CN117778447A - Rice OsVIP2 gene and application of encoding protein thereof in improving stress resistance of rice - Google Patents
Rice OsVIP2 gene and application of encoding protein thereof in improving stress resistance of rice Download PDFInfo
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Abstract
The invention discloses an application of a rice OsVIP2 gene and a coded protein thereof in improving stress resistance of rice, and relates to the technical field of genetic engineering. Research shows that the gene can be excessively expressed to improve drought resistance and salt tolerance of rice; the gene editing mutant is more sensitive to drought stress and salt stress, so that the gene can be combined with an over-expression promoter in a plant and then introduced into a proper expression vector and transformed into a plant host, has the functions of improving drought resistance and salt tolerance of rice, and has important significance for improving the abiotic stress resistance of the rice.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to an application of a rice OsVIP2 gene and a coded protein thereof in improving stress resistance of rice.
Background
Rice is one of the most important grain crops in China. Salt and drought stress are important limiting factors for inhibiting growth and development of rice and affecting seed germination, growth and yield. The area of the saline-alkali soil in China is 9913 ten thousand hectares, the area is relatively large, and the areas where the saline-alkali soil in China is distributed are more. Soil salinization has become one of the main factors restricting crop yield improvement. The rice is sensitive to abiotic stress, the yield in the saline environment is obviously reduced, coastline of China is long, large-area coastal offshore beach and farmland are difficult to realize high and stable yield in saline-alkali soil due to relatively high salt content of soil, and further expansion of rice planting area in China is limited. Meanwhile, in recent years, drought frequently occurs, and soil salinization is aggravated by drought.
Therefore, the development and identification of the salt tolerance and drought tolerance genes of the rice, the improvement of the stress tolerance of the rice, and the realization of high and stable yield in the saline-alkali soil for enlarging the planting area of the rice in the saline-alkali soil are the problems to be solved by the technicians in the field.
Disclosure of Invention
In view of the above, the invention provides an application of a rice OsVIP2 gene and a coded protein thereof in improving stress resistance of rice.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
application of enhancing protein or regulating expression level of protein coding gene expression substance in improving stress resistance of rice, wherein the amino acid sequence of the protein is shown as SEQ ID NO:2 is shown in the figure;
or a homologous sequence of the sequence shown in SEQ ID NO. 2;
or a conservative variant of the sequence depicted in SEQ ID NO. 2;
or an allelic variant of the sequence set forth in SEQ ID NO. 2;
or a natural mutant of the sequence described by SEQ ID NO. 2;
or an induced mutant of the sequence described in SEQ ID NO. 2.
Another object of the present invention is to provide an application of the protein-related biomaterial in improving stress resistance of rice, wherein the biomaterial is any one of the following:
a: a nucleic acid molecule encoding the protein of claim 1;
b: an expression cassette comprising over-expression of the nucleic acid molecule of a;
c: an over-expression vector comprising the nucleic acid molecule of A, or a recombinant vector comprising the expression cassette of B;
d: a recombinant microorganism comprising the nucleic acid molecule of A, a recombinant microorganism comprising the expression cassette of B, or a recombinant microorganism comprising the recombinant vector of C.
Preferably, the nucleic acid molecule has the nucleotide sequence set forth in SEQ ID NO:1 is shown in the specification;
or a sequence which is completely complementary to the sequence shown in SEQ ID NO. 1;
or a sequence which hybridizes with the sequence shown in SEQ ID NO. 1;
or a subfragment functionally equivalent to the sequence shown in SEQ ID NO. 1.
Another object of the present invention is to provide a biological material for improving stress resistance of rice, wherein the biological material is any one of the following:
a: an expression cassette comprising overexpression of a nucleic acid molecule as shown in SEQ ID NO. 1;
b: a recombinant vector comprising the expression cassette of a;
c: a recombinant microorganism comprising the expression cassette of a, or a recombinant microorganism comprising the recombinant vector of b.
Another object of the present invention is to provide a method for improving stress resistance of rice, which comprises enhancing the activity of the protein of claim 1 or increasing the expression level of the gene encoding the protein of claim 1.
Preferably, the nucleotide sequence of the protein coding gene is shown in SEQ ID NO: 1.
Preferably, the method for increasing the expression level of the protein coding gene in claim 1 is to construct an OsVIP2 gene overexpression vector, comprising the following steps:
(1) Designing and amplifying a primer of a complete coding reading frame according to the full-length sequence of the OsVIP2 gene;
(2) PCR amplification is carried out by taking the cDNA sequence of the OsVIP2 gene as a template and connecting the cDNA sequence to a cloning vector-Blunt Cloning Vector;
(3) Designing the primers SEQ ID NO.5 and 6 with the linker, and carrying out recombination reaction with a plant expression vector Ub08 containing a promoter and a terminator protein.
It is another object of the present invention to provide a high stress-tolerant transgenic rice transformed with any one of the following nucleic acids:
the nucleotide of the nucleic acid molecule is shown as SEQ ID NO:1 is shown in the specification;
or a sequence which is completely complementary to the sequence shown in SEQ ID NO. 1;
or a sequence which hybridizes with the sequence shown in SEQ ID NO. 1;
or a subfragment functionally equivalent to the sequence shown in SEQ ID NO. 1.
Preferably, said stress resistance in said application or said biological material or said method or said transgenic rice is drought and/or salt tolerance.
Preferably, said rice in said application or said biological material or said method or said transgenic rice is Nipponbare.
The beneficial effects are that: the invention utilizes the transgenic technology such as over-expression to create the OsVIP2 gene over-expression plant, which can improve the survival rate of the transgenic plant under drought stress and/or salt stress conditions and improve drought resistance and salt tolerance so as to cultivate high stress resistance rice.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of the overexpressing vector Ub08-OsVIP 2.
FIG. 2 is a schematic diagram of an OsVIP2 gene editing vector.
FIG. 3 is a graph showing the relative expression level of OsVIP2 gene in the leaf of over-expressed transgenic rice. Wherein the number of the horizontal axis is OsVIP2 transgenic rice line; the vertical axis represents: the ratio of the expression level of OsVIP2 of the transgenic strain relative to that of a wild control plant, and the internal reference gene is actin1.
FIG. 4 is a graph showing the results of editing and identifying OsVIP2 genes.
FIG. 5 is an OsVIP2 transgenic material drought tolerance evaluation; wherein, FIG. 5A is the phenotype of the gene editing mutant strain under drought stress, FIG. 5B is the result of survival rate of the gene editing mutant strain under drought stress, FIG. 5C is the phenotype of the over-expressed strain under drought stress, and FIG. 5D is the result of survival rate of the over-expressed strain under drought stress; WT represents non-transgenic wild type rice; OE1, OE2, OE3 represent the os vip2 gene-transgenic overexpressing strain; KO1, KO2 and KO3 represent gene editing mutant lines. * Represents P <0.05, and P <0.01.
FIG. 6 is an OsVIP2 transgenic material salt tolerance evaluation; wherein, FIG. 6A shows the phenotype of the over-expression strain under the normal culture condition, FIG. 6B shows the phenotype of the gene editing mutant strain under the normal culture condition, FIG. 6C shows the phenotype of the over-expression strain under the salt stress, FIG. 6D shows the phenotype of the gene editing mutant strain under the salt stress, and FIG. 6E shows the survival rate result of each genotype under the salt stress; WT represents wild type rice; osVIP2-OE1 OsVIP2-OE2, wherein OsVIP2-OE3 represents an OsVIP2 gene-transferred overexpression line; osVIP2-KO1, osVIP2-KO2 and KO3 represent gene editing mutant lines. * Represents P <0.05, and P <0.01.
Detailed Description
In order that the invention may be understood more fully, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended claims. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It will be appreciated that the experimental procedure, without specific conditions noted in the examples below, is generally followed by routine conditions, such as molecular cloning by Sambrook et al: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. The various reagents commonly used in the examples are all commercially available products.
As used herein, the terms "isolated", "purified" DNA refer to DNA or fragments that have been isolated from sequences that flank them in nature, as well as DNA or fragments that have been separated from components that accompany nucleic acids in nature, and from proteins that accompany them in cells.
The invention also includes variants of the open reading frame sequence of SEQ ID NO.1 encoding proteins having the same function as OsVIP 2. These variants include (but are not limited to): deletions, insertions and/or substitutions of several (typically 1-90, preferably 1-60, more preferably 1-20, most preferably 1-10) nucleotides, and additions of several (typically within 60, more preferably within 30, more preferably within 10, most preferably within 5) nucleotides at the 5 and/or 3 terminus.
In the present invention, a variant of SEQ ID NO.2 having the same function as OsVIP2 is also included. These variants include (but are not limited to): deletion, insertion and/or substitution of several (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal end. For example, in the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. As another example, the addition of one or more amino acids at the C-terminus and/or N-terminus typically does not alter the function of the protein.
The percent homology of proteins was determined by GAP (Needleman and Wunsh, 1970) analysis (GCG program), where the parameter GAP creation penalty = 5,gap extension penalty =0.3. Where the sequence being analyzed is at least 15 amino acids in length, the GAP analysis is performed over a region of at least 15 amino acids of the two sequences involved in the test. More preferably, the GAP analysis is performed over a region of at least 50 amino acids of the two sequences involved in the test when the sequence being analyzed is at least 50 amino acids in length. More preferably, the GAP analysis is performed over a region of at least 100 amino acids of the two sequences involved in the test when the sequence being analyzed is at least 100 amino acids in length. More preferably, the GAP analysis is performed over a region of at least 250 amino acids of the two sequences involved in the test when the sequence being analyzed is at least 250 amino acids in length. Even more preferably, the GAP analysis is performed over a region of at least 500 amino acids of the two sequences involved in the test when the sequence being analyzed is at least 500 amino acids in length.
Polynucleotides (DNA or RNA), vectors, transformants and organisms can be isolated and purified by methods known in the art.
Polynucleotides isolated according to the invention include, but are not limited to: a nucleotide sequence of SEQ ID NO.1 encoding an OsVIP2 gene; or the nucleotide sequence can be hybridized with the nucleotide sequence from 1 st to 2400 th nucleotide in SEQ ID NO. 1; or a subfragment functionally equivalent to the sequence shown in SEQ ID NO. 1.
The cloned OsVIP2 gene can be used as a probe, and the gene or homologous gene can be obtained by screening cDNA and genome libraries, or can be synthesized directly by adopting a gene synthesis method. The OsVIP2 gene of the present invention and any DNA fragment or a DNA fragment homologous thereto can be amplified from the genome or cDNA using PCR (polymerase chain reaction) techniques as well.
The vector used in the present invention may be, for example, a phage, plasmid, cosmid, minichromosome, viral or retroviral vector. Vectors useful for cloning and/or expressing polynucleotides of the invention are vectors capable of replicating and/or expressing polynucleotides in a host cell in which the polynucleotides are to be replicated and/or expressed. In general, the recombinant expression vector carrying the nucleic acid sequence of the present invention can be introduced into plant cells by conventional biotechnological methods such as Ti plasmid, plant viral vector, direct DNA transformation, microinjection, electroporation (Weissbach, 1998,Method for Plant Molecular Biology VIII,Academy Press,New York,pp.411-463;Geiserson and Corey,1998,Plant Molecular Biology (2 nd Edition).
Various methods have been developed for operably linking a polynucleotide to a vector via complementary cohesive ends. For example, complementary fragments of the homopolymer sequence may be added to the DNA segment to be inserted into the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymer tails to form a recombinant DNA molecule.
Synthetic linkers containing one or more restriction sites provide another method of linking a DNA segment to a vector. The DNA segment produced by restriction endonuclease digestion is treated with phage T4 DNA polymerase or e.coli DNA polymerase I, both of which remove the protruding γ -single stranded ends with their 3', 5' -exonuclease activity and fill in the 3' -concave ends with their polymerization activity. Thus, the combination of these activities produces blunt-ended DNA segments which are then incubated with a molar excess of linker molecules in the presence of an enzyme capable of catalyzing the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the reaction product is a DNA segment bearing a polymeric linker sequence at the end, and these DNA segments are then cleaved with an appropriate restriction enzyme and ligated into an expression vector that has been cleaved with an enzyme that produces ends compatible with the DNA segment. Synthetic linkers containing multiple restriction endonuclease sites are commercially available from a variety of merchants.
Other newly developed techniques utilize homologous recombination methods in which polynucleotides carrying specific sequence linkers or homologous sequence linkers are subjected to homologous recombination with a vector, and the DNA segment to be inserted into the vector DNA is reacted with a vector also carrying the specific sequence or homologous sequence by the action of a recombinase to form a recombinant DNA molecule.
The polynucleotide insert should be operably linked to a suitable promoter compatible with the host cell in which the polynucleotide is to be expressed, which may be a strong promoter and/or an inducible promoter. Examples of some of the promoters listed include phage PL promoter, e.coli lac, trP, phoA, tac promoter, SV40 early and late promoters, and retroviral LTR promoters; other suitable promoters are known to those skilled in the art. The expression recombinant vector further contains transcription initiation and termination sites, and a ribosome binding site for translation in the transcribed region. The coding portion of a transcript expressed by a recombinant vector may include a translation initiation codon at the start and a termination codon (UAA, UGA or UAG) suitably at the end of the polypeptide being translated.
As described above, the expression vector may include at least one selectable marker. The markers include resistance genes encoding antibiotics, such as: neomycin phosphotransferase (Neomycin phosphotransferase) gene nptII, hygromycin phosphotransferase (Hygromycin phosphotransferase) gene hpt and dihydrofolate reductase (Dihydrofolate reductase) gene dhfr; another class is the genes encoding herbicide resistance, e.g., the gene Bar for glufosinate acetyltransferase (Phosphinothricin acetyltransferase), the gene epsps for 5-enolpyruvyl oxalate-3-phosphate synthase (5-Enoylpyruvate shikimatr-3-phosphate). Representative examples of suitable hosts include, but are not limited to: protoplast cells and plant cells. Suitable media and culture conditions for the above-described host cells are known in the art.
A method for transforming a gene of interest or a polynucleotide of interest: one type is a vector-mediated transformation method, in which a target gene is inserted into a vector molecule such as a plasmid of agrobacterium or a DNA of a virus, and the target gene is introduced into a plant genome along with transfer of the vector DNA; agrobacterium-mediated and virus-mediated methods are among such methods. The second type is a direct gene transfer method, which refers to directly transferring an exogenous gene of interest into the genome of a plant by a physical or chemical method. Physical methods include gene gun transformation, electric excitation transformation, ultrasonic, microinjection, laser microbeam, and the like; the chemical method includes PEG-mediated transformation method, liposome method, etc. The third category is germplasm systems, which includes pollen tube channel, germ cell dip, embryo sac and ovary injection, and the like.
In the present invention, the term "transformant" (transformation), i.e.a host cell or organism carrying a heterologous DNA molecule, is used.
The invention also includes host cells comprising a nucleotide sequence of the invention operably linked to one or more heterologous control regions (e.g., promoters and/or enhancers) via techniques known in the art. Host strains can be selected which either modulate the expression of the inserted gene sequence or can modify and process the gene product in the particular manner desired. In the presence of certain inducers, expression from certain promoters may be elevated.
The successfully transformed cells, i.e.the cells or organisms containing the recombinant vectors of the nucleotide sequences according to the invention, can be identified by well known techniques.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The invention is described in detail below by way of examples:
example 1 isolation and cloning of Rice OsVIP2 Gene
Seedlings of rice (Nippon Temminck) grown for 4 weeks were salted for 1 hour, and then leaf pieces were used to extract total RNA, and TRNzol Universal reagent (Tiangen Biochemical Co., ltd.) was used to extract total RNA of rice. It was reverse transcribed into first strand cDNA using reverse transcriptase MLV (Tiangen, china).
The full-length cDNA encoding the gene was amplified using primer F (5'-ATGATGGAAGCGGACATGGAGAACG-3', SEQ ID No. 3) and primer R (5'-TCACAAGAATATTGGAGCCATCACT-3', SEQ ID No. 4). The PCR reaction conditions were: pre-denaturation at 94℃for 3min;98℃20sec,60℃30sec,72℃60sec for a total of 35 cycles; extending at 72℃for 5min.
Ligating the amplified PCR product into-Blunt Cloning Vector vector (Beijing full gold Biotech (TransGen Biotech) Co., ltd.), positive clones were screened and sequenced to obtain the cDNA sequence of the OsVIP2 gene (SEQ ID NO. 1).
The cDNA sequence of the OsVIP2 gene is:
ATGATGGAAGCGGACATGGAGAACGGGAGGTTGTATCCGGAGAGACCCAGAACTTTCTCTACCGTACGAACTAAATCATCTCTGCCACCGATCTTCCGTGTGTTGATGAGGATAAATCCCCGTGCTTTCATTGTTCTTCTTCTTTTGGTATTCAGTGGTGTGCTCTATGTAGGAGCCAGTACATCACCAATTGTGCTCTTTGTGTTCTGCATATGTACTCTAAGTTTATTCTTCTCTCTCTACCTCACAAAGTGGGTTCTTGCCAAAGATGAAGGTCCTCCAGAAATGTCTGAGATATCTGATGCCATAAGAGATGGTGCTGAAGGATTTTTTAGGACACAATATGGGACTATTTCTAAAATGGCTTGTATCCTGGCACTTGTCATTCTTGGCATCTATCTCTTTCGTTCCACCACCCCACAGCAGGAGGCATCCGGTGTTGGAAGGACAACATCGGCATATATTACTGTTGCTTCCTTTCTCCTTGGAGCTTTATGTTCTGGGATTGCTGGTTTTGTTGGGATGTGGGTATCCGTACGTGCAAATGTTAGAGTTTCAAGTGCTGCTCGGCGGTCTGCAAGAGAAGCACTGCAGATTGCTGTCCGCGCTGGTGGTTTCTCAGCCATTGTGGTTGTTGGCATGGCTGTTTTTGGTGTGGCAATACTGTATGCAACATTCTATGTTTGGCTTGAAGTAGATTCACCTGGTTCAATGAAGGTTACTGATCTGCCTCTTCTCCTTGTGGGATATGGTTTTGGTGCGTCTTTTGTCGCCCTTTTTGCTCAGTTGGGTGGTGGAATATACACCAAAGCTGCTGATGTCGGAGCTGATCTTGTGGGAAAGGTTGAGCAGGGAATACCAGAAGATGATCCTCGTAATCCTGCTGTCATTGCTGACTTGGTTGGTGACAATGTTGGAGATTGTGCCGCACGAGGGGCTGACCTTTTTGAGAGCATAGCAGCTGAAATTATCAGTGCAATGATACTCGGGGGAACAATGGCACAACGCTGCAAAATTGAAGATCCCTCAGGCTTTATACTGTTTCCTCTTGTTGTTCATTCATTTGATCTGGTGATCTCATCAGTTGGAATACTCTCAATTCGGGGAACACGTGACTCTGGGTTAATATCCCCTATTGAAGATCCGATGGCAATCATGCAGAAAGGATATTCTATCACTATACTGCTCGCTGTTGTTACCTTTGGAGTGTCTACCCGGTGGCTCCTGTACACTGAACAAGCACCTTCTGCATGGCTCAATTTTGCCTTGTGTGGCTTGGTGGGCATCATCACAGCATATGCTTTTGTTTGGATTTCTAAGTATTACACAGATTACAAACATGAGCCTGTCCGCCTTTTGGCTCTTTCAAGTTCCACAGGGCATGGAACTAATATTATTGCTGGAGTAAGCTTGGGACTGGAATCAACAGCGTTACCAGTTCTAGTGATAAGTGTAGCCATCATATCAGCATTTTGGTTGGGGCATACATCTGGATTAGTAGATGAATCTGGGAACCCAACTGGTGGTCTTTTTGGGACAGCTGTAGCTACAATGGGGATGCTTAGCACAGCAGCATATGTTCTCACCATGGACATGTTTGGTCCAATAGCTGACAATGCTGGTGGTATTGTGGAGATGAGTCAGCAGCCTGAAAGTGTAAGAGAAATCACAGACATTCTAGATGCTGTGGGCAATACAACAAAAGCTACTACAAAGGGATTTGCTATTGGGTCAGCAGCACTGGCTTCCTTTCTCCTGTTCAGTGCATATATGGATGAAGTAGCTGCTTTCGCACAATTGCCATTCAAAGAGGTTGACATAGCAATCCCAGAGGTTTTTGTTGGTGGTTTACTTGGTTCAATGCTTATATTCCTGTTTAGTGCATGGGCTTGTTCTGCTGTTGGCAGAACTGCACAAGAAGTTGTTAACGAGGTCAGGAGACAGTTTATTGAGAGGCCTGGCATTATGGACTACAACGAGAAGCCTGATTACGGTCGTTGTGTTGCAATTGTGGCATCTGCTTCCTTGAGGGAAATGATAAGGCCGGGGGCTTTAGCCATTATATCACCCATGGCTGTTGGTATTATCTTCCGTATGTTGGGTCACGCTACTGGCCGGCCTCTTCTTGGAGCCAAAGTTGTAGCCGCTATGCTTATGTTTGCAACTGTTTCTGGTATTCTCATGGCACTCTTCTTGAACACTGCTGGTGGTGCCTGGGATAATGCTAAGAAGTACATCGAGACTGGCGCTCTTGGTGGCAAAGGCAGTGAGTCTCACAAGGCTGCAGTTACTGGTGATACGGTTGGAGATCCATTTAAAGACACGGCGGGGCCTTCCATCCATGTTCTTATTAAGATGCTCGCTACAATCACATTAGTGATGGCTCCAATATTCTTGTGA,SEQ ID NO.1。
the amino acid sequence of the OsVIP2 protein is as follows:
MMEADMENGRLYPERPRTFSTVRTKSSLPPIFRVLMRINPRAFIVLLLLVFSGVLYVGASTSPIVLFVFCICTLSLFFSLYLTKWVLAKDEGPPEMSEISDAIRDGAEGFFRTQYGTISKMACILALVILGIYLFRSTTPQQEASGVGRTTSAYITVASFLLGALCSGIAGFVGMWVSVRANVRVSSAARRSAREALQIAVRAGGFSAIVVVGMAVFGVAILYATFYVWLEVDSPGSMKVTDLPLLLVGYGFGASFVALFAQLGGGIYTKAADVGADLVGKVEQGIPEDDPRNPAVIADLVGDNVGDCAARGADLFESIAAEIISAMILGGTMAQRCKIEDPSGFILFPLVVHSFDLVISSVGILSIRGTRDSGLISPIEDPMAIMQKGYSITILLAVVTFGVSTRWLLYTEQAPSAWLNFALCGLVGIITAYAFVWISKYYTDYKHEPVRLLALSSSTGHGTNIIAGVSLGLESTALPVLVISVAIISAFWLGHTSGLVDESGNPTGGLFGTAVATMGMLSTAAYVLTMDMFGPIADNAGGIVEMSQQPESVREITDILDAVGNTTKATTKGFAIGSAALASFLLFSAYMDEVAAFAQLPFKEVDIAIPEVFVGGLLGSMLIFLFSAWACSAVGRTAQEVVNEVRRQFIERPGIMDYNEKPDYGRCVAIVASASLREMIRPGALAIISPMAVGIIFRMLGHATGRPLLGAKVVAAMLMFATVSGILMALFLNTAGGAWDNAKKYIETGALGGKGSESHKAAVTGDTVGDPFKDTAGPSIHVLIKMLATITLVMAPIFL*,SEQ ID NO.2。
EXAMPLE 2 construction of OsVIP2 Gene overexpression and editing vector and genetic transformation
1. Construction of overexpression of OsVIP2 genes:
primers were designed to amplify the complete coding reading frame based on the full length sequence of the OsVIP2 gene (SEQ ID NO. 1), and adaptor primers were added to the upstream and downstream primers, respectively, to construct an expression vector. PCR amplification with high-fidelity KOD enzyme (Toyobo Co., ltd.) using the amplified product obtained in example 1 as a template, cloning the OsVIP2 gene cDNA into a cloning vectorBlunt Cloning Vector vector (Beijing full gold biotechnology (TransGen Biotech Co., ltd.) further transformed E.coli DH 5. Alpha. And intermediate vector was identified with the correct reading frame ensured, then plasmids were extracted, and adaptor-designed primer F (5'-gatgaactatacaaaactagtATGATGGAAGCGGACATGGAGAACG-3', SEQ ID No. 5) and primer R (5'-gggaaattcgagctggtcaccTCACAAGAATATTGGAGCCATCACT-3', SEQ ID No. 6) were used by Beijing full gold biotechnology (TransGen Biotech) Co., ltd.>The Basic Seamless Cloning and Assembly Kit recombinase reacts with the plant expression vector Ub08 containing the promoter and the terminator protein to form a complete expression unit (see figure 1), agrobacterium tumefaciens EHA105 is transformed, and finally rice callus transformation experiments are carried out.
2. Constructing a target gene editing expression vector:
for construction of an OsVIP2 gene vector for gene editing by using a Crispr/Cas9 system, a CRISPR-P2.0 tool (http:// Crispr. Hzau. Edu. Cn/CRISPR2 /) is used for designing sgRNA targets as follows: 5'-GAGCACAATTGGTGATGTACTGG-3' (SEQ ID NO. 7), then constructing a gene editing vector by referring to the method provided by the published Crispr/Cas9 gene editing system, and finally loading the U6 promoter and the sgRNA expression cassette into the expression vector pYLCRISPR/Cas9Pubi-H (see figure 2). Specific procedures are described in the references (MaX, zhang Q, zhu Q, liu W, chen Y, qia R, wang B, yang Z, li H, lin Y, xie Y, shen R, chen S, wang Z, chen Y, liu Y.A Robust CRISPR/Cas9 System for Convenient, high-Efficiency Multiplex Genome Editing in Monocot and Dicot plants.molecular Plant,2015, 8:1274-1284). And transforming the constructed plasmid into agrobacterium EHA105, and performing a rice callus transformation experiment.
3. Genetic transformation of rice
(1) Seed disinfection
Removing the shell of mature Nippon Rice seeds, placing into a sterile triangular flask, soaking in 75% alcohol for 1-2min, and washing with sterile water for 2 times; sterilizing with 3% NaClO for 30min, shaking, washing with sterile water for 3-4 times, sucking excessive water with sterile filter paper, inoculating the seeds onto callus induction medium (NB+2, 4-D3.0 mg/L), culturing about 30 grains per dish, and dark culturing at 28deg.C.
(2) Subculture
After induction for nearly 1 month, the rice grows yellow and enlarged callus, scutellum is removed, and the callus is transferred to a fresh callus induction culture medium (NB+2, 4-D2.0 mg/L) for subculture (28 ℃ C., dark culture). And 2-4 times of subculture is carried out every 2 weeks to obtain tender yellow granular embryogenic callus suitable for transgenosis. After 2 weeks of subculture, embryogenic particles were selected for genetic transformation.
(3) Cultivation of Agrobacterium
The Agrobacterium single colonies were picked on transformation plates and cultured in 1ml of Agrobacterium medium. 1ml of the above culture was added to 50ml of Agrobacterium medium (containing the corresponding antibiotic) and incubated at 200rpm and 28℃for 5-6h to OD 600 Acetosyringone (AS, acetosyringone, final concentration 100 uM) was added 2h before the end of the incubation at 0.6-1.0. Taking the bacterial liquid at room temperature, 4000rpm for 10min, discarding the supernatant, adding MS liquid culture medium (containing AS 100 uM) to resuspend bacterial cells, culturing under the same conditions for 2h to make OD of bacterial liquid 600 =0.5-1, which can be used to transform calli at this time.
(4) Co-cultivation
The embryogenic callus of rice is immersed in agrobacterium liquid for 20-30min, then the moisture is absorbed by sterile absorbent paper, the infected callus is placed on a co-culture medium (MS+2, 4-D2.0 mg/L+AS 100 uM), and the infected callus is subjected to dark culture at 28 ℃ for three days.
(5) Bacteria washing
The co-cultured callus is washed by sterile water for 3-5 times, then soaked in MS liquid culture medium containing Cef 400mg/L for 20-30min, and transferred to sterile filter paper for drying.
(6) Screening culture
The callus with the water absorbed was inoculated on a selection medium (NB+2, 4-D2.0 mg/L+Hyg30mg/L+Cef400 mg/L). After 3 weeks, the newly grown calli were selected and inoculated onto selection medium (NB+2, 4-D2.0 mg/L+Hyg50mg/L+Cef250mg/L) and selected for 2 weeks.
(7) Differentiation culture
The resistant callus obtained by 2 times of screening is transferred to a pre-differentiation culture medium (N6+KT2.0 mg/L+NAA 0.2mg/L+6-BA 2.0mg/L+Hyg 30 mg/L+Cef200mg/L+agar 9g/L+sucrose 45 g/L) for dark culture for about 10 days, and then transferred to a differentiation culture medium (N6+KT2.0 mg/L+NAA 0.2mg/L+6-BA 2.0mg/L+Hyg 30 mg/L+agar 4.5 g/L+sucrose 30 g/L) for illumination culture.
(8) Rooting culture
About 1-2 months, seedlings about 2cm high were transferred to rooting medium (1/2MS+Hyg 15 mg/L+agar 4.5 g/L+sucrose 20 g/L) to induce adventitious roots.
(9) Transplanting of transgenic seedlings
When the seedlings grow to 10cm high, the seedlings are taken out, the attached solid culture medium is washed by sterile water and is transferred into soil, the seedlings are covered by a glass cover for several days just before beginning, and the glass cover is taken down after the plants are strong, and the seedlings are cultivated in a greenhouse.
EXAMPLE 3 analysis of expression of OsVIP2 Gene in transgenic plants
(1) Material preparation
Transgenic T0 generation rice seedling, cutting leaf blade, fast storing in liquid nitrogen for extracting RNA.
(2) DNA-free total RNA preparation
Total RNA of rice was extracted according to the specification using TRNzol Universal reagent (Tiangen Biochemical technology (Beijing) Co., ltd.).
(3) Synthesis of first strand cDNA
By means ofThe One-Step gDNA Removal and cDNA Synthesis SuperMix (catalog number: AW311-02, beijing full-scale gold Biotechnology Co., ltd.) kit was reverse transcribed to form a first strand cDNA according to the instructions.
(4) Quantitative PCR
Specific primers were designed based on the sequence of gene OsVIP 2:
qOsVIP2F:5’-TGTATCCTGGCACTTGTCATTC-3’,SEQ ID NO.8,
qOsVIP2R:5’-ATCCCAGAACATAAAGCTCCAA-3’,SEQ ID NO.9。
for fluorescent quantitative PCR, specific primers were designed based on the cDNA sequence of the rice action gene (GenBank accession No. AY 212324):
Actin-F:5’-CTTCCTCATGCCATCCTGC-3’,SEQ ID NO.10,
Actin-R:5’-GCAAGCTTCTCCTTGATGTCC-3’,SEQ ID NO.11。
fluorescent quantitative PCR for reference genes.
PCR Using American ABI7000 quantitative PCR instrument, three replicates per PCR setup. The reaction system comprises->Top Green qPCR SuperMix (+DyeI) (2X) 10. Mu.L each of forward and reverse primers, 1. Mu.L of cDNA template, and water was added to make up the volume to 20. Mu.L. The reaction procedure is: and (3) cycling for 40 times at 95 ℃ for 30s, then cycling for 10s at 95 ℃ and 34s at 61 ℃, setting the fluorescent value to be read at 60 ℃ for 34s in each cycle, correcting the ROX value, and finally adding a melting curve analysis of a fluorescent PCR product, wherein other operations are shown in the instruction of instrument use. To detect the presence of DNA contamination in RNA samples, 3 samples were randomly selected, each with 1. Mu. LRNA as template for PCR, as described above.
(5) Analysis method
Ct is generated by 7000system SDS Version1.2.3 software after the fluorescence threshold of PCR is manually determined to be 0.2, and the data is input to EXCEL for computational analysis. The data analysis was performed using a 2- ΔΔCT method, and then using EXCEL to represent the difference histogram.
(6) Analysis results
The expression level of the over-expression material OsVIP2-OE is identified by taking a blank non-transgenic Japanese fine variety as a reference, and the strains such as the over-expression plants VIP2-OE1, VIP2-OE2, VIP2-OE3, VIP2-OE4 and the like are found to have higher expression levels (see figure 3), so that the over-expression transgenic plants are successfully obtained in the embodiment.
Example 4 identification of OsVIP2 Gene editing mutant
Identification was performed on the CRISPR/Cas9 edited OsVIP2 gene T0 generation individuals using a generation sequencing. The rapid extraction DNA method is utilized to extract the genomic DNA of the T0 generation single plant, primers (casOsVIP 2F:5'-TGTTTGATGCAGATCTTCCGT-3', SEQ ID NO.12; casOsVIP2R:5'-TGCAGGACAAAAGAAAAGAGCA-3', SEQ ID NO. 13) are designed aiming at the two sides of the editing site of the sgRNA, the amplified fragments cover the area of the editing site, and the PCR product is sequenced. And (3) performing multi-sequence comparison on the Japanese target site sequence serving as a reference sequence and all amplified editing site regions to determine a homozygous single plant subjected to genome editing.
According to the sequencing result, as shown in the multi-sequence alignment result in FIG. 4, the mutation types are respectively that 1 base C (OsVIP 2KO1 and OsVIP2KO3, nucleotide sequence SEQ ID NO. 14) is deleted at the target site acted by sgRNA, so that frame shift mutation is caused, the transcript is caused to terminate in advance, and a polypeptide (SEQ ID NO. 15) of 73 amino acids is formed; insertion of 1 base A (OsVIP 2KO2, SEQ ID NO. 16) resulted in frame shift mutation, leading to premature termination of the transcript, resulting in a 90 amino acid polypeptide (SEQ ID NO. 17). The mutant is inconsistent with a protein sequence (SEQ ID NO. 2) obtained by normal expression of the OsVIP2 gene.
The gene sequences of the OsVIP2KO1 and the OsVIP2KO3 are as follows:
ATGATGGAAGCGGACATGGAGAACGGGAGGTTGTATCCGGAGAGACCCAGAACTTTCTCTACCGTACGAACTAAATCATCTCTGCCACCGATCTTCCGTGTGTTGATGAGGATAAATCCCCGTGCTTTCATTGTTCTTCTTCTTTTGGTATTCAGTGGTGTGCTCTATGTAGGAGCCAGTAATCACCAATTGTGCTCTTTGTGTTCTGCATATGTACTCTAA,SEQ ID NO.14;
the OsVIP2KO1 and OsVIP2KO3 protein sequences are as follows:
MMEADMENGRLYPERPRTFSTVRTKSSLPPIFRVLMRINPRAFIVLLLLVFSGVLYVGASNHQLCSLCSAYVL*,SEQ ID NO.15。
the OsVIP2KO2 gene sequence is as follows:
ATGATGGAAGCGGACATGGAGAACGGGAGGTTGTATCCGGAGAGACCCAGAACTTTCTCTACCGTACGAACTAAATCATCTCTGCCACCGATCTTCCGTGTGTTGATGAGGATAAATCCCCGTGCTTTCATTGTTCTTCTTCTTTTGGTATTCAGTGGTGTGCTCTATGTAGGAGCCAGTAACATCACCAATTGTGCTCTTTGTGTTCTGCATATGTACTCTAAGTTTATTCTTCTCTCTCTACCTCACAAAGTGGGTTCTTGCCAAAGATGA,SEQ ID NO.16;
the OsVIP2KO2 protein sequence is as follows:
MMEADMENGRLYPERPRTFSTVRTKSSLPPIFRVLMRINPRAFIVLLLLVFSGVLYVGASNITNCALCVLHMYSKFILLSLPHKVGSCQR*,SEQ ID NO.17。
example 5 evaluation of drought tolerance in OsVIP2 Gene overexpressing plants and Gene editing mutant plants
Seedling drought tolerance evaluation was performed on the OsVIP2 gene over-expression transgenic T3 generation strains VIP2-OE1, VIP2-OE2, VIP2-OE3 of example 3 and the OsVIP2 gene editing mutant T3 generation strains OsVIP2KO1, osVIP2KO2 and OsVIP2KO3 of example 4.
The method comprises the following specific steps: immersing transgenic seeds and wild control seeds harvested by synchronous sowing, accelerating germination, sowing in small barrels (soil culture, half of wild plants are sown in each small barrel as a control), culturing the plants until the plants reach a 3-leaf one-heart period, then cutting off water, starting drought stress, recovering the culture for one week after about 10 days of treatment, and then counting the survival rate of each plant line.
The research shows that the survival rate of the over-expression strain of the OsVIP2 after drought stress is extremely higher than that of the wild type strain; the survival rate of the gene editing mutant plants is obvious or extremely lower than that of the wild type, which indicates that the OsVIP2 positively regulates drought tolerance in the seedling stage (see figure 5).
EXAMPLE 6 evaluation of salt tolerance of OsVIP2 Gene overexpressing plants and Gene editing mutant plants
Seedling salt tolerance evaluation was performed on the OsVIP2 gene over-expression transgenic T3 generation strains VIP2-OE1, VIP2-OE2, VIP2-OE3 of example 3 and the OsVIP2 gene editing mutant T2 generation strains OsVIP2KO1, osVIP2KO2 and OsVIP2KO3 of example 4.
The method comprises the following specific steps: immersing transgenic seeds and wild control seeds harvested by synchronous sowing, accelerating germination, sowing, culturing (water culture) to a period of 4 leaves and one heart by using a nutrient solution, treating experimental materials by using a nutrient solution containing 0.7% NaCl, changing into a normal nutrient solution for recovering and culturing for one week after treating for one week, and then counting the survival rate of each strain.
The research shows that the survival rate of the over-expression strain of the OsVIP2 after salt stress is extremely higher than that of the wild type strain; the survival rate of the mutant plants obtained by gene editing is obvious or extremely lower than that of the wild type, which indicates that the OsVIP2 positively regulates the salt tolerance in the seedling stage (see figure 6).
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. The application of enhancing protein or regulating the expression level of the protein coding gene expression substance in improving the stress resistance of rice is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO:2 is shown in the figure;
or a homologous sequence of the sequence shown in SEQ ID NO. 2;
or a conservative variant of the sequence depicted in SEQ ID NO. 2;
or an allelic variant of the sequence set forth in SEQ ID NO. 2;
or a natural mutant of the sequence described by SEQ ID NO. 2;
or an induced mutant of the sequence described in SEQ ID NO. 2.
2. Use of a biological material related to the protein of claim 1 for increasing stress resistance in rice, wherein the biological material is any of the following:
a: a nucleic acid molecule encoding the protein of claim 1;
b: an expression cassette comprising over-expression of the nucleic acid molecule of a;
c: an over-expression vector comprising the nucleic acid molecule of A, or a recombinant vector comprising the expression cassette of B;
d: a recombinant microorganism comprising the nucleic acid molecule of A, a recombinant microorganism comprising the expression cassette of B, or a recombinant microorganism comprising the recombinant vector of C.
3. The use according to claim 2, wherein the nucleic acid molecule has the nucleotide sequence set forth in SEQ ID NO:1 is shown in the specification;
or a sequence which is completely complementary to the sequence shown in SEQ ID NO. 1;
or a sequence which hybridizes with the sequence shown in SEQ ID NO. 1;
or a subfragment functionally equivalent to the sequence shown in SEQ ID NO. 1.
4. A biological material for improving stress resistance of rice, which is characterized by being any one of the following:
a: an expression cassette comprising overexpression of a nucleic acid molecule as shown in SEQ ID NO. 1;
b: a recombinant vector comprising the expression cassette of a;
c: a recombinant microorganism comprising the expression cassette of a, or a recombinant microorganism comprising the recombinant vector of b.
5. A method for improving stress resistance of rice, which comprises enhancing the activity of the protein of claim 1 or enhancing the expression level of the gene encoding the protein of claim 1 to thereby improve stress resistance of rice.
6. The method of claim 5, wherein the nucleotide sequence of the protein encoding gene is set forth in SEQ ID NO: 1.
7. The method according to claim 5, wherein the method for increasing the expression level of the protein-encoding gene according to claim 1 is to construct an OsVIP2 gene overexpression vector, comprising the steps of:
(1) Designing and amplifying a primer of a complete coding reading frame according to the full-length sequence of the OsVIP2 gene;
(2) PCR amplification is carried out by taking the cDNA sequence of the OsVIP2 gene as a template and connecting the cDNA sequence to a cloning vector-BluntCloningVector;
(3) Designing the primers SEQ ID NO.5 and 6 with the linker, and carrying out recombination reaction with a plant expression vector Ub08 containing a promoter and a terminator protein.
8. A transgenic rice plant with high stress resistance, which is transformed with any one of the following nucleic acids:
the nucleotide of the nucleic acid molecule is shown as SEQ ID NO:1 is shown in the specification;
or a sequence which is completely complementary to the sequence shown in SEQ ID NO. 1;
or a sequence which hybridizes with the sequence shown in SEQ ID NO. 1;
or a subfragment functionally equivalent to the sequence shown in SEQ ID NO. 1.
9. A use according to any one of claims 1 to 3 or a biomaterial according to claim 4 or a method according to any one of claims 5 to 7 or a transgenic rice according to claim 8, wherein the stress resistance is drought and/or salt tolerance.
10. The use according to any one of claims 1 to 3 or the biomaterial according to claim 4 or the method according to any one of claims 5 to 7 or the transgenic rice according to claim 8, characterized in that the rice is japan.
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