Bitter gourd low-temperature-resistant gene McADF09 and application thereof

Abstract

The invention provides a bitter gourd low-temperature-resistant gene McADF09 and application thereof, and belongs to the technical field of gene engineering. Research on expression rules in a bitter gourd cold-resistant material RM and a cold-sensitive material SM shows that the McADF09 gene plays an important role in low-temperature stress resistance of plants. The gene is over-expressed in a model organism arabidopsis thaliana, and the low-temperature stress resistance of an arabidopsis thaliana plant is enhanced. Therefore, the McADF09 gene and the protein coded by the McADF09 gene can regulate and control the cold resistance of the plant, and a new molecular target is provided for preparation of the cold-resistant plant.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, in particular to a low-temperature-resistant gene McADF09 of Momordica charantial L. and application thereof. BACKGROUND

[0002] Momordica charantial L. (Momordica charantial L.), also known as cool melon, leprosy grape or jade lychee, is an annual climbing herbaceous plant of the Cucurbitaceae family. Momordica charantial L. has outstanding advantages in nutritional value, insect and disease resistance, and medical care, and its cultivation area and consumption are large, and its planting and application potential are gradually valued by people. Momordica charantial L. is mainly distributed in subtropical and tropical regions, and is warm-loving and heat-tolerant but cold-intolerant. With global climate change, extreme weather events increase, and low-temperature disasters occur frequently, which leads to Momordica charantial L. often suffering from low temperature when sowing and planting in early spring in the south, thereby affecting seed germination and seedling growth, and greatly restricting the planting season range and market supply of Momordica charantial L. Research on low-temperature stress of Momordica charantial L. helps to cultivate Momordica charantial L. varieties that can adapt to climate change and maintain good growth and yield under low-temperature stress, thereby ensuring the stable supply of vegetables and having important significance for maintaining food security. Application of low-temperature-resistant Momordica charantial L. varieties can advance or delay the planting season of Momordica charantial L., extend the growth cycle and market supply period of Momordica charantial L., expand the planting range of Momordica charantial L., and enable Momordica charantial L. to be planted in colder regions, thereby improving land utilization and agricultural production efficiency. Therefore, at present, cultivating low-temperature-resistant Momordica charantial L. varieties has become one of the focuses of Momordica charantial L. breeding, and in-depth research on identifying low-temperature-resistant genes of Momordica charantial L. has important practical significance. SUMMARY

[0003] Therefore, the present application provides a low-temperature-resistant gene McADF09 of Momordica charantial L. and application thereof to solve the above problems.

[0004] In order to achieve the above application purposes, the present application provides the following technical solutions:

[0005] The present application provides a low-temperature-resistant gene McADF09 of Momordica charantial L., and the nucleotide sequence of the McADF09 gene is shown in SEQ ID NO. 1.

[0006] The present application also provides a protein encoded by the low-temperature-resistant gene McADF09, and the amino acid sequence of the protein is shown in SEQ ID NO. 2.

[0007] The present application also provides an overexpression vector containing the low-temperature-resistant gene McADF09 of Momordica charantial L.

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

[0009] Preferably, the basic plasmid is pCAMBIA1300.

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

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

[0012] S2. Digesting the plant overexpression vector pCAMBIA1300 and connecting the specific sequence of the McADF09 gene with homologous arms to obtain a recombinant plasmid;

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

[0014] S4. Transforming the recombinant Agrobacterium into Arabidopsis thaliana.

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

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

[0017] Preferably, the connection system is as follows: Biorun 2×EasyClone Mix 10 μL, McADF09 gene fragment 5 μL, pCAMBIA1300 linearized vector 5 μL; and the reaction is carried out at 37 ℃ for 30 min.

[0018] By adopting the above technical solution, the application has the following beneficial effects: the application studies the expression rules in the cold-tolerant material RM and the cold-sensitive material SM of Momordica charantia, and finds that the McADF09 gene plays an important role in the plant resistance to low-temperature stress. The application overexpresses the gene in the 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 thereby can regulate the cold resistance of plants, and provide a new molecular target for the preparation of cold-resistant plants. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1Phylogenetic tree of the AD F proteins of Momordica charantia, Arabidopsis thaliana, Oryza sativa, Zea mays and Triticum aestivum constructed by the AD F protein-adjacent method, with 1000 replicates. Family members are divided into eight sub-branches of different colors. The abbreviations used in the phylogenetic tree are as follows, Mc, Momordica charantia; At, Arabidopsis thaliana; Os, Oryza sativa; Zm, Zea mays; Ta, Triticum aestivum.

[0020] Figure 2 Conserved motifs and gene structures of McADFs.

[0021] Figure 3 Cis-acting elements in the promoter region of McADFs.

[0022] Figure 4 Relative expression patterns of 11 McADFs in RM and SM leaves under low temperature stress (0, 6, 12 and 24 h). Black columns represent RM, and gray columns represent SM. Data were analyzed by 2 -ΔΔCt

[0023] Figure 5 Phenotype of cold-intolerant materials under low temperature stress changed by exogenous salicylic acid (SA) treatment.

[0024] Figure 6 Expression of McADFs in cold-intolerant materials under low temperature stress changed by exogenous salicylic acid (SA) treatment. SM represents cold-intolerant materials.

[0025] Figure 7 PCR amplification product of McADF09 gene.

[0026] Figure 8 Bacterial liquid PCR electrophoresis result of McADF09 gene.

[0027] Figure 9 Phenotype of wild-type Arabidopsis thaliana and McADF09 overexpression lines before and after low temperature stress. DETAILED DESCRIPTION

[0028] The technical solutions provided by the present application are described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0029] The nucleotide sequence of the Momordica charantia low temperature tolerance gene McADF09 in the present application is shown in SEQ ID No. 1:

[0030] ​ATGGCAAATTCAGCATCAGGAATGGCAGTGCATGATGAGTGCAAGCTCAAGTTTCTGGATCTGAAGGCCAAGAGAAAATACAGGTTCATAGTGTTCAAGATTGAGGAGAATATCCAGCAGGTGACTGTAGATAAGGTCGGTGGCCACAACGAAACCTACGACGATTTCATCGCCTCCATCCCTGCCAACGAGTGTCGTTATGCTGTCTTTGATTTTGACTTCACAACCGATGAGAATTGCCAGAAGAGCAAGATTTTCTTCATTTCTTGGTCACCCGACACGTCGAAAATAAGAAGTAAAATGTTGTATGCGAGTTCCAAGGACAGATTCAAGAGAGAGCTGGATGGAATTCAAGTTGAATTACAAGCAACAGATCCCAGTGAGATGAGCTTTGACATCATCAAAGGAAGAGCTATCTGA (SEQ ID No. 1).

[0031] The amino acid sequence of the protein encoded by the Momordica charantia cold tolerance gene McADF09 is shown as SEQ ID No. 2:

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

[0033] Example 1. Identification of Momordica charantia cold tolerance gene

[0034] Based on the need for Momordica charantia cold tolerance variety improvement, the Momordica charantia cold tolerance gene was identified and analyzed, and the steps were as follows:

[0035] 1. Identification of ADF gene family at the whole genome level of Momordica charantia

[0036] The protein sequences and genome gff3 annotation files of Momordica charantia were obtained from NCBI (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF_001995035.1 / ), and the hidden Markov model (HMM) (PF00241) was downloaded from Pfam database (http: / / pfam.xfam.org / ). The HMMER3 was used to identify the possible McADF protein domain genes in M. charantia, and the threshold was set to E<1e−5. The integrity of the domain was further confirmed by online websites Pfam (https: / / pfam.xfam.org / ) and NCBI-CDD search (https: / / www.ncbi.nlm.nih.gov / cdd / ). Finally, it was determined that there were 11 members of M. charantia ADF gene family.

[0037] 2. Evolution analysis of M. charantia ADF family

[0038] In order to further understand the evolutionary relationship of ADF members in M. charantia, Arabidopsis thaliana, Oryza sativa, Zea mays and Triticum aestivum, the phylogenetic tree was constructed by MEGA7.0 and NJ method. Eleven McADF proteins were identified and divided into four main branches, respectively, I, II, III and IV, and each branch contained two sub-branches. The first, second and fourth branches contained 1, 3 and 7 McADF proteins, respectively. The IV branch contained the most McADF members, which was further divided into two independent sub-branches IVa and IVb, containing 4 and 3 McADF members, respectively. The Ib, IIa, IIIa and IIIb sub-branches did not contain members of M. charantia ADF gene family. Figure 1 ).

[0039] 3. Identification of M. charantia ADF family gene structure

[0040] The gene structure diversity of McADFs was further analyzed by the distribution of coding DNA sequences (CDS) and untranslated regions (UTRs). As shown in Fig. 2, the CDS of McADF1, McADF2, McADF3, McADF4, McADF5, McADF6, McADF7, McADF8, McADF9, McADF10 and McADF11 were 1, 1, 1, 1, 1, 1, 1, 1, 1, 1 and 1, respectively. The 5' UTR of McADF1, McADF2, McADF3, McADF4, McADF5, McADF6, McADF7, McADF8, McADF9, McADF10 and McADF11 was 1, 1, 1, 1, 1, 1, 1, 1, 1, 1 and 1, respectively. The 3' UTR of McADF1, McADF2, McADF3, McADF4, McADF5, McADF6, McADF7, McADF8, McADF9, McADF10 and McADF11 was 1, 1, 1, 1, 1, 1, 1, 1, 1, 1 and 1, respectively. Figure 2As shown, there are 2 members McADF07 and McADF08 without 5'-UTR, most of the McADFs gene family members (McADF01, McADF03, McADF04, McADF09, McADF10 and McADF11) contain two CDS, McADF06, McADF07 and McADF08 contain three CDS, and the CDS number of McADF02 is the most, containing 8. In order to further study the members of McADFs gene family, the motifs of McADFs were predicted using MEME online tool, and the results showed that there were 20 conserved motifs. Motifs 1 and 2 exist in all Momordica charantia McADFs protein sequences, and McADF02 has the most motifs, a total of 18. Except for McADF07 and McADF08, the remaining 9 McADF family members all have motif 4. McADF01, McADF03, McADF04, McADF06, McADF07, McADF08, McADF09 and McADF10 have 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 cis-acting elements of Momordica charantia ADF family genes

[0042] In order to obtain the cis-acting elements of McADF family genes, the promoter region was analyzed in the PlantCARE online website. The cis-acting elements are divided into four categories, which are light response, hormone response, stress response and growth and development response elements. Among the four categories of elements, the light response elements account for the largest proportion, including G-box, sp1, AE-box and other 13 elements. Most of the elements related to hormone response are ABRE, TGACG motif and CGTCA motif. The stress response elements include MBS (drought response), LTR (cold response) and TC-rich repeat (defense and stress response). Ten of the McADF genes have growth and development response elements, including CAT-box (related to meristem) and ARE (essential for anaerobic induction), and they are important and account for a large proportion. The response elements of the promoter region of Momordica charantia McADFs indicate that the family members can respond to the changes in the external growth and development Figure 3 ).

[0043] 5. Identification of Momordica charantia cold-tolerant key gene McADF09

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

[0055] Table 2 PCR reaction procedure

[0056] 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.

[0057] 2. Vector enzyme digestion

[0058] 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.

[0059] 3. Homologous recombination

[0060] 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.

[0061] 4. Escherichia coli transformation

[0062] Take 50 μL of E. coli on ice to melt, add 5 μL of ligation product to E. coli and mix well, ice bath for 20 min. Place the centrifuge tube in a 42°C water bath for 90 s, continue to ice for 2 min. Add 500 μL of liquid medium without antibody, place in 37°C, 200 rpm shaking bed for 45 min. Take 200 μL of cultured bacteria evenly on solid medium, 37°C biochemical incubator inverted culture for 12 h.

[0063] 5. Bacterial liquid PCR identification

[0064] Pick 10 colonies, and perform 1.5 mL EP tube inoculation and bacterial liquid PCR identification at the same time. The identification primers are: F: ctgccaacgagtgtcgttatg (SEQ ID NO. 5); R: gatcctgttgacgagggtgtc (SEQ ID NO. 6). The PCR reaction system and procedure are shown in Table 3 and Table 4:

[0065] Table 3 PCR reaction system

[0066] Reagent Volume Nuclease-free Water 20 μL Biorun Pfu PCR Mix 25 μL Upstream primer (100 mM) 2 μL Downstream primer (100 mM) 2 μL DNA template 1 μL Total volume 50 μL

[0067] Table 4 PCR reaction procedure

[0068] The bacterial liquid PCR electrophoresis result is shown in Figure 8 The target band is a fragment of about 617 bp. Take 100 μL of the three positive bands for sequencing, and the remaining 400 μL of bacterial liquid is inoculated into 10 mL of LB liquid medium containing kanamycin. After the sequencing results are obtained, one tube of plasmid is extracted for Agrobacterium transformation.

[0069] 6. Agrobacterium transformation

[0070] After the sequencing correct bacterial liquid is extracted for plasmid, Agrobacterium transformation is performed. Add 1 μg of plasmid to melted Agrobacterium (50 μL), mix well with a gun head, place on ice for 5 min; freeze rapidly in liquid nitrogen for 5 min; water bath in a 37°C water bath for 5 min; re-ice bath on ice for 5 min; under the condition of a clean bench, add 500 mL of liquid medium to the centrifuge tube, place in a 28°C shaking bed for 4 h, then take 200 μL of liquid and spread on solid medium plate, and culture in a 28°C biochemical incubator for 48 h. Pick 10 single colonies and inoculate in liquid LB containing rifampicin and kanamycin, and culture in a shaking bed at 28°C, 200 rpm for 2 d.

[0071] Example 3. Floral dip method for transforming Arabidopsis and identifying low-temperature-resistant phenotype

[0072] Wild-type Arabidopsis (Col-0) was sown in nutrient soil, and when the seedlings grew to the two-leaf stage, they were transplanted into small pots for further cultivation. The floral dip method was used for transformation 5 days after the Arabidopsis began to bloom, and the transformation steps were as follows:

[0073] 1. Preparation of Agrobacterium: Agrobacterium GV3101 containing the target vector was inoculated into LB liquid medium and cultured at 28°C and 200 rpm for 24 hours until the OD 600 =1.

[0074] Preparation of infection solution: 50 mL of sterile water + 5% sucrose, after stirring and dissolving, add surfactant to a final concentration of 0.05%, mix well and use immediately (freshly prepared).

[0075] 2. Infection operation

[0076] Agrobacterium treatment: Centrifuge the cultured Agrobacterium solution at 4000 rpm for 10 min, discard the supernatant, resuspend the precipitate with the prepared infection solution, and adjust the OD 600 =0.5.

[0077] Flower dip infection: Gently bend the Arabidopsis inflorescence, and immerse the unopened inflorescence (emphasis on the bud) in the infection solution for 20 seconds.

[0078] Auxiliary infection: Gently shake the container during soaking to ensure that the flower bud is fully exposed to the bacterial solution; if there are many flowers, they can be infected in multiple times to avoid missing.

[0079] Screening on 1 / 2 MS medium containing 50 mg / L hygromycin. Three independent transgenic lines of T3 homozygous generation were used for phenotype analysis. The transgenic Arabidopsis plants and wild-type plants were placed under 5°C stress for 3 days, and then cultured under normal conditions for 3 days. After observation, it was found that the overexpression plants of McADF09 gene grew better than the wild-type plants, and the leaves of the wild-type plants turned purple Figure 9 . This indicates that overexpression of McADF09 gene can improve the stress resistance of Momordica charantia to low temperature.

[0080] As can be seen from the above examples, the present application provides Momordica charantia low-temperature-resistant gene McADF09 and its application. Overexpression of McADF09 gene in plants can improve the cold resistance of plants.

[0081] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, which should also be considered within the scope of protection of the present application.

Claims

1. The bitter gourd cold-resistance gene McADF09, characterized by, The nucleotide sequence of the McADF09 gene is shown in SEQ ID NO.

1.

2. The protein encoded by the cryoresistance gene McADF09 as described in claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. An overexpression vector containing the bitter melon cold-resistance gene McADF09.

4. The overexpression vector according to claim 3, characterized in that, The overexpression vector comprises a basic plasmid and the nucleotide fragment shown in SEQ ID NO.

1.

5. The overexpression vector according to claim 4, characterized in that, The basic plasmid is pCAMBIA1300.

6. A method for enhancing the low-temperature resistance of Arabidopsis thaliana, characterized in that, Includes the following steps: S1. Design primers to amplify the McADF09 gene sequence with homologous arms; 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; 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.

7. The method according to claim 6, characterized in that, The sequences of the primers are shown in SEQ ID NO.3 and SEQ ID NO.

4.

8. The method according to claim 6, characterized in that, 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 °C for 24 h.

9. The method according to claim 6, 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.

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