Protein SaNRAMP5 related to cadmium absorption of solanum nigrum as well as coding gene and application of protein SaNRAMP5

Abstract

The invention relates to a protein SaNRAMP5 related to cadmium absorption of solanum nigrum as well as a coding gene and application of the protein SaNRAMP5. Cadmium accumulation of black nightshade can be remarkably reduced by knocking out the gene in black nightshade, accumulation of cadmium in arabidopsis thaliana root systems can be promoted by overexpressing the gene in arabidopsis thaliana, and sensitivity of a yeast strain to cadmium can be remarkably improved by expressing the gene in cadmium-sensitive yeast ycf1.

Description

Technical Field

[0001] The present application belongs to the field of genetic engineering technology, and specifically relates to a protein SaNRAMP5 related to Solanum nigrum cadmium absorption, and its encoding gene and application. Background Art

[0002] Cadmium (Cd) is a highly toxic heavy metal element. In recent years, with the process of large-scale industrialization and urbanization, many farmlands have been contaminated by cadmium to varying degrees. In places where cadmium pollution is low, plants may not show symptoms of poisoning. However, plants may accumulate cadmium in edible parts that exceeds the level allowed by humans, which may cause health problems once these plants enter the food chain. The huge harm of cadmium to the human body makes it crucial to use various methods to prevent cadmium from entering the food chain. There are usually two ways to achieve this goal. One is to change the existence form of cadmium in the soil through physical, chemical and biological methods, increase or reduce its effectiveness to plants, and thus use plants to repair or control cadmium from entering the food chain. The other is to conduct in-depth research on the molecular mechanism of cadmium absorption and transport in different crops, and use genetic engineering technology to reduce plant absorption of cadmium, or reduce the accumulation of cadmium in the edible parts of crops.

[0003] Solanum nigrum of the Solanaceae family is a cadmium hyperaccumulator. Most of the previous studies on Solanum nigrum's hyperaccumulation and cadmium resistance have focused on Solanum nigrum root secretions, cadmium distribution in Solanum nigrum cells, chelation and compartmentalization, antioxidant system, amino acid metabolism, maintenance of photosynthesis and respiration, and regulation of nitrogen metabolism. These studies are still at the physiological level, and research on the molecular mechanisms of Solanum nigrum's hyperaccumulation and cadmium resistance is still very weak. For example, there are few reports on the cloning and functional studies of genes that absorb and transport cadmium in Solanum nigrum. Summary of the invention

[0004] In order to solve the above technical problems, the present application provides a protein, the amino acid sequence of which is shown in 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 in SEQ ID NO.1.

[0005] The present application also provides a biological material related to the above protein, which is any one of the following A1) to A12):

[0006] A1) a nucleic acid molecule encoding the protein according to claim 1;

[0007] A2) an expression cassette containing the nucleic acid molecule described in A1);

[0008] A3) a recombinant vector containing the nucleic acid molecule described in A1);

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

[0010] A5) a recombinant microorganism containing the nucleic acid molecule described in A1);

[0011] A6) a recombinant microorganism containing the expression cassette described in A2);

[0012] A7) a recombinant microorganism containing the recombinant vector described in A3);

[0013] A8) a recombinant microorganism containing the recombinant vector described in A4);

[0014] A9) a transgenic plant cell line containing the nucleic acid molecule described in A1);

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

[0016] A11) a transgenic plant cell line containing the recombinant vector described in A3);

[0017] A12) A transgenic plant cell line 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 in A1) is the nucleic acid molecule shown in 1) or 2) or 3) below:

[0019] 1) A nucleic acid molecule having a sequence as shown in SEQ ID NO.2;

[0020] 2) a nucleic acid molecule having 90% or more identity with SEQ ID NO.2 and encoding a protein related to cadmium absorption in Solanum nigrum;

[0021] 3) A nucleic acid molecule that hybridizes with the nucleic acid molecule of 1) or 2) under stringent conditions and encodes a protein associated with cadmium absorption in Solanum nigrum.

[0022] The present application also provides a method for reducing the absorption of cadmium or manganese by Solanum nigrum, comprising the step of reducing the content or activity of the above protein in Solanum nigrum. In some embodiments, the step knocks out the gene encoding the above protein.

[0023] The present application also provides a method for increasing the accumulation of cadmium or manganese in plants or microorganisms, comprising the step of overexpressing the above protein in the plants or microorganisms.

[0024] The present application also provides a method for repairing cadmium-contaminated soil, comprising the step of extracting cadmium or manganese pollutants in the soil using plants or microorganisms that hyperaccumulate cadmium or manganese, wherein the plants or microorganisms overexpress the above-mentioned protein.

[0025] In some embodiments, the microorganism is a fungus or a bacterium, or the plant is a monocot or a dicot. In some embodiments, the fungus is a yeast. In some embodiments, the dicot is Solanum nigrum or Arabidopsis thaliana.

[0026] In some embodiments, the protein is overexpressed in the root, stem, leaf, flower, fruit or seed of the plant.

[0027] Beneficial effects: The present invention discloses for the first time that knocking out SaNRAMP5 in Solanum nigrum can significantly reduce the cadmium accumulation in the aboveground part and root system of Solanum nigrum, overexpressing this gene in Arabidopsis thaliana can promote the accumulation of cadmium in the root system of Arabidopsis thaliana, and expressing this gene in yeast can promote the accumulation of cadmium in yeast cells. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0030] Figure 3 : Knocking out SaNRAMP5 in Solanum nigrum significantly reduced cadmium accumulation in the shoots and roots.

[0031] Figure 4 :Overexpression of SaNRAMP5 in Arabidopsis significantly increased the accumulation of root tissue in Arabidopsis root system.

[0032] Figure 5 : Comparison of cadmium and manganese uptake capacity between SaNRAMP5 and OsNRAMP5. DETAILED DESCRIPTION

[0033] In order to specifically illustrate the universal design concept of the present application, specific experimental parameters are used as an example below, but this should not be used as a reason to limit the scope of protection of the present application.

[0034] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

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

[0036] Solanum americanum seeds were sterilized with 30% NaClO by mass ratio, germinated, and cultured for 2 weeks. Seedlings of uniform size were selected and cultured in Hoagland's complete nutrient solution for one week. The roots and leaves were quickly placed in liquid nitrogen for cryopreservation. About 0.1 g of the sample was weighed and crushed with liquid nitrogen. After grinding, it was fully added to a 1.5 ml centrifuge tube, 1 ml of Trizol reagent was quickly added, 0.2 ml of chloroform was added, and the supernatant was aspirated after centrifugation. 0.5 ml of isopropanol was added, and the supernatant was discarded after centrifugation. 70% ethanol was added to wash the precipitate. RNA was dissolved in DEPC water (volume ratio of 1‰), and the RNA quality was detected by agarose gel electrophoresis with a mass ratio of 1.7%, and the concentration and purity of the total RNA were detected by a spectrophotometer. 2 μg of each RNA sample was added with 50 μmol L-1 OligodT18, and 1‰ DEPC water was added to make up to 10 μL. The mixture was water bathed at 70°C for 5 min, placed on ice for 5 min, and RNaseinhibitor was added in sequence. 0.5μL and 5μL of 5xRT buffer, 2.5μL of 10mM dNTP, 1μL of M-MLV reverse transcriptase, make up to 25μL with 1‰ DEPC water, incubate in a 42℃ water bath for 60min, and terminate the reaction in a 70℃ water bath for 10min.

[0037] After the first chain of total cDNA was synthesized by reverse transcription, it was used as a template for PCR amplification. Reaction process: The cDNA amplification reaction of each tissue of Solanum nigrum was carried out in a 96-well PCR plate. The reaction system was 20μl: SYBRmix (2x), 10μl, ROX 0.4μl, 0.4μl of forward primer and reverse primer, 1μl of cDNA template, 7.8μl of sterile water, and a total volume of 20μl. The prepared mixture was mixed thoroughly and distributed in the PCR plate. The reaction conditions are as follows: 95℃5s, 94℃denaturation 30s, 65℃annealing 30s, 72℃extension 30s, 40 cycles, 72℃extension 5min.

[0038] Calculation method: The results were obtained by double ΔΔCt value calculation. By analyzing the expression of this gene in different parts of Solanum nigrum (roots, leaves, stems, flowers and fruits), it was found that the expression level of SaNRAMP5 in the root system was significantly higher than that in other tissues ( Figure 1 ).

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

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

[0041] The PCR program was as follows: 95°C pre-denaturation for 3 min, 95°C denaturation for 30 s, 56°C annealing and 72°C annealing and extension for 1 min 20 s, 35 cycles, 72°C 7 min, the amplified PCR product was detected by 1% agarose gel electrophoresis, and its size was 1605 bp fragment. The target PCR product was separated by agarose electrophoresis and then cut off the gel for recovery. The recovered fragment was mixed with P-easy The blunt vector was connected, and the total volume of the enzyme connection system was 5 μL, including 1 μL of vector and 4 μL of PCR purified product. After adding the sample, the mixture was evenly mixed, centrifuged to the bottom of the tube, placed at 28°C for 15 minutes, and then transformed; the cells were heat-shocked at 42°C and transferred into Escherichia coli DH5α competent cells, and 500 μL of LB liquid medium without antibiotics was added to shake the bacteria for 1 hour, and then centrifuged at low speed to enrich the bacteria, which were spread on LB solid medium containing 100 μg / mL of kana and grown for 12-14 hours. After that, the positive colonies were picked for DNA sequencing. The open reading frame (ORF) of SaNRAMP5 was 1605 bp in length, and the sequence was shown in SEQ ID NO.2; the sequence of its encoded protein was shown in SEQ ID NO.1, indicating the natural resistance-related macrophage protein SaNRAMP5 of Solanum nigrum. The correctly sequenced bacterial solution was added with an equal volume of 30% glycerol and stored at -70°C for use, and the P vector containing the open reading frame of SaNRAMP5 was named SaNRAMP5 inP.

[0042] Example 3 Transgenic yeast overexpressing SaNRAMP5

[0043] The yeast expression vector pDR196 was double-digested with EcoR1 and Xho1 and purified and recovered. The SaNRAMP5 inP plasmid was used as a template and the following primers were used to amplify and purify the PCR product. Finally, the linearized plasmid and the purified PCR product were subjected to homologous recombination reaction: the system was 5 μL, including 1 μL of linearized vector and 4 μL of PCR purified product, and then the reaction was carried out at 37 degrees for 1 hour for transformation, and a single clone was picked for sequencing. The correct single clone was preserved and this plasmid was named SaNRAMP5-pDR196.

[0044] Conclusion: Expression of SaNRAMP5 in the cadmium-sensitive yeast strain ycf1 can significantly increase the sensitivity of yeast cells to cadmium. The reason why yeast cells transformed with SaNRAMP5 are sensitive to cadmium is that excessive cadmium accumulates in yeast cells. Figure 2. Among them, (A) The yeast strain Δycf1 transformed with the empty vector pDR196 or SaNRAMP5 was spotted on SD-Ura medium containing different concentrations of cadmium. The initial concentration of yeast cells was adjusted to OD=0.8, and three concentration gradients were diluted 10 times. The plate was then inverted and cultured at 30°C for 3-5 days. (B) Cd content in yeast Δycf1 cells transformed with the pDR196 empty vector and SaNRAMP5. Data are mean ± SD, n = 5. The lowercase letters above each column indicate statistical differences (P < 0.05, ANOVA). (C) Growth curves of Δycf1 yeast transformed with the pDR196 empty vector and SaNRAMP5 at different time points under 0, 4 and 8 μM CdCl2. Data are mean ± SD, n = 3.

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

[0046] According to the CDS sequence of Solanum nigrum natural resistance-related macrophage protein SaNRAMP5, PCR primers were designed, and the PCR product contained the complete SaNRAMP5 reading frame (excluding the stop codon). The restriction sites Kpn1 and Sal1 were selected according to the final vector, and homologous recombination primers were designed.

[0047] The SaNRAMP5inP plasmid obtained above was used as a template. The PCR procedure was as follows: 95℃ pre-denaturation for 1min, 95℃ denaturation for 30s, 56℃ annealing and 72℃ renaturation extension for 1min20s. After 30 cycles, 72℃ for 5min. The amplified PCR product was detected by 1% agarose gel electrophoresis. The size of the PCR product was about 1600bp. The target PCR product was separated by agarose electrophoresis and then cut and purified. At the same time, the plant overexpression vector pCAMBIA1305 plasmid was double-digested with KpnI and Sal1, and then the purified vectors were recovered and the recovered linearized vector and the target gene fragment were subjected to homologous recombination reaction at 37℃ for one hour. Then the enzyme-linked product was transformed into Escherichia coli DH5a competent cells, smeared on LB solid medium containing kanamycin 50μg / mL and grown for 12h. The positive colonies were picked, the plasmid was extracted, and the fragment size was verified by KpnI and Sal1 digestion. The bacterial solution was subjected to DN A sequencing, add an equal volume of 30% glycerol to the bacterial solution containing the correctly sequenced clones and store it at -70°C, extract the positive clone plasmid and name it 35S:SaNRAMP5-GFP; finally, transform the 35S:SaNRAMP5-GFP plasmid into the competent cells of Agrobacterium tumefaciens EHA105 by electroporation, spread it on YEP solid culture medium containing 50 μg / mL of kanamycin and streptomycin, grow for 48 hours, pick the positive colonies, extract the plasmid, and after verification by double enzyme digestion with KpnI and Sal1, add an equal volume of 30% glycerol to the bacterial solution and store it at -70°C for genetic modification.

[0048] Example 5 Construction of CRISPR vector for knocking out SaNRAMP5 and transgenic Solanum nigrum 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 with the primer set SaNRAMP5TF / R, and then the PCR product was 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 the Agrobacterium-mediated transformation system. Young leaves of Solanum nigrum (SP1102) were used to induce callus tissue and co-cultivated with Agrobacterium. After infection, the callus was placed on a selective medium containing 50 mg / L hygromycin. The selected callus was then transferred to a regeneration medium for development into seedlings. The transgenic line was amplified using the primer set SaNRAMP5gF / R around the target region, and the derived PCR products were sequenced to determine the mutation site of SaNRAMP5. The T2 generation homozygous SaNRAMP5 mutant was used for further experiments.

[0050] The phenotype of the Solanum nigrum SaNRAMP5 knockout mutant is shown in Figure 3 . Among them, (A) Identification of SaNRAMP5 mutants. The upper figure shows the gene structure and mutation site of SaNRAMP5. The solid box and black line represent the exon and intron of SaNRAMP5, respectively. The 20 bp sequence of sgRNA and PAM motif (NGG) are underlined and highlighted in 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 in hydroponic medium containing 0 and 10 μM CdCl2 for 1 day. Cd content in roots and aboveground parts under 0 μM CdCl2 (DE) or 10 μM CdCl2 (FG). Data are the mean ± SD of five replicates. Different letters indicate (P < 0.05, ANOVA) comparison of cadmium content in wild type and transgenic lines. Conclusion: Knocking out this gene can significantly reduce the cadmium accumulation in the shoots and roots of Solanum nigrum.

[0051] Example 6 Transgenic Arabidopsis thaliana overexpressing SaNRAMP5

[0052] The Agrobacterium obtained above and transferred with the 35S: SaNRAMP5-GFP plasmid was used to infect the Arabidopsis Col inflorescence, and then co-cultured for 60 hours, and then the infected Arabidopsis plants were cultivated normally until the seeds were harvested, and the T1 generation seeds were germinated on a 1 / 2MS medium containing 50μg / mL hygromycin for screening. The single plants that can grow normally on the 1 / 2MS medium containing 50μg / mL hygromycin are positive seedlings, and the positive seedlings are tested for overexpression effects by RT-qPCR. The strains with overexpression effects were propagated multiple times, and finally stable genetic homozygous strains were obtained for experiments.

[0053] Figure 4The phenotypes of aNRAMP5-overexpressing transgenic Arabidopsis plants are shown. Among them, the overexpression effect of SaNRAMP5 overexpression lines was identified. The expression levels were relative to Col and set to 1, and AtActin8 was used as an internal reference. (BC) Phenotypes of Col and SaNRAMP5 overexpression lines (OV5, OV11, and OV20) grown in 1 / 2MS medium with 0 and 20 μM CdCl2 for 2 weeks. Bars = 1 cm. (DE) Root length of Col and SaNRAMP5 overexpression lines under 0 and 20 μM CdCl2. Cd content in roots and shoots of Col and SaNRAMP5 overexpression lines under 0 μM CdCl2 (FG) and 20 μM CdCl2 (HI). Data are the mean ± SD of five replicates. Different letters indicate significant differences in Cd content between wild-type and SaNRAMP5 transgenic plants (P < 0.05, ANOVA). Conclusion: SaNRAMP5 was overexpressed in Arabidopsis and three homozygous strains with overexpression effect (OV5, OV11 and OV20) were obtained. When cultured on a cadmium-free medium, there was no significant difference in the growth of the wild-type and overexpression strains. However, when cultured on a medium containing 20 μM CdCl2, the root growth of the transgenic strains was inhibited, and the root length was significantly shorter than that of the wild type. Root cadmium content determination showed that more cadmium accumulated in the roots of the transgenic strains, which led to the stunted root growth.

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

[0055] SaNRAMP5 affects the ability of organisms to absorb cadmium and manganese, especially has a significant effect on the ability of organisms to absorb cadmium. To prove this, this example compares the effects of SaNRAMP5 and OsNRAMP5 on the absorption capacity of cadmium and manganese. Figure 5As shown. Among them, (A) Δycf1 yeast cells expressing pDR196 empty vector, SaNRAMP5 or OsNRAMP5 were spotted on SD-Ura medium supplemented with different Cd concentrations. (B) Δsmf1 yeast cells expressing pDR196 empty 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 pDR196 empty vector, SaNRAMP5 or OsNRAMP5. Data are expressed as mean ± standard deviation, n = 5. Different letters indicate that there are significant differences in the contents of cadmium and manganese between SaNRAMP5, OsNRAMP5 and pDR196 empty vector (P < 0.05, ANOVA). Using GAPDH as an internal reference, the protein expression levels of SaNRAMP5 and OsNRAMP5 in yeast strains Δycf1 (E) and Δsmf1 (F) were detected, respectively.

[0056] It can be seen that although the effect of SaNRAMP5 on manganese absorption capacity is not as good as that of OsNRAMP5, the effect of SaNRAMP5 on cadmium absorption capacity is significantly higher than that of OsNRAMP5, indicating that there is functional differentiation between SaNRAMP5 and OsNRAMP5 in the selective absorption of cadmium and manganese.

[0057] Obviously, those skilled in the art can make various changes and modifications to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and variations.

Claims

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

1.

2. The biological material related to the protein according to claim 1, characterized by any one of the following A1) to A12): A1) a nucleic acid molecule encoding the protein according to claim 1; A2) an expression cassette containing the nucleic acid molecule described in A1); A3) a recombinant vector containing the nucleic acid molecule described in A1); A4) a recombinant vector containing the expression cassette described in A2); A5) a recombinant microorganism containing the nucleic acid molecule described in A1); A6) a recombinant microorganism containing the expression cassette described in A2); A7) a recombinant microorganism containing the recombinant vector described in A3); A8) a recombinant microorganism containing the recombinant vector described in A4); A9) a transgenic plant cell line containing the nucleic acid molecule described in A1); A10) a transgenic plant cell line containing the expression cassette described in A2); A11) a transgenic plant cell line containing the recombinant vector described in A3); A12) A transgenic plant cell line containing the recombinant vector described in A4).

3. The biomaterial according to claim 2, characterized in that A1) The nucleic acid molecule is the nucleic acid molecule shown in 1) or 2) or 3) below: 1) A nucleic acid molecule having a sequence as shown in SEQ ID NO.2; 2) a nucleic acid molecule having 90% or more identity with SEQ ID NO.2 and encoding a protein related to cadmium absorption in Solanum nigrum; 3) A nucleic acid molecule that hybridizes with the nucleic acid molecule of 1) or 2) under stringent conditions and encodes a protein associated with cadmium absorption in Solanum nigrum.

4. A method for reducing the absorption of cadmium or manganese by Solanum nigrum, characterized in that: The method comprises the step of reducing the content or activity of the protein according to claim 1 in Solanum nigrum.

5. The method according to claim 4, characterized in that: The step knocks out the gene encoding the protein according to claim 1.

6. A method for increasing the accumulation of cadmium or manganese in plants or microorganisms, characterized in that: The method comprises the step of overexpressing the protein according to claim 1 in the plant or microorganism.

7. A method for repairing cadmium-contaminated soil, characterized in that: The method comprises the steps of extracting cadmium or manganese pollutants in soil by using plants or microorganisms that hyperaccumulate cadmium or manganese, wherein the protein of claim 1 is overexpressed in the plants or microorganisms.

8. The method according to claim 6 or 7, characterized in that The microorganism is a fungus or a bacterium, or the plant is a monocotyledon or a dicotyledon.

9. The method according to claim 8, characterized in that The fungus is yeast, or the dicotyledonous plant is Solanum nigrum or Arabidopsis thaliana.

10. The method according to any one of claims 6 to 9, characterized in that: The method comprises overexpressing the protein of claim 1 in the root, stem, leaf, flower, fruit or seed of the plant.

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