A protein sa nramp5 related to cadmium absorption of nightshade solanum nigrum and its coding gene and application

Abstract

The application relates to a protein SaNRAMP5 related to cadmium absorption of nightshade, a coding gene and application thereof. Knocking out the gene in nightshade can significantly reduce cadmium accumulation in nightshade, overexpressing the gene in Arabidopsis can promote cadmium accumulation in the root system of Arabidopsis, and expressing the gene in a cadmium-sensitive yeast ycf1 can significantly improve the sensitivity of the yeast strain to cadmium.

Description

Technical Field

[0001] This application belongs to the field of genetic engineering technology, specifically relating to a protein SaNRAMP5 related to cadmium absorption from black nightshade, its encoding gene, and its applications. Background Technology

[0002] Cadmium (Cd) is a highly toxic heavy metal. In recent years, with large-scale industrialization and urbanization, many farmlands have been contaminated with cadmium to varying degrees. In areas with low cadmium pollution, plants may not show symptoms of poisoning. However, plants can accumulate cadmium in their edible parts beyond levels permissible for humans, and once these plants enter the food chain, they can cause health problems. The significant harm cadmium poses to humans makes it crucial to utilize various methods to prevent cadmium from entering the food chain. There are generally two methods to achieve this. One is to use physical, chemical, and biological methods to alter the form of cadmium in the soil, increasing or decreasing its availability to plants, thereby using phytoremediation or controlling cadmium entry into the food chain. The other is to conduct in-depth research into the molecular mechanisms of cadmium absorption and translocation in different crops, using genetic engineering techniques to reduce plant absorption of cadmium or reduce the accumulation of cadmium in the edible parts of crops.

[0003] Solanum nigrum, a member of the Solanaceae family, is a cadmium hyperaccumulator. Previous studies on its hyperaccumulation and tolerance have largely focused on its root exudates, cadmium distribution within cells, chelation and compartmentalization, antioxidant systems, amino acid metabolism, maintenance of photosynthesis and respiration, and regulation of nitrogen metabolism. These studies remain at the physiological level. However, research on the molecular mechanisms of cadmium hyperaccumulation and tolerance in Solanum nigrum is still very weak. For example, there are few reports on the cloning and functional studies of genes involved in cadmium absorption and transport in Solanum nigrum. Summary of the Invention

[0004] To address the aforementioned technical problems, this application provides a protein, the amino acid sequence of which is shown in SEQ ID NO.1; or a fusion protein obtained by attaching a tag substance to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO.1.

[0005] This application also provides biological materials related to the above-mentioned proteins, which are any one of A1) to A12) below:

[0006] A1) A nucleic acid molecule encoding the protein of claim 1;

[0007] A2) An expression cassette containing the nucleic acid molecules described in A1);

[0008] A3) A recombinant vector containing the nucleic acid molecules described in A1);

[0009] A4) A recombinant vector containing the expression cassette described in A2);

[0010] A5) Recombinant microorganisms containing the nucleic acid molecules described in A1);

[0011] A6) Recombinant microorganisms containing the expression cassette described in A2);

[0012] A7) Recombinant microorganisms containing the recombinant vector described in A3);

[0013] A8) Recombinant microorganisms containing the recombinant vector described in A4);

[0014] A9) Transgenic plant cell lines containing the nucleic acid molecules described in A1);

[0015] A10) A transgenic plant cell line containing the expression cassette described in A2);

[0016] A11) Transgenic plant cell lines containing the recombinant vector described in A3);

[0017] A12) Transgenic plant cell lines containing the recombinant vector described in A4).

[0018] In some embodiments, the transgenic plant cell line does not develop into a complete plant. In some embodiments, the nucleic acid molecule described in A1) is a nucleic acid molecule as shown in 1), 2), or 3) below:

[0019] 1) Nucleic acid molecules with sequences as shown in SEQ ID NO.2;

[0020] 2) A nucleic acid molecule that has 90% or more identity with SEQ ID NO.2 and encodes a protein related to cadmium absorption of solanum;

[0021] 3) Under strict conditions, it hybridizes with nucleic acid molecules of 1) or 2) and encodes nucleic acid molecules that encode proteins related to cadmium absorption of solanum.

[0022] This application also provides a method for reducing the absorption of cadmium or manganese by black nightshade, including the step of reducing the content or activity of the aforementioned protein in black nightshade. In some embodiments, the step involves knocking out the gene encoding the aforementioned protein.

[0023] This application also provides a method for increasing the accumulation of cadmium or manganese in plants or microorganisms, including the step of overexpressing the aforementioned proteins in the plants or microorganisms.

[0024] This application also provides a method for remediating cadmium-contaminated soil, comprising the step of extracting cadmium or manganese pollutants from the soil using plants or microorganisms that hyperaccumulate cadmium or manganese, wherein the plants or microorganisms overexpress the aforementioned proteins.

[0025] In some embodiments, the microorganism is a fungus or bacteria, or the plant is a monocotyledonous or dicotyledonous plant. In some embodiments, the fungus is yeast. In some embodiments, the dicotyledonous plant is black nightshade or Arabidopsis thaliana.

[0026] In some embodiments, the protein is overexpressed in the roots, stems, leaves, flowers, fruits, or seeds of the plant.

[0027] Beneficial effects: This invention discloses for the first time that knocking out SaNRAMP5 in black nightshade can significantly reduce cadmium accumulation in the aboveground parts and roots of black nightshade, overexpressing this gene in Arabidopsis thaliana can promote cadmium accumulation in Arabidopsis thaliana roots, and expressing this gene in yeast can promote cadmium accumulation in yeast cells. Attached Figure Description

[0028] Figure 1 Expression characteristics of SaNRAMP5 in different parts of black nightshade (roots, leaves, stems, flowers, and fruits).

[0029] Figure 2 The increased cadmium sensitivity of yeast by the Δycf1 yeast strain expressing SaNRAMP5 is caused by increased cadmium accumulation.

[0030] Figure 3 Knocking out SaNRAMP5 in black nightshade significantly reduced cadmium accumulation in the aboveground parts and roots of black nightshade.

[0031] Figure 4 Overexpression of SaNRAMP5 in Arabidopsis thaliana significantly increased root accumulation.

[0032] Figure 5 Comparison of cadmium and manganese absorption capacity between SaNRAMP5 and OsNRAMP5. Detailed Implementation

[0033] To illustrate the universal design concept of this application, specific experimental parameters are used as examples below, but this should not be used as a reason to limit the scope of protection of this application.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0035] Example 1: Total RNA extraction, cDNA synthesis, and quantitative PCR

[0036] Solanum americanum seeds were sterilized with 30% NaClO by mass, germinated, and cultured for 2 weeks. Seedlings of uniform size were selected and cultured in Hoagland's complete nutrient solution for one week. Roots and leaves were then quickly frozen in liquid nitrogen. Approximately 0.1g of sample was weighed, ground thoroughly with liquid nitrogen, and added to a 1.5ml centrifuge tube. 1ml Trizol reagent and 0.2mL chloroform were added immediately. After centrifugation, the supernatant was collected, and 0.5mL isopropanol was added. After centrifugation, the supernatant was discarded, and the precipitate was washed with 70% ethanol. RNA was dissolved in DEPC water (1‰ v / v). RNA quality was detected by 1.7% agarose gel electrophoresis, and the concentration and purity of total RNA were determined by spectrophotometry. For each RNA sample (2μg), 50μmol L-1 OligodT18 was added, and 1‰ DEPC water was added to make up to 10μL. The sample was incubated at 70℃ for 5min, placed on ice for 5min, and then RNase inhibitor was added sequentially. 0.5 μL of 5xRT buffer, 5 μL of 10 mM dNTP, 1 μL of M-MLV reverse transcriptase, and 1‰ DEPC water were added to make up to 25 μL. The reaction was incubated at 42°C for 60 min, and then incubated at 70°C for 10 min to terminate the reaction.

[0037] After reverse transcription to synthesize the first strand of total cDNA, it was used as a template for PCR amplification. Reaction procedure: The cDNA amplification reaction of various tissues of *Solanum nigrum* was performed in a 96-well PCR plate. The reaction mixture consisted of 20 μl: 10 μl of SYBR mix (2x), 0.4 μl of ROX, 0.4 μl each of forward and reverse primers, 1 μl of cDNA template, and 7.8 μl of sterile water, for a total volume of 20 μl. The mixture was thoroughly mixed and then dispensed into the PCR plate. The reaction conditions were as follows: 95℃ for 5 s, 94℃ denaturation for 30 s, 65℃ annealing for 30 s, 72℃ extension for 30 s, 40 cycles, followed by a final extension at 72℃ for 5 min.

[0038] Calculation method: The results were obtained using double ΔΔCt values. Expression analysis of this gene in different parts of *Solanum nigrum* (roots, leaves, stems, flowers, and fruits) revealed that the expression level of SaNRAMP5 in the roots was significantly higher than that in other tissues. Figure 1 ).

[0039] Example 2: Obtaining the full-length cDNA of the SaNRAMP5 gene

[0040] Using the obtained black nightshade root cDNA as a template, PCR primers were designed, and the PCR product contained the complete SaNRAMP5 reading frame (from start codon ATG to TAG).

[0041] The PCR program was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 s, 56℃ annealing, 72℃ annealing and extension for 1 min 20 s, 35 cycles, followed by 72℃ for 7 min. The amplified PCR product was detected by 1% agarose gel electrophoresis, and its size was a 1605 bp fragment. The target PCR product was separated by agarose gel electrophoresis, excised, and recovered. The recovered fragment was compared with P-easy... The blunt vector was ligated using a 5 μL enzyme ligation system, containing 1 μL of the vector and 4 μL of purified PCR product. After addition, the mixture was thoroughly mixed, centrifuged to the bottom of the tube, and incubated at 28°C for 15 min before transformation. The cells were then heat-transformed into *E. coli* DH5α competent cells at 42°C, and incubated with 500 μL of antibiotic-free LB broth for 1 h. After low-speed centrifugation, the cells were enriched and plated onto LB solid medium containing 100 μg / mL kanamycin. After incubation for 12-14 h, positive colonies were picked for DNA sequencing. The open reading frame (ORF) of SaNRAMP5 was 1605 bp in length, as shown in SEQ ID NO.2. The sequence of its encoded protein, as shown in SEQ ID NO.1, represents the *Solanum nigrum* natural resistance-associated macrophage protein SaNRAMP5. The correctly sequenced bacterial culture was added to an equal volume of 30% glycerol and stored at -70°C for later use. The P vector containing the SaNRAMP5 ORF was named *SaNRAMP5 inP*.

[0042] Example 3: Transgenic yeast overexpressing SaNRAMP5

[0043] The yeast expression vector pDR196 was digested and purified using EcoR1 and Xho1 enzymes. Using the SaNRAMP5 inP plasmid as a template, the PCR product was amplified and purified using the following primers. Finally, the linearized plasmid and the purified PCR product were subjected to homologous recombination: the system was 5 μL, containing 1 μL of linearized vector and 4 μL of purified PCR product. The mixture was then incubated at 37 degrees Celsius for 1 hour for transformation. Single clones were picked and sequenced. Correct single clones were preserved and the plasmid was named SaNRAMP5-pDR196.

[0044] Conclusion: Expression of SaNRAMP5 in the cadmium-sensitive yeast strain ycf1 significantly increases the sensitivity of yeast cells to cadmium. The reason for the cadmium sensitivity of yeast cells transformed with SaNRAMP5 is the accumulation of excessive cadmium in the yeast cells. Figure 2The study included: (A) Yeast strain Δycf1 transformed with empty vector pDR196 or SaNRAMP5 was spotted on SD-Ura medium containing different concentrations of cadmium. The initial yeast cell concentration was adjusted to OD = 0.8, and three concentration gradients were performed by 10-fold dilution. The plates were then incubated upside down at 30°C for 3–5 days. (B) Cd content in yeast Δycf1 cells transformed with pDR196 empty vector and SaNRAMP5. Data are presented as mean ± standard deviation, n = 5. The lowercase letter above each column indicates statistical difference (P < 0.05, ANOVA). (C) Growth curves of Δycf1 yeast transformed with pDR196 empty vector and SaNRAMP5 at different time points under 0, 4, and 8 μM CdCl2. Data are presented as mean ± SD, n = 3.

[0045] Example 4: Construction of the overexpression vector 35S:SaNRAMP5-GFP

[0046] Based on the CDS sequence of the black nightshade natural resistance-associated macrophage protein SaNRAMP5, PCR primers were designed, and the PCR product contained the complete SaNRAMP5 reading frame (with the stop codon removed). Homologous recombination primers were designed based on the restriction enzyme sites Kpn1 and Sal1 selected according to the final vector.

[0047] Using the obtained SaNRAMP5inP plasmid as a template, the PCR program was as follows: 95℃ pre-denaturation for 1 min, 95℃ denaturation for 30 s, 56℃ annealing, 72℃ annealing and extension for 1 min 20 s, 30 cycles, followed by 72℃ for 5 min. The amplified PCR product was detected by 1% agarose gel electrophoresis, and the PCR product size was approximately 1600 bp. The target PCR product was separated by agarose gel electrophoresis and purified by gel extraction. Simultaneously, the plant overexpression vector pCAMBIA1305 plasmid was digested with KpnI and Sal1, and the vector was then purified. The purified linearized vector and the target gene fragment were subjected to homologous recombination at 37℃ for one hour. The enzyme-linked product was then transformed into *E. coli* DH5α competent cells, plated on LB solid medium containing 50 μg / mL kanamycin, and grown for 12 h. Positive colonies were picked, and the plasmid was extracted and verified by KpnI and Sal1 digestion to confirm the fragment size. The bacterial culture was then subjected to DNA... Sequencing was performed, and the bacterial culture containing the correctly sequenced clone was added to an equal volume of 30% glycerol and stored at -70℃. The positive clone plasmid was extracted and named 35S:SaNRAMP5-GFP. Finally, the 35S:SaNRAMP5-GFP plasmid was transformed into competent cells of Agrobacterium tumefaciens EHA105 by electroporation. After growing on YEP solid medium containing 50 μg / mL kanamycin and streptomycin for 48 h, positive colonies were picked, plasmids were extracted, and after double digestion with KpnI and Sal1 to confirm that the bacterial culture was correct, an equal volume of 30% glycerol was added to the bacterial culture and stored at -70℃ for transgenic use.

[0048] Example 5: Construction of CRISPR vector with SaNRAMP5 knockout and transgenic black nightshade seedlings

[0049] Using GT-bone (Wuhan Boyuan) as a template, the target sequence of SaNRAMP5 was selected, and the fragment containing the target sequence was amplified using the primer pair SaNRAMP5TF / R. The PCR product was then recombined into the pHK2-AtCas9-U6 vector (Wuhan Boyuan), named SaNRAMP5-CRISPR. The transgenic process of *Solanum nigrum* was completed by Wuhan Boyuan Biotechnology Co., Ltd. using an Agrobacterium-mediated transformation system. Young leaves of *Solanum nigrum* (SP1102) were used to induce callus tissue, which was then co-cultured with *Agrobacterium*. After infection, the callus was placed on a selective medium containing 50 mg / L hygromycin. The selected callus tissue was then transferred to a regeneration medium for seedling development. The transgenic line was amplified using the primer pair SaNRAMP5gF / R around the target region, and the derived PCR product was sequenced to determine the mutation site of SaNRAMP5. The T2 generation homozygous SaNRAMP5 mutant was used for further experiments.

[0050] The phenotype of the black nightshade SaNRAMP5 knockout mutant is shown in the figure. Figure 3 (A) Identification of SaNRAMP5 mutants. The figure above shows the gene structure and mutation sites of SaNRAMP5. Solid boxes and black lines represent exons and introns of SaNRAMP5, respectively. The 20bp sequences of sgRNA and PAM motif (NGG) are highlighted with underline and red, respectively. (B) Phenotypes of wild-type (WT) and knockout lines (m1, m2, and m3). Bar = 20 cm. (C) Plant height (cm). (DG) 21-day-old seedlings of wild-type (WT) and knockout lines (m1, m2, and m3) were treated for 1 day in hydroponic media containing 0 and 10 μM CdCl2. Cd content in roots and aerial parts of 0 μM CdCl2 (DE) or 10 μM CdCl2 (FG). Data are the mean ± SD of five replicates. Different letters represent (P < 0.05, ANOVA) comparisons of cadmium content in wild-type and transgenic lines. Conclusion: Knocking out this gene significantly reduced cadmium accumulation in the aboveground parts and roots of black nightshade.

[0051] Example 6: Transgenic Arabidopsis thaliana overexpressing SaNRAMP5

[0052] Agrobacterium, transformed with the 35S:SaNRAMP5-GFP plasmid obtained above, was used to infect Arabidopsis thaliana Col inflorescences. The inflorescences were then co-cultured for 60 hours. The infected Arabidopsis plants were then cultured normally until seed harvest. T1 generation seeds were germinated on 1 / 2 MS medium containing 50 μg / mL hygromycin for selection. Individual plants that grew normally on 1 / 2 MS medium containing 50 μg / mL hygromycin were considered positive seedlings. The overexpression effect of positive seedlings was detected by RT-qPCR. Lines showing overexpression were repeatedly propagated to obtain stable, genetically homozygous lines for experimental use.

[0053] Figure 4The phenotypes of transgenic Arabidopsis plants overexpressing aNRAMP5 are shown. The overexpression effect of the saNRAMP5 overexpression lines was identified. Expression level relative to Col was set as 1, with AtActin8 as an internal control. (BC) Phenotypes of Col and saNRAMP5 overexpression lines (OV5, OV11, and OV20) grown for 2 weeks in 1 / 2 MS medium with 0 and 20 μM CdCl2. Bars = 1 cm. (DE) Root lengths of Col and saNRAMP5 overexpression lines at 0 and 20 μM CdCl2. Cd content in roots and shoots of Col and saNRAMP5 overexpression lines at 0 μM CdCl2 (FG) and 20 μM CdCl2 (HI). Data are mean ± SD of five replicates. Different letters indicate significant differences in cadmium content between wild-type and saNRAMP5 transgenic plants (P < 0.05, ANOVA). Conclusion: Overexpression of SaNRAMP5 in Arabidopsis thaliana yielded three homozygous lines (OV5, OV11, and OV20) with overexpression effect. When cultured on cadmium-free medium, there was no significant difference in growth between wild-type and overexpressing lines. However, when cultured on medium containing 20 μM CdCl2, root growth of the transgenic lines was inhibited, with root lengths significantly shorter than those of the wild-type. Cadmium content analysis of the roots indicated that the transgenic lines accumulated more cadmium, thus hindering root growth.

[0054] Example 7: Absorption capacity of SaNRAMP5 for cadmium and manganese

[0055] SaNRAMP5 affects the bioavailability of cadmium and manganese, especially cadmium. To demonstrate this, this example compares the effects of SaNRAMP5 and OsNRAMP5 on cadmium and manganese absorption capacity. Figure 5As shown in the figure. (A) Δycf1 yeast cells expressing the empty pDR196 vector, SaNRAMP5, or OsNRAMP5 were spotted on SD-Ura medium supplemented with different Cd concentrations. (B) Δsmf1 yeast cells expressing the empty pDR196 vector, SaNRAMP5, or OsNRAMP5 were spotted on SD-Ura medium supplemented with different EGTA concentrations. (C) Cd and (D) Mn contents in yeast cells expressing the empty pDR196 vector, SaNRAMP5, or OsNRAMP5. Data are expressed as mean ± standard deviation, n = 5. Different letters indicate significant differences in cadmium and manganese contents among SaNRAMP5, OsNRAMP5, and the empty pDR196 vector (P < 0.05, ANOVA). Using GAPDH as an internal control, the protein expression levels of SaNRAMP5 and OsNRAMP5 in yeast strains Δycf1(E) and Δsmf1(F) were detected.

[0056] It is evident that although SaNRAMP5 has a less significant effect on manganese absorption than OsNRAMP5, it has a significantly greater effect on cadmium absorption than OsNRAMP5, indicating that SaNRAMP5 and OsNRAMP5 exhibit functional differentiation in the selective absorption of cadmium and manganese.

[0057] Obviously, those skilled in the art can make various modifications and variations to this application without departing from the spirit and scope of this application. Therefore, if such modifications and variations fall within the scope of the claims of this application and their equivalents, this application also intends to include such modifications and variations.

Claims

1. A protein, characterized in that, The amino acid sequence of the protein is shown as SEQ ID NO.

1. Or, a fusion protein obtained by connecting a tag substance to the N terminus and / or C terminus of the protein shown as SEQ ID NO.

1.

2. A biological material related to the protein of claim 1, characterized by any one of the following A1) to A8): A1) a nucleic acid molecule encoding the protein of claim 1; A2) an expression cassette containing the nucleic acid molecule of A1); A3) a recombinant vector containing the nucleic acid molecule of A1); A4) a recombinant vector containing the expression cassette of A2); A5) a recombinant microorganism containing the nucleic acid molecule of A1); A6) a recombinant microorganism containing the expression cassette of A2); A7) a recombinant microorganism containing the recombinant vector of A3); A8) a recombinant microorganism containing the recombinant vector of A4).

3. The biomaterial of claim 2, wherein, The sequence of the nucleic acid molecule of A1) is shown as SEQ ID NO.

2.

4. A method of reducing cadmium or manganese uptake by Solanum nigrum, comprising: A step of reducing the content of the protein of claim 1 in a Solanum nigrum.

5. The method of claim 4, wherein: The step knocks out a gene encoding the protein of claim 1.

6. A method for remediation of cadmium-contaminated soil, characterized in that, A step of extracting cadmium or manganese pollutants from soil using Solanum nigrum or yeast that overexpresses the protein of claim 1, the Solanum nigrum or yeast accumulating cadmium or manganese.

7. The method of claim 6, wherein, A step of overexpressing the protein of claim 1 in roots, stems, leaves, flowers, fruits, or seeds of the Solanum nigrum.

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