A soybean non-receptor tyrosine kinase binding protein gene GmABIL2 and its application
Through the overexpression and function study of the soybean non-receptor tyrosine kinase binding protein gene GmABIL2, the response mechanism of this gene under soybean saline and alkali stress was clarified, and the problem of reduced yield in soybean under saline and alkali conditions was solved, providing a theoretical basis for the study of soybean saline and alkali regulation network.
Patent Information
- Application Number
- CN202411977492.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-04-04
- Estimated Expiration
- 2044-12-31
AI Technical Summary
Soybean yield can be severely reduced under saline-alkali stress conditions. There are few studies on soybean saline-alkali-related genes in the prior art, especially the functional studies of soybean saline-alkali tolerance have not been reported.
It proposes the gene GmABIL2, a soybean non-receptor tyrosine kinase binding protein gene, and its application, which reduces the ability of plants tolerate saline and alkali stress by overexpressing GmABIL2, and it is also clear through Arabidopsis and soybean root systems that the protein is induced by saline and alkali stress and has a biological function of reducing saline and alkali tolerance in the soybean seedling stage.
In order to further enrich the mechanism of the soybean saline-alkali regulation network in my country, it has been preliminarily proved that GmABIL2 has the function of reducing saline-alkali tolerance in Arabidopsis thaliana during the germination period, and has shown an effect of reducing saline-alkali tolerance in soybean roots.
Smart Images
Figure CN119391766B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of genetic engineering technology, and in particular to a soybean non-receptor tyrosine kinase binding protein gene. GmABIL2 and its applications. Background Art
[0002] Soil salinization is one of the important factors affecting global agricultural production, ecological environment and food security. According to the Food and Agriculture Organization (FAO), the area of saline-alkali land in the world is 954 million hm2. 2 Among them, the distribution area of saline soil and potential salinized soil in my country is about 99.13 million hm2. 2 . Saline-alkali soil contains high concentrations of Na + 、CO3 2- and HCO3 - Plasma has a direct toxic effect on plant roots, making it difficult for root cells to absorb water from the soil, which leads to plant death, greatly reducing the yield of crops in my country and bringing great impact to agricultural production. Therefore, it is of great significance to improve the salt-alkali tolerance of crops and cultivate new varieties of salt-alkali tolerant crops.
[0003] Soybean originated in my country and is an important dual-purpose crop for food, oil and feed in my country. It plays an important role in the development of the national economy and the guarantee of national food and food security. Soybean is a moderately salt-alkali sensitive crop; under salt-alkali stress conditions, soybean yield can be severely reduced by 50%, which has a serious impact on my country's soybean yield. Therefore, it is of great research significance to explore important soybean salt-alkali related genes, analyze their regulatory network and genetic basis, and cultivate new salt-alkali tolerant soybean varieties (lines) to promote the development of my country's soybean industry.
[0004] Non-receptor tyrosine kinase binding protein gene GmABIL2 It can regulate plant growth and development by affecting root growth. However, there are few studies on the role of this gene in plant adversity, and no studies on its function in soybean salt-alkali tolerance have been reported. Summary of the invention
[0005] In view of the above-mentioned defects of the prior art, the present invention proposes a soybean non-receptor tyrosine kinase binding protein gene GmABIL2 and its application to solve the problems raised by the above background technology.
[0006] In order to achieve the above object, the present invention provides the following technical solutions:
[0007] Non-receptor tyrosine kinase binding protein gene GmABIL2 Application in regulating plant tolerance to salt and alkali stress, the GmABIL2 The nucleotide sequence is shown in SEQ ID NO:1.
[0008] Preferably, the GmABIL2 The amino acid sequence is shown in SEQ ID NO:2.
[0009] Preferably, by overexpressing the non-receptor tyrosine kinase binding protein gene GmABIL2 Reduce the plant's tolerance to salt-alkali stress.
[0010] Preferably, the plant is one of soybean or Arabidopsis thaliana.
[0011] Preferably, the non-receptor tyrosine kinase binding protein gene GmABIL2 Localized in mitochondria.
[0012] Preferably, GmABIL2 It can inhibit soybean root growth and Arabidopsis germination.
[0013] Preferably, the condition of saline-alkali stress is one of 148mM NaHCO3+2mM NaCl or 146mM NaHCO3+4mM NaCl or 100mM NaCl+95mM NaHCO3+5mM Na2CO3.
[0014] Preferably, a method for cultivating soybeans that are intolerant to salt-alkali stress comprises the following steps:
[0015] (1) Non-receptor tyrosine kinase binding protein gene GmABIL2 Connecting to a vector to obtain an expression vector;
[0016] (2) The expression vector obtained in step (1) is transferred into Agrobacterium by electrophoresis to obtain recombinant bacteria;
[0017] (3) The recombinant bacteria obtained in step (2) are used to infect soybeans using a high-efficiency transient infection technique to obtain soybeans that are intolerant to salt-alkali stress.
[0018] Preferably, the vector in step (1) is pCAMBIA3301 vector.
[0019] Preferably, the Agrobacterium in step (2) includes Agrobacterium EHA105 or Agrobacterium rhizogenes K599.
[0020] Compared with the prior art, the present invention has the following beneficial effects:
[0021] The applicant previously used the salt-alkali tolerance phenotype identification of cultivated soybean seedlings and whole genome association analysis to dig out soybean salt-alkali related candidate genes and identify whether they have the function of improving plant salt-alkali tolerance. On this basis, the present invention discloses a soybean non-receptor tyrosine kinase binding protein gene GmABIL2, and used Arabidopsis and soybean rooting systems to clarify that this protein is induced by salt-alkali stress and has the biological function of reducing salt-alkali tolerance in the soybean seedling stage, providing a theoretical basis for further enriching the mechanism of action of my country's soybean salt-alkali regulatory network. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 soybean non-receptor tyrosine kinase binding protein gene GmABIL2 PCR detection electrophoresis band diagram; M is DL2000; 1 is GmABIL2 Purpose fragment;
[0023] Figure 2 soybean non-receptor tyrosine kinase binding protein gene GmABIL2 Construction of expression vector; M is DL2000; 1-3 is pCAMBIA3301-GmABIL2 bacterial solution PCR detection; - is negative control;
[0024] Figure 3 Expression of soybean non-receptor tyrosine kinase binding protein gene under saline-alkali stress GmABIL2 Analysis of expression levels in leaves and roots; the left picture shows the expression level in leaves, and the right picture shows the expression level in roots;
[0025] Figure 4 soybean non-receptor tyrosine kinase binding protein gene GmABIL2 Subcellular localization analysis;
[0026] Figure 5 Overexpression under saline-alkali stress GmABIL2 Phenotypic identification of wild-type Arabidopsis thaliana during the germination period;
[0027] Figure 6 Overexpression under saline-alkali stress GmABIL2 Statistical analysis of germination rate of wild-type Arabidopsis thaliana at 2 0.5 s
[0028] Figure 7 Overexpression under saline-alkali stress GmABIL2 Functional identification of soybean hairy roots. DETAILED DESCRIPTION
[0029] In order to enable those skilled in the art to better understand the technical content of the present invention, the technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.
[0030] Example 1
[0031] Williams 82 was selected as the gene cloning material. When the plant grew the first set of trifoliate leaves, samples were taken, and total RNA from the leaves was extracted and reverse transcribed into the first chain of cDNA. GmABIL2Gene CDS region sequence, using Primer 5 software to design gene cloning primers, cDNA as a template, using TOYOBO brand KOD-Plus-Neo high-fidelity PCR enzyme to amplify the target gene (for details of the electrophoresis bands, see Figure 1 ); PCR reaction system and procedure are shown in Table 1 and Table 2:
[0032] Table 1 PCR reaction system
[0033]
[0034] Table 2 PCR reaction procedure
[0035]
[0036] The plant expression vector pCAMBIA3301 plasmid was extracted and double-digested. The digested product was connected to the successfully amplified target fragment using homologous recombination, and transformed into E. coli DH5α competent cells. Single clones were picked and identified by bacterial liquid PCR and sequenced (see Figure 2 ), the construction of the recombinant plasmid pCAMBIA3301-GmABIL2 was completed. The recombinant plasmid was further transformed into Agrobacterium EHA105 competent cells, and the reaction system and procedure for PCR identification of Agrobacterium liquid were the same as above, and the Agrobacterium liquid with correct PCR was retained.
[0037] Example 2 Soybean non-receptor tyrosine kinase binding protein gene GmABIL2 Expression analysis
[0038] Design of soybean non-receptor tyrosine kinase binding protein gene by fluorescence quantitative PCR GmABIL2 The specific quantitative primers were selected, and the soybean housekeeping gene EF1A was selected as the internal reference gene and primers were designed. Williams 82 was used as the experimental material. When it grew to three compound leaves, it was subjected to salt-alkali stress. At the same time, the material that was not subjected to stress was set as the control group. Plant leaves and root tissues were selected at 0h, 1h, 3h, 6h, 9h, 12h, 24h, 36h, 48h, and 72h after stress, and RNA was extracted for fluorescence quantitative analysis to clarify the effect of salt-alkali stress on soybean. GmABIL2 expression of.
[0039] The results showed that in leaves and roots after stress GmABIL2 The genes were up-regulated (see Figure 3 ), indicating soybean GmABIL2 Response to saline-alkali stress in soybean.
[0040] Example 3 Soybean non-receptor tyrosine kinase binding protein gene GmABIL2 Subcellular localization analysis
[0041] To further clarify soybean GmABIL2 The green fluorescent protein fusion expression vector pCAMBIA2300-GmABIL2 was constructed by using Agrobacterium GV3101 (pSoup-p19) with good transient transformation performance, and then transformed into Nicotiana benthamiana for transient expression. The fluorescence signal was detected under a laser confocal microscope, and the transformed mesophyll cells were observed using a laser confocal microscope with an excitation wavelength of 488 nm and an emission wavelength of 507 nm. The results showed that GmABIL2 Localized in mitochondria (see Figure 4 ).
[0042] Example 4 Overexpression under saline-alkali stress conditions GmABIL2 Identification of germination phenotypes of wild-type Arabidopsis
[0043] (1) Construction of pCAMBIA3301-GmABIL2 expression vector
[0044] The pCAMBIA3301-GmABIL2 plasmid and the pCAMBIA3301 plasmid were double-digested with Spe I and NCO I, respectively, and the target gene and the large vector fragment were recovered, respectively. The recombinant expression vector pCAMBIA3301-GmABIL2 was obtained by homologous recombination. The empty vectors pCAMBIA3301 and pCAMBIA3301-GmABIL2 were transformed into EHA105 and infected with Arabidopsis thaliana, respectively, so as to be used for identification of soybean GmABIL2 Function of salt-alkaline tolerance in Arabidopsis thaliana.
[0045] (2) Overexpression under saline-alkali stress GmABIL2 Phenotypic identification of Arabidopsis thaliana during germination
[0046] In this study, overexpression GmABIL2 and wild-type Arabidopsis seeds were planted on 1 / 2MS basal medium (PhytoTech, M519 Murashige&Skoog Basal Medium with Vitamins) and saline-alkali medium (1 / 2MS+148mM NaHCO3+2mM NaCl, 1 / 2MS+146mM NaHCO3+4mM NaCl), and their germination rates were observed and counted.
[0047] The results show that GmABIL2 The germination rate of wild-type Arabidopsis seeds on 1 / 2MS basal medium remained the same, but in saline-alkali stress medium, the overexpression GmABIL2 The germination rate of Arabidopsis thaliana strains with the gene was significantly weaker than that of wild-type Arabidopsis thaliana, which preliminarily proved that GmABIL2 It has the function of reducing the salt-alkali tolerance of Arabidopsis during the germination period (see Figure 5 , Figure 6 ).
[0048] Example 5 Overexpression GmABIL2 Phenotypic identification of soybean root growth under saline-alkali stress
[0049] (1) Construction of plant expression vector
[0050] The pUBGFP-GmABIL2 plasmid and the pUBGFP plasmid were double-digested with Xba I and Kpn I, respectively, the target gene and the large vector fragment were recovered, and the recombinant expression vector pUBGFP-GmABIL2 was obtained by homologous recombination. The empty vectors, i.e., pUBGFP and pCAMBIA-GmABIL2, were transformed into Agrobacterium rhizogenes K599, respectively, for soybean hairy root induction and transformation.
[0051] (2) Soybean GmABIL2 Identification of genes involved in salt-alkali tolerance in soybean hairy roots
[0052] 1) Seed germination: Select Williams 82 varieties of uniform size, full growth, and free of pests and diseases, sow them evenly in vermiculite to a depth of 2 cm, and cultivate them in a climate chamber for about 4-6 days until the soybean seeds germinate;
[0053] 2) Infection: Use a sterile scalpel to cut the soybean embryonic stem obliquely (keep all soybean seedlings of the same length), dip the wound in bacterial paste (apply the plate one and a half days in advance), make sure to cover the entire surface of the cut, wrap the seedlings with filter paper soaked in 1 / 2 Hoagland solution (1 / 2 Hoagland), with the cotyledons spread out outside the paper, and place the wrapped seedlings in a bouquet shape into the bacterial solution resuspended in 1 / 2 Hoagland solution (just scrape a little of the bacterial paste);
[0054] 3) Co-cultivation: Place the above step into a black tray filled with water, keep it away from light for two days, expose it to light for two days, and cultivate it for four days. (The optimum temperature is 28°, and the humidity is kept between 60-80. This stage is the most important);
[0055] 4) Transfer to hydroponics: When the wound forms a callus-like structure, that is, the wound swells, it can be moved to a hydroponic box filled with 1 / 2 Hoagland solution to induce hairy roots;
[0056] 5) Remove non-positive roots: After 2-4 days of hydroponic growth, remove the first roots that grow. (The first roots that grow are not positive roots, and any roots that grow from higher parts of the plant are not genetically modified);
[0057] 6) Continue hydroponics: In the later stage, the roots grow fast and absorb more water, so check every other day to see if there is water shortage, add 1 / 2 Hoagland solution in time, and change the Hoagland solution every two days to avoid moss. This stage grows for 6-8 days;
[0058] 7) Soil culture: In the dark, use a handheld excitation light source (model: 3415GR, serial number: DJ2738) to illuminate the hairy roots with a green fluorescent excitation light source, select soybean seedlings with high transformation efficiency, consistent root quantity and seedling growth, and move them into 10*10 black pots filled with vermiculite. (Put 9 black pots in each black tray, pour 3L of 1 / 2 Hoagland solution, and when the black pots are fully watered, pour out the excess water in the black tray in time to prevent the roots from growing out). After 3 days of culture, stress treatment was started. The stress treatment conditions were 100mM NaCl+95mM NaHCO3+5mM Na2CO3, and 1 / 2Hoagland was used as the control. The growth of soybeans under different stress conditions was observed and recorded.
[0059] The results showed that the root growth of soybean transformed with empty vector was significantly better than that of soybean transformed with overexpression vector. GmABIL2 Genetic soybean root material, soybean GmABIL2 The gene has the function of reducing the salt-alkali tolerance of soybean seedlings (see Figure 7 ).
[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. Non-receptor tyrosine kinase binding protein gene GmABIL2 The application of the method in regulating the salt-alkali stress tolerance of plants is characterized in that: Said GmABIL2 The nucleotide sequence is shown in SEQ ID NO: 1; The plant is one of soybean or Arabidopsis thaliana; The regulation is through overexpression of the non-receptor tyrosine kinase binding protein gene GmABIL2 Reduce the plant's tolerance to salt-alkali stress.
2. The use according to claim 1, characterized in that: Said GmABIL2 The amino acid sequence is shown in SEQ ID NO:
2.
3. The use according to claim 1, characterized in that: The condition of saline-alkali stress was one of 148mM NaHCO3+2mM NaCl or 146mM NaHCO3+4mM NaCl or 100mM NaCl+95mM NaHCO3+5mM Na2CO3.
4. A method for cultivating soybeans that are intolerant to salt-alkali stress, characterized in that: The following steps are involved: (1) The non-receptor tyrosine kinase binding protein gene of claim 1 GmABIL2 Connecting to a vector to obtain an expression vector; (2) The expression vector obtained in step (1) is transferred into Agrobacterium by electrophoresis to obtain recombinant bacteria; (3) The recombinant bacteria obtained in step (2) are used to infect soybeans using a high-efficiency transient infection technique to obtain soybeans that are intolerant to salt-alkali stress.
5. The method according to claim 4, characterized in that The vector in step (1) is pCAMBIA3301 vector.
6. The method according to claim 4, characterized in that The Agrobacterium in step (2) includes Agrobacterium EHA105 or Agrobacterium rhizogenes K599.