Populus davidiana PdPapERF098 transcription factor and application thereof in improving cadmium sensitivity of yeast

Abstract

The application discloses a Populus davidiana PdPapERF098 transcription factor and application thereof to improving cadmium sensitivity of yeast, and belongs to the technical field of genetic engineering. The application screens a cadmium stress-induced transcription factor PdPapERF098 from Populus davidiana, the amino acid sequence of which is shown as SEQ ID NO. 2, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1. It is found through experiments that overexpression of PdPapERF098 in a cadmium stress-sensitive Saccharomyces cerevisiae strain can reduce the cadmium concentration in the strain, and significantly improve the cadmium stress sensitivity of the Saccharomyces cerevisiae. The application provides a new genetic material for genetic engineering improvement of the cadmium sensitivity of an engineering strain, and provides technical support for cultivation of a new functional strain.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of a *Populus alba* PdPapERF098 transcription factor and its encoding gene in improving the cadmium sensitivity of yeast. Background Technology

[0002] Cadmium (Cd) is a highly toxic heavy metal environmental pollutant. Cadmium pollution in soil and water can disrupt the physiological metabolism of plants and animals, accumulate along the food chain, and induce kidney and bone diseases in humans, posing serious ecological and health risks. Current environmental cadmium detection methods have two major shortcomings: instrumental detection (atomic absorption, ICP mass spectrometry) equipment is expensive and has a long detection cycle, making on-site batch screening impossible; existing biosensor yeast strains have low cadmium response sensitivity, showing only slight differences in growth under high cadmium stress, resulting in high detection limits. Therefore, identifying novel plant genes that can significantly amplify the toxic effects of cadmium and make eukaryotic cells sensitive to cadmium, and developing highly sensitive biosensor strains, has significant application value. ERF (ethylene response factor) is a family of transcription factors unique to plants. Existing publicly available research reports that heterologous transformation of plant ERF genes into yeast can mostly improve yeast's tolerance to cadmium stress; however, there are no reports of heterologous expression of ERF transcription factors significantly enhancing yeast's cadmium sensitivity for rapid heavy metal detection. Summary of the Invention

[0003] The purpose of this invention is to provide an application of the *Populus spp.* PdPapERF098 transcription factor and its encoding gene in improving the cadmium sensitivity of yeast, in order to solve the problems existing in the prior art. By overexpressing the PdPapERF098 gene, the sensitivity of *Saccharomyces cerevisiae* to cadmium stress is significantly improved. This provides new genetic material for the genetic engineering improvement of engineered bacteria to cadmium sensitivity and provides technical support for the cultivation of new functional strains.

[0004] To achieve the above objectives, the present invention provides the following solution: This invention provides the application of the Populus alba PdPapERF098 transcription factor in any of the following: (1) Improve the cadmium sensitivity of brewer's yeast; (2) Cultivate highly cadmium-sensitive Saccharomyces cerevisiae strains; (3) Prepare products that improve the cadmium sensitivity of Saccharomyces cerevisiae; The cadmium sensitivity of Saccharomyces cerevisiae was improved by overexpressing the gene encoding the Populus alba PdPapERF098 transcription factor in Saccharomyces cerevisiae. The amino acid sequence of the Populus alba PdPapERF098 transcription factor is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0005] This invention also provides the application of the gene encoding the *Populus alba* PdPapERF098 transcription factor in any of the following: (1) Improve the cadmium sensitivity of brewer's yeast; (2) Cultivate highly cadmium-sensitive Saccharomyces cerevisiae strains; (3) Prepare products that improve the cadmium sensitivity of Saccharomyces cerevisiae; The cadmium sensitivity of Saccharomyces cerevisiae was improved by overexpressing the gene encoding the Populus alba PdPapERF098 transcription factor in Saccharomyces cerevisiae. The amino acid sequence of the Populus alba PdPapERF098 transcription factor is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0006] This invention also provides the use of the recombinant vector in any of the following: (1) Improve the cadmium sensitivity of brewer's yeast; (2) Cultivate highly cadmium-sensitive Saccharomyces cerevisiae strains; (3) Prepare products that improve the cadmium sensitivity of Saccharomyces cerevisiae; The recombinant vector contains the encoding gene of the *Populus spp.* PdPapERF098 transcription factor, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1. The cadmium sensitivity of Saccharomyces cerevisiae was improved by overexpressing the gene encoding the PdPapERF098 transcription factor in Saccharomyces cerevisiae.

[0007] This invention also provides the use of recombinant bacteria in any of the following: (1) Improve the cadmium sensitivity of brewer's yeast; (2) Cultivate highly cadmium-sensitive Saccharomyces cerevisiae strains; (3) Prepare products that improve the cadmium sensitivity of Saccharomyces cerevisiae; The recombinant bacteria contains the encoding gene of the *Populus salina* PdPapERF098 transcription factor, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1. The cadmium sensitivity of Saccharomyces cerevisiae was improved by overexpressing the gene encoding the PdPapERF098 transcription factor in Saccharomyces cerevisiae.

[0008] The present invention also provides a method for improving the cadmium sensitivity of Saccharomyces cerevisiae, comprising overexpressing the encoding gene of the Populus alba PdPapERF098 transcription factor in Saccharomyces cerevisiae; wherein the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0009] The present invention also provides a method for cultivating a highly cadmium-sensitive Saccharomyces cerevisiae strain, comprising overexpressing the encoding gene of the Populus alba PdPapERF098 transcription factor in Saccharomyces cerevisiae to obtain a highly cadmium-sensitive Saccharomyces cerevisiae strain; wherein the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0010] The present invention discloses the following technical effects: This invention screened a transcription factor, PdPapERF098, from *Populus alba* that is induced to express under cadmium stress. Experiments showed that heterologous expression of PdPapERF098 significantly enhanced yeast cadmium sensitivity, completely opposite to the function of existing techniques that use ERF transcription factors to enhance cadmium tolerance in yeast. The recombinant yeast heterologously expressing PdPapERF098 exhibits significantly improved cadmium response sensitivity; 80 μM cadmium is sufficient to produce visible growth differences, eliminating the need for large instruments. Detection plates can be prepared in batches for rapid on-site screening of cadmium pollution in farmland soil and livestock wastewater. This method is low-cost, easy to operate, and has clear industrial application value. Attached Figure Description

[0011] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0012] Figure 1 The image shows the full-length CDS PCR amplification electrophoresis of the PdPapERF098 gene, with the target fragment being 447 bp. Figure 2 Image of the recombinant yeast expression vector pYES2-PdPapERF098 plasmid; Figure 3 This is a comparison of the growth of Saccharomyces cerevisiae strain Δycf1 after transformation with the empty vector pYES2 and the recombinant vector pYES2-PdPapERF098 under 80 μM CdCl2 stress. Figure 4 Bar chart showing cadmium accumulation in two yeast cells after treatment with 30 μM Cd. Detailed Implementation

[0013] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0014] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0015] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0016] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0017] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0018] This invention first cloned the full-length cDNA reading frame sequence of the Populus alba ERF transcription factor gene PdPapERF098. Using young Populus alba samples collected from Nanjing Botanical Garden, total RNA was extracted and reverse transcribed into cDNA. Using the cDNA as a template, the full-length cDNA reading frame of PdPapERF098 was amplified using primer pairs PdPapERF098F (SEQ ID NO.3) and PdPapERF098R (SEQ ID NO.4), and then inserted into the Saccharomyces cerevisiae expression vector pYES2 using the InFusion method. This recombinant vector was then transformed into Saccharomyces cerevisiae, resulting in overexpression of the PdPapERF098 gene in yeast, which enhanced the yeast's sensitivity to the heavy metal cadmium.

[0019] Example 1: RT-PCR amplification of the PdPapERF098 gene cDNA sequence Young poplar plant samples were collected from Nanjing Zhongshan Botanical Garden. Total RNA was extracted from the leaves according to the instructions of the polysaccharide and polyphenol plant RNA extraction kit from Beijing Huayueyang Co., Ltd. The concentration of the extracted total RNA from the leaves was detected using a NanoDrop 1000 nucleic acid and protein analyzer.

[0020] The first strand of cDNA was obtained by reverse transcription using total RNA as a template in a two-step process. The synthesis of the cDNA single strand was performed according to the instructions of PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) Co., Ltd. by Baori Biotechnology (Beijing) Co., Ltd.

[0021] Using single-stranded cDNA from *Populus tomentosa* leaves as a template, the high-fidelity DNA polymerase Probest was employed. TM DNA Polymerase amplification of the PdPapERF098 gene. The primers used were PdPapERF098F and PdPapERF098R, with the following sequences: PdPapERF098F: 5'-ATGGAAGAGCCTGATAAAGGGA-3', SEQ ID NO.3; PdPapERF098R: 5'-TCAATCTTGCTGCCGTTTCT-3', SEQ ID NO. 4.

[0022] The PCR amplification reaction system (total volume 25 μL) consisted of: 12.5 μL 10×PCR Buffer, 0.5 μL 10 mM dNTP Mix, 1 μL 10 μM PdPapERF098F, 1 μL 10 μM PdPapERF098R, 0.5 μL DNA Polymerase, 1 μL cDNA template, and 8.5 μL ddH2O. All components were thoroughly mixed and placed in a PCR instrument for the reaction.

[0023] The reaction program was as follows: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 56℃ for 45 s, extension at 72℃ for 60 s, for 30 cycles; and final extension at 72℃ for 10 min.

[0024] PCR products were detected by 1% agarose gel electrophoresis, such as... Figure 1 As shown, a single DNA band (approximately 447 bp) was obtained, which is the PdPapERF098 gene fragment obtained by PCR amplification. The PdPapERF098 fragment was recovered by agarose gel electrophoresis according to the Magen HiPureGel Pure DNA Kits instructions.

[0025] Example 2: Construction of a yeast recombinant expression vector for the PdPapERF098 gene The Saccharomyces cerevisiae expression vector pYES2 was recovered by double digestion with KpnI and EcoRI.

[0026] The concentrations of the recovered PdPapERF098 fragment and the linearized pYES2 vector were determined using a NanoDrop UV spectrophotometer, and homologous recombination ligation of the DNA fragment and vector was performed using the HD Cloning Kit from Baori Biotechnology (Beijing) Co., Ltd.

[0027] The reaction product was transformed into competent Escherichia coli JM109 strain according to the instructions from Baori Biotechnology (Beijing) Co., Ltd. Single clones were picked, plasmids were extracted, and after sequencing identification, the plasmids were preserved for later use.

[0028] After sequencing analysis, the nucleotide sequence of the cDNA reading frame of PdPapERF098 is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2. The constructed Saccharomyces cerevisiae recombinant expression vector pYES2-PdPapERF098 is shown in... Figure 2 As shown.

[0029] SEQ ID NO.1: ATGGAAGAGCCTGATAAAGGGAAGGGAAATGAGGAGAAAGGGAGAGGAGAGGTTCGATACCGCGGTGTTCGAAGGAGACCTTGGGGAAAATTTGCGGCGGAGATACGAGACCCGTCAAGGCAAGGAGGAAGGTTATGGCTAGGGACATTTGATACGGCAGAAGAGGCAGCTCGCGCTTATGATAAAGCTGCATTTAACTTGAGACGTCATCTTGCAGTCCTCAA CTTTCTAGTGATTATATCTCTCAAGTGACGGAGTTTTCTCCTCGTCCTCGATCATACTCACCATCTTGCAGTAATGTTGGTGCAACTCAGACTTTTGAGAGGGGAAGCTCATCAAGTGCCGGACATGGAAACCAAGTTATCGAGTTTGAGTGCTTGGATGATAAGATTTTGGAGGAGCTTCTTGAAACAGAAGAGGAGAAGAAGAAACGGCAGCAAGATTGA.

[0030] SEQ ID NO.2: MEEPDKGKGNEEKGRGEVRYRGVRRRRPWGKFAAEIRDPSRQGGRLWLGTFDTAEEAARAYDKAAFNLRRHLAVLNFPSDYISQVTEFSPRPRSYSPSCSNVGATQTFERGSSSSAGHGNQVIEFECLDDKILEELLETEEEKKKRQQD*.

[0031] Example 3: PdPapERF098 gene enhances yeast's sensitivity to the heavy metal cadmium. Saccharomyces cerevisiae strains WT and Δycf1 (Shanghai Weidi Biotechnology Co., Ltd., strain numbers CAT#: YC1060 and CAT#: YC1081, respectively) were cultured and transformed using the pYES2 plasmid and the recombinant plasmid pYES2-PdPapERF098. Since the PdPapERF098 gene is regulated by the yeast galactose-inducible promoter pGAL1, transforming Saccharomyces cerevisiae with pYES2-PdPapERF098 and culturing it on a galactose-added synthetic growth selective medium (Synthetic Dropout Medium plus Galactose, SC-ura medium) can induce heterologous overexpression of PdPapERF098 in yeast.

[0032] The yeast conversion method used is the lithium acetate conversion method, and the specific steps are as follows: 1. Pretreatment of Carrier DNA: Insert the Carrier DNA into a 95°C metal bath for 5 min, then heat and quickly insert it into ice.

[0033] 2. Take 100 µL of WT and Δycf1 competent Saccharomyces cerevisiae cells thawed on ice, add 1 µg of pre-chilled PdPapERF098 plasmid, 10 µL of pretreated Carrier DNA, and 500 µL of PEG / LiAc, and mix by pipetting several times. Incubate at 30°C for 30 min (invert 6-8 times at 15 min to mix).

[0034] 3. Transfer competent cells to a 42°C water bath for 15 min (twirl 6-8 times at 7.5 min to mix). Centrifuge at 5000 rpm for 40 s, discard the supernatant, resuspend in 400 µL ddH2O, centrifuge for 30 s, and discard the supernatant.

[0035] 4. Resuspend in 50 µL ddH2O, spread on SC-ura solid medium plates containing 2% galactose, and incubate at 30°C for 48-96 h until transformants appear.

[0036] The liquid YPD medium used in this embodiment has the following formulation: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L; the liquid SC-ura medium has the following formulation: yeast nitrogen source without added amino acids (YNB, amino acid-free nitrogen source medium) 6.7 g / L, galactose 20 g / L, DO Supplement-ura 0.78 g / L; the corresponding solid medium is the liquid medium with 15 g / L agar powder added, autoclaved at 115°C for 20 minutes.

[0037] Single clones of yeast strain Δycf1, transformed with the empty vector pYES2 and the PdPapERF098 gene overexpression vector pYES2-PdPapERF098, were selected and inoculated into 2 mL of SC-ura liquid medium supplemented with galactose. The cultures were incubated at 30°C in a shaker (200 rpm) until the bacterial culture reached OD. 600 The value reached 0.9. The bacterial culture was serially diluted at ratios of 1:1, 1:10, 1:100, and 1:1000. 2 μL of each serially diluted culture was then dropped onto SC-ura solid medium plates with and without 80 μM CdCl2. The plates were incubated at 30°C for 7 days, and yeast growth was observed.

[0038] like Figure 3 As shown, the results indicated that there were no significant differences in colony size and growth density between the two groups of *Saccharomyces cerevisiae* in cadmium-free medium, proving that PdPapERF098 expression does not affect the normal physiological growth of yeast. Under 80 μM CdCl2 stress, *Saccharomyces cerevisiae* Δycf1 transformed with the empty vector could form clear colonies; however, the transgenic *Saccharomyces cerevisiae* overexpressing PdPapERF098 had extremely small colonies, severely inhibited growth, and significantly reduced biomass.

[0039] Conclusion: Heterologous expression of the PdPapERF098 gene significantly improves the sensitivity of Saccharomyces cerevisiae to cadmium ions and can be used to construct highly sensitive engineered strains for cadmium detection.

[0040] Example 4: Determination of cadmium content in brewer's yeast Saccharomyces cerevisiae strain Δycf1 was cultured and transformed into the empty yeast expression vector pYES2 and the PdPapERF098 gene overexpression vector pYES2-PdPapERF098 using the method described in Example 3. In a clean bench, two yeast transformants (saccharomyces cerevisiae Δycf1 transformed into pYES2 and pYES2-PdPapERF098 respectively) were picked with a sterile toothpick and added to 10 mL of SC-ura liquid medium supplemented with 2% galactose. The culture was then incubated at 30°C on a shaker (200 rpm) until the culture reached OD500. 600The value reached approximately 2. Then, the two yeasts were inoculated at a volume ratio of 1:500 into 600 mL of SC-ura liquid medium supplemented with 2% galactose, and cultured at 30°C on a shaker (200-250 rpm) for 12 h until the OD value reached approximately 2. 600 The value reached 0.9, and then CdCl2 was added to a final concentration of 30 μM.

[0041] After culturing for another hour, the bacterial cells were collected by centrifugation. The bacterial cells were washed three times with double-distilled water and CaCl2 (25 mmol / L, pH 5.0), and then dried at 65°C for 3-5 days. The dried yeast blocks were then microwave-digested, and the amount of cadmium accumulated in the yeast cells was determined by flame atomic absorption spectrometry.

[0042] like Figure 4 As shown, the yeast strain transformed with pYES2-PdPapERF098 had a lower intracellular cadmium accumulation than the yeast strain transformed with the empty pYES2 vector, but its growth inhibition was more severe. This indicates that PdPapERF098 does not cause toxicity by increasing intracellular cadmium accumulation, but rather by amplifying cadmium oxidation damage by regulating the cellular cadmium response pathway. This further proves the unique cadmium-sensitive regulatory function of this gene, which is different from existing heavy metal transport genes.

[0043] Example 5: Application of cadmium-sensitive recombinant yeast strains According to Example 4, a recombinant Saccharomyces cerevisiae transformed into strain Δycf1 with pYES2-PdPapERF098 (experimental strain), a cadmium-sensitive recombinant yeast strain, and a yeast strain transformed with empty vector pYES2 (control strain) were prepared. Prepare a solid screening medium of 80 μmol / L CdCl2. Spread the water or soil extract to be tested on the surface of the medium. After serially diluting the two groups of bacterial solutions, spot them onto the screening medium and incubate at 30℃. Compare the differences in colony growth: if the growth of the experimental strain is significantly inhibited while the control strain can grow normally, then the sample to be tested is determined to contain a high concentration of cadmium pollutants.

[0044] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of the *Populus spp.* PdPapERF098 transcription factor in any of the following: (1) Improve the cadmium sensitivity of brewer's yeast; (2) Cultivate highly cadmium-sensitive Saccharomyces cerevisiae strains; (3) Prepare products that improve the cadmium sensitivity of Saccharomyces cerevisiae; The cadmium sensitivity of Saccharomyces cerevisiae was improved by overexpressing the gene encoding the Populus alba PdPapERF098 transcription factor in Saccharomyces cerevisiae. The amino acid sequence of the Populus alba PdPapERF098 transcription factor is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

2. The application of the gene encoding the *Populus spp.* PdPapERF098 transcription factor in any of the following: (1) Improve the cadmium sensitivity of brewer's yeast; (2) Cultivate highly cadmium-sensitive Saccharomyces cerevisiae strains; (3) Prepare products that improve the cadmium sensitivity of Saccharomyces cerevisiae; The cadmium sensitivity of Saccharomyces cerevisiae was improved by overexpressing the gene encoding the Populus alba PdPapERF098 transcription factor in Saccharomyces cerevisiae. The amino acid sequence of the Populus alba PdPapERF098 transcription factor is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

3. Application of recombinant vectors in any of the following: (1) Improve the cadmium sensitivity of brewer's yeast; (2) Cultivate highly cadmium-sensitive Saccharomyces cerevisiae strains; (3) Prepare products that improve the cadmium sensitivity of Saccharomyces cerevisiae; in, The recombinant vector contains the encoding gene of the *Populus spp.* PdPapERF098 transcription factor, the nucleotide sequence of which is shown in SEQ ID NO.

1. The cadmium sensitivity of Saccharomyces cerevisiae was improved by overexpressing the gene encoding the PdPapERF098 transcription factor in Saccharomyces cerevisiae.

4. Application of recombinant bacteria in any of the following: (1) Improve the cadmium sensitivity of brewer's yeast; (2) Cultivate highly cadmium-sensitive Saccharomyces cerevisiae strains; (3) Prepare products that improve the cadmium sensitivity of Saccharomyces cerevisiae; in, The recombinant bacteria contains the encoding gene of the *Populus salina* PdPapERF098 transcription factor, the nucleotide sequence of which is shown in SEQ ID NO.1; The cadmium sensitivity of Saccharomyces cerevisiae was improved by overexpressing the gene encoding the PdPapERF098 transcription factor in Saccharomyces cerevisiae.

5. A method for improving the cadmium sensitivity of Saccharomyces cerevisiae, characterized in that, The invention includes a gene encoding the transcription factor PdPapERF098 overexpressed in *Saccharomyces cerevisiae*; wherein the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

6. A method for cultivating a highly cadmium-sensitive Saccharomyces cerevisiae strain, characterized in that, The invention includes overexpressing the encoding gene of the *Populus salsa* PdPapERF098 transcription factor in *Saccharomyces cerevisiae* to obtain a *Saccharomyces cerevisiae* strain with high cadmium sensitivity; wherein the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

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