A soybean cytochrome b5 gene and its application
By overexpressing the soy cytochrome b5 gene GmCytb5-2, the problem of soybean production decreased under saline-alkali stress conditions was solved, significantly improving the saline-alkali tolerance of soybeans and promoting its growth and germination.
Patent Information
- Application Number
- CN202411898683.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-23
- Publication Date
- 2025-03-18
- Estimated Expiration
- 2044-12-23
AI Technical Summary
Soybean production can be seriously reduced under saline and alkali stress conditions, affecting food security. The existing technology has not yet effectively solved the problem of improving soybean saline and alkali resistance.
By overexpressing the soy cytochrome b5 gene GmCytb5-2, the salt-tolerant function of soybeans is regulated and the growth and germination ability of soybeans under saline-alkali stress is improved.
It significantly improves the saline-alkali tolerance of soybeans, promotes the growth of soybeans in the seedling and germination stages, and enhances the resistance of soybeans to saline-alkali stress.
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Figure CN119331886B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of genetic engineering, and in particular to a soybean cytochrome b5 gene and application thereof. Background Art
[0002] Soil salinization is one of the important factors affecting 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 , accounting for 1 / 9 of the world's saline soil area, and less than 27 million hm2 from China's arable land bottom line. 2 . Saline-alkali soil contains high concentrations of Na + 、CO3 2- and HCO3 - Plasma has a direct toxic effect on the roots of plants, making it difficult for root cells to absorb water from the soil, causing a serious decline in crop yield and quality, and even leading to plant death. This has greatly reduced the yield of crops in my country and has had a huge impact on agricultural production.
[0003] Soybean is an important crop for food, oil and feed in my country, and plays an important role in the national economy. More than 80% of China's soybeans need to be imported, and the import has exceeded 100 million tons for five consecutive years, while my country's own production is only about 10 million tons, which has greatly affected my country's food security. Compared with major crops such as wheat and rice, soybeans are moderately salt- and alkali-sensitive crops; under salt- and alkali stress conditions, soybean yields can be severely reduced by 50%, which has a serious impact on my country's soybean production. Therefore, how to use the resources of resistance genes to improve the salt- and alkali tolerance of soybeans, increase the yield of soybeans in saline-alkali land, increase the soybean self-sufficiency rate, and better ensure national food security is of great significance to the efficient and sustainable development of the soybean industry.
[0004] Cytochrome B5 is a membrane-bound protein with an amino acid content of about 130-150. It is a type of heme protein that exists in plants, animals and fungi and is located in the endoplasmic reticulum. Plant cytochrome b5 is a hemoglobin anchored in the endoplasmic reticulum or chloroplast envelope that protrudes into the cytoplasm through the C-terminal hydrophobic domain and their hydrophilic N-terminal domain. It participates in various redox reactions in cells as an intermediate electron transporter, thereby regulating the balance of reactive oxygen species (ROS) in plants, reducing the damage to cells caused by adversity, and participating in the response of plants to various biological and abiotic adversities; however, there have been no reports on the study of this gene in soybean salt-alkali tolerance. Summary of the invention
[0005] In view of the above-mentioned defects of the prior art, the present invention proposes a soybean cytochrome b5 gene and its application to solve the problems raised by the above-mentioned background technology.
[0006] In order to achieve the above object, the present invention provides the following technical solutions:
[0007] Cytochrome b5 gene GmCytb5-2 Cytochrome b5 gene GmCytb5-2 Application of the encoded protein in regulating soybean salt-alkali stress tolerance, the cytochrome b5 gene GmCytb5-2 The nucleotide sequence is shown in SEQ ID NO:1.
[0008] Preferably, the amino acid sequence of the protein is as shown in SEQ ID NO:2.
[0009] Preferably, by overexpressing the cytochrome b5 gene GmCytb5-2 Cytochrome b5 gene GmCytb5-2 The encoded protein improves soybean salt-alkali tolerance.
[0010] Preferably, GmCytb5-2 Under saline-alkali stress conditions, it can promote soybean growth in the seedling stage and soybean seed germination in the germination stage.
[0011] Preferably, GmCytb5-2 The encoded protein is localized in the endoplasmic reticulum.
[0012] Preferably, the condition of saline-alkali stress at the seedling stage is 120 mM NaCl + 30 mM NaHCO3.
[0013] Preferably, the saline-alkali stress condition during the germination period is 100 mM NaCl + 97.5 mM NaHCO3 + 1.25 mM Na2CO3.
[0014] Preferably, a method for cultivating soybeans tolerant to salt-alkali stress comprises the following steps:
[0015] (1) Gene GmCytb5-2 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 resistant to salt-alkali stress.
[0018] Preferably, the vector in step (1) comprises a pYES2 vector or a pCAMBIA vector.
[0019] Preferably, the Agrobacterium in step (2) comprises one of Agrobacterium EHA105 or Agrobacterium rhizogenes K599.
[0020] Compared with the prior art, the present invention has the following beneficial effects:
[0021] The present invention uses 303 cultivated soybeans as experimental materials to accurately identify the salt-alkali tolerance phenotype at the seedling stage, and evaluates its salt-alkali tolerance level based on the root-crown ratio and the degree of leaf damage. At the same time, genome-wide association analysis is used to mine and identify the candidate gene for soybean salt-alkali tolerance, the cytochrome b5 gene. GmCytb5-2 , further using yeast, soybean hairy root transient transformation system and stable transformation overexpression GmCytb5 Soybean material, it is determined that the gene has the function of improving the salt-alkali tolerance of soybean during the germination and seedling stages. GmCytb5-2 The role of the gene in the process of soybean tolerance to salt-alkali stress was studied. It was found that the protein was strongly induced by salt-alkali stress and had strong salt-alkali tolerance. On the other hand, it provided candidate genes and new ideas for cultivating new salt-alkali tolerant soybean germplasm and related breeding work. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 Soybean cytochrome b5 gene GmCytb5-2 PCR detection, where M is DL2000; - is the negative control; 1 is the target fragment of GmCytb5-2;
[0023] Figure 2 Soybean cytochrome b5 gene GmCytb5-2 Construction of yeast expression vector; M is DL2000; - is negative control; 1-5 is PCR detection of pYES2-GmCytb5-2 bacterial solution; 6-10 is PCR detection of pCAMBIA3301-GmCytb5-2 bacterial solution;
[0024] Figure 3 Expression of soybean cytochrome b5 gene after saline-alkali stress GmCytb5-2 Expression analysis of ; the left picture is leaves, and the right picture is roots;
[0025] Figure 4 Soybean GmCytb5-2 Subcellular localization analysis;
[0026] Figure 5 For overexpression GmCytb5-2 Functional analysis of yeast under salinity and alkali stress;
[0027] Figure 6 Verification for transient systems GmCytb5-2 It has the function of improving the salt-alkali tolerance of soybean at the seedling stage;
[0028] Figure 7 For overexpression GmCytb5-2 Identification of salt-alkali tolerance function of soybean during germination period. 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 GmCytb5-2 Gene cloning and construction of plant expression vector
[0031] William Mus 82 was selected as the gene cloning material. When the plant grew the first set of three compound leaves, samples were taken, and the total RNA of the leaves was extracted and reverse transcribed into the first chain of cDNA. GmCytb5-2 Gene 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] Extract the plasmid of plant expression vector pYES2 or pCAMBIA3301 and perform double restriction digestion. Connect the digested product with the successfully amplified target fragment using homologous recombination, transform E. coli DH5α competent cells, pick single clones and perform bacterial liquid PCR identification and sequencing verification (see Figure 2 ), complete the construction of the recombinant plasmid pYES2-GmCytb5-2 or pCAMBIA3301-GmCytb5-2, further transform the recombinant plasmid into Agrobacterium EHA105 competent cells, and the PCR identification reaction system and procedure of Agrobacterium liquid are the same as above, and the Agrobacterium liquid with correct PCR is retained.
[0037] Example 2 Soybean cytochrome b5 gene GmCytb5-2 Expression analysis
[0038] Design of soybean cytochrome b5 gene by fluorescence quantitative PCR GmCytb5-2Specific quantitative primers were developed, and the soybean housekeeping gene Actin was selected as the internal reference gene to design primers. 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 expression of soybean cytochrome b5 gene after salt-alkali stress. GmCytb5-2 expression of.
[0039] The results showed that in leaves and roots after stress GmCytb5-2 The genes were up-regulated (see Figure 3 ), indicating that soybean cytochrome b5 gene GmCytb5-2 Response to saline-alkali stress in soybean.
[0040] Example 3 Soybean cytochrome b5 gene GmCytb5-2 Subcellular localization analysis
[0041] To further clarify the soybean cytochrome b5 gene GmCytb5-2 The green fluorescent protein fusion expression vector pGDG-GmCytb5-2 was constructed and transformed into Nicotiana benthamiana for transient expression using Agrobacterium GV3101 with good transient transformation performance. The fluorescence signal was detected under a laser confocal microscope. 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 GmCytb5-2 The encoded protein is localized in the endoplasmic reticulum (see Figure 4 ).
[0042] Example 4 Soybean cytochrome b5 gene GmCytb5-2 Identification of salt-alkaline tolerance phenotype in yeast
[0043] (1) Construction of yeast vector
[0044] The pEASY-GmCytb5-2 plasmid and the pYES2 plasmid were double-digested with Sac I and BamH I, respectively, and the target gene and the large vector fragment were recovered, respectively, and connected under the action of T4 DNA ligase to obtain the expression vector pYES2-GmCytb5-2; the empty vector pYES2 and pYES2-GmCytb5-2 were transformed into Saccharomyces cerevisiae INVSc1, respectively, for gene function identification;
[0045] (2) Soybean cytochrome b5 gene GmCytb5-2 Identification of salt tolerance and salt-alkali tolerance in yeast
[0046] The pYES2-GmCytb5-2 vector and its empty vector pYES2 were transferred into the yeast strain (INVSc1), and then cultured in the basal medium, salt stress medium and saline-alkali stress medium to observe the growth of the yeast strain. GmCytb5-2 The yeast expressing the gene was cultured in basal medium (SD-Ura), salt stress medium (700 mM NaCl) and saline-alkali stress medium (695 mM NaCl and 5 mM Na2CO3), and the OD values were measured at 0 h, 24 h, 36 h, 48 h, 60 h and 72 h, respectively. 600 The absorbance value was measured and the growth curve was drawn.
[0047] The results showed that in the basal medium (see Figure 5 ), the growth potential of yeast transformed with pYES2 empty vector and pYES2-GmCytb5-2 vector was basically the same; however, under saline-alkali stress conditions, the OD value of yeast transformed with pYES2-GmCytb5-2 vector was significantly higher than that of yeast transformed with pYES2, confirming that soybean GmCytb5-2 The gene has the biological function of enhancing the salt-alkali tolerance of yeast cells.
[0048] Example 5 Overexpression GmCytb5-2 Phenotypic identification under saline-alkali stress
[0049] (1) Construction of plant expression vector
[0050] The pCAMBIA-GmCytb5-2 plasmid and the pCAMBIA-Flag plasmid were double-digested with Nco I and Spe I, respectively, the target gene and the large vector fragment were recovered, and the plant expression vector pCAMBIA-GmCytb5-2 was constructed by homologous recombination; the empty vectors, i.e., pCAMBIA-Flag and pCAMBIA-GmCytb5-2, were transformed into Agrobacterium rhizogenes K599, respectively, for soybean hairy root induction and transformation.
[0051] (2) Overexpression of soybean cytochrome b5 gene GmCytb5-2 Phenotypic identification of salt-alkali tolerance at the seedling stage
[0052] Soybean seeds were sterilized overnight with chlorine gas using a mixture of sodium hypochlorite and concentrated hydrochloric acid in a ratio of 1:1. The soybeans were then washed with sterile water and planted in vermiculite sterilized at high temperature and high pressure, and kept moist with 1 / 4 Hoagland solution. The seeds were cultured in an artificial climate chamber at 25°C and 16 hours of light for 6 days. Agrobacterium rhizogenes K599 was cultured in a 28°C incubator overnight. The bacteria were collected by scraping with a blade. Agrobacterium rhizogenes K599 was injected into the hypocotyl near the cotyledon. Water was maintained for 5 to 6 days after the injection. The wound was covered with sterilized vermiculite and kept moist. The true roots were removed after 15 days of culture, and the seedlings were moved to Hoagland culture solution after 3 days of culture. After 3 days of culture, stress treatment was started. The stress treatment conditions were 120 mM NaCl + 30 mM NaHCO3. The untreated plant was used as the control group. The growth status of each plant under different stress conditions was observed and recorded (see Figure 6 ).
[0053] The results showed that after salt-alkali stress, the soybean cytochrome b5 gene was transferred into GmCytb5-2 The growth of soybean materials in bacterial solution was significantly better than that of soybean materials transferred with empty vector, indicating that soybean cytochrome b5 gene has the function of improving soybean salt-alkali tolerance.
[0054] (3) Overexpression of soybean cytochrome b5 gene GmCytb5-2 Identification of salt-alkaline tolerance phenotype during germination
[0055] Soybean seeds were sterilized overnight with chlorine gas using a mixture of 96 mL of sodium hypochlorite and 4 mL of concentrated hydrochloric acid, and then washed with sterile water. Then, the treated soybean seeds were placed in an incubator at 25 ° C, and the stress treatment experiment was carried out under light-proof conditions. Among them, the experimental group set up 3 replicates, each containing 15 seeds, and a mixture of 100 mM NaCl + 97.5 mM NaHCO3 + 1.25 mM Na2CO3 was used as the stress solution. 10 mL of stress solution was injected into each dish for saline-alkali stress treatment; the control group also set up 3 replicates and used clean water treatment (CK). During the experiment, the stress solution and water were replaced every 2 days, and the germination rate and hypocotyl length of seeds under different stress conditions were observed and recorded every day.
[0056] The experimental results showed that after saline-alkali stress, the germination rate of seeds overexpressing soybean cytochrome b5 gene (OE-B5) was significantly higher than that of wild-type soybean seeds (WT), and the hypocotyl length of seeds overexpressing soybean cytochrome b5 gene was longer under saline-alkali stress (see Figure 7 ). This shows that soybean cytochrome b5 gene has the function of improving soybean salt-alkali tolerance.
[0057] 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. Cytochrome b5 gene GmCytb5-2 Cytochrome b5 gene GmCytb5-2 The use of the encoded protein in regulating the salt-alkali stress tolerance of soybean is characterized in that: The cytochrome b5 gene GmCytb5-2 The nucleotide sequence of the protein is shown in SEQ ID NO: 1, and the amino acid sequence of the protein is shown in SEQ ID NO: 2; By overexpressing cytochrome b5 gene GmCytb5-2 Cytochrome b5 gene GmCytb5-2 The encoded protein improves soybean's tolerance to salt-alkali stress; GmCytb5-2 Under saline-alkali stress conditions, it can promote soybean growth in the seedling stage and soybean seed germination in the germination stage; The conditions of saline-alkali stress in the seedling stage were 120mM NaCl+30mM NaHCO3, and those in the germination stage were 100mMNaCl+97.5mM NaHCO3+1.25mM Na2CO3.
2. The use according to claim 1, characterized in that: GmCytb5-2 The encoded protein is localized in the endoplasmic reticulum.
3. A method for cultivating soybeans tolerant to salt and alkali stress, characterized in that: The following steps are involved: (1) The gene according to claim 1 GmCytb5-2 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 resistant to salt-alkali stress.
4. The method according to claim 3, characterized in that The vector in step (1) comprises a pYES2 vector or a pCAMBIA vector.
5. The method according to claim 3, characterized in that: The Agrobacterium in step (2) includes one of Agrobacterium EHA105 and Agrobacterium rhizogenes K599.