A cotton MAP4K13 gene and its application in salt and drought tolerance

Abstract

The invention belongs to the technical field of molecular biology plant breeding and provides a cotton MAP4K13 gene and application thereof in salt and drought resistance. GhMAP4K13 The amino acid sequence encoded by the gene is shown in SEQ ID NO: 1. Under drought and salt stress, Arabidopsis seeds overexpressing the gene have a higher germination rate and good growth conditions, which improves the salt tolerance and drought resistance of Arabidopsis. The above gene is applied to the breeding of salt-tolerant and drought-resistant plants to improve the drought and salt resistance of the plants, and effectively provide an alternative for the molecular breeding of drought-resistant and salt-alkali-tolerant crops.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology plant breeding, and particularly relates to a cotton MAP4K13 gene and an application thereof in salt and drought resistance. Background Art

[0002] The disclosure of this background information is intended to enhance understanding of the general background of the invention and should not necessarily be taken as an acknowledgment or any form of suggestion that this information constitutes the prior art already known to a person skilled in the art.

[0003] Cotton is one of the important fiber and oil crops, an important source of natural fiber and cottonseed oil, and an important raw material for the chemical and defense industries. Cotton has strong salt tolerance and is considered one of the pioneer crops for saline-alkali land improvement. Studying the salt tolerance mechanism of cotton at the molecular level to improve cotton salt tolerance is a current research hotspot. The introduction of exogenous genes or overexpression of endogenous related genes through genetic engineering can improve plant salt tolerance. The rapid development of biotechnology has gradually clarified the corresponding salt stress mechanism in plants, and more and more salt tolerance-related genes have been successfully cloned and transformed, which is of great significance to the study of cotton salt tolerance and the breeding of new salt-tolerant varieties.

[0004] Mitogen-activated protein kinase kinase kinase kinase (MAP4K) is a class of serine / threonine protein kinases that are widely involved in the growth and development of plants. MAP4K The number of genes in the genome of higher plants is relatively small, generally around 5-20, and they are mainly divided into four subfamilies. MAP4K The gene is involved in the growth and development of various plants and plays a regulatory role in abiotic stresses such as heat, salt and drought. Summary of the invention

[0005] In view of the problems in the prior art, the present invention provides a cotton MAP4K13 gene, which can improve the salt tolerance and drought resistance of Arabidopsis thaliana when expressed in Arabidopsis thaliana.

[0006] Another object of the present invention is to provide the application of the above gene in salt-tolerant and drought-resistant plant breeding, and to provide an alternative solution for the molecular breeding of drought-resistant and salt-alkali-tolerant crops.

[0007] To achieve the above purpose, the present invention adopts the following technical solution.

[0008] A cotton MAP4K13 gene ( GhMAP4K13 ), the amino acid sequence encoded by it is shown in SEQ ID NO: 1. In order to facilitate the expression of the gene in non-homologous plants, synonymous codons can also be converted according to specific crop varieties. Preferably, the encoding nucleotide sequence of the gene is shown in SEQ ID NO: 2.

[0009] A method comprising the above GhMAP4K13 The expression cassette, vector or cell of the invention can be a plasmid, a virus or a bacteriophage. The cell can be derived from a bacterial, fungal, animal or plant cell line. The expression cassette, vector or cell can be used to express MAP4K13 protein in a target species.

[0010] An application of the cotton MAP4K13 gene in crop drought resistance or salinity resistance.

[0011] Specifically, the above application is to GhMAP4K13 Expression in crops.

[0012] A method for screening drought-resistant or salt-alkali-resistant crops comprises the following steps:

[0013] (1) Construct GhMAP4K13 recombinant vector;

[0014] (2) Include GhMAP4K13 Transform cells with the recombinant vector and screen to obtain positive transformants;

[0015] (3) Transfect wild-type crops with positive transformants and screen for drought-resistant or salt-alkali-resistant transgenic crops.

[0016] Preferably, the above screening process also includes:

[0017] (4) Sexual reproduction of transgenic crops and selection of sexually reproduced offspring GhMAP4K13 Homozygotes are retained to obtain drought-resistant or saline-alkali-resistant crops.

[0018] Specifically, the above method comprises the following steps:

[0019] (1) Obtain GhMAP4K13 The gene was connected to the expression vector plasmid to construct GhMAP4K13 recombinant vector;

[0020] (2) Include GhMAP4K13 The recombinant vector was transformed into Agrobacterium tumefaciens competent cells by freeze-thaw method, and positive transformants were obtained by screening;

[0021] (3) Transfect wild-type crops with bacterial solution containing positive transformants, harvest the T0 generation after the crops mature, and screen the T0 generation to obtain drought-resistant or salt-alkali-resistant transgenic crops;

[0022] (4) Propagate the transgenic crops sexually or asexually, screen and harvest plants or seeds with stable traits; select the offspring with stable traits GhMAP4K13 The homozygotes are retained to obtain drought-resistant or saline-alkali-resistant crops.

[0023] The present invention has the following advantages:

[0024] The invention provides a cotton MAP4K13 Gene, under drought and salt stress, Arabidopsis seeds overexpressing this gene have a higher germination rate and good growth conditions, which improves the salt tolerance and drought resistance of Arabidopsis. The MAP4K gene family is evolutionarily conserved and widely participates in and regulates plant growth and development processes such as photoperiod, flower organ growth, plant hormone signal transduction and other biological processes. Applying the above genes to the breeding of salt-tolerant and drought-resistant plants can improve the drought and salt resistance of plants, and effectively provide alternatives for the molecular breeding of drought-resistant and salt-alkali-tolerant crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The pBI121-GhMAP4K13 expression vector spectrum (A) and verification electrophoresis (B); M represents marker, and lanes 1-3 represent target genes GhMAP4K13 Fragment;

[0026] Figure 2 yes GhMAP4K13 Phylogenetic tree of genes;

[0027] Figure 3 Seed growth of WT and OE-GhMAP4K13 Arabidopsis under drought stress (A) and salt stress (B). DETAILED DESCRIPTION

[0028] The present invention is further described below with reference to the examples and drawings, but the present invention is not limited by the following examples. The various reagents mentioned in the examples but not listed are prepared according to the method of the Molecular Cloning Experiment Guide, and the biochemical reagents are analytical grade or higher.

[0029] Example 1 Amplification of cotton GhMAP4K13 gene

[0030] 1. Extraction of Total RNA

[0031] Upland cotton TM-1 was planted in the experimental field of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences and managed as a normal field. The parts taken included roots, stems, leaves, petals, ovules, and fibers at different developmental stages. The materials were quickly frozen in liquid nitrogen and stored in a -80℃ refrigerator for later use. The total RNA of cotton was extracted using a kit from Aidlab biotechnologies.

[0032] 2. Reverse transcription of RNA into cDNA

[0033] RNA was reverse transcribed into cDNA using the TransGen Biotech reverse transcription kit, and the RT reaction solution was prepared on ice according to the components shown in Table 1.

[0034] Table 1 First-strand DNA synthesis system

[0035]

[0036] The reverse transcription reaction conditions were as follows: 42°C for 30 min (reverse transcription reaction); 85°C for 5 s (reverse transcriptase inactivation reaction)

[0037] 3. PCR amplification of target gene

[0038] The cDNA solution of the reverse transcription product was diluted 10 times with Vazyme high-fidelity enzyme as a PCR reaction template, and the following nucleotide sequences were used as primers:

[0039] Upstream primer:

[0040] 5'-ctcaagatcagaagtactATGGGAGGAAATCGGAGTTACTCAG-3', (SEQ ID NO:3);

[0041] Downstream primer:

[0042] 5'-gaccacccggggatccCTAATCCCACTCTTCCCCACCAG-3', (SEQ ID NO:4);

[0043] Table 2 Target gene PCR system

[0044]

[0045] The target gene PCR amplification procedure is as follows:

[0046]

[0047] The PCR amplification products were sequenced and compared with the sequences of the MAP4K family in Genbank. Figure 2 As shown: The MAP4K family is divided into four subgroups, of which GhMAP4K13 is the largest, indicating that large-scale expansion and variation occurred during evolution. GhMAP4K13 is located on chromosome A12 of upland cotton and has a unique gene structure and conserved sequence with Arabidopsis MAP4K.

[0048] Example 2 GhMAP4K13 Application of genes

[0049] 1. Construction of overexpression vector

[0050] The PCR amplification product sequenced correctly in Example 1 was connected into the plant expression vector pBI121 using the In-Fusion ligation technology, and the above PCR amplification product and the pBI121 linearized vector were configured according to the system shown in Table 3.

[0051] Table 3 Construction system of overexpression vector

[0052]

[0053] After mixing the reagents, incubate at 50°C for 15 min, then place on ice, transform DH5α competent cells, pick single clones, and sequence to verify the correctness of the sequence. The sequencing results showed that the obtained plasmid was a GhMAP4K13 exogenous gene fragment inserted into the BamHI / SacI restriction site of the pBI121 vector, and the plasmid was named pBI121-35S:: GhMAP4K13.

[0054] 2. Obtaining GhMAP4K13 transgenic Arabidopsis

[0055] (1) Use the freeze-thaw method to transform Agrobacterium tumefaciens LBA4404 competent cells. The specific transformation process is as follows:

[0056] Take the competent Agrobacterium stored at -80℃ and wait for it to partially melt at room temperature. When it is in the state of ice-water mixing, insert it into ice; add 1 µg of plasmid for every 100 µL competent state, stir the bottom of the tube by hand to mix, and then stand on ice for 5 min, liquid nitrogen for 5 min, 37℃ water bath for 5 min, and ice bath for 5 min; add 700 µL of LB liquid medium without antibiotics, shake and culture at 28℃ for 2-3 h; centrifuge at 6000 rpm for 1 min to collect bacteria, keep about 100 µL of supernatant, gently blow and resuspend the bacteria block, and apply it on the LB plate containing kanamycin (50 µg / mL) and rifampicin (25 µg / mL), invert and culture in a 28℃ incubator for 48-72 h. Pick a single colony and culture it in 1 mL of LB medium containing dual antibodies for about 16 h until it becomes turbid; perform colony PCR and enzyme digestion identification to screen out positive Agrobacterium strains. The plasmid of the positive recombinant bacteria was extracted and sent for sequencing, and the recombinant bacteria containing the plasmid pBI121-35S:: GhMAP4K13 that was sequenced correctly was named LBA4404 / pBI121-35S:: GhMAP4K13.

[0057] (2) The recombinant Agrobacterium was transformed into Arabidopsis thaliana using the inflorescence infection method. The specific transformation steps are as follows:

[0058] Inoculate 20 µL of Agrobacterium culture stored at -20℃ into 1mL LB liquid medium, and culture overnight at 28℃ and 180rpm. Take 1mL of activated culture and add it into 50mL LB liquid medium, and culture at 28℃ and 180rpm. 600 When the value is about 1.2-1.6, centrifuge the bacterial solution at 5000 rpm to collect the cells; suspend the cells in transformation medium (1 / 2 MS salt, 5% sucrose, pH=5.8) and adjust the OD 600 After the concentration reaches 0.8, add silwet-L-77 (0.02%) to obtain the impregnation solution; place the Arabidopsis inflorescence in the impregnation solution for 50 seconds, wrap the Arabidopsis with plastic wrap after impregnation, culture in the dark for 24 hours, and then culture under normal conditions. To increase the transformation rate, the seeds can be impregnated again after 7 days, and the transgenic T0 generation seeds can be harvested after maturity.

[0059] (3) Identification of transgenic Arabidopsis thaliana plants expressing the GhMAP4K13 gene. The specific process is as follows:

[0060] After the harvested seeds are dried, they are disinfected: 1 min with alcohol, 10 min with 50% sodium hypochlorite, and 5 times with sterile water. They are then spotted on 1 / 2MS containing kanamycin and vernalized at 4°C for 3 days. After culturing for about 10-15 days (16 h daytime / 8 h dark, 21°C), the difference between positive and negative plants can be observed. Those that can grow normally may be positive plants. Transplant Arabidopsis that can grow normally to the culture room and detect whether the gene has been transferred into the plant when it grows to 7-8 leaves. GhMAP4K13 ;

[0061] PCR was performed on living leaves using the TransDirect Plant Tissue PCR Kit according to the instructions, with LBA4404 / pBI121-35S:: GhMAP4K13 recombinant Agrobacterium solution as a positive control, ddH2O as a blank control, and non-transgenic Arabidopsis DNA as a negative control.

[0062] The primer sequences for PCR amplification are:

[0063] GhMAP4K13F: 5'-GTAAAGCTCCGGCCAAGTCA-3', (SEQ ID NO:5),

[0064] GhMAP4K13R: 5'- AATCATTGTGGCGTAGGGGT-3', (SEQ ID NO: 6);

[0065] Verification of the PCR amplification system is shown in Table 4.

[0066] Table 4 Verification of PCR amplification system

[0067]

[0068] Verification PCR reaction procedure is as follows:

[0069]

[0070] The amplified products were respectively electrophoresed on 1.2% agarose gel. The test results are shown in Figure 1 :Correct bands were detected in the selected transgenic plants with normal growth and the positive control group, while no corresponding DNA molecular fragments were detected in the blank control and the wild-type Arabidopsis negative control group, indicating that GhMAP4K13 The gene has been integrated into the Arabidopsis genome mentioned above.

[0071] (4) Identification of salt tolerance and drought resistance of transgenic Arabidopsis plants

[0072] Under sterile conditions, transfer GhMAP4K13 Gene Arabidopsis thaliana (OE- GhMAP4K13 ) T1 generation seeds and wild-type Arabidopsis seeds were sterilized and sown on MS solid medium containing 100 mM NaCl and 200 mM mannitol to simulate saline-alkali environment and drought environment, respectively. Vernalization was carried out at 4°C without light for 3 days, and then placed in an incubator for germination and culture for about 2 weeks to observe OE- GhMAP4K13 Growth conditions of Arabidopsis thaliana and wild-type Arabidopsis thaliana.

[0073] The results are as follows Figure 3 As shown: Under salt and drought stress, OE- GhMAP4K13 There are significant differences in the phenotypes of Arabidopsis thaliana and wild-type Arabidopsis thaliana. Transgenic Arabidopsis thaliana exhibits salt tolerance and drought resistance, that is, it can grow normally in high-salt and drought environments, while wild-type Arabidopsis thaliana cannot grow normally or does not grow. In stress media containing mannitol and stress media containing NaCl, OE- GhMAP4K13 Arabidopsis thaliana has larger cotyledons and longer roots than wild-type Arabidopsis thaliana, showing significant differences.

[0074] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A kind of cotton MAP4K13 The application of genes in crop drought resistance or salt-alkali resistance is characterized in that: Cotton MAP4K13 Genes are expressed in crops; The cotton MAP4K13 The encoded amino acid sequence is shown in SEQ ID NO: 1; The crop is Arabidopsis thaliana or cotton.

2. A method for screening drought-resistant or salt-alkali-resistant crops, characterized in that: The following steps are involved: (1) Construct GhMAP4K13 recombinant vector; (2) Include GhMAP4K13 Transform cells with the recombinant vector and screen to obtain positive transformants; (3) Transfecting wild-type crops with positive transformants to obtain drought-resistant or salt-alkali-resistant transgenic crops; Said GhMAP4K13 The encoded amino acid sequence is shown in SEQ ID NO: 1; The crop is Arabidopsis thaliana or cotton.

3. The method according to claim 2, characterized in that The screening process includes: Sexual reproduction of transgenic crops and selection of sexually reproduced offspring GhMAP4K13 Homozygotes are retained to obtain drought-resistant or saline-alkali-resistant crops.

4. The method according to claim 2, characterized in that: The following steps are involved: (1) Obtain GhMAP4K13 The gene was connected to the expression vector plasmid to construct GhMAP4K13 recombinant vector; (2) Include GhMAP4K13 The recombinant vector was transformed into Agrobacterium tumefaciens competent cells by freeze-thaw method, and positive transformants were obtained by screening; (3) Transfect wild-type crops with bacterial solution containing positive transformants, harvest the T0 generation after the crops mature, and screen the T0 generation to obtain drought-resistant or salt-alkali-resistant transgenic crops; (4) Propagate the transgenic crops sexually or asexually, screen and harvest plants or seeds with stable traits; select the offspring with stable traits GhMAP4K13 The homozygotes are retained to obtain drought-resistant or saline-alkali-resistant crops.

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