CN111171125A

CN111171125A - Application of protein IbCAF1 in regulation and control of salt and drought resistance of plants

Info

Publication number: CN111171125A
Application number: CN202010095692.8A
Authority: CN
Inventors: 翟红; 刘庆昌; 何绍贞; 赵宁; 陈杉彬
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2020-02-17
Filing date: 2020-02-17
Publication date: 2020-05-19
Anticipated expiration: 2040-02-17
Also published as: CN111171125B

Abstract

The invention discloses an application of protein IbCAF1 in regulation and control of salt and drought resistance of plants. The invention firstly discloses the application of protein in improving the salt and drought resistance of plants; the protein is a protein with an amino acid sequence shown in SEQ ID NO.1 or a fusion protein obtained by connecting protein labels at the N end or/and the C end of the amino acid sequence shown in SEQ ID NO. 1. The invention further discloses the protein-related biomaterial and application thereof. The invention discovers the IbCAF1 protein and the coding gene thereof, and introduces the coding gene of the IbCAF1 protein into tobacco to obtain a transgenic tobacco plant of the IbCAF 1. The transgenic plant is subjected to drought and salt stress treatment, the salt and drought resistance of the transgenic plant is enhanced, the transgenic plant plays an important role in the processes of salt and drought resistance of the plant, and has wide application space and market prospect in the agricultural field.

Description

Application of protein IbCAF1 in regulation and control of salt and drought resistance of plants

Technical Field

The invention relates to the field of biotechnology. In particular to application of protein IbCAF1 in regulation and control of plant salt and drought resistance.

Background

Sweet potato is an important crop for grain, feed and industrial raw materials, and is a novel energy plant in the world, and the position of the sweet potato is particularly important. China is the biggest sweet potato producing country in the world, and the annual planting area is 348.2 kilohm²43.0% of the total planting area in the world, 7336.1 ten thousand tons of annual output and 71.1% of the total world output. The saline-alkali soil in China is about 2000 kilohm²The salt content is 0.6-10% and the yield and quality of sweet potato are seriously affected. Although sweet potatoes are drought-enduring crops, the growth, development, physiology and yield of sweet potatoes are affected in the absence of water, as in other crops. By deeply researching the salt and drought resistance mechanism of plants, the salt and drought resistance gene resources are excavated, and the cultivation of new salt and drought resistant sweet potato varieties is one of the most economic and effective measures for utilizing saline-alkali soil and arid and semi-arid resources.

Therefore, the gene with the functions of drought resistance and salt tolerance is obtained by cloning and identifying through a genetic engineering means, and the gene is transferred into a plant body to improve the drought resistance and salt tolerance of the plant, so that the gene has important research and application values.

Disclosure of Invention

The invention aims to solve the technical problem of how to regulate and control the salt and drought resistance.

The invention firstly provides a protein, which is derived from any one of proteins shown in sweet potatoes (Ipomoea batatas), is named IbCAF1 protein or protein IbCAF1, and is A1) or A2) or A3):

A1) protein with amino acid sequence shown as SEQ ID NO. 1;

A2) the N end or/and the C end of the amino acid sequence shown in SEQ ID NO.1 is connected with a protein label to obtain a fusion protein;

A3) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.1, has more than 90 percent of identity with the protein shown in A1), and has the same function.

Wherein SEQ ID NO.1 consists of 281 amino acid residues.

The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

Among the above proteins, protein-tag (protein-tag) refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate the expression, detection, tracking and/or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

In the above proteins, identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11,1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

In the above protein, the 90% or more identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

The invention also provides the application of any one of the following proteins IbCAF 1:

B1) improving the salt tolerance of the plants;

B2) preparing a product for improving the salt tolerance of plants;

B3) improving the drought resistance of the plants;

B4) preparing a product for improving the drought resistance of plants;

B5) improving the rooting condition of the plants under drought and/or salt stress conditions;

B6) preparing a product for improving the rooting condition of the plant under the drought and/or salt stress condition;

B7) increasing the growth vigor of the plant under drought and/or salt stress conditions;

B8) preparing a product of the growth vigor of the plant under drought and/or salt stress conditions;

B9) increasing the proline content of plants under drought and/or salt stress conditions;

B10) preparing a product for improving the proline content of plants under drought and/or salt stress conditions;

B11) reducing the malondialdehyde content of plants under drought and/or salt stress conditions;

B12) preparing a product for reducing the malondialdehyde content of a plant under drought and/or salt stress conditions;

B13) improving the SOD activity of plants under drought and/or salt stress conditions;

B14) preparing the product for improving the SOD activity of plants under drought and/or salt stress conditions.

The biological material related to the protein IbCAF1 also belongs to the protection scope of the invention.

The related biomaterial is any one of the following C1) -C10):

C1) a nucleic acid molecule encoding the protein IbCAF 1;

C2) an expression cassette comprising the nucleic acid molecule of C1);

C3) a recombinant vector comprising the nucleic acid molecule of C1), or a recombinant vector comprising the expression cassette of C2);

C4) a recombinant microorganism containing C1) the nucleic acid molecule, or a recombinant microorganism containing C2) the expression cassette, or a recombinant microorganism containing C3) the recombinant vector;

C5) a transgenic plant cell line comprising C1) the nucleic acid molecule, or a transgenic plant cell line comprising C2) the expression cassette, or a transgenic plant cell line comprising C3) the recombinant vector;

C6) transgenic plant tissue comprising C1) the nucleic acid molecule, or transgenic plant tissue comprising C2) the expression cassette, or transgenic plant tissue comprising C3) the recombinant vector;

C7) a transgenic plant organ containing C1) said nucleic acid molecule, or a transgenic plant organ containing C2) said expression cassette, or a transgenic plant organ containing C3) said recombinant vector;

C8) a transgenic plant containing C1) the nucleic acid molecule, or a transgenic plant containing C2) the expression cassette, or a transgenic plant containing C3) the recombinant vector;

C9) a tissue culture produced from regenerable cells of the transgenic plant of C8);

C10) protoplasts produced by the tissue culture of C9).

Wherein 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, etc.

In the biological material, the C1) nucleic acid molecule for encoding the protein IbCAF1 can be specifically any one of the following D1) or D2) or D3):

D1) DNA molecule shown in SEQ ID NO. 2;

D2) the coding sequence is DNA molecule shown in SEQ ID NO. 2;

D3) a DNA molecule which hybridizes with the DNA molecule defined by D1) or D2) under stringent conditions and codes for the protein IbCAF 1.

Wherein, SEQ ID NO.2 consists of 846 nucleotides, the Open Reading Frame (ORF) thereof is from 1 st to 846 nd from the 5' end, and the encoded amino acid sequence is the protein shown in SEQ ID NO. 1.

The stringent conditions are hybridization and washing of the membrane 2 times 5min at 68 ℃ in a solution of 2 XSSC, 0.1% SDS and 2 times 15min at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS.

In the above-mentioned related biological materials, the expression cassette described in C2) refers to DNA capable of expressing the protein IbCAF1 in a host cell, which DNA may include not only a promoter for initiating transcription of IbCAF1 gene, but also a terminator for terminating transcription of IbCAF1 gene. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters and inducible promoters. Examples of promoters include, but are not limited to: the constitutive promoter of cauliflower mosaic virus 35S; to comeThe wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al (1999) Plant Physiol120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1(PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with jasmonic acid ester); heat shock promoters (U.S. patent 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128(CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin, and soybean beta conglycin (Beach et al (1985) EMBO J.4: 3047-3053.) they can be used alone or in combination with other plant promoters⁹⁸⁵) Nature 313: 810; rosenberg et al (1987) Gene,56: 125; guerineau et al (1991) mol.gen.genet,262: 141; proudfoot (1991) Cell,64: 671; sanfacon et al Genes Dev.,5: 141; mogen et al (1990) plant cell,2: 1261; munroe et al (1990) Gene,91: 151; ballad et al (1989) Nucleic Acids Res.17: 7891; joshi et al (1987) Nucleic Acid Res, 15: 9627).

In the related biological material, C3) the recombinant vector can contain a DNA molecule shown in SEQ ID NO.2 and used for encoding the protein IbCAF 1.

Plant expression vectors can be used for constructing recombinant vectors containing the IbCAF1 coding gene expression cassettes. The plant expression vector can be a Gateway system vector or a binary agrobacterium vector and the like, such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1300, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pBI121, pCAMBIA1391-Xa or pCAMBIA 1391-Xb. When IbCAF1 is used to construct recombinant vector, any one of enhanced, constitutive, tissue-specific or inducible promoters such as cauliflower mosaic virus (CAMV)35S promoter, ubiquitin gene Ubiqutin promoter (pUbi) and the like can be added before the transcription initiation nucleotide, and can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct plant expression vectors, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.

In order to facilitate the identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound which can produce a color change (GUS gene, luciferase gene, etc.), an antibiotic marker having resistance (gentamicin marker, kanamycin marker, etc.), or a chemical-resistant marker gene (e.g., herbicide-resistant gene), etc., which can be expressed in plants.

In a specific embodiment of the invention, the recombinant vector is a recombinant plasmid pCAMBIA1300-IbCAF 1. The recombinant plasmid pCAMBIA1300-IbCAF1 is a recombinant vector obtained by replacing a fragment between Kpn I and Sal I enzyme cutting sites of the vector pCAMBIA1300 with a DNA molecule shown in SEQ ID NO.2 and keeping other sequences unchanged.

In the related biological material, the recombinant microorganism C4) can be yeast, bacteria, algae and fungi.

In the above-mentioned related biological materials, C7) the transgenic plant organ may be a root, a stem, a leaf, a flower, a fruit, and a seed of the transgenic plant.

In the above-mentioned related biomaterials, C9) the tissue culture may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.

In the related biological material, the transgenic plant cell line, the transgenic plant tissue and the transgenic plant organ do not comprise propagation materials.

The application of any one of the following biological materials related to the protein IbCAF1 is also in the protection scope of the invention:

B1) improving the salt tolerance of the plants;

B2) preparing a product for improving the salt tolerance of plants;

B3) improving the drought resistance of the plants;

B4) preparing a product for improving the drought resistance of plants;

In the above application, the rooting condition is specifically the number and/or length of roots.

The application of the protein IbCAF1 or the related biological materials in plant breeding is also within the protection scope of the invention.

Among the above applications, the plant breeding application can be specifically that a plant containing the protein IbCAF1 or the related biological material (such as the protein IbCAF1 encoding gene IbCAF1) is crossed with other plants to perform plant breeding.

The invention further provides a method for cultivating the transgenic plant with high drought resistance and/or salt tolerance.

The method for cultivating the transgenic plant with high drought resistance and/or salt tolerance comprises the steps of improving the expression quantity of a gene of a protein IbCAF1 in a target plant, and/or the content of the protein IbCAF1 and/or the activity of the protein IbCAF1 to obtain the transgenic plant; the drought resistance and/or salt tolerance of the transgenic plant is higher than that of the target plant.

In the method, the method for improving the expression level of the gene of the protein IbCAF1 and/or the content of the protein IbCAF1 and/or the activity of the protein IbCAF1 in the target plant is to express or over-express the protein IbCAF1 in the target plant.

In the above method, the expression or overexpression is carried out by introducing a gene encoding the protein IbCAF1 into a plant of interest.

In the above method, the gene encoding the protein IbCAF1 can be introduced into a plant of interest by a plant expression vector carrying the IbCAF1 gene of the invention. The plant expression vector carrying the gene IbCAF1 of the invention can be used for transforming plant cells or tissues by using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, conductance, Agrobacterium mediation and other conventional biological methods, and culturing the transformed plant cells or tissues into plants.

In the method, the nucleotide sequence of the coding gene of the protein IbCAF1 is a DNA molecule shown in SEQ ID NO. 2.

In a specific embodiment of the invention, the plant expression vector carrying the gene IbCAF1 of the invention can be pCAMBIA1300-IbCAF 1. Specifically, pCAMBIA1300-IbCAF1 was obtained by inserting the DNA molecule shown by SEQ ID NO.2 into pCAMBIA1300 vector using the restriction enzymes Sal I and Kpn I.

In the method, the drought resistance and/or salt tolerance is mainly embodied in that the number and the length of the roots of the plants are increased, the proline content is increased, the SOD activity is increased, and the H is reduced₂O₂Content and reduction of malondialdehyde content.

In the present invention, the plant is any one of the following E1) to E7):

E1) a dicotyledonous plant;

E2) a monocot plant;

E3) a plant of the genus nicotiana;

E4) tobacco;

E5) a plant of the family Convolvulaceae;

E6) a plant of the genus Ipomoea;

E7) sweet potatoes (Ipomoea batatas).

The invention discovers the IbCAF1 protein and the coding gene thereof, and introduces the coding gene of the IbCAF1 protein into tobacco to obtain a transgenic tobacco plant of the IbCAF 1. The transgenic plant is subjected to drought and salt stress treatment, compared with a control, the salt and drought resistance of the transgenic plant is enhanced, and the specific embodiment is that the proline content and the SOD activity are increased, and the malonaldehyde content and the H content are reduced₂O₂And (4) content. Therefore, the IbCAF1 gene and the protein coded by the same play an important role in the process of plant salt and drought resistance, have important application values in the research of improving plant salt and drought resistance, and have wide application space and market prospect in the agricultural field.

Drawings

FIG. 1 shows the PCR detection result of transgenic plants; wherein M is marker, and W is negative control water; p is positive control (recombinant plasmid pCAMBIA1300-IbCAF 1); WT is wild type W38 tobacco plant; L1-L18 is a tobacco pseudotransgenic plant.

FIG. 2 is the expression analysis of IbCAF1 in transgenic tobacco.

FIG. 3 shows the identification of salt tolerance and drought resistance of transgenic tobacco plants overexpressing IbCAF1 and wild type W38 tobacco plants; wherein, WT is a wild type W38 tobacco plant; l1, L9 and L13 are transgenic tobacco plants overexpressing IbCAF 1.

FIG. 4 shows proline content determination of transgenic tobacco plants overexpressing IbCAF1 and wild type W38 tobacco plants; wherein, WT is a wild type W38 tobacco plant; l1, L9 and L13 are transgenic tobacco plants overexpressing IbCAF 1.

FIG. 5 shows the measurement of physiological and biochemical indicators of transgenic tobacco plants overexpressing IbCAF1 and wild type W38 tobacco plants; wherein, A is SOD activity determination; b is MDA content determination; WT is wild type W38 tobacco plant; l1, L9 and L13 are transgenic tobacco plants overexpressing IbCAF 1.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1, obtaining of salt-tolerant drought-resistant related protein IbCAF1 and coding gene thereof of sweet potato and functional verification I, obtaining of salt-tolerant drought-resistant related protein IbCAF1 and coding gene thereof of sweet potato

Cloning of IbCAF1 Gene cDNA of Ipomoea batatas

Experimental materials: sweet potato variety Lushu No. 3 (non-patent documents for recording the material are Zhai red, Shanli, Liu Qingchang. sweet potato stem nematode induced inhibition hybrid cDNA library construction and expression sequence label analysis. agricultural biotechnology bulletin 2010,18 (1): 141-148. public after the author agrees, can be obtained from Chinese agriculture university sweet potato genetic breeding research laboratory to repeat the experiment, can not be used as other purposes)

1. Extraction of total RNA from sweet potato

Grinding 0.1g of young and tender leaves of sweet potato into powder in liquid nitrogen, adding into a 2mL centrifuge tube, and extracting the total RNA of the sweet potato by using a TIANGEN RNAPREPPure plant total RNA extraction kit (catalog number: DP432), wherein the kit comprises: lysis solution RL, deproteinization solution RW1, rinsing solution RW, RNase-Free ddH₂O, RNase-Free adsorption column CR3, RNase-Free filtration column CS, DNase I, buffer RDD, RNase-Free centrifuge tube, RNase-Free collection tube. Collecting 1 μ L, performing 1.2% agarose gel electrophoresis to detect its integrity, diluting 2 μ L to 500 μ L, and detecting its quality (OD) with ultraviolet spectrophotometer₂₆₀) And purity (OD)₂₆₀/OD₂₈₀) The extracted Luma 3 total RNA is detected by non-denaturing gel agarose gel electrophoresis, 28S and 18S bands are clear, and the brightness ratio of the two bands is 1.5-2: 1, which shows that the total RNA is not degraded, and the obtained mRNA conforms to the standardThe experimental requirements can be used for cloning the full length of the IbCAF1 protein cDNA.

2. full-Length cloning of IbCAF1 Gene cDNA

Primers were designed for full-length cloning of IbCAF1 cDNA.

The primer sequences are as follows:

primer 1: 5'-ATGGGTGTACAAGAAGATGTTTTG-3'

Primer 2: 5'-CTAAAAAACTTCTAGTCCGTACAATACT-3'

The total RNA extracted in the step 1 is subjected to reverse transcription by a QuantScript RT Kit (TIANGEN, Beijing) to be used as a template, and high-fidelity LA enzyme is used for PCR amplification. And detecting the PCR amplification product by agarose gel electrophoresis to obtain an amplification fragment with the length of 846 bp.

After sequencing, the PCR product has a nucleotide sequence shown in SEQ ID NO.2, a gene shown in the sequence is named as IbCAF1 gene, the coding region of the gene is the 1 st to 846 th nucleotides from the 5' end of the SEQ ID NO.2, the protein coded by the gene is named as IbCAF1 protein or protein IbCAF1, the amino acid sequence is SEQ ID NO.1, and the protein consists of 281 amino acid residues.

Second, application of IbCAF1 protein of sweet potato in improving salt tolerance and drought resistance of plants

1. Construction of plant expression vectors

Designing and amplifying a primer sequence of a complete coding sequence according to the coding sequence of the cDNA of the IbCAF1 gene of the sweet potato, respectively introducing Kpn I and Sal I enzyme cutting sites into forward and reverse primers (a primer 3 and a primer 4), wherein the primer sequences are as follows:

primer 3: 5' -CGGGATCCATGGGTGTACAAGAAGATGTTTTG-3' (the underlined part represents the Kpn I cleavage site),

primer 4: 5' -ACGCGTCGACAAAAACTTCTAGTCCGTACAATACT-3' (Sal I cleavage site is underlined).

The artificially synthesized SEQ ID NO.2 is used as a template, after PCR amplification, a product is connected to a pMD19-T vector (purchased from Beijing Zeping science and technology Limited liability company, product catalog number is A1360) and named as a pGIbCAF1 vector, sequencing is carried out, and the correctness of the reading frame and the restriction enzyme cutting site of the sweet potato IbCAF1 gene cDNA is ensured.

The vector pCAMBIA1300 (purchased from Wuhan transduction biology laboratories, the product catalog number is VT4001) is cut by Sal I and Kpn I, the large fragment of the vector is recovered, meanwhile, the vector pGIbCAF1 is cut by Sal I and Kpn I, the intermediate fragment of about 1.0kb is recovered, and the recovered large fragment of the vector is connected with the intermediate fragment of about 1.0kb to obtain the target plasmid. The target plasmid is transformed into escherichia coli DH5a (purchased from Beijing Quanyujin biotechnology limited, product catalog number is CD201-01), cultured for 20h at 37 ℃, subjected to PCR analysis and enzyme digestion identification of the recombinant vector, and subjected to sequencing verification. The sequencing result shows that the sequence shown by 1 st to 846 nd from the 5' end of SEQ ID NO.2 is inserted between the Kpn I and Sal I enzyme cutting sites of the vector pCAMBIA1300, so that the construction of the recombinant vector is correct, and the recombinant vector is named as pCAMBIA1300-IbCAF 1.

2. Plant expression vector transformation agrobacterium tumefaciens

(1) 200 μ L of EHA105 competent cells (purchased from Beijing Byledy Biotechnology Co., Ltd.) were taken out from a low temperature refrigerator at-80 ℃ and thawed on ice, and 1 μ g of the plant expression vector pCAMBIA1300-IbCAF1 obtained in the above step 1 was added and mixed well.

(2) Freezing with liquid nitrogen for 1min, and incubating at 37 deg.C for 5 min.

(3) Adding 800 μ L LB liquid culture medium, and culturing at 28 deg.C for 2-6 h.

(4) mu.L of the resulting suspension was applied to LB solid medium (containing 100. mu.g/mL rifampicin (Rif) and 25. mu.g/mL kanamycin (Kan)), and the applied solution was spread uniformly, followed by sealing the petri dish. The plates were inverted and incubated at 28 ℃ for 2 d.

(5) Taking a single colony which is positive in PCR identification, inoculating the single colony into LB liquid culture medium containing 100 mu g/mL Rif and 25 mu g/mL Kan, culturing at 28 ℃ for 30h to logarithmic phase, taking a proper amount of agrobacterium, and diluting by 30 times with a liquid MS culture medium for later use, thus obtaining the agrobacterium liquid introduced with pCAMBIA1300-IbCAF 1.

3. Genetic transformation and regeneration of tobacco

The coding sequence of the IbCAF1cDNA was introduced into W38 tobacco (described in Jiang T, ZHai H, Wang FB, Zhou HN, Si ZZ, He SZ, Liu QC. cloning and haractionato f a Salt hierarchy-Associated Gene Encoding Trehalose-6-Phosphonate Synthesis tobacco pototo, Journal of Integrated Agriculture 2014,13(8):1651-1661) by Agrobacterium-mediated method. The specific method comprises the following steps:

w38 tobacco leaves growing for about 4 weeks in culture medium were selected and placed in sterilized glass petri dishes. The main vein and the leaf edge are cut off, and a tobacco leaf disc of 1X 1cm is cut. The tobacco leaf discs are placed on a pre-culture medium (1.0mg/L6-BA and 0.1mg/L NAA in MS) with the front faces upward, and grow for 2-3 days under the dark condition at the temperature of 28 ℃. Soaking the pre-cultured tobacco leaf disc in the OD prepared in the step 2₆₀₀0.4-0.6 of agrobacterium is soaked for 5-10 min. And (4) sucking off redundant bacterial liquid on the tobacco leaf disc, placing the tobacco leaf disc on the co-culture medium at the front side, and co-culturing for 2-3 days at 28 ℃ under a dark condition. 150, 300 and 600mg/L CS are respectively added into the liquid MS culture medium, and the co-cultured tobacco leaf discs are washed for 3 times according to the concentration from high to low. The excess liquid on the leaf disks was blotted with clean filter paper sterilized at high temperature and high pressure, and placed on a regeneration medium (MS of 15mg/L hygromycin, 400mg/L CS, 1.0mg/L6-BA and 0.1mg/L NAA) and cultured for 30 days at 28 ℃ under 3000lux of light until the shoots differentiated. 1cm of the regenerated bud is cut off and subcultured on a rooting medium (1/2 MS solid medium of 1.0mg/L6-BA, 0.1mg/L NAA, 400mg/L cephalexin and 15mg/L hygromycin) until a complete plant is grown, and a pseudotransgenic plant is obtained.

The identification of the transgenic plants uses a method combining PCR detection and qRT-PCR detection.

A. PCR detection

Extracting genome DNA of a pseudotransgenic plant and a wild type W38 tobacco plant by a CTAB method. PCR detection is carried out by a conventional method, and the used primers of the IbCAF1 gene are as follows: primer 1: 5 'ATGGGTGTACAAGAAGATGTTTTG 3' and primer 2: 5 'CTAAAAAACTTCTAGTCCGTACAATACT 3'. To a 0.2ml Eppendorf centrifuge tube were added 2. mu.l of 10 XPCR buffer, 1. mu.l of 4dNTP (10mol/L), 1. mu.l of each primer (10. mu. mol/L), 2. mu.l of template DNA (50ng/ul), 1ul of Taq DNA polymerase, and H₂O to a total volume of 20. mu.l. The reaction program is denaturation at 94 ℃ for 4min, renaturation at 57 ℃ for 1.5min, and extension at 72 ℃ for 1min30s, and the total number of 36 cycles. The pCAMBIA1300-IbCAF1 carrier plasmid is used as a positive control, water and a wild type W38 tobacco plant are used as negative controls, and then electricity is performedAnd (4) carrying out electrophoresis detection.

The results are shown in FIG. 1, from which it can be seen that the 846bp target band is amplified by the pseudotransgenic plant L1-L18 and the positive control, which indicates that the IbCAF1 gene has been integrated into the genome of tobacco W38, and these plants are proved to be transgenic plants; no 846bp target band was amplified from water and wild type W38 tobacco plants. The method obtains transgenic tobacco positive plants L1-L18.

B、qRT-PCR

And extracting RNA of the positive transgenic tobacco plant, carrying out reverse transcription to obtain cDNA, and carrying out qRT-PCR (quantitative reverse transcription-polymerase chain reaction) by taking the wild W38 tobacco plant as a control CK.

Taking a tobacco Actin (Actin) gene as an internal reference, wherein the primer sequence is as follows:

NtActin-F：5’-GAGGAATGCAGATCTTCGTG-3’

tActin-R：5’-TCCTTGTCCTGGATCTTAGC-3’

the sequence of the IbCAF1 primer is as follows:

IbCAF1-qRT-F：5’-GGCGGTGGAGACATATCTGG-3’

IbCAF1-qRT-R：5’-TGTGACATCAGAGCCGGAAG-3’

the qRT-PCR result is shown in figure 2, and the result shows that the IbCAF1 gene is expressed in different degrees in transgenic plants. And selecting three transgenic IbCAF1 tobacco plants L1, L9 and L13 with the highest expression level for expanding propagation to carry out subsequent experiments.

4. Identification of salt and drought resistance of transgenic plant

4.1 phenotypic characterization

The transgenic tobacco strains L1, L9, L13 and the wild type W38 of the IbCAF1 are respectively cultured on an MS culture medium, an MS culture medium containing NaCl (with the concentration of 200mM) and an MS culture medium containing polyethylene glycol (with the concentration of 10%), the temperature is 27 +/-1 ℃, the illumination is carried out for 13h and 3000lux every day, and the growth state and the rooting condition are observed after the stress culture is carried out for 4 weeks.

As shown in FIG. 3, the wild type W38 tobacco plant (shown as "CK" in the figure) was weakly grown and hardly rooted on the MS medium of NaCl (concentration 200mM) and the MS medium of polyethylene glycol (concentration 10%); the growth states and rooting conditions of 3 transgenic tobacco strains L1, L9 and L13 of the IbCAF1 are better than those of wild W38 tobacco in different degrees, and the result shows that the salt tolerance and drought resistance of the transgenic tobacco strains are improved by over-expressing the IbCAF 1.

4.2 proline content determination

Under normal conditions, the content of free proline in plants is very low, but when the plants are stressed by drought, low temperature, salt and the like, a large amount of free amino acid is accumulated, and the accumulation index is related to the stress resistance of the plants. Therefore, proline can be used as a biochemical index of plant stress resistance.

The proline content of tobacco plants was determined by reference to the method of He et al (He SZ, Han YF, Wang YP, Zhai H, Liu QC. in vitro selection and differentiation of sweet potatoo (Ipomoea bases (L.)) plants tolerant ToNaCl. plant Cell Tissue Organ Cult,2009,96: 69-74).

The transgenic tobacco strains L1, L9 and L13 and the wild W38 tobacco strains are respectively subcultured on an MS culture medium containing NaCl (with the concentration of 200mM) and an MS solid culture medium containing polyethylene glycol (with the concentration of 10%) for drought stress and normal culture on the MS solid culture medium at the temperature of 27 +/-1 ℃ under the illumination of 13h and 3000lux every day for 4 weeks, and then the leaves are taken for proline content determination and repeated for 3 times.

1) Main reagent and formula

(1)6M phosphoric acid: measuring 102.5mL of 85% phosphoric acid in a 250mL volumetric flask, and fixing the volume to the scale;

(2) 2.5% ninhydrin acid: 5.0g of ninhydrin were weighed, 120mL of glacial acetic acid and 80mL of 6M phosphoric acid were added, and the mixture was dissolved by water bath at 70 ℃ and stored in a brown bottle as soon as possible after cooling. The product can be stored for 3 days in a refrigerator at 4 ℃.

2) Measurement method

(1) Weighing 10mg of proline, dissolving with a small amount of absolute ethyl alcohol, transferring into a 100mL volumetric flask, and diluting to constant volume with distilled water to prepare a mother solution of 100 mug/mL;

(2) respectively putting 0mL, 0.625 mL, 1.25 mL, 2.5mL, 3.75 mL, 5.0 mL, 6.25 mL and 7.5mL of the mother liquor into 8 volumetric flasks with 25mL, respectively adding distilled water to a constant volume to scale, fully and uniformly mixing to prepare proline solutions with the series concentrations of 0, 1.25, 2.5, 5, 7.5, 10, 12.5 and 15 mu g/mL;

(3) respectively adding 2mL of the above solutions into 2mL of glacial acetic acid and 2mL of acidic ninhydrin (not contacting with skin), mixing, developing with boiling water bath for 15min, cooling, and measuring absorbance at 520 nm;

(4) the absorbance value is taken as the abscissa and the proline content is taken as the ordinate. The results are shown in FIG. 2.

Extraction and determination of plant samples:

(1) weighing 1.0g of plant leaves, shearing, adding 5mL of 80% ethanol, and grinding to homogenate;

(2) transferring the homogenate liquid into a test tube, adding water to supplement 25mL, fully and uniformly mixing, and carrying out water bath at 80 ℃ for 20 min;

(3) respectively adding 0.5g of artificial zeolite and 0.2g of activated carbon, oscillating on a vortex oscillator for 1min, mixing, and filtering with a layer of filter paper;

(4) detecting the content of 1mL proline of a detected sample from the prepared proline standard curve, and finally calculating the average content of free proline according to the following formula:

proline content (. mu.g/g) ═ C.times.V 1/V2)/W

C-Curve finding C value (. mu.g);

V1-Total volume of extract (mL);

v2 — volume of assay solution (mL);

w-sample mass (g).

The results of measuring the proline content of the plants of the transgenic tobacco strains L1, L9 and L13 and the wild-type W38 tobacco plant are shown in FIG. 4, and the results show that the proline content of 3 transgenic strains is higher than that of the wild-type W38 tobacco plant on an MS culture medium containing NaCl (at a concentration of 200mM) and an MS culture medium containing polyethylene glycol (at a concentration of 10%); the difference significance analysis shows that the proline content is obviously higher than that of wild type W38 tobacco plants.

4.3SOD Activity assay

SOD activity is an important physiological and biochemical index for identifying the salt tolerance of plants. SOD activity of tobacco plants was measured by the method described in He et al (2009).

Transgenic tobacco strains L1, L9 and L13 and wild W38 tobacco strains are respectively subcultured on an MS culture medium containing NaCl (with the concentration of 200mM) and an MS solid culture medium containing polyethylene glycol (with the concentration of 10%) for drought stress and normal culture on the MS solid culture medium at the temperature of 27 +/-1 ℃ under the illumination of 13h and 3000lux every day for 4 weeks, and then the leaves are taken for SOD activity determination and repeated for 3 times.

1) Main reagent and formula

(1)0.1M sodium phosphate (Na)₂HPO4-NaH₂PO4) buffer (pH 7.8)

Solution A (0.1M Na)₂HPO4 solution): weighing Na₂HPO4·2H₂O7.163 g was dissolved in a small amount of distilled water, transferred to a 200mL volumetric flask to a constant volume, and mixed well. Storing in a refrigerator at 4 ℃ for later use;

liquid B (0.1M NaH)₂PO4 solution): weighing NaH₂PO4·2H₂0.780g of O, dissolved in a small amount of distilled water, transferred into a 50mL volumetric flask to a constant volume, and mixed well. Storing in a refrigerator at 4 ℃ for later use;

183mL of the solution A and 17mL of the solution B were mixed well to obtain 0.1M sodium phosphate buffer (pH 7.8). Storing in a refrigerator at 4 ℃ for later use.

(2)0.026M methionine (Met) sodium phosphate buffer

Weighing methionine (C)₅H₁₁NO₂S)0.388g, dissolved in a small amount of 0.1M sodium phosphate buffer (pH 7.8), transferred into a 100mL volumetric flask, and then added with the same concentration of sodium phosphate buffer to a constant volume, and mixed well. The storage time of the product in a refrigerator at 4 ℃ can be 1-2 d.

(3) 7.5X 10-4M NBT solution

Weighing NBT (C)₄OH₃OCl₂N₁₀O₆)0.153g, dissolved in a small amount of distilled water, transferred to a 250mL volumetric flask, and added to a constant volume with distilled water, and mixed well. The storage time of the product in a refrigerator at 4 ℃ can be 2-3 d.

(4) 2X 10 with 1.0. mu.M EDTA^-5M Riboflavin solution

Solution A: weighing EDTA0.003g, and dissolving with a small amount of distilled water;

and B, liquid B: weighing 0.075g of riboflavin, and dissolving with a small amount of distilled water;

and C, liquid C: and combining the solution A and the solution B, transferring the combined solution A and solution B into a 100mL volumetric flask, and fixing the volume by using distilled water to obtain a solution, namely a 2mM riboflavin solution containing 0.1mM EDTA, and storing the solution in a dark place (the brown bottle containing the solution can be wrapped by black paper). The storage time of the product in a refrigerator at 4 ℃ can be 8-10 days. When measuring SOD enzyme activity, diluting the solution C by 100 times to obtain 2 × 10-5M riboflavin solution containing 1.0 μ M EDTA.

(5) 0.05M sodium phosphate buffer (pH 7.8) containing 2% polyvinylpyrrolidone (PVP)

50mL of 0.1M sodium phosphate buffer (pH 7.8) is taken, 2g of PVP is added, the mixture is fully dissolved and then is transferred into a 100mL volumetric flask, the volume is determined by distilled water, and the mixture is fully and uniformly mixed. Storing in a refrigerator at 4 ℃ for later use.

2) Measurement method

(1) Weighing 1.0g of sample blade, placing the sample blade in a precooled mortar, adding precooled 4mL of 0.05M sodium phosphate buffer solution (pH 7.8) containing 2% PVP, grinding in ice bath to homogenate, transferring the homogenate into a10 mL centrifuge tube, and fixing the volume to 5 mL;

(2) centrifuging at 4 deg.C and 10,000rpm for 10min to obtain supernatant as enzyme solution sample;

(3) a10 mL centrifuge tube with good clarity was taken, each strain was replicated 3 times, and reagents were added as follows:

(4) setting 3 controls CK1, CK2 and CK3, wrapping CK1 with aluminum foil and keeping out of the sun, placing the aluminum foil and other sample tubes (including CK2 and CK3) under 4500lux daylight lamp at 28 deg.C, and immediately shielding with black cloth to stop reaction after 25 min;

(5) SOD activity determination and calculation: the SOD activity (SOD activity unit is 50% inhibiting NBT photochemical reduction as an enzyme activity unit) is calculated by using a light-shielded control tube CK1 as a blank for zero adjustment and measuring the absorbance of each tube at a wavelength of 560nm and an average value of CK2 and CK3 as a control according to the following formula:

SOD activity (U/g) ═ ODC-ODS). times.V 1/ODC.times.0.5 XFW.times.V 2

Wherein the SOD activity is expressed in enzyme units per g fresh weight;

ODC-light absorption value of light control;

ODS-light absorption value of sample tube;

v1 — total volume of sample fluid (mL);

FW-sample fresh weight (g);

v2-sample volume (mL) at the time of assay.

The SOD activity measurement results of the plants of the transgenic tobacco strains L1, L9 and L13 and the wild type W38 tobacco plant are shown in A in figure 5, and the results show that the SOD activity of 3 transgenic strains is higher than that of the wild type W38 tobacco plant on an MS culture medium containing NaCl (with the concentration of 200mM) and an MS culture medium containing polyethylene glycol (with the concentration of 10%); the analysis of difference significance indicates that the SOD activity of the tobacco plants is obviously higher than that of wild type W38 tobacco plants.

4.4 assay of Malondialdehyde (MDA)

When plant organs are aged or damaged under stress, membrane lipid peroxidation often occurs, and Malondialdehyde (MDA) is a final decomposition product of membrane lipid peroxidation, and the content of Malondialdehyde (MDA) can reflect the degree of stress damage to plants. After release from the membrane-derived sites, MDA can react with proteins, nucleic acids, change the conformation of these macromolecules, or cause cross-linking reactions that result in loss of function or inhibition of protein synthesis. Thus, the accumulation of MDA may cause some damage to membranes and cells.

The MDA content of tobacco plants was measured by the method described in Gao et al (Gao S, Yuan L, ZHai H, Liu CL, He SZ, et al. Transgenic sweet potato plants expressing an LOS5 gene area tall to salt stress. plant cell Tissue Organ Cult,2011,107: 205-213).

The transgenic tobacco strains L1, L9 and L13 and the wild W38 tobacco strains are respectively subcultured on an MS culture medium containing NaCl (with the concentration of 200mM) and an MS solid culture medium containing polyethylene glycol (with the concentration of 10%) for drought stress and normal culture on the MS solid culture medium at the temperature of 27 +/-1 ℃ under the illumination of 13h and 3000lux every day for 4 weeks, and then the whole strain is taken to measure the MDA content and repeated for 3 times.

1) Main reagent and formula

(1) 5% trichloroacetic acid (TCA): weighing 5g of trichloroacetic acid, dissolving with a small amount of distilled water, transferring into a 100mL volumetric flask for constant volume, and fully and uniformly mixing;

(2) 0.5% thiobarbituric acid (TBA): weighing 0.5g of thiobarbituric acid, dissolving with a small amount of 5% TCA, transferring into a 100mL volumetric flask for constant volume, and mixing uniformly;

(3) and (4) quartz sand.

2) Extraction and determination method

(1) Weighing 1.0g of material, adding 10mL of 5% TCA and a small amount of quartz sand, and grinding to homogenate;

(2) centrifuging at 3,000rpm for 10min, and collecting supernatant as malonaldehyde extractive solution;

(3) taking 1.5mL of the above extract (1.5 mL of 5% TCA in a control tube), adding 2.5mL of 0.5% TBA, mixing, reacting in a boiling water bath for 15min, and rapidly cooling in an ice bath;

(4) centrifuging at 1,800g for 10 min;

(5) adjusting to zero with distilled water, and measuring absorbance of the supernatant at wavelength of 532nm and 600 nm;

(6) and (3) calculating the content:

MDA content (nM/g) ═ (OD532-OD600) times V1 times V2/(0.155 times FW times V3)

OD 532-light absorption at 532nm for the sample tube;

OD 600-the light absorption of the sample tube at 600 nm;

V1-Total volume of reaction solution (mL);

V2-Total volume of extract (mL);

FW-sample fresh weight (g);

V3-Total volume of solution for determination (mL).

The results of the MDA content determination of the plants of transgenic tobacco lines L1, L9, L13 and the wild type W38 tobacco plant are shown in fig. 5B, which shows that the MDA content of 3 transgenic lines is lower than that of the wild type W38 tobacco plant on both the MS medium containing NaCl (concentration 200mM) and the MS medium containing polyethylene glycol (concentration 10%); the analysis of difference significance indicates that the MDA content is significantly lower than that of the wild type W38 tobacco plants.

The determination results of the proline content, SOD activity and MDA content of the transgenic tobacco plant show that compared with a wild W38 tobacco plant, the salt tolerance and drought resistance of the transgenic tobacco plant over-expressing IbCAF1 are remarkably improved, and the protein IbCAF1 and the coding gene thereof can be used for regulating and controlling the stress tolerance of the plant, particularly improving the salt tolerance and drought resistance of the plant.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

SEQUENCE LISTING

<110> university of agriculture in China

Application of <120> protein IbCAF1 in regulation and control of salt and drought resistance of plants

<130>GNCFY200079

<160>2

<170>PatentIn version 3.5

<210>1

<211>281

<212>PRT

<213> sweet potato (Ipomoea batatas)

<400>1

Met Gly Val Gln Glu Asp Val Leu Asp Ala Asn Pro Ile Lys Ile Arg

1 5 10 15

Glu Val Trp Ala Asp Asn Leu Glu Ser Glu Phe Gln Leu Ile Ser Tyr

20 25 30

Leu Ile Asp Asp Tyr Pro Tyr Ile Ser Met Asp Thr Glu Phe Pro Gly

35 40 45

Val Val Phe Lys Pro Glu Ser Arg Arg Arg Gly Pro Leu Ser Ala Pro

50 55 60

Asp Cys Ser Ala Asp Ser Tyr Arg Leu Leu Lys Ser Asn Val Asp Ala

65 7075 80

Leu Asn Leu Ile Gln Leu Gly Leu Thr Leu Ser Asp Ala Ser Gly Asn

85 90 95

Leu Pro Val Leu Gly Ser Asp Gly His Arg Phe Ile Trp Gln Phe Asn

100 105 110

Phe Ala Asp Phe Asp Val Gln Arg Asp Leu Tyr Ala Pro Asp Ser Val

115 120 125

Glu Leu Leu Arg Arg Gln Gly Ile Asp Phe Asp Lys Asn Arg Asp Tyr

130 135 140

Gly Ile Asp Ser Ala Arg Phe Ala Glu Leu Met Met Ser Ser Gly Leu

145 150 155 160

Val Cys Asn Glu Ser Val Ser Trp Val Thr Phe His Ser Ala Tyr Asp

165 170 175

Phe Gly Tyr Leu Val Lys Ile Leu Thr Arg Arg Ser Leu Pro Gly His

180 185 190

Leu Glu Asp Phe Leu Glu Ile Leu Lys Ile Phe Phe Gly Asp Arg Val

195 200 205

Tyr Asp Val Lys His Leu Met Lys Phe Cys His Ser Leu Tyr Gly Gly

210 215 220

Leu Asp Arg Leu Ala Ser Thr Leu Glu Val Asp Arg Val Val Gly Lys

225 230235 240

Cys His Gln Ala Gly Ser Asp Ser Leu Leu Thr Trp His Thr Phe Gln

245 250 255

Lys Met Arg Asp Val Tyr Phe Leu Asn Glu Glu Pro Glu Lys Tyr Ala

260 265 270

Gly Val Leu Tyr Gly Leu Asp Val Phe

275 280

<210>2

<211>846

<212>DNA

<213> sweet potato (Ipomoea batatas)

<400>2

atgggtgtac aagaagatgt tttggatgca aatcctatta agattaggga agtttgggcg 60

gacaacctgg aatcggagtt ccagctcatc agctacctca tcgacgacta tccgtacatc 120

tccatggata cggagtttcc tggggtggtg ttcaagccgg agagtcgccg gcgagggcct 180

ttgtcggctc ccgattgctc tgccgattct tacaggttgc tcaagtcaaa cgtggacgct 240

ctgaatctga ttcagctcgg gttgactttg tcggacgcta gcgggaacct ccccgtgctc 300

ggatctgacg gccaccgctt tatctggcag ttcaacttcg ccgatttcga cgtgcagcgt 360

gacctctacg cgccggattc cgtcgagctg ctccggcgtc agggaattga ttttgacaag 420

aaccgggact acgggattga ctcggcccgg ttcgccgagt tgatgatgtc ttccggtctg 480

gtctgtaacg agtccgtcag ttgggtcacc ttccacagcg cgtacgactt tggatacctc 540

gtcaagatcc taacgcgtcg ctccttgccc ggacatctgg aagacttctt ggaaattctc 600

aagattttct tcggagaccg ggtttatgat gtcaaacacc ttatgaaatt ctgtcacagc 660

ctctatggcg ggctagatcg gttggccagc acgctggagg tagaccgggt cgtcggaaaa 720

tgccatcagg ccggttcaga tagcctgctg acgtggcaca cattccaaaa aatgagagat 780

gtgtatttct taaatgaaga accagaaaaa tatgccggag tattgtacgg actagatgtt 840

ttttag 846

Claims

1. Use of a protein in any one of:

B1) improving the salt tolerance of the plants;

B2) preparing a product for improving the salt tolerance of plants;

B3) improving the drought resistance of the plants;

B4) preparing a product for improving the drought resistance of plants;

B14) preparing a product for improving the SOD activity of plants under drought and/or salt stress conditions;

the protein is B1) or B2) or B3) as follows:

A1) protein with amino acid sequence shown as SEQ ID NO. 1;

2. Use of the protein-related biomaterial of claim 1 in any one of B1) -B14) of claim 1, wherein the protein-related biomaterial is any one of the following C1) -C4):

C1) a nucleic acid molecule encoding the protein of claim 1;

C2) an expression cassette comprising the nucleic acid molecule of C1);

C4) a recombinant microorganism containing C1) the nucleic acid molecule, or a recombinant microorganism containing C2) the expression cassette, or a recombinant microorganism containing C3) the recombinant vector.

3. Use according to claim 2, characterized in that: C1) the nucleic acid molecule is shown in any one of the following D1) or D2) or D3):

D1) DNA molecule shown in SEQ ID NO. 2;

D2) the coding sequence is DNA molecule shown in SEQ ID NO. 2;

D3) a DNA molecule which hybridizes under stringent conditions with a DNA molecule defined in D1) or D2) and which encodes a protein as claimed in claim 1.

4. Use of a protein as claimed in claim 1, or a related biological material as claimed in claim 2, in plant breeding.

5. A method for cultivating a transgenic plant with high drought resistance and/or salt tolerance, which is characterized by comprising the following steps: the method comprises increasing the expression level of a gene of the protein of claim 1 and/or the content of the protein and/or the activity of the protein in a target plant to obtain a transgenic plant; the drought resistance and/or salt tolerance of the transgenic plant is higher than that of the target plant.

6. The method of claim 5, wherein: the method for increasing the expression level of the gene of the protein of claim 1 and/or the content of the protein and/or the activity of the protein in a target plant is to express or overexpress the protein of claim 1 in the target plant.

7. The method of claim 6, wherein: the method for expression or overexpression is to introduce a gene encoding the protein of claim 1 into a plant of interest.

8. The method of claim 7, wherein: the nucleotide sequence of the coding gene is a DNA molecule shown in SEQ ID NO. 2.

9. The protein of claim 1, or the related biological material of claim 2.

10. The use according to any one of claims 1 to 4, or the method according to any one of claims 5 to 8, wherein: the plant is any one of the following plants:

E1) a dicotyledonous plant;

E2) a monocot plant;

E3) a plant of the genus nicotiana;

E4) tobacco;

E5) a plant of the family Convolvulaceae;

E6) a plant of the genus Ipomoea;

E7) sweet potato.