A yeast gene ScFIT3 for regulating biological cadmium resistance and its application
By constructing and transforming the expression vector of the yeast gene ScFIT3, the problem of cadmium contamination in plants is solved, the host organisms are enhanced, the cadmium accumulation and absorption are reduced, and the safer cadmium treatment is achieved.
Patent Information
- Application Number
- CN202211483455.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-11-24
- Publication Date
- 2025-08-26
- Estimated Expiration
- 2042-11-24
AI Technical Summary
In the prior art, the problem of cadmium pollution in plants has not been effectively solved, the potential anti-cadmium capacity of the yeast gene has not been fully utilized, and the existing cadmium transporters only isolated cadmium ions, which poses a risk of food chain pollution.
The yeast gene ScFIT3 is used to construct an expression vector and transform it into a host organism to enhance the anti-cadmium ability of the organism. The specific method includes extracting RNA from budding yeast, performing reverse transcription PCR amplification, ligation to the pRS416 plasmid, constructing an expression vector and transforming it into a host organism.
It significantly enhances the resistance to cadmium stress of host organisms, reduces cadmium accumulation and absorption, and improves the resistance to heavy metal cadmium in organisms.
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Abstract
Description
Technical Field
[0001] The invention relates to a yeast gene ScFIT3 for regulating biological cadmium resistance and application thereof, belonging to the field of plant genetic engineering. Background Art
[0002] In recent years, due to the irrational application of agricultural chemicals such as pesticides and fertilizers, the accumulation of heavy metal pollutants in the soil of cultivated land and agricultural facilities has steadily increased, posing a serious threat to people's lives and health. Heavy metal pollution is insidious, long-lasting, and irreversible, and compared to pesticide residues, its severity has not yet received significant attention. Cadmium, classified as a Class I carcinogen by the World Health Organization, is the primary element of heavy metal pollution in soil. The national rate of exceedance at designated sites reached 7.0%, far exceeding that of other measured inorganic pollutants (National Soil Pollution Survey Bulletin, 2014).
[0003] At present, the use of molecular biological methods to improve plants is an effective means to deal with plant cadmium pollution. In the past decade, botanists and crop scientists have screened many cadmium transporters in different plants, such as the ABC protein family, the HMA protein family, the NRAMP protein family, etc., and applied them to the process of plant and crop improvement. However, most of these proteins transport cadmium ions to the plant's vacuole or a certain part of the plant, and only isolate the cadmium ions, which poses a risk of cadmium pollution to the food chain. Yeast is the simplest eukaryote and the common ancestor of other eukaryotes. It contains a large number of genes with unknown functions waiting to be explored. Further research is urgently needed to discover new genes that affect plant cadmium stress and cadmium accumulation. Summary of the Invention
[0004] In view of the deficiencies of the prior art, the present invention provides a yeast gene ScFIT3 for regulating the cadmium resistance of organisms and its application.
[0005] The technical solutions of the present invention are as follows:
[0006] A yeast gene ScFIT3 for regulating the cadmium resistance of organisms and an application thereof. The nucleotide sequence of the yeast gene ScFIT3 for regulating the cadmium resistance of organisms is shown in SEQ ID NO.1.
[0007] The invention discloses an expression vector containing the yeast gene ScFIT3 for regulating the cadmium resistance of plants.
[0008] According to the present invention, the method for constructing the expression vector preferably comprises the following steps:
[0009] (1) RNA is extracted from budding yeast and then reverse transcribed to obtain cDNA;
[0010] (2) PCR amplification was performed using cDNA as a template to obtain the yeast gene ScFIT3 sequence. The PCR primer sequences are as follows:
[0011] Forward primer: 5'-aatctagaactagtgATGAAATTCTCTTCCGCTTT-3',
[0012] Reverse primer:
[0013] 5'-gcagcccgggggatcTTCCTCCTCCTCCTCCTCCTTACAATAA-CATGACGGCAG-3';
[0014] (3) The yeast gene ScFIT3 was connected to the pRS416 plasmid to obtain an expression vector containing the yeast gene ScFIT3.
[0015] Application of the yeast gene ScFIT3 in enhancing biological cadmium resistance.
[0016] Preferably, according to the present invention, the application approach comprises: transforming an expression vector containing the yeast gene ScFIT3 into a host organism.
[0017] According to the present invention, preferably, the host organism includes plants and microorganisms.
[0018] More preferably, the microorganism is yeast.
[0019] Beneficial effects:
[0020] This paper discloses for the first time the use of the yeast gene ScFIT3 in enhancing biological cadmium resistance. The yeast gene ScFIT3 is linked to an expression vector and transformed into budding yeast to construct cadmium-resistant yeast. The growth of budding yeast transformed with the ScFIT3 gene is significantly better than that of wild-type budding yeast, indicating that the budding yeast transformed with the ScFIT3 gene has excellent resistance to cadmium stress, significantly enhanced resistance to heavy metal cadmium, and reduced cadmium accumulation and absorption. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 The growth results of transgenic ScFIT3 yeast and JRY472 yeast on SC solid screening medium containing CdCl2 (50 μM) and normal SC solid screening medium;
[0022] In the figure, the triangle symbol indicates a 10-fold decrease in concentration (the starting concentration is 0.3 OD600).
[0023] Figure 2The Cd content in yeast JRY472 transformed with the yeast gene ScFIT3 after 14 hours of growth under the same cadmium treatment conditions. The error bars represent the results of three independent replicates. ± SD statistics. T-test P value: Comparison of cadmium content between transgenic yeast and wild-type yeast, **P < 0.01. DETAILED DESCRIPTION
[0024] The present invention will be further described below in conjunction with the examples. However, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that, without departing from the spirit and scope of the present invention, various changes and modifications may be made to the present invention. The present invention provides general and / or specific descriptions of the materials and test methods used in the test. Although many materials and operating methods used to achieve the purpose of the present invention are well known in the art, the present invention is still described in as much detail as possible here.
[0025] The budding yeast JRY472 described in the examples has been disclosed in the document A Mitochondrial Pyruvate Carrier Required for Pyruvate Uptake in Yeast, Drosophila, and Humans, 2012.
[0026] Example 1
[0027] 1. Extract RNA from budding yeast JRY472. The specific method is as follows:
[0028] Weigh 5 g of budding yeast JRY472 cell powder and suspend it in 30 mL of 0.04 M NaOH solution. Grind it evenly in a mortar. Transfer the suspension to a conical flask, heat it in a boiling water bath for 30 minutes, cool it, transfer it to a centrifuge tube, and centrifuge it at 3000 rpm for 15 minutes. Then, slowly pour the supernatant into 10 mL of acidic ethanol with stirring. After the addition, let it stand for RNA precipitation. Centrifuge it at 3000 rpm for 3 minutes, discard the supernatant, wash the precipitate twice with 95% ethanol and once with ether, then transfer it to a Buchner funnel and filter it with ether. Air-dry the precipitate and dissolve the total RNA sample in 50 μL of DEPC-treated ddH2O. Store the prepared total RNA sample in a -80°C freezer until needed.
[0029] 2. Reverse transcription PCR
[0030] Reverse transcription PCR amplification (two-step method) was performed using the total RNA sample as a template. The specific reverse transcription PCR amplification system and conditions are as follows:
[0031] The reaction steps are as follows:
[0032] Step 1:
[0033]
[0034] Reaction conditions: incubate at 42°C for 2 min and store at 4°C.
[0035] Step 2:
[0036]
[0037] Reaction conditions: incubate at 37°C for 15 min, then at 85°C for 5 s.
[0038] The total cDNA of budding yeast JRY472 was obtained by the above method. The reagents in Step 1 and Step 2 were from PrimeScript of TAKALA. TM II 1st Strand cDNA Synthesis Kit.
[0039] Example 2
[0040] 1. Construct an expression vector containing the yeast gene ScFIT3, which regulates plant cadmium resistance. The specific method is as follows:
[0041] (1) PCR amplification was performed using yeast cDNA as a template to obtain the yeast gene ScFIT3 sequence (SEQ ID NO. 1). The primers for the PCR amplification were as follows:
[0042] Forward primer: 5'-aatctagaactagtgATGAAATTCTCTTCCGCTTT-3',
[0043] Reverse primer:
[0044] 5'-gcagcccgggggatcTTCCTCCTCCTCCTCCTCCTTACAATAA-CATGACGGCAG-3';
[0045] The PCR system is as follows:
[0046]
[0047] The PCR conditions were as follows: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s; annealing at 55-65°C for 30 s; extension at 72°C for 1 min (35 cycles); termination of extension at 72°C for 5-10 min; and finally, incubation at 4°C.
[0048] (2) The pRS416 plasmid (containing the pGPD promoter) was digested with the restriction endonuclease BamH1, and the yeast gene ScFIT3 sequence was ligated into the pRS416 plasmid using the homologous recombinase (In-Fusion) to obtain an expression vector containing the yeast gene ScFIT3;
[0049] The connection system is as follows:
[0050]
[0051] Ligation reaction conditions: 50°C, 15 min; storage at 4°C.
[0052] 2. Preparation of transgenic yeast
[0053] The budding yeast JRY472 stock solution stored at -80°C was streaked on YPDA solid medium and inverted at 30°C for 4 days; a single colony of budding yeast JRY472 was picked and transferred to 5 mL of YPDA liquid medium and cultured on a shaker at 30°C and 250 rpm for 10 h; 3 mL of budding yeast JRY472 bacterial solution was taken to 50 mL of YPDA liquid medium for expansion culture and cultured on a shaker at 30°C and 250 rpm until OD600 ≤ 0.5; the budding yeast JRY472 bacterial solution was then centrifuged at 3000 rpm and 25°C for 8 min, the supernatant was removed, and the solution was resuspended with sterile water; the solution was centrifuged at 3000 rpm and 25°C for 3 min, and the solution was resuspended with sterile water; the solution was centrifuged at 6000 rpm and 25°C for 2 min, the supernatant was removed, and the solution was resuspended with TE-LiAC buffer; the solution was centrifuged at 6000 rpm and 25°C for 2 min, remove the supernatant, and resuspend with TE-LiAC solution; draw 100 μL of the resuspension, mix with 2 μL of vector DNA (heated at 100° C. for 5 min, placed on ice, repeated twice) and 10 μL of an expression vector containing the yeast gene ScFIT3, mix well, and place at 25° C. for 10 min; add 260 μL of a 40% PEG / TE-LiAC solution, mix well, water bath at 30° C. for 1 h, add 43 μL of DMSO preheated at 37° C., mix well, heat shock at 42° C. for 5 min, centrifuge the heat-shocked mixture at 12000 rpm for 30 s, and resuspend with sterile water; centrifuge the resuspended solution at 12000 rpm for 30 s, resuspend with sterile water, take 100 μL of the resuspension and apply it to SC solid screening medium, and culture it upside down at 30° C. for 6 days to obtain budding yeast JRY472 transformed with the yeast gene ScFIT3.
[0054] Example 3
[0055] 1. A single colony of the ScFIT3-transgenic yeast JRY472 (WT) from Example 2 was placed in 5 mL of SC liquid screening medium, dispersed and mixed, and cultured on a shaker at 250 rpm and 30°C for 10 h until OD600 > 0.3 to obtain a ScFIT3-transgenic yeast JRY472 bacterial solution. The ScFIT3-transgenic yeast JRY472 bacterial solution was diluted to OD600 = 0.1 and cultured on a shaker at 250 rpm and 30°C for 6 h. The ScFIT3-transgenic yeast JRY472 bacterial solution was then diluted to OD600 = 0.3. This ScFIT3-transgenic yeast JRY472 bacterial solution was used as a stock solution. The stock solution was diluted to a concentration gradient of 1 / 10, 1 / 100, and 1 / 1000, respectively. The wild-type budding yeast JRY472 culture liquid was cultured to OD600=0.3 and diluted according to the same ratio.
[0056] Take 3 μL of the mother solution and the gradient dilution of the mother solution, and use the wild-type budding yeast JRY472 bacterial solution and the gradient dilution of the wild-type budding yeast JRY472 bacterial solution as controls, drop them onto the SC solid screening medium containing CdCl2 (75 μM) and the normal SC solid screening medium, respectively, and culture them upside down at 30°C for 4 days. The results are as follows Figure 1 shown.
[0057] Depend on Figure 1 It can be seen that the growth of budding yeast with the yeast gene ScFIT3 is significantly better than that of wild-type budding yeast, indicating that the budding yeast with the yeast gene ScFIT3 has good resistance to cadmium stress, significantly enhanced the ability to resist heavy metal cadmium, and reduced cadmium accumulation and cadmium absorption.
[0058] 2. The budding yeast JRY472 transgenic with the yeast gene ScFIT3 and the wild-type budding yeast JRY472 in Example 2 were inoculated into YPDA liquid culture medium, shaken at 250 rpm and 30°C until OD600 = 0.3, and then CdCl2 was added to a final concentration of 50 μM (EV group and FIT3 group, respectively). Samples were taken 14 hours after the addition of CdCl2, and the Cd in the transgenic yeast JRY472 transgenic with the yeast gene ScFIT3 and the wild-type budding yeast JRY472 cells was measured. 2+ Content, the results are as follows Figure 2 shown.
[0059] Depend on Figure 2It can be seen that the cadmium content in the wild-type budding yeast JRY472 is more than three times that of the budding yeast JRY472 with the transformed yeast gene ScFIT3, indicating that the budding yeast with the transformed yeast gene ScFIT3 has good resistance to cadmium stress, significantly enhanced the ability to resist heavy metal cadmium, and reduced cadmium accumulation and cadmium absorption.
Claims
1. Application of gene ScFIT3 in enhancing cadmium resistance in yeast, characterized in that: The nucleotide sequence of the gene ScFIT3 is shown in SEQ ID NO.
1.
2. The use according to claim 1, characterized in that The application approach includes: transforming the expression vector containing the gene ScFIT3 into yeast.
3. The use according to claim 2, characterized in that The method for constructing the expression vector containing the gene ScFIT3 comprises the following steps: (1) Extract RNA from budding yeast and then reverse transcribe the RNA to obtain cDNA; (2) PCR amplification was performed using cDNA as a template to obtain the gene ScFIT3 sequence. The PCR primer sequences are as follows: Forward primer: 5'-aatctagaactagtgATGAAATTCTCTTCCGCTTT-3', Reverse primer: 5'-gcagcccgggggatcTTCCTCCTCCTCCTCCTCCTTACAATAA-CATGACGGCAG-3'; (3) The ScFIT3 gene was connected to the pRS416 plasmid to obtain an expression vector containing the ScFIT3 gene.