Populus sutchuenensis PdPapWRKY49 transcription factor and application of coding gene thereof in improvement of cadmium resistance of yeast

Abstract

The invention discloses a Populus sutchuenensis PdPapWRKY49 transcription factor and application of a coding gene of the Populus sutchuenensis PdPapWRKY49 transcription factor in improvement of cadmium resistance of yeast, and belongs to the field of gene engineering. The transcription factor PdPapWRKY49 which is subjected to cadmium stress induced expression is screened from populus pinnatifida, the amino acid sequence of the transcription factor PdPapWRKY49 is as shown in SEQ ID NO. 2, and the nucleotide sequence of the coding gene of the transcription factor PdPapWRKY49 is as shown in SEQ ID NO. 1. Experimental results show that the PdPapWRKY49 gene is expressed in the cadmium stress sensitive saccharomyces cerevisiae strain, so that the strain can normally grow, and the cadmium concentration in the strain is reduced; the PdPapWRKY49 gene can be used for remarkably improving the tolerance of the saccharomyces cerevisiae to cadmium stress. According to the invention, a new gene material is provided for genetic engineering improvement of engineering bacteria aiming at cadmium tolerance, and a technical support is provided for culturing a new functional strain.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to the application of a *Populus alba* PdPapWRKY49 transcription factor and its encoding gene in improving cadmium tolerance in yeast. Background Technology

[0002] Cadmium (Cd) is one of the most dangerous environmental pollutants, posing significant risks to ecosystems and human health. Cadmium emissions have led to a substantial increase in soil pollution, creating widespread ecological pressure. Cadmium pollution disrupts plant physiology, inhibits soil microbial activity, and reduces agricultural productivity. Its bioaccumulation in staple crops further propels it into the human food chain, causing kidney dysfunction, bone diseases, and even cancer, threatening food safety and human health.

[0003] Heavy metal pollution is a global environmental problem. Phytoremediation, as a sustainable method, has received much attention in recent years due to its low cost, safety, and environmental friendliness, especially in remediating heavy metal-contaminated soils, where it has shown unique advantages. Phytoremediation is the process of using plants to remove pollutants from the environment or transform them into non-toxic or less toxic substances.

[0004] Populus davidiana × Populus alba var. pyramidalis is a fast-growing hybrid poplar widely used for afforestation and urban greening due to its cold resistance, drought tolerance, and rapid growth. It is also recommended for phytoremediation of heavy metal-contaminated soils. WRKY transcription factors are important transcriptional regulatory proteins in plants, playing a crucial role in plant responses to abiotic stress. However, research on the regulation of cadmium stress by WRKY genes in the Populus davidiana genome has not been reported. Summary of the Invention

[0005] The purpose of this invention is to provide an application of the *Populus spp.* PdPapWRKY49 transcription factor and its encoding gene in improving cadmium tolerance in yeast, thereby solving the problems existing in the prior art. The PdPapWRKY49 gene significantly improves the tolerance of *Saccharomyces cerevisiae* to cadmium stress. This invention provides new genetic material for the genetic engineering improvement of engineered bacteria to cadmium tolerance and provides technical support for the cultivation of new functional strains.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides an application of the Populus alba PdPapWRKY49 transcription factor in any of the following:

[0008] (1) Improve the cadmium tolerance of yeast;

[0009] (2) Cultivate highly cadmium-resistant yeast strains;

[0010] (3) Prepare products that improve the cadmium resistance of yeast;

[0011] The amino acid sequence of the *Populus spp.* PdPapWRKY49 transcription factor is shown in SEQ ID NO.2.

[0012] The present invention also provides an application of the gene encoding the above-mentioned Populus alba PdPapWRKY49 transcription factor in any of the following:

[0013] (1) Improve the cadmium tolerance of yeast;

[0014] (2) Cultivate highly cadmium-resistant yeast strains;

[0015] (3) Prepare products that improve the cadmium resistance of yeast;

[0016] The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0017] Furthermore, yeast cadmium tolerance was improved by upregulating the expression of the encoded gene in yeast.

[0018] The present invention also provides an application of a recombinant vector, wherein the recombinant vector comprises the gene encoding the PdPapWRKY49 transcription factor of Populus tomentosa;

[0019] The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1;

[0020] The application is any one of the following:

[0021] (1) Improve the cadmium tolerance of yeast;

[0022] (2) Cultivate highly cadmium-resistant yeast strains;

[0023] (3) Prepare products that improve the cadmium resistance of yeast.

[0024] The present invention also provides the use of engineered bacteria comprising the above-described recombinant vector in any of the following:

[0025] (1) Improve the cadmium tolerance of yeast;

[0026] (2) Cultivate highly cadmium-resistant yeast strains;

[0027] (3) Prepare products that improve the cadmium resistance of yeast.

[0028] The present invention also provides a method for improving the cadmium tolerance of yeast, including the step of overexpressing the expression level of the gene encoding the Populus alba PdPapWRKY49 transcription factor in yeast to improve the cadmium tolerance of yeast;

[0029] The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0030] This invention also provides a method for cultivating a highly cadmium-tolerant yeast strain, comprising the following steps:

[0031] A recombinant vector encoding the PdPapWRKY49 transcription factor of *Populus salsa* was constructed and transformed into yeast cells to obtain the highly cadmium-tolerant yeast strain.

[0032] The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0033] Furthermore, the skeletal carrier of the recombinant vector is pYES2.

[0034] The present invention also provides a highly cadmium-tolerant yeast strain obtained according to the above-described cultivation method.

[0035] The present invention also provides an application of the above-mentioned highly cadmium-tolerant yeast strain in the remediation of cadmium-contaminated environments.

[0036] The present invention discloses the following technical effects:

[0037] This invention screened a transcription factor, PdPapWRKY49, from *Populus alba* that is induced to express under cadmium stress. Its amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.1. Experimental results showed that expression of the PdPapWRKY49 gene in a cadmium-stress-sensitive *Saccharomyces cerevisiae* strain enabled normal growth of the strain and reduced the cadmium concentration within the strain. The PdPapWRKY49 gene significantly improved the tolerance of *Saccharomyces cerevisiae* to cadmium stress. This invention provides new genetic material for the genetic engineering improvement of strains to cadmium tolerance and provides technical support for the cultivation of new functional strains. Attached Figure Description

[0038] 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.

[0039] Figure 1 Figure 1 shows the PCR amplification and electrophoresis analysis results of the cDNA reading frame sequence of the PdPapWRKY49 gene.

[0040] Figure 2 A schematic diagram of the structure of the Saccharomyces cerevisiae recombinant expression vector PdPapWRKY49-pYES2;

[0041] Figure 3 Figure 1 shows the experimental results of cadmium tolerance of the transgenic yeast mutant strain Δycf1 transformed with PdPapWRKY49-pYES2.

[0042] Figure 4 Figure shows the results of cadmium ion content determination in cells of the mutant strain Δycf1 transformed by the recombinant expression vector PdPapWRKY49-pYES2 and the empty vector pYES2 of Saccharomyces cerevisiae. Detailed Implementation

[0043] 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.

[0044] 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 within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] This invention first cloned the full-length cDNA reading frame sequence of the Populus alba WRKY transcription factor gene PdPapWRKY49. Using young Populus alba samples collected from Nanjing Botanical Garden (32°3′N, 118°49′E), total RNA was extracted and reverse transcribed into cDNA. Using the cDNA as a template, the full-length cDNA reading frame of PdPapWRKY49 was amplified using primer pairs PdPapWRKY49F (SEQ ID NO.3) and PdPapWRKY49R (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 PdPapWRKY49 gene, which improved the yeast's tolerance to the heavy metal cadmium and increased the accumulation of cadmium pollution from the external environment.

[0049] Example 1: RT-PCR amplification of the PdPapWRKY49 gene cDNA sequence

[0050] Young poplar plant samples were collected from Nanjing Zhongshan Botanical Garden (32°3′N, 118°49′E). 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.

[0051] 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., Baori Biotechnology (Beijing) Co., Ltd.

[0052] Using single-stranded cDNA from *Populus tomentosa* leaves as a template, the high-fidelity DNA polymerase Probest was employed. TM DNA Polymerase was used to amplify the PdPapWRKY49 gene. The primers used were PdPapWRKY49F (SEQ ID NO.3) and PdPapWRKY49R (SEQ ID NO.4), and the PCR reaction volume was 25 μL.

[0053] PdPapWRKY49F:ATGGAAGGAGAAGTAATCAGTAGT, SEQ ID NO.3;

[0054] PdPapWRKY49R:TCACCCAATGCTAGTGTTTAAG, SEQ ID NO.4.

[0055] The PCR amplification reaction system consisted of: 12.5 μL of 10×PCR Buffer, 0.5 μL of 10 mM dNTP Mix, 1 μL of 10 μM dPapWRKY49F, 1 μL of 10 μM PdPapWRKY49R, 0.5 μL of DNA Polymerase, 1 μL of cDNA template, and 8.5 μL of ddH2O. All components were thoroughly mixed and then placed in a PCR instrument for the reaction.

[0056] 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.

[0057] PCR products were detected by 1% agarose gel electrophoresis, such as... Figure 1 As shown, a single DNA band (approximately 930 bp) was obtained, which is the PdPapWRKY49 gene fragment obtained by PCR amplification. The PdPapWRKY49 fragment was recovered by agarose gel electrophoresis according to the Magen HiPureGel Pure DNA Kits instructions.

[0058] Example 2 Construction of yeast recombinant expression vector for PdPapWRKY49 gene

[0059] The Saccharomyces cerevisiae expression vector pYES2 was recovered by double digestion with KpnI and EcoRI.

[0060] The concentrations of the recovered PdPapWRKY49 PCR fragment and 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.

[0061] 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.

[0062] After sequencing analysis, the nucleotide sequence of the cDNA reading frame of PdPapWRKY49 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 PdPapWRKY49-pYES2 is shown in [image / description]. Figure 2 As shown.

[0063] SEQ ID NO.1:

[0064] ATGGAAGGAGAAGTAATCAGTAGTAGTTGGTTTAGTGAGAGTGAAGAAGACGAGCTCGTGAGAGAGCTCCTTGATGATGTATCCCCTTTCTTTTTCTTGCCGGAGGAAAGAAACCAATCCAAAGCAGCAAGTCCCACTCCAAGGAATGAAGAAGCTATGAACCAGATCATTTCCAAAGTCTATTCAGGACCAACAATGGAAGATATTGAGAATGCTTTGTCGATGACAAGCCGAAGAGACCAACCCCAGCCAGTTTCACAAGCCAGATTCTCATTGTTGCAAAAGGGTTTGAGTAAGATTGAGAACAACAGGTACACTGTAAAACTGAAGAGCTGTGACAATGGTGTTGCTGGTGATGGTTATAAATGGAGGAAATACGGGCAGAAGTCTATCAAGAATAGCACACATCCCAGAAGCTATTACAGGTGCACGAACCCACGGTGCGGTGCAAAAAAGCAGGTGGAGAGGTCCGGCGAGGATCCAGACACGCTCGTCATCACCTACGAAGGGCTCCATTTACACTTTTCTCACCCATTTTTCTTATCGAACCAGCCACAACATGTTGATCCACCTTCCAAGAAACCTAAGAGGACCATTTCAGAGGACGAGTTTCAAGCCCATGAAACCCGACAACCACCAGAACAAGGCCAAGAATGCTCCACACATGCGACCAGTCCTGGTTCCCTGCCTAGCTCAAGCACAGCTGATGATTACATGCAAGAATCGGATCTGGAAGCGATGGGTCCTCGAGGGTTGCTTGAAGATGTGGTGCCTTTTATGATTCGAAACCCATCAAGCTACAATGTGTCTTCTTATTCTTCATCGTCGTCCCATCGTTCTCCCCCTACCTCACCATCTTCCTCCTTGTCTTGGTCTCCCAATCTTTCGCATTCGTGTTTCGATGTTGGCTTAAACACTAGCATTGGGTGA。

[0065] SEQ ID NO.2:

[0066] MEGEVISSSWFSESEEDELVRELLDDVSPFFFLPEERNQSKAASPTPRNEEAMNQIISKVYSGPTMEDIENALSMTSRRDQPQPVSQARFSLLQKGLSKIENNRYTVKLKSCDNGVAGDGYKWRKYGQKSIKNSTHPRSYYRCTNPRCGAKKQVE RSGEDPDTLVITYEGLHLHFSHPFFLSNQPQHVDPPSKKPKRTISEDEFQAHETRQPPEQGQECSTHATSPGSLPSSSTADDYMQESDLEAMGPRGLLEDVVPFMIRNPSSYNVSSYSSSSSHRSPPTSPSSSLSWSPNLSHSCDVGLNTSIGF.

[0067] Example 3: PdPapWRKY49 gene enhances yeast's tolerance to the heavy metal cadmium.

[0068] 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 PdPapWRKY49-pYES2. Since the PdPapWRKY49 gene is regulated by the yeast galactose-inducible promoter pGAL1 (e.g., ...), ... Figure 2 As shown in the figure, PdPapWRKY49-pYES2 can be transformed into Saccharomyces cerevisiae and cultured on a growth-selective synthetic medium (Synthetic Dropout Medium plus Galactose, SC-ura medium) to induce heterologous overexpression of PdPapWRKY49 in yeast.

[0069] The yeast conversion method used is the lithium acetate conversion method, and the specific steps are as follows:

[0070] 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.

[0071] 2. Take 100 µL of WT and Δycf1 Saccharomyces cerevisiae competent cells thawed on ice, add 1 µg of pre-cooled PdPapWRKY49 plasmid, 10 µL of pretreated Carrier DNA, and 500 µL of PEG / LiAc, and mix by pipetting several times. Incubate in a 30℃ water bath for 30 min (invert 6-8 times at 15 min to mix).

[0072] 3. Transfer the competent cells to a 42℃ water bath for 15 min (invert 6-8 times at 7.5 min to mix thoroughly). Centrifuge at 5000 rpm for 40 s, discard the supernatant, resuspend in 400 µL of ddH2O, centrifuge for 30 s, and discard the supernatant.

[0073] 4. Resuspend in 50 µL of ddH2O, spread on SC-ura solid medium plates supplemented with 2% galactose, and incubate at 30°C for 48-96 h until transformants appear.

[0074] 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 (YNB) without added amino acids 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.

[0075] Single clones of yeast strain Δycf1 transformed with the empty vector pYES2 and the PdPapWRKY49 gene overexpression vector PdPapWRKY49-pYES2 were selected and inoculated into 2 mL of SC-ura liquid medium supplemented with galactose, and cultured 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.

[0076] Growth status as follows Figure 3 As shown, Δycf1 is a cadmium-sensitive mutant strain of *Saccharomyces cerevisiae*, which readily accumulates cadmium and exhibits toxicity. It struggles to grow on media supplemented with 80 μM CdCl2, indicating its low tolerance to cadmium. Clearly, transgenic yeast overexpressing the PdPapWRKY49 gene (transformed into the recombinant vector PdPapWRKY49-pYES2) can grow on SC-ura solid medium plates supplemented with 80 μM CdCl2, compared to the Δycf1 yeast mutant strain transformed with the empty vector pYES2. In contrast, yeast without PdPapWRKY49 gene expression (transformed into the empty vector pYES2) struggles to grow on SC-ura solid medium plates supplemented with 80 μM CdCl2. This suggests that expression of the PdPapWRKY49 gene in yeast can reduce the toxicity of cadmium to the Δycf1 yeast strain and improve its tolerance to the heavy metal cadmium.

[0077] Example 4: Expression of the PdPapWRKY49 gene in Saccharomyces cerevisiae reduces intracellular cadmium accumulation in yeast cells.

[0078] Saccharomyces cerevisiae strain Δycf1 was cultured and transformed into the empty yeast expression vector pYES2 and the PdPapWRKY49 overexpression vector PdPapWRKY49-pYES2 using the method described in Example 3. In a clean bench, two yeast transformants (saccharomyces Δycf1 transformed into pYES2 and PdPapWRKY49-pYES2, 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. 600 The 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.

[0079] 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.

[0080] like Figure 4 As shown, the cadmium accumulation in the yeast strain Δycf1 transformed with PdPapWRKY49-pYES2 in a medium supplemented with 30 μM CdCl2 was significantly lower than that in the yeast strain transformed with the empty vector pYES2. This indicates that the expression of the PdPapWRKY49 gene in yeast cells reduces the enrichment of the heavy metal cadmium in yeast and inhibits the absorption of cadmium by yeast.

[0081] 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. The application of a *Populus alba* PdPapWRKY49 transcription factor in any of the following: (1) Improve the cadmium tolerance of yeast; (2) Cultivate highly cadmium-resistant yeast strains; (3) Prepare products that improve the cadmium resistance of yeast; The amino acid sequence of the *Populus spp.* PdPapWRKY49 transcription factor is shown in SEQ ID NO.

2.

2. The use of the gene encoding the *Populus alba* PdPapWRKY49 transcription factor as described in claim 1 in any of the following: (1) Improve the cadmium tolerance of yeast; (2) Cultivate highly cadmium-resistant yeast strains; (3) Prepare products that improve the cadmium resistance of yeast; The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

3. The application as described in claim 2, characterized in that, The cadmium tolerance of yeast was improved by upregulating the expression of the encoded gene in yeast.

4. An application of a recombinant vector, characterized in that, The recombinant vector includes the gene encoding the *Populus spp.* PdPapWRKY49 transcription factor. The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1; The application is any one of the following: (1) Improve the cadmium tolerance of yeast; (2) Cultivate highly cadmium-resistant yeast strains; (3) Prepare products that improve the cadmium resistance of yeast.

5. The use of an engineered bacterium comprising the recombinant vector of claim 4 in any of the following: (1) Improve the cadmium tolerance of yeast; (2) Cultivate highly cadmium-resistant yeast strains; (3) Prepare products that improve the cadmium resistance of yeast.

6. A method for improving cadmium tolerance in yeast, characterized in that, This includes steps to improve yeast cadmium tolerance by overexpressing the gene encoding the PdPapWRKY49 transcription factor in yeast. The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

7. A method for cultivating a highly cadmium-tolerant yeast strain, characterized in that, Includes the following steps: A recombinant vector encoding the PdPapWRKY49 transcription factor of *Populus salsa* was constructed and transformed into yeast cells to obtain the highly cadmium-tolerant yeast strain. The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

8. The cultivation method as described in claim 7, characterized in that, The backbone vector of the recombinant vector is pYES2.

9. A highly cadmium-tolerant yeast strain obtained by the cultivation method according to claim 7 or 8.

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