Application of a tumorous stem mustard BjuA017664 gene in improving plant resistance to drought stress
By overexpressing the BjuA017664 gene in stem tumor mustard, the drought resistance of plants is improved by genetic transformation technology, and the problem of fewer drought resistance genes in the existing technology is solved, achieving a significant effect of improving the drought resistance of plants.
Patent Information
- Application Number
- CN202210853962.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-07-12
- Publication Date
- 2025-02-07
- Estimated Expiration
- 2042-07-12
AI Technical Summary
In the prior art, the anti-drought gene of the stem tumor mustard is obtained less, which cannot meet the needs of improved plant yield and quality, limiting its widespread use in agricultural production and commercial applications.
By extracting and overexpressing the BjuA017664 gene of stalk mustard, recombinant DNA and genetic transformation techniques were used to introduce plants to the crops, thereby increasing the resistance of plants to drought stress.
The drought resistance of plants has been significantly improved, and the survival rate of transgenic plants under drought conditions reaches 75% or above, which is better than the wild type and has significant economic value and application potential.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to an application of a tumorous stem mustard BjuA017664 gene in improving the drought stress resistance of plants. Background Art
[0002] In recent years, with global warming and the intensification of human activities, the frequency and number of extreme weather events such as drought, floods, high temperatures and frost have gradually increased, and the threat to agricultural production has become increasingly severe. Currently, drought and high temperatures are considered to be the two main abiotic stress factors that lead to crop yield reduction and failure. Excessively dry air or low soil moisture content can cause plants to develop drought stress due to lack of water, which has a serious inhibitory effect on plant growth and development.
[0003] As one of the main constraints in plant growth, drought will hinder plant respiration, photosynthesis and stomatal movement, thereby affecting plant growth and physiological metabolism. Therefore, it is of great significance to explore drought resistance-related genes, explore their drought resistance mechanisms, and cultivate new drought-resistant varieties to ensure the stability and safety of my country's crops. In recent years, people have conducted a lot of research on the mechanism of plant response to drought and other adverse stresses from the perspectives of physiology, biochemistry, metabolism, ecology, genetics, and evolution. Due to the complexity of plant drought resistance traits, it is very difficult to improve plant drought resistance using traditional breeding methods. With the development of molecular biology, people can understand the mechanism of plant resistance to drought stress at the molecular level such as gene composition, expression regulation and signal transduction, which has opened up new ways to improve plant stress resistance using genetic engineering methods. At present, some progress has been made in plant genetic transformation research, so the drought resistance of plants can be improved by transgenic methods. Transgenic is an important means for plants to control their protein components by molecular means under stress conditions. This method has the advantages of fast breeding, easy to obtain resistant plants, and good genetic stability, and has become the main means of obtaining resistant plants. Therefore, using genetic engineering to improve the drought resistance of crops and improve the adaptability of crops and cash crops to adversity is a key and major issue that needs to be solved in the breeding of new varieties. However, the isolation of efficient drought-resistant genes has become the main factor limiting plant stress-resistant genetic engineering.
[0004] Tumor mustard (Brassica juncea var.tumida Tsen et Lee), also known as green cabbage head, is a plant of the genus Brassica in the family Cruciferae. It is the main raw material of mustard and one of the main economic crops in Chongqing, Sichuan, Zhejiang and other regions in my country. For many years, the relevant research on tumor mustard has mainly focused on genetic breeding, breeding of improved varieties, cultivation technology, quality and safety, and only in recent years have there been reports on biological research. Previous studies have shown that tumor mustard has strong resistance to abiotic stress, and multiple adversity stress-related genes have been obtained from tumor mustard. However, so far, the drought-resistant genes obtained are still too few, and they cannot meet the needs of molecular design for improving plant yield and quality, so the application of drought-resistant genetic engineering of tumor mustard in agricultural production and commerce is greatly limited. Therefore, it is still urgent to obtain efficient and broad-spectrum drought-resistant genes in tumor mustard. Summary of the invention
[0005] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide an application of the stem tumor mustard BjuA017664 gene in improving plant resistance to drought stress, providing a new gene target for improving drought-resistant crop varieties and enriching the gene pool.
[0006] To achieve the above-mentioned object, the present invention adopts the following technical scheme: an application of the tumorous stem mustard BjuA017664 gene in improving plant resistance to drought stress, wherein the nucleotide sequence of the BjuA017664 gene is as shown in SEQ ID NO.1 or a nucleotide sequence with the same function obtained by replacing, deleting or inserting one or more nucleotides of the nucleotide sequence shown in SEQ ID NO.1.
[0007] Furthermore, the amino acid sequence encoded by the BjuA017664 gene is as shown in SEQ ID NO.2 or an amino acid sequence having the same function obtained by replacing, deleting or inserting one or more amino acids in the amino acid sequence shown in SEQ ID NO.2.
[0008] Another object of the present invention is to provide a biological material containing the tumorous stem mustard BjuA017664 gene, wherein the biological material is a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector or an engineered bacterium.
[0009] Another object of the present invention is to provide the use of the biological material in improving the drought stress resistance of plants.
[0010] Furthermore, the plant is a dicotyledonous plant or a monocotyledonous plant, preferably Brassica juncea, Arabidopsis thaliana, Chinese cabbage, tobacco or rapeseed.
[0011] Another object of the present invention is to provide a method for improving plant resistance to drought stress, comprising: increasing the expression level and / or activity of BjuA017664 protein in the plant; the amino acid sequence of the BjuA017664 protein is as shown in SEQ ID NO.2 or has an amino acid sequence with the same function obtained by replacing, deleting or inserting one or more amino acids in the amino acid sequence shown in SEQ ID NO.2.
[0012] Furthermore, the plant is a dicotyledonous plant or a monocotyledonous plant, preferably Brassica juncea, Arabidopsis thaliana, Chinese cabbage, tobacco or rapeseed.
[0013] Furthermore, the method specifically includes the following steps: transforming the prepared or provided expression vector containing the BjuA017664 gene into Agrobacterium to obtain engineered Agrobacterium bacteria, then infecting the engineered Agrobacterium bacteria into stem tumor mustard explants, and using hypocotyl genetic transformation technology to overexpress the BjuA017664 gene, thereby obtaining transgenic plants resistant to drought stress.
[0014] Compared with the prior art, the present invention has the following beneficial effects:
[0015] 1. The present invention reports for the first time the application of the stem tumor mustard BjuA017664 gene in enhancing plant drought resistance. By constructing an overexpression vector of the BjuA017664 gene, it is overexpressed in the stem tumor mustard and subjected to drought stress treatment. The study found that after drought stress treatment, normal growth conditions were restored, and a large number of transgenic Arabidopsis plants could resume normal growth. The survival rate of the transgenic strains reached 75% or more, which was significantly higher than that of the wild type. This indicates that the stem tumor mustard BjuA017664 gene can be introduced into crops through biological means such as transgenic technology and overexpressed to significantly improve the drought stress resistance of plants, and is a high-quality candidate gene for breeding drought-resistant crop varieties.
[0016] 2. The tumorous stem mustard BjuA017664 gene of the present invention can significantly enhance plant drought resistance, is conducive to maintaining crop yield under drought adversity conditions, can reduce the losses caused by drought adversity to cash crops, and has significant economic value. In addition, the present invention also provides new gene targets and resources for genetic improvement of crop drought resistance, can be widely used in plant genetic breeding, germplasm resource improvement and cultivation, and is of great significance for promoting stable crop yield. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 This is a semi-quantitative expression analysis of the BjuA017664 gene in the overexpression transgenic strain; WT is the wild-type BjuA017664 plant, and #2, #3, #4 and #5 are transgenic BjuA017664 plants.
[0018] Figure 2Figure 2 is the phenotype of the transgenic strain overexpressing BjuA017664 under drought stress; WT is the wild-type Brassica juncea strain, and #2, #3, #4 and #5 are transgenic Brassica juncea strains.
[0019] Figure 3 is the survival rate of transgenic lines overexpressing BjuA017664 after drought stress; WT is the wild-type Brassica juncea line, and #2, #3, #4 and #5 are transgenic Brassica juncea lines. DETAILED DESCRIPTION
[0020] The present invention is further described in detail below with reference to specific examples and accompanying drawings. The raw materials described in the examples are common commercial products unless otherwise specified. The experimental methods described in the examples are operated according to conventional molecular biology experimental methods unless otherwise specified.
[0021] Example 1 Cloning of the gene sequence of Tumor Brassica juncea BjuA017664
[0022] In order to verify the full-length gene of B. juncea BjuA017664, specific primers SEQ ID NO.3 (BjuA017664-F) and SEQ ID NO.4 (BjuA017664-R) were designed for PCR amplification analysis.
[0023] The leaf tissue of tumorous mustard at the budding stage was used as the material and TRIzol TM Plus RNA Purification Kit (12183555, Invitrogen TM ), total RNA was extracted according to the instructions, and DNase I (18047019, Invitrogen TM ) Remove the residual trace DNA and measure the concentration of RNA using a spectrophotometer for later use.
[0024] About 2.0 μg of total RNA from the leaves of Brassica juncea was taken and the first-strand cDNA was synthesized according to the instructions of PrimeScript II first-strand cDNA synthesis kit (6210A, Takara).
[0025] The PCR amplification system was high-fidelity amplification enzyme Prime STAR HS (R010A, TaKaRa) 0.25 μL, 5× PrimeSTAR Buffer (Mg 2+ Plus) 5μL, forward primer (Bju-F, 10μM) 0.5μL, reverse primer (Bju-R, 10μM) 0.5μL, template (DNA) 1μL, dNTP (2.5mM) 2μL, and sterile ddH2O to 25μL.
[0026] The sequences of the forward and reverse primers are as follows:
[0027] BjuA017664-F:ATGGCTACCGGAGAGGAGAAAACCTG
[0028] BjuA017664-R: TCAGTGCTTGATCTTAGGCTTCTTC
[0029] The PCR reaction program was as follows: pre-denaturation at 95°C for 5 min; 95°C for 30 s; 58°C for 30 s; 72°C for 30 s, 35 cycles; 72°C for 10 min.
[0030] The PCR product was analyzed by agarose gel electrophoresis, and a specific amplification band was observed at about 500 bp under ultraviolet light. It was purified using a gel recovery kit (9672, Takara) and used for later use.
[0031] The purified DNA fragment was A-added using a blunt end A reagent and connected to the pMD20-T vector (6019, Takara) through TA cloning. The ligation product was transformed into Escherichia coli DH5a, and positive clones were picked from LB culture plates containing ampicillin (100 mg / L) for sequencing and analysis. The results showed that the CDS sequence of the B.juncea BjuA017664 gene was shown in SEQ ID NO.1, containing an open reading frame of 489 bp, and the encoded protein contained 162 amino acids (as shown in SEQ ID NO.2).
[0032] Example 2 Construction and transformation of recombinant expression vector pTF101-BjuA017664-GFP
[0033] (1) Construction of pTF101-BjuA017664-GFP expression vector
[0034] According to the full-length gene sequence SEQ ID NO.1 of stem tumor mustard BjuA017664, primers Bju-gfp-F and Bju-gfp-R were designed, and restriction site sequences were introduced into the primers. The positive clone plasmid connected by TA in Example 1 was used as a template, and Bju-gfp-F (forward primer) and Bju-gfp-R (reverse primer) were used as primers to specifically amplify the gene BjuA017664.
[0035] The primer sequences are as follows:
[0036] Bju-gfp-F: CCCGGG ATGGCTACCGGAGAGGAGAAACCTG
[0037] Bju-gfp-R: GGATCC TCAGTGCTTGATCTTAGGCTTCTTC
[0038] Among them, the underlined sequence of Bju-gfp-F is the SmaI restriction site, and the underlined sequence of Bju-gfp-R is the BamHI restriction site.
[0039] PCR reaction system: high-fidelity amplification enzyme PrimeSTAR HS (R010A, TaKaRa) 0.5 μL, 5x PrimeSTAR Buffer (Mg 2+ Plus) 10 μL, forward primer (10 μM) 1 μL, reverse primer (10 μM) 1 μL, template (50-fold diluted plasmid) 1 μL, dNTP (2.5 mM) 4 μL, and sterile ddH2O to 50 μL.
[0040] PCR reaction conditions: pre-denaturation at 95°C for 5 min; 95°C for 30 s; 58°C for 30 s; 72°C for 30 s, 35 cycles; 72°C for 10 min.
[0041] The PCR amplification product was detected by agarose gel electrophoresis. The amplified target fragment had the same size as the expected fragment, and was recovered and purified according to the instructions of the gel recovery kit (9672, Takara) to obtain the target gene fragment.
[0042] The pTF101-GFP expression vector was treated with double enzyme digestion of SmaI and BamHI. The enzyme digestion system was: pTF101-GFP vector 5μL; SmaI 0.5μL; BamHI 0.5μL; Buffer 10XK 2μL; sterile ddH2O to 20μL; 37℃ for 3h. After the enzyme digestion, the pTF101-GFP vector fragment was recovered using the Takara agarose gel recovery kit.
[0043] The pTF101-BjuA017664-GFP expression vector was constructed using T4 DNA Ligase (FL101, Trans).
[0044] The ligation reaction system is:
[0045] Purified PCR fragment (recovered BjuA017664 target fragment) 50ng; Linearized vector (pTF101-GFP vector) 100ng; 5x T4 DNA Ligase Buffer 2μL; T4 DNA Ligase 0.5μL; Sterile ddH2O supplemented to 10μL; 25℃ reaction for 30min. According to the molecular cloning experimental guide, the above recombinant reaction system was transformed into Escherichia coli DH5a and coated on a screening culture plate containing spectinomycin resistance (75mg / L). The correct recombinant expression vector pTF101-BjuA017664-GFP containing the BjuA017664 gene fragment was obtained by sequencing the positive clones. After the reporter gene GFP in the recombinant expression vector was fused with the 5' end of the target gene BjuA017664, it was located downstream of the constitutive promoter P35S to form a fusion expression; the 3' end of BjuA017664 was assembled with the NOS terminator, which can effectively terminate the transcription of the fusion gene. The reporter gene GFP can emit green fluorescence after being excited by blue light without the need for auxiliary factors and substrates. As a reporter gene, it can be used to detect the expression of the target gene.
[0046] (2) Agrobacterium-mediated genetic transformation of Brassica juncea
[0047] The constructed recombinant expression vector pTF101-BjuA017664-GFP was transferred into Agrobacterium strain GV3101 by conventional freeze-thaw method, and positive clones were screened by PCR. Then, Agrobacterium carrying pTF101-BjuA017664-GFP vector was introduced into Tuber mustard using the stable genetic transformation technology of hypocotyls. The expression level of BjuA017664 gene in the transgenic strains (#2, #3, #4 and #5) with good phenotype overexpressing BjuA017664 and the wild type was identified by semi-quantitative RT-PCR. Trizol reagent (Invitrogen TM ), and total RNA was extracted from the leaves of Brassica juncea according to the instructions. TM ) was used to remove residual DNA, and the first-strand cDNA was synthesized using cDNA reverse transcription reagent (Takara) according to the instructions.
[0048] The primers for detecting the target gene are:
[0049] BjuA017664-F:ATGGCTACCGGAGAGGAGAAAACCTG
[0050] BjuA017664-R: TCAGTGCTTGATCTTAGGCTTCTTC
[0051] The Arabidopsis internal reference primers are:
[0052] RT-AtACTIN3-F:GGCTACTCTTTCACCACGAC
[0053] RT-AtACTIN3-R:GGATACCAGCATTCTCCATAC
[0054] The results are as follows Figure 1 As shown, the target gene BjuA017664 of Brassica juncea was up-regulated in the four transgenic lines (#2, #3, #4 and #5), while the expression of BjuA017664 was almost undetectable in the wild-type plant (WT) (the expression level was too low), indicating that BjuA017664 had been introduced into the Brassica juncea genome and successfully transcribed and expressed.
[0055] Example 3 Phenotypic observation and analysis of transgenic tumorous stem mustard
[0056] The wild-type WT seeds and the seeds of the obtained transgenic lines #2, #3, #4 and #5 were disinfected, sterilized and vernalized, and then germinated on filter paper soaked in water. The seedlings 4 days after germination were transferred to a water-saturated culture matrix flowerpot (vermiculite: nutrient soil = 3:1) and placed in a plant culture room for culture. The light cycle of the culture room was day: night = 16h:8h, and the temperature was 22°C. When the seedlings grew to the stage of 3 pairs of leaves, the transgenic and wild-type stem mustard plants were subjected to drought stress (i.e., no watering) for 8 days, the growth status of the plants was observed, the phenotype was analyzed, and then the plants were rehydrated by watering. After 2 days, the number of plants that recovered to normal growth in each group was observed and counted, and the survival rate was calculated. The transgenic plants and wild-type plants were tested three times each, and 25 plants were taken from each test group for stress treatment. The results are shown in the figure. Figure 2 and Figure 3 shown.
[0057] The results showed that before drought treatment, the transgenic strains overexpressing BjuA017664 and the wild type grew normally, with no significant differences ( Figure 2 a). After drought treatment, the stems of the wild-type plants were completely unable to stand upright, while the leaves of the transgenic lines overexpressing BjuA017664 wilted but were still mostly able to remain upright ( Figure 2 b). After rehydration, only a very small number of wild-type plants recovered to normal, while most of the transgenic plants recovered to normal ( Figure 2 c) According to calculations, about 75% or more of the transgenic BjuA017664-transgenic stem mustard strains recovered normal growth, among which the transgenic #3 strains recovered normal growth with a survival rate of about 100%; while only about 25% of the wild-type stem mustard plants survived ( Figure 3). The results showed that overexpression of the BjuA017664 gene in Tuber mustard can significantly improve the drought resistance of plants.
[0058] The sequences described in the above embodiments are specifically as follows:
[0059] Sequence 1: SEQ ID NO.1BjuA017664 nucleotide sequence, 489 bp long
[0060] ATGGCTACCGGAGAGGGAGAAACCTGTTATGGTCGTCGGTATGGACGAAAGCGAGCAGAGCACTTACGCCTTGGAGTGGACCCTCGATCGTTTCTTCGCTCCTTACGCTCCTAATTTTCCTTT CAAGCTCTTCATCGTCCACGCCAAACCTAACGCCGTCTCTGCCGTTGGTCTTGCTGGTCCCGGAGCTGCGGAGGTTCTGCCGTATGTTGATACCGATTTGAAGCATATTGCTGCCAGGGGTTA TCGAGATGGCTAAAGGTATCTGTCAGAGCAAATCGGTTGATGGCGCTATGTTCGAAGTTTTTGAAGGTGATGCAAGAAGTATACTGTGCGATGTTGTGGATAAACACCATGCTTCTCTTCTTGTCGTGGGAAGCCATGGTTATGGAGCTATCAAGAGGGCGGTTCTAGGGAGCGTGAGCGACTACTGTGCTCATCATGCTCATTGCTCGGTGATGATCGTGAAGAAGCCTAAGATCAAGCACTGA
[0061] Sequence 2: SEQ ID NO.2 protein BjuA017664 amino acid sequence, 162 amino acids long
[0062] MATGEEKPVMVVGMDESEQSTYALEWTLDRFFAPYAPNFPFKLFIVHAKPNAVSAVGLAGPGAAEVLPYVDTDLKHIAARVIEMAKGICQSKSVDGAMFEVFEGDARSILCDVVDKHHASLLVVGSHGYGAIKRAVLGSVSDYCAHHAHCSVMIVKKPKIKH*
[0063] Sequence 3: SEQ ID NO.3BjuA017664-F nucleotide sequence, 25 bp long
[0064] ATGGCTACCGGAGAGGAGAAACCTG
[0065] Sequence 4: SEQ ID NO.4BjuA017664-R nucleotide sequence, 25 bp long
[0066] TCAGTGCTTGATCTTAGGCTTCTTC
[0067] Sequence 5: SEQ ID NO.5 RT-AtACTIN3-F nucleotide sequence, 20 bp long
[0068] GGCTACTCTTTCACCACGAC
[0069] Sequence 6: SEQ ID NO.6 RT-AtACTIN3-R nucleotide sequence, 21 bp long
[0070] GGATACCAGCATTCTCCATAC
[0071] Sequence 7: SEQ ID NO.7 Bju-gfp-F nucleotide sequence, 31 bp long
[0072] CCCGGGATGGCTACCGGAGAGGAGAAAACCTG
[0073] Sequence 8: SEQ ID NO.8 Bju-gfp-R nucleotide sequence, 31 bp long
[0074] GGATCCTCAGTGCTTGATCTTAGGCTTCTTC
[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.
Claims
1. A tumorous stem mustard BjuA017664 Application of genes in improving the drought stress resistance of tumorous stem mustard, the BjuA017664 The nucleotide sequence of the gene is shown in SEQ ID NO.
1.
2. Containing the tumorous stem mustard according to claim 1 BjuA017664 The invention discloses an application of a gene-containing biological material in improving the drought stress resistance of tumorous stem mustard. The biological material is a recombinant DNA, an expression box, a transposon, a plasmid vector, a virus vector or an engineering bacterium.
3. A method for improving the drought stress resistance of Tumor Brassica juncea, characterized in that: include: Increase the BjuA017664 The expression level of the protein; BjuA017664 The amino acid sequence of the protein is shown in SEQ ID NO.
2.
4. The method according to claim 3, characterized in that: The method specifically comprises the following steps: preparing or providing the BjuA017664 The gene expression vector is transformed into Agrobacterium to obtain Agrobacterium engineering bacteria, and then the Agrobacterium engineering bacteria is infecting plants to make BjuA017664 Gene overexpression can produce transgenic plants resistant to drought stress.