Application of pvfip37 protein and related biological materials in regulating cadmium tolerance of plants

Abstract

The application discloses application of a PvFIP37 protein and related biological materials thereof in regulating plant cadmium tolerance. The application clones a PvFIP37 gene from switchgrass and successfully constructs a PvFIP37 overexpression switchgrass. Through analysis of a phenotype of the PvFIP37 overexpression switchgrass under cadmium stress, it is found that, compared with a wild type switchgrass, the increase of the PvFIP37 gene expression amount can enhance the cadmium tolerance of the switchgrass, which indicates that the PvFIP37 has a function of regulating plant cadmium tolerance. The switchgrass PvFIP37 gene provided by the application can be used as an excellent gene resource and is widely applied to the genetic breeding field of the gramineous grasses or other crops, and has important significance for understanding of the plant tolerance under heavy metal stress.

Description

TECHNICAL FIELD

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

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

[0003] Panicum virgatum L. is a perennial C4 grass for cellulosic ethanol conversion, which has a clear genetic structure and heterologous tetraploid genome, and a comprehensive multi-omics database and stable genetic transformation system have been established. Notably, it has strong abiotic stress tolerance, and compared with food crops, it reduces the risk of pollutants entering the food chain. Previous studies have shown the ability to maintain yield in the presence of several metals, including zinc, lead, copper and cadmium, however, higher concentration stress still leads to yield reduction, which affects overall productivity. SUMMARY

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

[0005] To solve the above technical problem, the present application first provides a new use of PvFIP37 protein.

[0006] The present application provides application of PvFIP37 protein in any one of the following A1) to A3):

[0007] A1) regulating plant cadmium tolerance;

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

[0009] A3) plant breeding;

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

[0011] B1) the amino acid sequence is the protein shown in sequence 2;

[0012] B2) a fusion protein with the same function obtained by connecting a tag to the N terminus and / or C terminus of the amino acid sequence shown in sequence 2;

[0013] B3) a protein having the same function as that of the amino acid sequence shown in SEQ ID NO: 2, obtained by substitution and / or deletion and / or addition of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 2;

[0014] B4) a protein having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 2 and having the same function.

[0015] In the protein of B2) above, the tag refers to a polypeptide or protein fused and expressed together with the protein of interest by using DNA in vitro recombination technology, so as to facilitate the expression, detection, tracing and / or purification of the protein of interest. The tag includes but is not limited to: GST (glutathione S-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 of B3) above, the substitution and / or deletion and / or addition of one or several amino acid residues is no more than 10 or 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 amino acid residue substitution and / or deletion and / or addition.

[0017] In the protein of B4) above, the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using the homology search site on the Internet, such as the BLAST webpage of the NCBI homepage. For example, the identity of a pair of amino acid sequences can be calculated by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and performing a search in Advanced BLAST 2.1, and then the value of the identity (%) can be obtained. The identity includes an amino acid sequence having 80% or more, or having 85% or more, or having 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 to the amino acid sequence shown in SEQ ID NO: 2.

[0018] The proteins of B1) to B4) above can be artificially synthesized, or the encoding gene thereof can be synthesized first and then expressed biologically to obtain.

[0019] To solve the above technical problems, the present application also provides a new use of biological material related to the above-mentioned PvFIP37 protein.

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

[0021] A1) regulating the cadmium tolerance of plants;

[0022] A2) cultivating 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 use, the nucleic acid molecule is any one of the following:

[0026] F1) a DNA molecule as shown in SEQ ID NO: 1 or SEQ ID NO: 3;

[0027] F2) a DNA molecule having 75% or more identity with the nucleotide sequence defined in F1) and encoding the above-mentioned PvFIP37 protein.

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

[0029] Those artificially modified nucleotides having 75% or more identity with the nucleotide sequence encoding the PvFIP37 protein and having the same function are derived from the nucleotide sequence of the present application and are equivalent to the sequence of the present application.

[0030] The term "identity" used herein refers to sequence similarity with the natural nucleic acid sequence. "Identity" includes nucleotide sequences having 75% or more identity with the nucleotide sequence of the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 of the present application. The identity can be evaluated by naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed in percentage (%), which can be used to evaluate the identity between related sequences.

[0031] The above-mentioned 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.

[0032] In the above-mentioned application, the expression cassette refers to a DNA capable of expressing PvFIP37 protein in a host cell, which can include not only a promoter for initiating transcription of PvFIP37, but also a terminator for terminating transcription of PvFIP37. Further, the expression cassette can also include an enhancer sequence. The promoters that can be used in the present application 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), CaMV 35S terminator of Cauliflower mosaic virus, tml terminator, pea rbcS E9 terminator, and nopaline and opine synthase terminator.

[0033] In the above-mentioned application, the vector refers to a vector capable of carrying the above-mentioned nucleic acid molecule into a host cell for amplification and expression, which can be a cloning vector or an expression vector, including but not limited to: plasmid, bacteriophage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e. cos plasmid), 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-mentioned nucleic acid molecule with the vector in vitro. The recombinant vector containing the expression cassette of the PvFIP37 gene can be constructed using the existing plant expression vectors. The plant expression vectors include binary Agrobacterium vectors and vectors 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 can also contain the 3' untranslated region of the exogenous gene, that is, it contains a polyadenylation signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylation signal can guide the addition of polyadenylation to the 3' end of the mRNA precursor, such as the untranslated region of the 3' end of the Agrobacterium crown gall tumor-inducing (Ti) plasmid gene (such as the nopaline synthase gene Nos), the plant gene (such as the soybean storage protein gene). The function of the untranslated region of the 3' end of the transcription is similar. When using the gene of the application to construct a plant expression vector, enhancers, including translation enhancers or transcription enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent regions of start codons, but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The source of the translation control signal and the start codon is wide, which can be natural or synthetic. The translation initiation region can come from the transcription initiation 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 to produce color-changing enzymes or luminescent compounds (GUS gene, luciferase gene, etc.), marker genes of antibiotics (such as nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to herbicide phosphine, hph gene conferring resistance to antibiotic hygromycin, and dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate), or chemical reagent-resistant marker genes (such as herbicide-resistant genes), mannose-6-phosphate isomerase genes that provide the ability to metabolize mannose. For the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened under stress.

[0035] In the above applications, 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 obtained by operating and modifying the genes of a target microorganism, so that the function of the recombinant microorganism is changed. For example, the recombinant microorganism obtained after introducing the above-mentioned recombinant vector into the target microorganism. The recombinant microorganism can be understood as not only referring to a specific recombinant microorganism, but also referring to the offspring of such cells, and due to natural, accidental or intentional mutations and / or changes, the offspring can not necessarily be completely consistent with the original parent cell, but is still included in the scope of the recombinant microorganism.

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

[0038] In some embodiments, the improvement of the cadmium tolerance of the plant is specifically embodied in that when the expression amount of the PvFIP37 gene in the plant is improved, the leaf growth of the plant after cadmium stress is better, the root tip cell death or damage of the plant is lighter, the superoxide dismutase activity in the leaf of the plant is higher, the peroxidase activity in the leaf of the plant is higher, the catalase activity in the leaf of the plant is higher, and the malondialdehyde content in the leaf of the plant is lower.

[0039] In the above-mentioned application, the purpose of the plant breeding is to breed a cadmium-tolerant plant variety.

[0040] In order to solve the above-mentioned technical problems, the present application finally provides a method for breeding a transgenic plant with improved cadmium tolerance.

[0041] The method for breeding a transgenic plant with improved cadmium tolerance provided by the present application comprises the following steps: improving the content and / or activity of the above-mentioned PvFIP37 protein in a target plant to obtain a transgenic plant; and the cadmium tolerance of the transgenic plant is higher than that of the target plant.

[0042] In the above-mentioned 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) to N6):

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

[0044] N2) Under cadmium stress, the death or damage of the root tip cell of the transgenic plant is lighter than that of the target plant;

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

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

[0047] N5) the hydrogen peroxidase activity in the leaves of the transgenic plant under cadmium stress is higher than that of the plant of interest;

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

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

[0050] Still further, the condition of the cadmium stress can be CdCl2 stress for 10 weeks.

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

[0052] Further, the method for overexpression is to introduce the gene encoding the PvFIP37 protein into the plant of interest.

[0053] Still further, the nucleotide sequence of the gene encoding the PvFIP37 protein is shown in SEQ ID NO: 1.

[0054] In any of the above applications or methods, the transgenic plant is understood to include not only the first generation transgenic plant obtained by transforming the PvFIP37 gene into a receptor plant, but also the offspring thereof. For the transgenic plant, 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, particularly including commercial varieties. The transgenic plant includes seeds, calli, whole plants and cells.

[0055] In any of the above applications or methods, the plant is a monocotyledonous plant or a dicotyledonous plant. Further, the monocotyledonous plant can be switchgrass. Still further, the switchgrass is switchgrass Alamo.

[0056] The present application clones the PvFIP37 gene from switchgrass and successfully constructs a PvFIP37 overexpression switchgrass. By analyzing the phenotype of the PvFIP37 overexpression switchgrass under cadmium stress, it is found that, compared with the wild type switchgrass, the leaf growth of the PvFIP37 overexpression switchgrass is better, the 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 hydrogen peroxidase activity in the leaves is higher, and the malondialdehyde content in the leaves is lower. This indicates that the cadmium tolerance mediated by PvFIP37 has a strong active oxygen scavenging ability, and reduces oxidative stress through hydrogen peroxide scavenging. The switchgrass PvFIP37 gene provided by the present application can be used as an excellent genetic resource and widely applied to the genetic breeding field of grasses or other crops, and has important significance for the phytoremediation of cadmium contaminated soil. BRIEF DESCRIPTION OF DRAWINGS

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

[0058] Figure 2 is an identification electrophoresis map of the PvFIP37 overexpression vector.

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

[0060] Figure 4 is a gene expression level detection map of the PvFIP37 overexpression switchgrass strain.

[0061] Figure 5 is a m 6 A modification level detection map.

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

[0063] Figure 7 is a cell potassium iodide (PI) staining phenotype map of the PvFIP37 overexpression switchgrass strain.

[0064] Figure 8 is a detection map of superoxide dismutase, peroxidase, catalase and malondialdehyde indicators of the PvFIP37 overexpression switchgrass strain. DETAILED DESCRIPTION

[0065] The present application will be further described in conjunction with the specific embodiments. The examples given are only to illustrate the present application, and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the present application.

[0066] The experimental methods in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[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 examples 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 YEP liquid medium in the following examples is prepared as follows: 5 g of yeast extract, 10 g of tryptone, and 10 g of sodium chloride are mixed with 1 L of deionized water.

[0070] The MP liquid medium in the following examples is prepared 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 medium pH is adjusted to 5.4 with NaOH, and the medium is sterilized at 121°C for 20 min.

[0071] The MP recovery medium in the following examples is prepared 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 phytagel are mixed with 1 L of deionized water, the medium pH is adjusted to 5.8 with NaOH, and the medium is sterilized at 121°C for 20 min; when the medium is cooled to about 60°C, sterile temilatin is added to make its concentration 250 mg / L, and the mixture is thoroughly mixed and poured into sterile culture dishes for use.

[0072] The preparation method of MP1 screening medium in the following examples is as follows: uniformly mix Ms519 4.428 g, maltose 30 g, 2,4-D 5 mg, 6-BA 1 mg, plant gel 4 g and 1 L deionized water, adjust the pH of the medium to 5.8 with NaOH, sterilize at 121°C for 20 min, when the medium is cooled to about 60°C, add sterile hygromycin and timentin to make their concentrations 50 mg / L and 150 mg / L respectively, mix thoroughly, pour into sterile culture dishes for standby.

[0073] The preparation method of MP2 screening medium in the following examples is as follows: uniformly mix Ms519 4.428 g, maltose 30 g, 2,4-D 5 mg, 6-BA 1 mg, plant gel 4 g and 1 L deionized water, adjust the pH of the medium to 5.8 with NaOH, sterilize at 121°C for 20 min, when the medium is cooled to about 60°C, add sterile hygromycin and timentin to make their concentrations 100 mg / L and 150 mg / L respectively, mix thoroughly, pour into sterile culture dishes for standby.

[0074] The preparation method of REG differentiation medium in the following examples is as follows: uniformly mix Ms519 4.428 g, maltose 30 g, 2,4-D 5 mg, 6-BA 1 mg, plant gel 4 g and 1 L deionized water, adjust the pH of the medium to 5.8 with NaOH, sterilize at 121°C for 20 min, when the medium is cooled to about 60°C, add sterile hygromycin and timentin to make their concentrations 20 mg / L and 150 mg / L respectively, mix thoroughly, pour into sterile culture dishes for standby.

[0075] The preparation method of MS rooting medium in the following examples is as follows: uniformly mix Ms513 (PhytoTech Lab, Lot: HYW0513016A) 4.428 g, maltose 30 g, 2,4-D 5 mg, 6-BA 1 mg, plant gel 4 g and 1 L deionized water, adjust the pH of the medium to 5.8 with NaOH, sterilize at 121°C for 20 min, when the medium is cooled to about 60°C, add sterile hygromycin and timentin to make their concentrations 50 mg / L and 150 mg / L respectively, mix thoroughly, pour into sterile culture dishes for standby.

[0076] Example 1, cloning of switchgrass PvFIP37 gene

[0077] 1. Obtaining of cDNA

[0078] The total RNA of switchgrass was extracted by HiPure Plant RNA Mini Kit (double column method), and the total RNA was reversely transcribed into cDNA by PrimeScript TMRT reagent Kit with gDNA Eraser (Perfect RealTime) kit (Takara, Japan).

[0079] 2. PCR amplification

[0080] The switchgrass cDNA obtained in step 1 was diluted 10 times as a template, and primers PvFIP37-F1 and PvFIP37-R1 were used for PCR amplification to obtain a PCR product, i.e. the ORF region of PvFIP37 gene, and 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 of template cDNA, 25 μL of 2xGflex PCR Buffer, 1 μL of Tks Gflex DNA Polymerase, 1.5 μL of upstream primer, 1.5 μL of downstream primer, and 16 μL of ddH2O.

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

[0085] 3. Electrophoretic detection of PCR product

[0086] The PCR product was detected by 1% agarose gel electrophoresis (120 V, 20 min), and then the band condition was observed by using a gel imaging system. The results are shown in Figure 1 .

[0087] 4. Sequencing of PCR product

[0088] 1) The agarose gel containing the target band was cut by a blade under a UV transmission gel cutting table and placed in a previously prepared centrifuge tube, and the gel recovery was performed according to the steps in the Magen kit instruction manual.

[0089] 2) Take 5 μL gel recovery product and 1 μL 6xLoading Buffer, then use 1% agarose gel electrophoresis to detect the recovery. If the band brightness and size are 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 is as follows: 1 μL gel recovery product, 1 μL pTOPO001 Blunt Simple Vector, 1 μL 10xTopo Buffer, and 7 μL ddH2O.

[0091] 3) Transform the ligation product into E. coli DH5a, and the specific operation steps are as follows: add 10 μL reaction solution to 100 μL E. coli competent cells, mix gently, then place in ice for 5 min; place in a 42°C water bath for 45 s, then quickly transfer into ice for 2 min; add 500 μL LB liquid medium (5 g / L Yeast extract, 10 g / L Tryptone, and 10 g / L NaCl), and place in a constant temperature culture shaker at 37°C, 220 rpm, and shake culture for 20 min to obtain the transformation solution.

[0092] 4) Centrifuge the transformation solution, remove a small amount of LB culture solution from the upper layer, mix the remaining transformation solution by blowing and sucking with a gun head, take 100 μL transformation solution, and spread on LB solid medium (5 g / L Yeast extract, 10 g / L Tryptone, 10 g / L NaCl, and 15 g / L Agar) containing Amp, seal with a sealing film, and invert in a 37°C constant temperature incubator for overnight culture.

[0093] 5) The next day, select 6 positive clones on the LB solid plate, and shake culture in 3 mL LB liquid medium containing Amp resistance at 37°C, 220 rpm for 16 h; then extract the plasmid according to the Magen kit instruction manual.

[0094] 6) Use the extracted plasmid as a template, and use the universal primers M13F and M13R of the T vector to perform PCR amplification to obtain the PCR product, and verify the size of the inserted target gene through the PCR reaction.

[0095] The PCR reaction system is 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 2xTaq PCR StarMix (Dye).

[0096] PCR reaction program as follows: 94℃ 2 min; 94℃ 30 s, 60℃ 30 s, 72℃ 90 s, cycle 35 times; 72℃ 5 min; 16℃ + ∞.

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

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

[0099] 7) After the PCR reaction, the PCR product was spotted on a 1% agarose gel, electrophoresis (120V, 15min) detection, whether the inserted target band is correct was observed on the gel imaging instrument, and the correct plasmid band was extracted 10 μL and sent to a biological company for sequencing. According to the sequencing results, the correct plasmid was stored at -20℃ refrigerator.

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

[0101] Example 2, preparation of PvFIP37 overexpression switchgrass

[0102] I. Construction of PvFIP37 overexpression vector

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

[0104] To further verify the connection of the PvFIP37 overexpression vector pCAMBIA1307-PvFIP37, the primer on the vector was selected for PCR verification, and the verification result 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 is as follows: 0.5 μL recombinant plasmid template, 8.7 μL ddH2O, 0.4 μL upstream ZmUbiF primer, 0.4 μL downstream Cas9R primer, 10 μL 2×Taq PCR StarMix (Dye).

[0106] The PCR identification reaction procedure is as follows: 94℃ for 2 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 90 s, 35 cycles; 72℃ for 5 min; 16℃ ± ∞.

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

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

[0109] II. Preparation of Transgenic Plants

[0110] 1. Preparation of recombinant Agrobacterium

[0111] The PvFIP37 overexpression vector pCAMBIA1307-PvFIP37 was transformed into Agrobacterium EHA105 competent cells via freeze-transfer, resulting in recombinant Agrobacterium pCAMBIA1307-PvFIP37 / EHA105. The specific steps are as follows:

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

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

[0114] 3) Spread 200 μL of bacterial culture onto a YEP plate containing 100 mg / L kanamycin and 50 mg / L rifampin. Incubate at 28°C in the dark for 2 days. Pick a single colony for colony PCR detection. The result was positive, indicating that pCAMBIA1307-PvFIP37 had been successfully introduced into Agrobacterium. Preserve the Agrobacterium culture.

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

[0116] Agrobacterium-mediated genetic transformation of switchgrass callus yielded regenerated seedlings transformed with pCAMBIA1307-PvFIP37. Figure 3 The specific method is as follows:

[0117] 1) Select plants that are growing well and are approximately 2×2mm in size. 2The Alamo callus was placed in a 50 mL sterile centrifuge tube, 10 mL 3% maltose solution containing 300 μM glutamine was added, and the tube was placed in an ice bath for 20 min. The maltose solution was discarded, and 10 mL of prepared Agrobacterium solution was added to the centrifuge tube, along with 100 μM acetosyringone and 300 μM glutamine. The tube was vacuumed for 10 min, and then placed in a 28°C shaker for 20 min. The bacterial solution was discarded, and the callus was placed on 3 layers of sterile filter paper in a clean bench and dried for 2 h. The callus was then transferred to 2 layers of sterile filter paper, 500 μL of MP liquid medium containing 300 μM glutamine and 100 μM acetosyringone was added, and the callus was cultured in the dark at 26°C for 2 d.

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

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

[0120] 4) The callus was transferred to MP2 selection medium containing 100 mg / L hygromycin and 150 mg / L phosphinothricin, 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 phosphinothricin, 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 phosphinothricin for rooting, and further selection of resistant regenerated seedlings.

[0123] 7) The roots were longer than 1 cm, and the seedlings were transplanted to a flowerpot containing a mixture of humus, black soil, and vermiculite at a ratio of 1:1:1, and cultured in a greenhouse.

[0124] III. Identification of Transgenic Plants

[0125] 1. Transgenic Positive Plants

[0126] Genomic DNA was extracted from the transgenic plants, and PCR verification was performed using the screening marker gene Hpt and the gene PvFIP37 primers on the vector, respectively, 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. Approximately 100 mg of uniformly growing leaves from transgenic positive plants were selected, ground in liquid nitrogen, and total RNA was extracted using the Trizol method. Agarose gel electrophoresis confirmed the acquisition of high-quality 28S and 18S intact RNA. The RNA concentration and purity were determined using Nanodrop 2000; OD260 / OD280 were both between 1.8 and 2.0, and OD260 / OD230 were both >2.0, demonstrating the high quality of the obtained RNA suitable for cDNA first-strand synthesis. Then, using 1 μg of the above RNA sample as a template, cDNA was synthesized using the TaKaRa PrimeScript RT reagent Kit. qRT-PCR analysis was then performed using the cDNA as a template. Simultaneously, UBI was used as an internal control gene to detect the expression level of the PvFIP37 gene in different plants. 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 analysis results of RT-qPCR are as follows: Figure 4 As shown, the results indicate that the expression level of PvFIP37 in the transgenic switchgrass positive lines OE#4 and OE#5 transformed with pCAMBIA1307-PvFIP37 was significantly higher than that in wild-type switchgrass, and can be used for the following phenotypic observations.

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

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

[0137] 2) 2 μL of RNA solution was dropped onto the surface of the nylon membrane. Then, the membrane was placed in the UV crosslinker and treated with 245 nm UV light for a total of three times.

[0138] 3) The nylon membrane was washed 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% skim milk for 1 hour.

[0140] 5) The blocking solution was discarded, and the membrane was mixed with diluted m 6 The primary antibody (1:2000) was mixed and then incubated overnight at 4°C in the chromatography cabinet.

[0141] 6) The next day, the membrane was washed with TBST solution for 10 minutes for three times.

[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 with TBST solution for 10 minutes for three times. When the last washing was performed, the luminescence solution was prepared.

[0144] 9) After washing, the ECL working solution was evenly covered on the surface of the membrane. Then, the membrane was exposed and photographed using the chemiluminescence imager.

[0145] The results showed that the m 6 A modification level of transgenic switchgrass lines OE#4 and OE#5 was significantly improved, further proving that PvFIP37 is a switchgrass m 6 A methyltransferase. Figure 5

[0146] Example 3, Phenotype analysis of PvFIP37 overexpressing switchgrass under cadmium stress

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

[0148] ​Experimental method: the seeds of the test material are planted according to the conventional method, and when they grow to the flowering stage, they are subjected to 250 μM cadmium stress treatment (CdCl2). The specific treatment method is as follows: the plants at the flowering stage are treated in nutrient soil (humus, black soil, vermiculite ratio of 1:1:1) containing 250 μM CdCl2 for 10 weeks. At the same time, plants in nutrient soil containing 0 μM CdCl2 are used as controls. After 10 weeks of treatment, the phenotype is observed and the following traits are statistically analyzed: root tip cell potassium iodide (PI) staining, superoxide dismutase activity, peroxidase activity, catalase activity and malondialdehyde content. 2的 The plants are cultured in nutrient soil for 10 weeks as controls. After 10 weeks of treatment, the phenotype is observed and the following traits are statistically analyzed: root tip cell potassium iodide (PI) staining, superoxide dismutase activity, peroxidase activity, catalase activity and malondialdehyde content.

[0149] The method of root tip cell potassium iodide staining includes the following steps: cutting 5 mm long root tips of control group (CK) and cadmium (Cd) treated group of switchgrass, soaking in 10 μg / mL potassium iodide (PI) solution (Solarbio, CA1630) for 15 minutes, and then rinsing with distilled water three times. The potassium iodide (PI) fluorescence signal is observed using a confocal laser scanning microscope (Nikon TE2000-E). Generally, 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 nucleus are stained (the cell outline can be outlined), and in severely damaged cells, only the nucleus is stained, and the complete cell outline cannot be seen.

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

[0151] The results of the phenotype observation are shown in Figure 6 As shown in the results, after 10 weeks of cadmium stress treatment, compared with WT plants, the PvFIP37 overexpression switchgrass lines OE#4 and OE#5 showed better leaf growth and less wilting and yellowing.

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

[0153] The results of the detection of superoxide dismutase activity, peroxidase activity, catalase activity and malondialdehyde content are shown in Table 2. Figure 8 As shown in Table 2, the results show that under normal growth conditions, there is no significant difference in superoxide dismutase, peroxidase, catalase activity and malondialdehyde content between WT and PvFIP37 overexpression switchgrass lines, but after 10 weeks of cadmium stress treatment, the superoxide dismutase, peroxidase and catalase activity of the PvFIP37 overexpression switchgrass lines are significantly increased compared with WT, and the malondialdehyde content is significantly lower than WT. This indicates that the cadmium tolerance mediated by PvFIP37 has strong active 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 the PvFIP37 protein can regulate the m 6 A modification level in plants to regulate cadmium stress tolerance of plants, providing new insights into the adaptability of switchgrass and other gramineous plants to cadmium toxicity, and having important significance for the breeding of vegetation repair candidate species.

[0155] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under equivalent parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In summary, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which are outside the scope disclosed in the present application. Some basic features can be applied within the scope of the following attached claims.

Claims

1. A method for cultivating transgenic plants with enhanced cadmium tolerance, comprising the following steps: overexpressing PvFIP37 protein in a target plant to obtain a transgenic plant; wherein the transgenic plant exhibits higher cadmium tolerance than the target plant; and wherein the PvFIP37 protein is any one of the following B1)-B2): B1) The amino acid sequence of the protein is shown in sequence 2; B2) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 2; The plant in question is *Symplocos serrata*.

2. The method according to claim 1, characterized in that: The transgenic plant exhibits higher cadmium tolerance than the target plant as evidenced by any one of the following N1)-N6): N1) Under cadmium stress, the leaves of the transgenic plant showed better growth than those of the target plant; N2) Under cadmium stress, the degree of death or damage to the root tip cells of the transgenic plant was less than that of the target plant; N3) Under cadmium stress, the superoxide dismutase activity in the leaves of the transgenic plant was higher than that in the target plant; N4) Under cadmium stress, the peroxidase activity in the leaves of the transgenic plant was higher than that in the target plant; N5) Under cadmium stress, the catalase activity in the leaves of the transgenic plant was higher than that in the target plant; (N6) Under cadmium stress, the malondialdehyde content in the leaves of the transgenic plant is lower than that in the target plant.

3. The method according to claim 1, characterized in that: The overexpression method involves introducing the gene encoding the PvFIP37 protein into the target plant.

4. The method according to any one of claims 1-3, characterized in that: The nucleotide sequence of the gene encoding the PvFIP37 protein is shown in Sequence 1.

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