Application of PvFIP37 protein and related biological materials thereof in regulation and control of cadmium tolerance of plants

Abstract

The invention discloses application of PvFIP37 protein and related biological materials thereof in regulation and control of cadmium tolerance of plants. According to the invention, the PvFIP37 gene is cloned from switchgrass, and the PvFIP37 overexpressed switchgrass is successfully constructed. The analysis on the phenotype of the PvFIP37 overexpressed switchgrass under cadmium stress finds that compared with wild type switchgrass, the cadmium tolerance of the switchgrass can be enhanced by increasing the expression quantity of the PvFIP37 gene, which indicates that the PvFIP37 has the function of regulating and controlling the cadmium tolerance of plants. The switchgrass PvFIP37 gene provided by the invention can be used as an excellent gene resource, is widely applied to the field of genetic breeding of gramineous grass or other crops, and has important significance on understanding of tolerance of plants under heavy metal stress.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to application of PvFIP37 protein and related biological materials in regulating plant cadmium tolerance. Background Art

[0002] N 6 -methyladenine (m 6 A) is the most common post-transcriptional modification of mRNA in eukaryotes. The marker exists in the 5'UTR, 3'UTR and coding region of eukaryotes. It can directly affect the abundance and function of RNA without changing the nucleotide sequence. It plays a key role in plant growth and development and in response to various environmental stresses.

[0003] Switchgrass (Panicum virgatum L.) is a perennial C4 herbaceous plant for cellulosic ethanol conversion. It has a clear genetic structure and an allotetraploid genome. A comprehensive multi-omics database and a stable genetic transformation system have been established. It is worth noting that it has strong tolerance to abiotic stresses. Compared with food crops, switchgrass reduces the risk of pollutants entering the food chain. Studies have shown the ability to maintain yield in the presence of several metals (including zinc, lead, copper and cadmium). However, higher concentrations of stress will still lead to a decrease in its yield, affecting overall productivity. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to regulate plant cadmium tolerance and cultivate cadmium-resistant plant varieties.

[0005] In order to solve the above technical problems, the present invention first provides a new use of PvFIP37 protein.

[0006] The present invention provides the use of PvFIP37 protein in any of the following A1)-A3):

[0007] A1) Regulating plant cadmium tolerance;

[0008] A2) breeding transgenic plants with improved cadmium tolerance;

[0009] A3) Plant breeding;

[0010] The PvFIP37 protein is any one of the following B1)-B4):

[0011] B1) The amino acid sequence is the protein shown in SEQ ID NO: 2;

[0012] B2) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 2;

[0013] B3) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 2;

[0014] B4) A protein that has 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and has the same function.

[0015] In the protein described in B2) above, the tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The tag includes but is not limited to: GST (glutathione sulfhydryl transferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0016] In the protein described in B3) above, the substitution and / or deletion and / or addition of one or several amino acid residues is a substitution and / or deletion and / or addition of no more than 10 or 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 amino acid residues.

[0017] In the protein described in B4) above, the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as a matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default value) respectively, and searching for the identity of a pair of amino acid sequences for calculation, the value of the identity (%) can be obtained. The identity includes amino acid sequences having 80% or more, or 85% or more, or 90% or more, or 91% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more homology with the amino acid sequence shown in Sequence 2 of the present invention.

[0018] The proteins described in B1)-B4) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0019] In order to solve the above technical problems, the present invention also provides new uses of biomaterials related to the above PvFIP37 protein.

[0020] The present invention provides the use of the biological material related to the above-mentioned PvFIP37 protein in any of the following A1)-A3):

[0021] A1) Regulating plant cadmium tolerance;

[0022] A2) breeding transgenic plants with improved cadmium tolerance;

[0023] A3) Plant breeding;

[0024] The biological material is a nucleic acid molecule encoding the above-mentioned PvFIP37 protein or an expression cassette, a recombinant vector or a recombinant microorganism containing the nucleic acid molecule;

[0025] In the above application, the nucleic acid molecule is any one of the following:

[0026] F1) The DNA molecule shown in Sequence 1 or Sequence 3;

[0027] F2) A DNA molecule that has 75% or more identity with the nucleotide sequence defined in F1) and encodes the above-mentioned PvFIP37 protein.

[0028] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0029] A person skilled in the art can easily mutate the nucleotide sequence encoding the PvFIP37 protein of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequence encoding the PvFIP37 protein are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the PvFIP37 protein and have the same function.

[0030] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or higher identity to the nucleotide sequence of a protein consisting of the amino acid sequence shown in the coding sequence 2 of the present invention. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0031] The above 75% or more identity can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity.

[0032] In the above application, the expression cassette refers to a DNA capable of expressing the PvFIP37 protein in a host cell, and the DNA may include not only a promoter for initiating transcription of PvFIP37, but also a terminator for terminating transcription of PvFIP37. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to, constitutive promoters; tissue, organ and development-specific promoters and inducible promoters. Suitable transcription terminators include, but are not limited to, Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and nopaline and octopine synthase terminators.

[0033] In the above application, the vector refers to a vector that can carry the above nucleic acid molecule into the host cell for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmid, phage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e., cosmid), Ti plasmid, viral vector (such as retrovirus (including lentivirus), adenovirus, adeno-associated virus, etc.).

[0034] The recombinant vector refers to a recombinant DNA molecule constructed by connecting the above nucleic acid molecule and the vector in vitro. The recombinant vector containing the PvFIP37 gene expression cassette can be constructed using an existing plant expression vector. The plant expression vector includes a binary Agrobacterium vector and a vector that can be used for plant microprojectile bombardment, etc. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb, etc. The plant expression vector may also contain the 3′ non-translated region of the foreign gene, i.e., a polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor, such as the non-translated region transcribed at the 3' end of the Agrobacterium crown gall induction (Ti) plasmid gene (such as the nopaline synthase gene Nos) and the plant gene (such as the soybean storage protein gene) all have similar functions. When using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive and can be natural or synthetic. The translation start region can come from the transcription start region or the structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as nptII gene that confers resistance to kanamycin and related antibiotics, bar gene that confers resistance to the herbicide phosphinothricin, hph gene that confers resistance to the antibiotic hygromycin, dhfr gene that confers resistance to methotrexate, EPSPS gene that confers resistance to glyphosate) or chemical resistance marker genes (such as herbicide resistance genes), mannose-6-phosphate isomerase gene that provides the ability to metabolize mannose. Considering the safety of transgenic plants, no selective marker gene can be added, and transformed plants can be directly screened by adversity.

[0035] In the above application, the microorganism can be yeast, bacteria, algae or fungi. The bacteria can be Agrobacterium, such as Agrobacterium EHA105.

[0036] The recombinant microorganism refers to a recombinant microorganism with changed functions obtained by operating and modifying the genes of the target microorganism. For example, the recombinant microorganism obtained by introducing the above-mentioned recombinant vector into the target microorganism. The recombinant microorganism can be understood to refer not only to a specific recombinant microorganism, but also to the offspring of such a cell, and due to natural, accidental or intentional mutations and / or changes, the offspring may not be completely identical to the original parent cell, but is still included in the scope of the recombinant microorganism.

[0037] In the above application, the regulation of plant cadmium tolerance is to improve plant cadmium tolerance. The improvement of plant cadmium tolerance is reflected in that when the content and / or activity of PvFIP37 protein in the plant is increased, the cadmium tolerance of the plant is improved.

[0038] In some embodiments, the improvement of plant cadmium tolerance is specifically manifested as: when the expression level of the PvFIP37 gene in the plant is increased, the leaves of the plant grow better after cadmium stress, the degree of root tip cell death or damage is lighter, the superoxide dismutase activity in the leaves is higher, the peroxidase activity in the leaves is higher, the catalase activity in the leaves is higher, and the malondialdehyde content in the leaves is lower.

[0039] In the above application, the purpose of plant breeding is to cultivate cadmium-resistant plant varieties.

[0040] In order to solve the above technical problems, the present invention finally provides a method for cultivating transgenic plants with improved cadmium tolerance.

[0041] The method for cultivating transgenic plants with improved cadmium tolerance provided by the present invention comprises the following steps: increasing the content and / or activity of the PvFIP37 protein in the target plant to obtain a transgenic plant; the cadmium tolerance of the transgenic plant is higher than that of the target plant.

[0042] In the above method, the cadmium tolerance of the transgenic plant is higher than that of the target plant, which is embodied in any one of the following N1)-N6):

[0043] N1) under cadmium stress, the growth of leaves of the transgenic plant is better than that of the target plant;

[0044] N2) under cadmium stress, the death or damage degree of the root tip cells of the transgenic plant is less than that of the target plant;

[0045] N3) under cadmium stress, the superoxide dismutase activity in the leaves of the transgenic plant is higher than that of the target plant;

[0046] N4) under cadmium stress, the peroxidase activity in the leaves of the transgenic plant is higher than that of the target plant;

[0047] N5) under cadmium stress, the catalase activity in the leaves of the transgenic plant is higher than that of the target plant;

[0048] N6) Under cadmium stress, the malondialdehyde content in the leaves of the transgenic plant is lower than that of the target plant.

[0049] Furthermore, the cadmium stress is CdCl2 stress.

[0050] Furthermore, the cadmium stress condition may be CdCl2 stress for 10 weeks.

[0051] In the above method, the method for increasing the content and / or activity of the above PvFIP37 protein in the target plant is to overexpress the above PvFIP37 protein in the target plant.

[0052] Furthermore, the overexpression method is to introduce the coding gene of the above-mentioned PvFIP37 protein into the target plant.

[0053] Furthermore, the nucleotide sequence of the gene encoding the PvFIP37 protein is shown in Sequence 1.

[0054] In any of the above applications or methods, the transgenic plants are understood to include not only the first generation transgenic plants obtained by transforming the PvFIP37 gene into a recipient plant, but also its progeny. For transgenic plants, the gene can be propagated in the species, or the gene can be transferred into other varieties of the same species using conventional breeding techniques, especially including commercial varieties. The transgenic plants include seeds, callus, complete plants and cells.

[0055] In any of the above applications or methods, the plant is a monocot or a dicot. Further, the monocot may be switchgrass. Further, the switchgrass is Alamo.

[0056] The present invention cloned the PvFIP37 gene from switchgrass and successfully constructed PvFIP37 overexpression switchgrass. By analyzing the phenotype of PvFIP37 overexpression switchgrass under cadmium stress, it was found that compared with wild-type switchgrass, the PvFIP37 overexpression switchgrass strain had better leaf growth, less death or damage to root tip cells, higher superoxide dismutase activity in the leaves, higher peroxidase activity in the leaves, higher catalase activity in the leaves, and lower malondialdehyde content in the leaves. This shows that the cadmium tolerance mediated by PvFIP37 has a strong reactive oxygen scavenging ability, and reduces oxidative stress through the scavenging effect of hydrogen peroxide. The switchgrass PvFIP37 gene provided by the present invention can be used as an excellent gene resource, widely used in the genetic breeding field of grasses or other crops of the Gramineae family, and is of great significance for plant restoration of cadmium-contaminated soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is the agarose gel electrophoresis diagram of the cloning of the switchgrass PvFIP37 gene.

[0058] Figure 2 This is the electrophoresis diagram for the identification of the PvFIP37 overexpression vector.

[0059] Figure 3 This is a diagram of genetic transformation and screening of transgenic switchgrass.

[0060] Figure 4 This is a graph showing the gene expression level detection in the PvFIP37 overexpressing switchgrass strain.

[0061] Figure 5 PvFIP37 overexpression switchgrass strain m 6 A Modification level detection diagram.

[0062] Figure 6 The phenotype of PvFIP37 overexpressing switchgrass strains under cadmium stress. Among them, WT is the wild-type plant, and OE#4 and OE#5 are PvFIP37 overexpressing switchgrass strains.

[0063] Figure 7 This is the potassium iodide (PI) staining phenotype of cells of the PvFIP37 overexpressing switchgrass strain.

[0064] Figure 8 This is the detection chart of superoxide dismutase, peroxidase, catalase and malondialdehyde indicators in the PvFIP37 overexpressing switchgrass strain. DETAILED DESCRIPTION

[0065] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0066] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0067] The switchgrass (Alamo) in the following examples is described in the document “Liu Y, Wang K, Li D, Yan J, Zhang W. Enhanced Cold Tolerance and Tillering in Switchgrass (Panicum virgatum L.) by Heterologous Expression of Osa-miR393a. Plant Cell Physiol. 2017 Dec 1; 58(12): 2226-2240. doi: 10.1093 / pcp / pcx157. PMID: 29069481.”

[0068] The plant binary expression vector pCAMBIA1307 in the following example is described in the document “(Yu, Q., Liu, S., Yu, L. et al. RNA demethylation increases the yield and biomass of rice and potato plants in field trials. Nat Biotechnol. 39, 1581–1588 (2021)”.

[0069] The preparation method of the YEP liquid culture medium in the following examples is as follows: 5 g of yeast extract, 10 g of tryptone, 10 g of sodium chloride and 1 L of deionized water are mixed.

[0070] The preparation method of the MP liquid culture medium in the following example is as follows: 4.428 g of Ms519 (PhytoTech Lab, Lot: HMW0519379A), 30 g of maltose, 5 mg of 2,4-D, and 1 mg of 6-BA are mixed with 1 L of deionized water, the pH of the culture medium is adjusted to 5.4 with NaOH, and sterilized at 121°C for 20 min.

[0071] The preparation method of the MP recovery medium in the following example is as follows: 4.428 g of Ms519, 30 g of maltose, 5 mg of 2,4-D, 1 mg of 6-BA, and 4 g of plant gel are mixed with 1 L of deionized water, the pH of the medium is adjusted to 5.8 with NaOH, and sterilized at 121°C for 20 min. After the temperature of the medium is cooled to about 60°C, sterile timentin is added to a concentration of 250 mg / L, and the mixture is fully mixed and poured into a sterile culture dish for later use.

[0072] The preparation method of the MP1 screening medium in the following example is as follows: 4.428 g of Ms519, 30 g of maltose, 5 mg of 2,4-D, 1 mg of 6-BA, and 4 g of plant gel are mixed with 1 L of deionized water, the pH of the medium is adjusted to 5.8 with NaOH, and sterilized at 121 ° C. for 20 min. After the culture medium is cooled to about 60 ° C, sterile hygromycin and timentin are added to a concentration of 50 mg / L and 150 mg / L, respectively, and the mixture is fully mixed and poured into a sterile culture dish for use.

[0073] The preparation method of the MP2 screening medium in the following example is as follows: 4.428 g of Ms519, 30 g of maltose, 5 mg of 2,4-D, 1 mg of 6-BA, 4 g of plant gel and 1 L of deionized water are mixed, the pH of the medium is adjusted to 5.8 with NaOH, and sterilized at 121°C for 20 min. After the temperature of the medium is cooled to about 60°C, sterile hygromycin and timentin are added to make their concentrations 100 mg / L and 150 mg / L, respectively, and the mixture is fully mixed and poured into a sterile culture dish for later use.

[0074] The preparation method of the REG differentiation medium in the following embodiment is as follows: 4.428g of Ms519, 30g of maltose, 5mg of 2,4-D, 1mg of 6-BA, 4g of plant gel and 1L of deionized water are mixed, the pH of the medium is adjusted to 5.8 with NaOH, and sterilized at 121°C for 20min. After the temperature of the medium is cooled to about 60°C, sterile hygromycin and timentin are added to make their concentrations 20mg / L and 150mg / L, respectively, and the mixture is fully mixed and poured into a sterile culture dish for later use.

[0075] The preparation method of the MS rooting medium in the following embodiment is as follows: 4.428g of Ms513 (PhytoTech Lab, Lot: HYW0513016A), 30g of maltose, 5mg of 2,4-D, 1mg of 6-BA, 4g of plant gel and 1L of deionized water are mixed, the pH of the medium is adjusted to 5.8 with NaOH, and sterilized at 121°C for 20min. After the culture medium is cooled to about 60°C, sterile hygromycin and timentin are added to their concentrations of 50mg / L and 150mg / L, respectively, and the mixture is fully mixed and poured into a sterile culture dish for later use.

[0076] Example 1: Cloning of the switchgrass PvFIP37 gene

[0077] 1. Obtaining cDNA

[0078] HiPure Plant RNA Mini Kit (dual-column method) was used to extract total RNA from switchgrass, and the total RNA was reverse transcribed into cDNA using PrimeScript TMRT reagent Kit with gDNA Eraser (Perfect RealTime) (Takara, Japan).

[0079] 2. PCR amplification

[0080] The switchgrass cDNA obtained in step 1 was diluted 10 times and used as a template. PCR amplification was performed using primers PvFIP37-F1 and PvFIP37-R1 to obtain a PCR product, which is the ORF region of the PvFIP37 gene. The primer sequences are as follows:

[0081] PvFIP37-F1: 5'-ATGAACACGGATCCAGGCGAGA-3';

[0082] PvFIP37-R1: 5'-TCAACTTTCCACTTTGACTTCCA-3'.

[0083] The PCR reaction system was as follows: 5 μL template cDNA, 25 μL 2×Gflex PCR Buffer, 1 μL Tks GflexDNA Polymerase, 1.5 μL upstream primer, 1.5 μL downstream primer, and 16 μL ddH2O.

[0084] The PCR reaction program was as follows: 94°C for 1 min; 98°C for 10 s, 60°C for 15 s, 68°C for 45 s, 35 cycles; 16°C+∞.

[0085] 3. Electrophoresis detection of PCR products

[0086] The PCR products were detected by 1% agarose gel electrophoresis (120V, 20min), and then the bands were observed using a gel imaging system. Figure 1 shown.

[0087] 4. PCR product sequencing

[0088] 1) Use a blade to cut the agarose gel containing the target band under the UV transmission gel cutting table and place it in a pre-prepared centrifuge tube, and refer to the steps in the Magen kit manual to recover the gel.

[0089] 2) Take 5 μL of gel recovery product and mix it with 1 μL 6× Loading Buffer, then use 1% agarose gel electrophoresis to detect the recovery. If the band brightness is appropriate, prepare the ligation reaction system according to the pTOPO001 Blunt Simple Cloning Kit (TC602) of Jinsha, react, and obtain the ligation product.

[0090] The ligation reaction system was as follows: 1 μL gel recovery product, 1 μL pTOPO001 Blunt Simple Vector, 1 μL 10×Topo Buffer, and 7 μL ddH2O.

[0091] 3) The ligation product was transformed into E. coli DH5α. The specific steps are as follows: add 10 μL of the reaction solution to 100 μL of E. coli competent cells, mix gently, and then place in ice for 5 minutes; place in a 42°C water bath for 45 seconds, and then quickly transfer to ice for 2 minutes; add 500 μL LB liquid culture medium (5 g / L Yeast extract, 10 g / L Tryptone, 10 g / L NaCl), place on a constant temperature culture shaker, 37°C, 220 rpm, shake and culture for 20 minutes to obtain the transformation solution.

[0092] 4) Centrifuge the transformation solution, remove a small amount of LB culture medium on the top layer, use a pipette to blow and mix the remaining transformation solution, take 100 μL of the transformation solution and apply it on LB solid culture medium containing Amp (5 g / L Yeast extract, 10 g / L Tryptone, 10 g / L NaCl, 15 g / L Agar), seal it with sealing film, and invert it in a 37°C constant temperature incubator for overnight culture.

[0093] 5) The next day, select 6 monoclonal plaques of positive clones on the LB solid plate and place them in 3 mL of LB liquid culture medium containing Amp resistance, and culture them at 37°C with shaking at 220 rpm for 16 h; then extract the plasmid. For specific steps, refer to the instructions of the Magen kit.

[0094] 6) Using the extracted plasmid as a template, PCR amplification was performed using the universal primers M13F and M13R of the T vector to obtain a PCR product, and the size of the inserted target gene was verified by PCR reaction.

[0095] The PCR reaction system was as follows: 0.5 μL plasmid template, 8.7 μL ddH2O, 0.4 μL upstream M13 primer, 0.4 μL downstream M13 primer, and 10 μL 2×Taq PCR StarMix (Dye).

[0096] The PCR reaction program was as follows: 94°C for 2 min; 94°C for 30 s, 60°C for 30 s, 72°C for 90 s, 35 cycles; 72°C for 5 min; 16°C+∞.

[0097] M13F: 5'-GTTGTAAAACGACGGCCAG-3';

[0098] M13R: 5'-CAGGAAACAGCTATGAC-3'.

[0099] 7) After the PCR reaction is completed, the PCR product is spotted on a 1% agarose gel and detected by electrophoresis (120V, 15min). The inserted target band is observed on a gel imager to see if it is correct. 10μL of the correct plasmid in the band is taken and sent to a biological company for sequencing. According to the sequencing results, the correct plasmid is stored in a -20℃ refrigerator.

[0100] The sequencing results show that the nucleotide sequence of the ORF region of the switchgrass PvFIP37 gene is shown in Sequence 1 in the sequence listing, and the amino acid sequence of the PvFIP37 protein encoded by the switchgrass PvFIP37 gene is shown in Sequence 2 in the sequence listing.

[0101] Example 2. Preparation of PvFIP37-overexpressing switchgrass

[0102] 1. Construction of PvFIP37 overexpression vector

[0103] Using homologous recombination method, the ORF region of switchgrass PvFIP37 gene was constructed into the plant binary expression vector pCAMBIA1307 between PstI and BamHI restriction sites using Uniclone One Step Seamless Cloning Kit (SC612) to obtain the PvFIP37 overexpression vector pCAMBIA1307-PvFIP37.

[0104] To further verify the connection of the PvFIP37 overexpression vector pCAMBIA1307-PvFIP37, PCR verification was performed using primers on the vector. The verification results showed that the target fragment had been connected to the vector and the band position was correct ( Figure 2 ), indicating that the plant expression vector connected with the switchgrass PvFIP37 gene was successfully constructed.

[0105] The PCR identification reaction system was as follows: 0.5 μL recombinant plasmid template, 8.7 μL ddH2O, 0.4 μL upstream ZmUbiF primer, 0.4 μL downstream Cas9R primer, and 10 μL 2×Taq PCR StarMix (Dye).

[0106] The PCR identification reaction program was as follows: 94°C for 2 min; 94°C for 30 s, 55°C for 30 s, 72°C for 90 s, 35 cycles; 72°C for 5 min; 16°C+∞.

[0107] ZmUbiF: 5'-AGCCCTGCCTTCATACGCTA-3';

[0108] Cas9R: 5'-CCGATCTAGTAACATAGATGACACC-3'.

[0109] 2. Preparation of transgenic plants

[0110] 1. Preparation of recombinant Agrobacterium

[0111] The PvFIP37 overexpression vector pCAMBIA1307-PvFIP37 was transferred into the competent cells of Agrobacterium EHA105 by freeze transfer to obtain the recombinant Agrobacterium pCAMBIA1307-PvFIP37 / EHA105. The specific steps are as follows:

[0112] 1) Take out the Agrobacterium EHA105 competent cells stored in a -80°C refrigerator, thaw them on ice, gently add the recombinant plasmid (pCAMBIA1307-PvFIP37) to the center of the freshly thawed competent cells, place them in an ice bath for 30 minutes, quickly freeze them in liquid nitrogen for 1 minute, heat shock the competent cells in a water bath at 37°C for 5 minutes, and quickly transfer them to ice and let them stand for 2 minutes.

[0113] 2) Add 500 μL YEP liquid medium to the transformed competent cells, mix thoroughly by inversion, and place on a shaker at 28°C and 220 rpm for 2 h.

[0114] 3) Take 200 μL of the bacterial solution and spread it on a YEP plate containing 100 mg / L kanamycin and 50 mg / L rifampicin, invert and culture at 28°C in the dark for 2 days, pick a single colony for colony PCR detection, and the test result is positive, indicating that pCAMBIA1307-PvFIP37 has been successfully introduced into Agrobacterium, and the Agrobacterium bacterial solution is preserved.

[0115] 2. Agrobacterium-mediated genetic transformation of switchgrass callus

[0116] Agrobacterium-mediated genetic transformation of switchgrass callus tissue was used to obtain regenerated seedlings transformed with pCAMBIA1307-PvFIP37 ( Figure 3 ), the specific method is as follows:

[0117] 1) Select the best growing area with a size of about 2×2mm 2The switchgrass (Alamo) callus tissue was placed in a 50 mL sterile centrifuge tube, and 10 mL of 3% maltose solution containing 300 μM glutamine was added, and the tube was placed in an ice bath for 20 min, and the maltose solution was discarded; 10 mL of the prepared Agrobacterium bacterial solution was added to the centrifuge tube, and 100 μM acetosyringone and 300 μM glutamine were added accordingly; vacuum was evacuated for 10 min, and the culture was gently shaken for 20 min at 28°C; the bacterial solution was discarded, and the infected callus tissue was placed on 3 layers of sterile filter paper and dried in a clean bench for 2 h; the callus tissue was moved to 2 layers of sterile filter paper, and 500 μL of MP liquid culture medium containing 300 μM glutamine and 100 μM acetosyringone was added, and the culture was darkly cultured at 26°C for 2 d.

[0118] 2) The co-cultured callus tissue was transferred to MP recovery medium containing 250 mg / L timentin and cultured in the dark at 26°C for 1 week.

[0119] 3) The callus was transferred to MP1 screening medium containing 50 mg / L hygromycin and 150 mg / L timentin and cultured in the dark at 26°C for 2 weeks.

[0120] 4) The callus tissue was transferred to MP2 screening medium containing 100 mg / L hygromycin and 150 mg / L timentin and cultured in the dark at 26°C for 2 weeks.

[0121] 5) The resistant callus was transferred to REG differentiation medium containing 20 mg / L hygromycin and 150 mg / L timentin and cultured at 26°C under light for 4 weeks.

[0122] 6) The differentiated seedlings were transferred to MS rooting medium containing 50 mg / L hygromycin and 150 mg / L timentin for rooting, and the resistant regenerated seedlings were further selected.

[0123] 7) Transplantation can be carried out when the roots grow to more than 1 cm. After hardening the seedlings in the culture room for 2 days, transplant them into pots filled with mixed nutrient soil with humus, black soil and vermiculite in a ratio of 1:1:1 and culture them in the greenhouse.

[0124] 3. Identification of transgenic plants

[0125] 1. Transgenic positive plants

[0126] The genomic DNA of the transgenic plants was extracted, and PCR verification was performed using primers for the selection marker gene Hpt and gene PvFIP37 on the vector to obtain transgenic positive plants. The primer sequences are as follows:

[0127] PvFIP37-F3: 5'-ATGAACACGGATCCAGGCG-3';

[0128] PvFIP37-R3: 5'-GGATCAAGCAACAATCTCCTGT-3'.

[0129] 2. Select about 100 mg of leaves of transgenic positive plants with consistent growth, grind them in liquid nitrogen, extract total RNA according to the Trizol method, and verify them by agarose gel electrophoresis to obtain 28S and 18S complete high-quality RNA. The RNA concentration and purity were measured by Nanodrop2000. OD260 / OD280 were both between 1.8-2.0, and OD260 / OD230 were both >2.0, which proved that the obtained RNA was of good quality and could be used for the synthesis of the first chain of cDNA. Then, 1 μg of the above RNA sample was used as a template to synthesize cDNA using the PrimeScript RT reagent Kit reverse transcription kit of TaKaRa Company, and then qRT-PCR analysis was performed using cDNA as a template. At the same time, UBI was used as an internal reference gene to detect the expression level of the PvFIP37 gene in different plants. The primer sequences are as follows:

[0130] PvFIP37-F: 5'-GCAAGAACGCAATGCTGGTGTG-3';

[0131] PvFIP37-R: 5'-CTGAGGAGGAGAGCGAGGATGAC-3';

[0132] qPvUBI-F: 5'-TTCGTGGTGGCCAGTAAG-3';

[0133] qPvUBI-R: 5'-AGAGACCAGAAGACCCAGGTACAG-3'.

[0134] The results of RT-qPCR analysis are shown in Figure 4 As shown, the results showed that the expression levels of PvFIP37 in the transgenic switchgrass positive lines OE#4 and OE#5 transformed with pCAMBIA1307-PvFIP37 were significantly higher than those in the wild-type switchgrass, and could be used for the following phenotypic observations.

[0135] 3. Extract RNA from wild-type switchgrass Alamo and transgenic switchgrass lines OE#4 and OE#5, and detect m 6 A modification level. The specific steps are as follows:

[0136] 1) Extract the total RNA sample from the -80℃ freezer and dilute it in an appropriate ratio. Then, incubate the diluted RNA in a 65℃ metal bath for 5 minutes to release the secondary structure of the RNA. After the treatment, quickly transfer the RNA sample to an ice box for cooling.

[0137] 2) 2 μL RNA solution was added dropwise onto the surface of the nylon membrane. Next, the membrane was placed in a UV crosslinker and subjected to 245 nm UV crosslinking treatment for three times.

[0138] 3) Wash the nylon membrane with Tris-buffered saline and Tween 20 (TBST) solution for 5 minutes to remove unbound RNA residues.

[0139] 4) The membrane was blocked with TBST solution containing 5% skimmed milk powder for 1 hour.

[0140] 5) Discard the blocking solution and mix the membrane with the diluted m 6 A primary antibody (1:2000) and then incubated overnight at 4°C in a chromatography cabinet.

[0141] 6) On the next day, the membrane was washed three times with TBST solution, each washing time was 10 minutes.

[0142] 7) The membrane was mixed with horseradish peroxidase (HRP)-labeled secondary antibody (1:5000) and then incubated at room temperature for 1 hour.

[0143] 8) The membrane was washed three times with TBST solution again, each washing time was 10 minutes. During the last washing, a luminescent solution was prepared.

[0144] 9) After washing, evenly cover the membrane surface with ECL working solution. Then, use a chemiluminescence imager to expose and photograph the membrane.

[0145] The results showed that the m 6 The A modification level was significantly increased, further proving that PvFIP37 is a switchgrass m 6 A methyltransferase ( Figure 5 ).

[0146] Example 3: Phenotypic analysis of PvFIP37 overexpressing switchgrass under cadmium stress

[0147] Test materials: wild-type switchgrass Alamo WT#1 and WT#2, and PvFIP37 overexpressing switchgrass lines OE#4 and OE#5.

[0148] Experimental method: The seeds of the test materials were planted according to the conventional method, and 250 μM cadmium stress (CdCl2) was applied when they grew to the flowering stage. The specific treatment method was as follows: the switchgrass in the flowering stage was treated in the nutrient soil (humus, black soil, vermiculite ratio of 1:1:1) containing 250 μM CdCl2 for 10 weeks, and at the same time, it was treated in the nutrient soil containing 0 μM CdCl2 for 10 weeks. 2的 The cultured plants were cultured in nutrient soil for 10 weeks as a control. After 10 weeks of treatment, phenotypic observations were made and the following traits were statistically analyzed: potassium iodide (PI) staining of root tip cells, superoxide dismutase activity, peroxidase activity, catalase activity and malondialdehyde content.

[0149] The method of potassium iodide staining of root tip cells includes the following steps: 5 mm long root tips of switchgrass from the control group (CK) and the cadmium (Cd) treatment group were cut, immersed in a 10 μg / mL potassium iodide (PI) solution (Solarbio, CA1630) for 15 minutes, and then rinsed three times with distilled water. Potassium iodide (PI) fluorescence signals were observed using a confocal laser scanning microscope (Nikon TE2000-E). Generally speaking, when potassium iodide (PI) staining is performed, only the cell wall of living cells can be stained, while in slightly damaged cells, both the cell wall and the cell nucleus are stained (the cell outline can be outlined), while severely damaged cells usually only have the cell nucleus stained, and the complete cell outline cannot be seen.

[0150] The detection method of superoxide dismutase activity, peroxidase activity, catalase activity and malondialdehyde content comprises the following steps: taking 0.1 g of switchgrass leaves from the control group (CK) and the cadmium (Cd) treatment group plants, respectively, and using a superoxide dismutase activity detection kit (Solarbio, BC0175), a peroxidase activity detection kit (Solarbio, BC0095), a catalase activity detection kit (Solarbio, BC0205) and a malondialdehyde content detection kit (Solarbio, BC0020), respectively, to perform detection and analysis of each index according to the manufacturer's instructions.

[0151] Phenotypic observations Figure 6 As shown, the results showed that after 10 weeks of cadmium stress treatment, PvFIP37 overexpressing switchgrass lines OE#4 and OE#5 showed better leaf growth and less wilting and yellowing compared with WT plants.

[0152] The results of potassium iodide staining of root tip cells are as follows Figure 7 As shown, the results showed that after 10 weeks of cadmium stress treatment, cell death in WT plants after cadmium treatment increased and damage was more severe compared with the PvFIP37 overexpressing switchgrass lines.

[0153] The test results of superoxide dismutase activity, peroxidase activity, catalase activity and malondialdehyde content are as follows Figure 8 The results showed that under normal growth conditions, there were no significant differences in superoxide dismutase, peroxidase, catalase activities and malondialdehyde content between WT and PvFIP37 overexpressing switchgrass strains, but after 10 weeks of cadmium stress treatment, the superoxide dismutase, peroxidase and catalase activities of PvFIP37 overexpressing switchgrass strains were significantly increased compared with WT, while the malondialdehyde content was significantly lower than that of WT. This indicates that PvFIP37-mediated cadmium tolerance has a strong reactive oxygen scavenging ability, reduces oxidative stress through hydrogen peroxide scavenging, and plays an active regulatory role in cadmium stress.

[0154] The above results show that PvFIP37 protein can regulate the m 6 A modification level is used to regulate plant cadmium stress tolerance, providing new insights into the adaptability of switchgrass and other grasses to cadmium toxicity, which is of great significance for the breeding of candidate species for vegetation restoration.

[0155] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Use of PvFIP37 protein in any of the following A1)-A3): A1) Regulating plant cadmium tolerance; A2) breeding transgenic plants with improved cadmium tolerance; A3) Plant breeding; The PvFIP37 protein is any one of the following B1)-B4): B1) The amino acid sequence is the protein shown in SEQ ID NO: 2; B2) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 2; B3) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 2; B4) A protein that has 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and has the same function.

2. Use of biological materials related to PvFIP37 protein in any of the following A1)-A3): A1) Regulating plant cadmium tolerance; A2) breeding transgenic plants with improved cadmium tolerance; A3) Plant breeding; The biological material is a nucleic acid molecule encoding the PvFIP37 protein or an expression cassette, a recombinant vector or a recombinant microorganism containing the nucleic acid molecule; The PvFIP37 protein is any one of the following B1)-B4): B1) The amino acid sequence is the protein shown in SEQ ID NO: 2; B2) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 2; B3) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 2; B4) A protein that has 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and has the same function.

3. The use according to claim 2, characterized in that: The nucleic acid molecule is any of the following: F1) The DNA molecule shown in Sequence 1 or Sequence 3; F2) A DNA molecule that has 75% or more identity with the nucleotide sequence defined in F1) and encodes the PvFIP37 protein.

4. The use according to any one of claims 1 to 3, characterized in that: The regulating plant cadmium tolerance is to improve the plant cadmium tolerance.

5. The use according to any one of claims 1 to 4, characterized in that: The plant is a monocotyledon or a dicotyledon.

6. A method for cultivating transgenic plants with improved cadmium tolerance, comprising the following steps: increasing the content and / or activity of PvFIP37 protein in a target plant to obtain a transgenic plant; the transgenic plant has higher cadmium tolerance than the target plant.

7. The method according to claim 6, characterized in that: The cadmium tolerance of the transgenic plant is higher than that of the target plant, which is embodied in any one of the following N1)-N6): N1) under cadmium stress, the growth of leaves of the transgenic plant is better than that of the target plant; N2) under cadmium stress, the death or damage degree of the root tip cells of the transgenic plant is less than that of the target plant; N3) under cadmium stress, the superoxide dismutase activity in the leaves of the transgenic plant is higher than that of the target plant; N4) under cadmium stress, the peroxidase activity in the leaves of the transgenic plant is higher than that of the target plant; N5) under cadmium stress, the catalase activity in the leaves of the transgenic plant is higher than that of the target plant; N6) Under cadmium stress, the malondialdehyde content in the leaves of the transgenic plant is lower than that of the target plant.

8. The method according to claim 6 or 7, characterized in that: The method for increasing the content and / or activity of the PvFIP37 protein in the target plant is to overexpress the PvFIP37 protein in the target plant.

9. The method according to claims 6-8, characterized in that: The overexpression method is to introduce the gene encoding the PvFIP37 protein into the target plant; Alternatively, the nucleotide sequence of the gene encoding the PvFIP37 protein is as shown in Sequence 1.

10. The method according to any one of claims 6 to 9, characterized in that: The plant is a monocotyledon or a dicotyledon.

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