0037] 2. Evolutionary analysis of the ADF family of bitter melon

[0038] To gain a deeper understanding of the evolutionary relationships among ADF members in bitter melon, Arabidopsis thaliana, rice, maize, and wheat, a phylogenetic tree was constructed using MEGA 7.0 and the NJ method. Eleven McADF proteins were identified and divided into four main branches: I, II, III, and IV, each containing two subbranches. Branches I, II, and IV contained 1, 3, and 7 McADF proteins, respectively. Branch IV contained the most McADF members, further subdividing into two independent subbranches, IVa and IVb, with 4 and 3 McADF members, respectively. Subbranches Ib, IIa, IIIa, and IIIb did not contain any members of the bitter melon ADF gene family. Figure 1 ).

[0039] 3. Identification of the ADF family gene structure of bitter melon

[0040] The genetic structural diversity of McADFs was further analyzed by examining the distribution of coding DNA sequences (CDS) and untranslated regions (UTRs). Figure 2As shown, two members, McADF07 and McADF08, lack a 5′-UTR. Most members of the McADF gene family (McADF01, McADF03, McADF04, McADF09, McADF10, and McADF11) contain two CDS, while McADF06, McADF07, and McADF08 contain three CDS. McADF02 has the most CDS, with eight. To further investigate the members of the McADF gene family, the motifs of McADFs were predicted using the MEME online tool, revealing 20 conserved motifs. Motifs 1 and 2 are present in all bitter melon McADF protein sequences, with McADF02 having the most motifs (18). Except for McADF07 and McADF08, the remaining nine members of the McADF family all possess motif 4. McADF01, McADF03, McADF04, McADF06, McADF07, McADF08, McADF09, and McADF10 share the same number and distribution of motifs. Different gene structures and motifs may lead to different biological functions, and the above analysis provides a basis for the functional classification of McADFs. Figure 2 ).

[0041] 4. Analysis of cistropic elements in the ADF family genes of bitter melon

[0042] To obtain the cis-regulatory elements of the McADF family genes, promoter regions were analyzed in the PlantCARE online database. Cis-regulatory elements were classified into four categories: light-responsive, hormone-responsive, stress-responsive, and growth-responsive elements. Among these four categories, light-responsive elements constituted the largest proportion, including G-box, sp1, AE-box, and 13 other elements. Most hormone-responsive elements were ABRE, TGACG, and CGTCA motifs. Stress-responsive elements included MBS (drought response), LTR (cold response), and TC-rich repetitive sequences (defense and stress responses). Ten McADF genes possessed growth-responsive elements, including CAT-box (associated with meristems) and ARE (essential for anaerobic induction), and these were important and constituted a significant proportion. The responsive elements in the promoter regions of bitter melon McADFs indicate that family members can respond to changes in external growth and development. Figure 3 ).

[0043] 5. Identification of the key cold-resistance gene McADF09 in bitter melon

[0044] To investigate the expression patterns of McADF family members in the cold-resistant material RM and the cold-sensitive material SM, qRT-PCR was used to detect 11 McADF family members. The results showed that at 0 h, the expression levels of all McADFs in RM were significantly higher than those in SM, indicating that McADFs may be involved in regulating the low-temperature tolerance mechanism of bitter gourd. McADF09 expression in RM was higher than that in SM at 6 h, 12 h, and 24 h under 4℃ low-temperature stress, suggesting that McADF09 plays an important role in low-temperature stress and may enhance cold resistance. Figure 4 ).

[0045] Salicylic acid (SA) is a phenolic compound that not only participates in defense responses but also regulates plant growth and development in response to various abiotic stresses. SA has great agronomic potential in improving the stress resistance of important crops and plays an important role in low temperature, salt stress, and drought stress.

[0046] The experiment was divided into two groups: 10 cold-sensitive bitter gourd (SM) plants and 10 cold-tolerant bitter gourd (RM) plants in each group. The experimental group was sprayed with 1.5 mM salicylic acid until the leaves dripped with water, while the control group was sprayed with the same amount of water. Low-temperature treatment was applied 24 h after the initial treatment. The results showed that under low-temperature stress, bitter gourd leaves exhibited defects in the wrinkled plant shape, while seedlings treated with salicylic acid before low-temperature treatment showed a normal phenotype. This indicates that salicylic acid treatment can effectively alleviate the damage of low temperature to seedlings. The expression levels of McADF0 in bitter gourd leaves before and after low-temperature treatment and salicylic acid treatment were analyzed at 0 h, 6 h, 12 h, and 24 h. The results (…) Figure 6 The results showed that after low-temperature stress, the expression level of McADF09 in salicylic acid-treated SM was consistently higher than that in untreated SM, indicating that McADF09 may regulate its cold resistance through the SA treatment pathway. Ultimately, the McADF09 gene was selected as the research object to explore its specific cold-resistance function.

[0047] Example 2. Construction of the McADF09 overexpression vector from bitter melon and its transformation with Agrobacterium.

[0048] 1. Cloning of the McADF09 gene from bitter melon

[0049] Using bitter gourd leaf tissue (a cold-resistant material) as material, RNA was extracted according to the MiniBEST Plant RNA Extraction Kit (TaKaRa) procedure, and cDNA was reverse transcribed according to the PrimeScript™ RT reagent RT Kit with gDNA Eraser (TaKaRa) procedure. Based on the bitter gourd McADF09 gene sequence, primer sequences for PCR amplification with homologous arms were designed:

[0050] F: caagctgactctagcaatctATGGCAAATTCAGCATCAGGAATGGCAG (SEQ ID NO.3);

[0051] R: tcctttgcccatggctctagaTCAGATAGCTCTTCCTTTGATGATGTCAAAGC (SEQ IDNO.4);

[0052] Using bitter melon cDNA as a template, PCR amplification was performed using the primers described above. The PCR reaction system and procedure are shown in Tables 1 and 2.

[0053] Table 1 PCR reaction system

[0054] reagents volume Nuclease-free Water 20μL Biorun Pfu PCR Mix 25μL Upstream primer (100mM) 2μL Downstream primer (100mM) 2μL DNA template 1μL Total volume 50μL

[0055] Table 2 PCR reaction procedure

[0056]

[0057] The PCR amplification products were subjected to 1.5% agarose gel electrophoresis (5V / cm, 20min). The agarose gel electrophoresis image is shown below. Figure 7 As shown in the figure. The amplification product was recovered by gel extraction under UV light, and the DNA was dissolved and recovered in 40 μL of water. After successful sequencing, it was recombined with the vector.

[0058] 2. Vector enzyme digestion

[0059] The plant overexpression vector pCAMBIA1300 was digested with BglII and XbaI. The digestion system was: 12 μL Nuclease-free Water, 2 μL 10× Buffer, 1 μL BglII, and 1 μL XbaI. The reaction was carried out at 37℃ for 24 h.

[0060] 3. Homologous recombination

[0061] The digested product and the gel-recovered product of the target fragment McADF09 were ligated using homologous recombinase. The ligation system was: 10 μL Biorun 2×EasyClone Mix, 5 μL McADF09 gene fragment, and 5 μL pCAMBIA1300 linearized vector. The reaction was carried out at 37 °C for 30 min.

[0062] 4. Escherichia coli transformation

[0063] Thaw 50 μL of *E. coli* on ice. Add 5 μL of the ligation product to the *E. coli* and mix thoroughly. Incubate on ice for 20 min. Heat shock the centrifuge tube in a 42°C water bath for 90 s, then continue to incubate on ice for 2 min. Add 500 μL of antibiotic-free liquid culture medium and incubate at 37°C with a shaker at 200 rpm for 45 min. Spread 200 μL of the cultured bacterial solution evenly onto a solid culture medium and incubate upside down in a 37°C biochemical incubator for 12 h.

[0064] 5. Bacterial culture PCR identification

[0065] Ten colonies were selected and simultaneously inoculated into 1.5 mL EP tubes for bacterial culture and culture PCR identification. The identification primers were: F: ctgccaacgagtgtcgttatg (SEQ ID NO.5); R: gatcctgttgacgagggtgtc (SEQ ID NO.6). The PCR reaction system and procedure are shown in Tables 3 and 4.

[0066] Table 3 PCR reaction system

[0067] reagents volume Nuclease-free Water 20μL Biorun Pfu PCR Mix 25μL Upstream primer (100mM) 2μL Downstream primer (100mM) 2μL DNA template 1μL Total volume 50μL

[0068] Table 4 PCR reaction procedure

[0069]

[0070] The results of bacterial culture PCR electrophoresis are shown in the figure below. Figure 8 As shown, the target band is a fragment of about 617 bp. Take 100 μL of the bacterial culture corresponding to the 3 positive bands and send it for sequencing. The remaining 400 μL of bacterial culture is inoculated into LB liquid medium containing 10 mL of kanamycin resistance. Shake the test tubes and wait for the sequencing results. Take the tube with the correct sequencing to extract the plasmid and perform Agrobacterium transformation.

[0071] 6. Agrobacterium-mediated transformation

[0072] After extracting plasmids from the correctly sequenced bacterial culture, Agrobacterium transformation was performed. 1 μg of plasmid was added to thawed Agrobacterium (50 μL), mixed thoroughly with a pipette tip, and placed on ice for 5 min; then rapidly frozen in liquid nitrogen for 5 min; incubated in a 37°C water bath for 5 min; and then placed back on ice for 5 min. Under laminar flow hood conditions, 500 mL of liquid culture medium was added to centrifuge tubes, and the mixture was shaken at 28°C for 4 h. 200 μL of the liquid was then transferred to solid culture plates and incubated upside down in a 28°C biochemical incubator for 48 h. Ten single colonies were picked and inoculated into liquid LB containing rifampicin and kanamycin resistance, and cultured on a shaker at 28°C and 200 rpm for 2 days.

[0073] Example 3. Transformation of Arabidopsis thaliana by inflorescence staining and identification of low-temperature tolerance phenotype.

[0074] Wild-type Arabidopsis thaliana (Col-0) was sown in nutrient soil. After the seedlings emerged and grew to the two-leaf stage, they were transplanted into small pots for further cultivation. The transformation was carried out by the flower-dipping method 5 days after the Arabidopsis thaliana bolted. The transformation steps are as follows:

[0075] 1. Agrobacterium preparation: Agrobacterium GV3101 containing the target vector was inoculated into LB liquid medium and cultured at 28°C with shaking at 200 rpm until OD. 600 =1.

[0076] Preparation of infection solution: 50mL sterile water + 5% sucrose, stir to dissolve, then add surfactant to a final concentration of 0.05%, mix well and set aside (prepare and use immediately).

[0077] 2. Infection procedures

[0078] Agrobacterium treatment: Centrifuge the cultured Agrobacterium tumefaciens solution at 4000 rpm for 10 min, discard the supernatant, resuspend the precipitate in the prepared infection solution, and adjust the OD value. 600 =0.5.

[0079] Flower inoculation: Gently bend the Arabidopsis flower stalk and immerse the unopened inflorescence (especially the flower buds) in the inoculation solution for 20 seconds.

[0080] Assisted infection: Gently shake the container during soaking to ensure that the flower buds are fully in contact with the bacterial solution; if there are many flowers, multiple infections can be carried out to avoid omissions.

[0081] Screening was performed on 1 / 2 MS medium containing 50 mg / L hygromycin. Three independent transgenic lines of the T3 homozygous generation were used for phenotypic analysis. Transgenic Arabidopsis plants and wild-type plants were subjected to 5°C temperature stress for 3 days, and then cultured under normal conditions for 3 days. Phenotypic observation was performed, and it was found that the McADF09 overexpressing plants had better growth vigor than wild-type plants, and the leaves of wild-type plants turned purple. Figure 9 This indicates that overexpression of the McADF09 gene can improve the resistance of bitter gourd to low-temperature stress.

[0082] As can be seen from the above embodiments, the present invention provides the bitter gourd cold-resistant gene McADF09 and its application. Overexpression of the McADF09 gene in plants can improve the cold resistance of plants.

[0083] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for enhancing the low-temperature resistance of Arabidopsis thaliana, characterized in that, Includes the following steps: S1. Design primers to amplify molecules with homologous arms. McADF09 Gene sequence; the McADF09 The nucleotide sequence of the gene is shown in SEQ ID NO.1; S2. Plant overexpression vector pCAMBIA1300 Enzyme digestion, and those with homologous arms McADF09 Specific sequences of genes are linked together to obtain recombinant plasmids; S3. Introduce the recombinant plasmid into Agrobacterium, and then culture the recombinant Agrobacterium for 36-48 h; S4. Transform the recombinant Agrobacterium into Arabidopsis thaliana to obtain the final product.

2. The method according to claim 1, characterized in that, The sequences of the primers are shown in SEQ ID NO.3 and SEQ ID NO.

4.

3. The method according to claim 1, characterized in that, The ligation system consisted of: 10 μL Biorun 2×EasyCloneMix, 5 μL McADF09 gene fragment, and 5 μL pCAMBIA1300 linearized vector; the reaction was carried out at 37 °C for 30 min.

Images