CN111793119A

CN111793119A - Protein for regulating and controlling plant drought resistance, coding gene and application thereof

Info

Publication number: CN111793119A
Application number: CN201910270834.7A
Authority: CN
Inventors: 张劲松; 陈受宜; 阴翠翠; 何锶洁; 张万科; 韦伟; 陶建军; 林晴
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2019-04-04
Filing date: 2019-04-04
Publication date: 2020-10-20

Abstract

The invention discloses a protein for regulating and controlling plant drought resistance, and a coding gene and application thereof. The protein for regulating and controlling the drought resistance of the plant disclosed by the invention is A1), A2) or A3) as follows: A1) the amino acid sequence is the protein of sequence 2; A2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 2 in the sequence table and has the same function; A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2). Experiments prove that the protein for regulating and controlling the drought resistance of the plant and the coding gene thereof can regulate and control the drought resistance of the plant and have wide application prospect.

Description

Protein for regulating and controlling plant drought resistance, coding gene and application thereof

Technical Field

The invention relates to a protein for regulating and controlling plant drought resistance, a coding gene and application thereof in the field of biotechnology.

Background

The change of physical and chemical factors in the environment, such as drought, saline alkali, low temperature and other stress factors, has important influence on the growth and development of plants, can cause large-scale yield reduction of crops in severe cases, and the cultivation of stress-tolerant crops is one of the main targets of the planting industry. At present, genetic engineering breeding has become one of the important methods for enhancing the stress tolerance of crops. Higher plant cells respond to various stresses in the environment in multiple ways, with transcription factors playing a role in regulating the expression of stress-tolerance-related effector genes. Several classes of transcription factors have been found to be associated with plant stress tolerance in plants, for example: DREB class in EREBP/AP2, bZIP, MYB, WRKY, etc.

Ethylene, a specific hormone in plants, is known to be an important plant hormone and is involved in regulating and controlling many aspects of plant growth and development, including disease resistance, stress tolerance and the like.

Disclosure of Invention

The invention aims to provide any one of the following applications of a protein derived from rice or a substance for regulating the activity or content of the protein:

D1) regulating and controlling the drought resistance of the plant;

D2) preparing a product for regulating and controlling the drought resistance of plants;

D3) cultivating drought-resistant enhanced plants;

D4) preparing and cultivating drought resistance enhancing plant products;

D5) cultivating drought-resistance-reduced plants;

D6) preparing and cultivating plant products with reduced drought resistance;

D7) plant breeding;

the protein is named as OsEIL1 and is A1), A2) or A3) as follows:

A1) the amino acid sequence is the protein of sequence 2;

A2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 2 in the sequence table and has the same function;

A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2).

In order to facilitate the purification of the protein of A1), the amino terminal or the carboxyl terminal of the protein consisting of the amino acid sequence shown in sequence 1 in the sequence listing may be labeled as shown in the following table.

Table: sequence of tags

The OsEIL1 protein in A2) is a protein having identity of 75% or more than 75% with the amino acid sequence of the protein shown in the sequence 1 and having the same function. The identity of 75% or more than 75% is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.

The OsEIL1 protein in A2) can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

The gene encoding the OsEIL1 protein in a2) above may be obtained by deleting one or several amino acid residues from the DNA sequence shown in sequence No. 2, and/or by performing missense mutation of one or several base pairs, and/or by attaching to its 5 'end and/or 3' end a coding sequence of the tag shown in the above table. Wherein the DNA molecule shown in the sequence 2 encodes OsEIL1 protein shown in the sequence 1.

It is another object of the present invention to provide any one of the following uses of a biomaterial related to OsEIL 1:

D1) regulating and controlling the drought resistance of the plant;

D3) cultivating drought-resistant enhanced plants;

D4) preparing and cultivating drought resistance enhancing plant products;

D5) cultivating drought-resistance-reduced plants;

D6) preparing and cultivating plant products with reduced drought resistance;

D7) plant breeding;

the biomaterial is any one of the following B1) to B9):

B1) a nucleic acid molecule encoding OsEIL 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic plant cell line comprising B1) the nucleic acid molecule or a transgenic plant cell line comprising B2) the expression cassette;

B6) transgenic plant tissue comprising the nucleic acid molecule of B1) or transgenic plant tissue comprising the expression cassette of B2);

B7) a transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2);

B8) a nucleic acid molecule which reduces the expression level of OsEIL 1;

B9) an expression cassette, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ comprising the nucleic acid molecule according to B8).

In the above application, the nucleic acid molecule of B1) may be B11) or B12) or B13) or B14) as follows:

b11) the coding sequence is cDNA molecule or DNA molecule of sequence 1 in the sequence table;

b12) a cDNA molecule or a DNA molecule of a sequence 1 in a sequence table;

b13) a cDNA molecule or DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by b11) or b12) and codes OsEIL 1;

b14) hybridizes with the nucleotide sequence defined by b11) or b12) or b13) under strict conditions and encodes a cDNA molecule or a DNA molecule of OsEIL 1.

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.

The nucleotide sequence encoding the OsEIL1 protein of the invention can be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence of the OsEIL1 protein isolated in the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as they encode the OsEIL1 protein and have the function of the OsEIL1 protein.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 1 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

In the above application, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: hybridization in a solution of 6 XSSC, 0.5% SDS at 65 ℃ followed by washing the membrane once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS; can also be: hybridization and washing of membranes 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and hybridization and washing of membranes 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; can also be: 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS at 65 ℃ and washing the membrane.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

In the above applications, the expression cassette containing a nucleic acid molecule encoding OsEIL1 protein (OsEIL1 gene expression cassette) described in B2) refers to a DNA capable of expressing OsEIL1 protein in a host cell, and the DNA may include not only a promoter that initiates transcription of OsEIL1 gene, but also a terminator that terminates transcription of OsEIL1 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; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al (1999) plantaPhysiol 120: 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 methyl jasmonate); heat shock promoters (U.S. patent 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5,057,422); seed-specific promoters, e.g. the millet seed-specific promoter pF128

(CN101063139B (Chinese patent 200710099169.7)), seed storage protein specific promoters (e.g., phaseolin, napin, oleosin andthe promoter of soybean beta conglycin (Beachy et al (1985) EMBOJ.4: 3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminators (see, e.g., Odell et al (I)⁹⁸⁵) Nature313: 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).

The recombinant vector containing the OsEIL1 gene expression cassette can be constructed by using the existing expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, PSN1301, or pCAMBIA1391-Xb (CAMBIA Corp.), etc. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can lead poly A to be added to the 3 'end of mRNA precursor, and the untranslated regions transcribed at the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions. When the gene of the present invention is used to construct a plant expression vector, 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 correct 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, the plant expression vector to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound capable of producing a color change (GUS gene, luciferase gene, etc.), a marker gene for antibiotics (e.g., nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to phosphinothricin as an herbicide, hph gene conferring resistance to hygromycin as an antibiotic, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or a marker gene for chemical resistance (e.g., herbicide resistance), a mannose-6-phosphate isomerase gene providing the ability to metabolize mannose, which can be expressed in plants. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

In the above application, the vector may be a plasmid, a cosmid, a phage, or a viral vector. The plasmid can be pCAMBIA2300 vector.

B3) The recombinant vector can be pCAMBIA2300-OsEIL 1. The pCAMBIA2300-OsEIL1 is a recombinant vector obtained by replacing a DNA fragment between BamHI and XbaI recognition sequences of the vector pCAMBIA2300 with OsEIL1 gene shown in 1 st-1923 rd position from the 5' end of the sequence 1.

B9) The expression cassette and the recombinant vector containing the nucleic acid molecule for reducing the expression quantity of the OsEIL1 can be an expression cassette and a recombinant vector for reducing the expression quantity of the OsEIL1 or knocking out the OsEIL1 gene by using an RNA interference (RNAi) method or a Clustered regularly spaced short palindromic repeats (CRISPR) method.

In the above application, the microorganism may be yeast, bacteria, algae or fungi. Wherein the bacteria can be Agrobacterium, such as Agrobacterium AGL 1.

In the above application, the transgenic plant cell line, the transgenic plant tissue and the transgenic plant organ do not comprise propagation material.

Above, the plant may be M1) or M2) or M3):

m1) monocotyledonous or dicotyledonous plants;

m2) gramineous plants;

m3) rice, wheat, corn, turf grass, cucumber, tomato or alfalfa.

It is a further object of the present invention to provide any of the following methods:

x1), comprising expressing OsEIL1 in a receptor plant, or increasing the content of OsEIL1 in the receptor plant, or increasing the activity of OsEIL1 in the receptor plant, to obtain a target plant with reduced drought resistance;

x2), comprising the steps of enabling a receptor plant to express OsEIL1, or improving the content of OsEIL1 in the receptor plant, or improving the activity of OsEIL1 in the receptor plant, obtaining a target plant with reduced drought resistance, and realizing the reduction of the drought resistance of the plant;

x3), reducing the content of OsEIL1 in a receptor plant, or reducing the activity of OsEIL1 in the receptor plant, or inhibiting the expression of an encoding gene of OsEIL1, or knocking out the encoding gene of OsEIL1 in the receptor plant to obtain a target plant with enhanced drought resistance;

x4), which comprises reducing the content of OsEIL1 in a receptor plant, or reducing the activity of OsEIL1 in the receptor plant, or inhibiting the expression of the coding gene of OsEIL1, or knocking out the coding gene of OsEIL1 in the receptor plant, so as to obtain a target plant with enhanced drought resistance, and realize the enhancement of the drought resistance of the plant.

In the method, the content of the OsEIL1 in the receptor plant can be increased in both X1) and X2) by introducing a coding gene of OsEIL1 into the receptor plant and expressing the coding gene.

The encoding gene may be B1) the nucleic acid molecule.

In the method, the encoding gene of OsEIL1 can be modified as follows and then introduced into a receptor plant to achieve better expression effect:

1) modifying and optimizing according to actual needs to enable the gene to be efficiently expressed; for example, according to the codon preference of the recipient plant, the codon can be changed to conform to the plant preference while maintaining the amino acid sequence of the gene encoding OsEIL1 of the present invention; during the optimization, it is desirable to maintain a GC content in the optimized coding sequence to best achieve high expression levels of the introduced gene in plants, wherein the GC content can be 35%, more than 45%, more than 50%, or more than about 60%;

2) modifying the sequence of the gene adjacent to the initiating methionine to allow efficient initiation of translation; for example, modifications are made using sequences known to be effective in plants;

3) linking with promoters expressed by various plants to facilitate the expression of the promoters in the plants; such promoters may include constitutive, inducible, time-regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the time and space requirements of expression, and will also depend on the target species; for example, tissue or organ specific expression promoters, depending on the stage of development of the desired receptor; although many promoters derived from dicots have been demonstrated to be functional in monocots and vice versa, desirably, dicot promoters are selected for expression in dicots and monocot promoters for expression in monocots;

4) the expression efficiency of the gene of the present invention can also be improved by linking to a suitable transcription terminator; tml from CaMV, E9 from rbcS; any available terminator which is known to function in plants may be linked to the gene of the invention;

5) enhancer sequences, such as intron sequences (e.g., from Adhl and bronzel) and viral leader sequences (e.g., from TMV, MCMV, and AMV) were introduced.

The coding gene of OsEIL1 can be introduced into a receptor plant by using a recombinant vector containing the coding gene of OsEIL 1. The recombinant vector can be specifically the pCAMBIA2300-OsEIL 1.

X3) and X4) can be realized by an RNA interference (RNAi) method or a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) method, or by treating plant seeds containing the gene or homologous genes thereof by using chemical mutagens such as EMS, azide compounds and the like and physical mutagenesis methods such as radiation and the like to obtain mutants, and identifying plants with reduced expression of the target gene, thereby obtaining the target plant with improved drought tolerance.

When the OsEIL1 is used for constructing a recombinant plant interference expression vector, any promoter, such as cauliflower mosaic virus (CAMV)35S promoter and maize Ubiquitin promoter (Ubiquitin), can be added in front of the transcription initiation nucleotide, and can be used alone or in combination with other plant promoters.

The recombinant vector can be introduced into a plant cell or tissue by using a conventional biological method such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, agrobacterium-mediated transformation, etc., and the transformed plant tissue can be cultured into a plant. The plant host to be transformed may be either a monocotyledonous or dicotyledonous plant, such as: rice, wheat, corn, lawn grass, cucumber, tomato, alfalfa, etc.

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 genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic markers having resistance (gentamicin marker, kanamycin marker, etc.), or chemical-resistant marker genes (e.g., herbicide-resistant gene), etc., which can be expressed in plants. From the safety of transgenic plants, the drought treatment can be directly used for screening transformed plants without adding any selective marker gene.

The plant of interest is understood to include not only the first generation plant in which the OsEIL1 protein or the gene encoding it is altered, but also its progeny. For the plant of interest, the gene may be propagated in the species, or transferred into other varieties of the same species, including commercial varieties in particular, using conventional breeding techniques. The plant of interest includes seeds, callus, whole plants and cells.

In the above method, the recipient plant may be M1) or M2) or M3):

m1) monocotyledonous or dicotyledonous plants;

m2) gramineous plants;

m3) rice, wheat, corn, turf grass, cucumber, tomato or alfalfa.

It is a further object of the invention to provide a product having the function of D1) or D2) as follows, said product containing OsEIL1 or said biological material:

D1) regulating and controlling the drought resistance of the plant;

D2) reducing the drought resistance of the plants.

The product can take OsEIL1 or the biological material as its active ingredient, and can also take OsEIL1 or the biological material and substances with the same function as their active ingredients.

In the above product, the plant may be M1) or M2) or M3):

m1) monocotyledonous or dicotyledonous plants;

m2) gramineous plants;

m3) rice, wheat, corn, turf grass, cucumber, tomato or alfalfa.

In the present invention, the drought tolerance may specifically be tolerance to drought stress among abiotic stresses.

Experiments prove that the OsEIL1 and the coding gene thereof can regulate and control the drought tolerance of plants: after the coding gene OsEIL1 is introduced into a plant, the drought resistance of the obtained transgenic plant is reduced, and OsEIL1 can be genetically transformed to construct a drought-intolerant model plant and further used for drought-resistant research and screening of drought-resistant genes, drought-resistant medicaments and the like; the drought resistance of the mutant with the OsEIL1 coding gene mutated is improved compared with that of a wild plant, and a drought resistant plant can be constructed by knocking out the OsEIL1 coding gene or inhibiting the expression of the OsEIL1 coding gene. The OsEIL1 and the coding gene thereof have wide application prospect.

Drawings

FIG. 1 is a schematic diagram of the upstream and downstream structure of OsEIL1 encoding gene in pCAMBIA2300-OsEIL 1.

FIG. 2 is T₀And detecting the relative expression of the OsEIL1 gene in a rice plant with the transgenic OsEIL1 gene.

FIG. 3 shows the results of the relative expression levels of OsEIL1 gene in two mutants and OX 4-4.

FIG. 4 shows the results of drought resistance tests. A is appearance, in the rice marked by eil1, the left column is eil1-1, and the right column is eil 1-2; b is the survival rate statistic result, and the survival rate of eil1 is the average survival rate of eil1-1 and eil 1-2.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

All plant materials were grown at 22 ℃ with 16h/8h light per day (light/dark).

Plant expression vector pCAMBIA2300 described in "Zhi Q, Wang S, Chai M, Zhang F, Li Q, Li S, Sun M., Transgenic mini-distance and protection against alcohol-induced growth J, J Genet genomics.2007 Aug; 34(8):756-63 ″, which is publicly available from institute of genetics and developmental biology, academy of sciences of china.

Agrobacterium AGL1 is described in "He Y, Jones HD, Chen S, Chen XM, Wang DW, Li KX, Wang DS, Xia LQ, Agrobacterium-mediated transformation of durum were (Triticum turgidumL. var. durum cv Stewart) with improved efficacy, J Exp bot.2010, 61(6): 1567-81" publicly available from the institute of genetics and developmental biology, Chinese academy of sciences.

Example 1 obtaining of OsEIL1 Gene

2 phenotypically similar mutants were obtained from the Nipponbare mutant pool of rice. The mutant and wild Nipponbare are used as parents to construct a mapping population, and a mutant gene for controlling the phenotype of the mutant is cloned by using a map-based cloning method. The gene controlling the phenotype in Nipponbare is recorded as an OsEIL1 gene, the length of a coding region of the OsEIL1 gene is 1923bp, the OsEIL1 gene does not contain an intron, the sequence is a sequence 1 in a sequence table, an OsEIL1 protein containing 640 amino acids is coded, and the protein sequence is a sequence 2 in the sequence table. The two mutants were named eil1-1 and eil1-2, respectively. Sequencing OsEIL1 gene in the mutant shows that the OsEIL1 gene in eil1-1 has 8bp deletion in 68 th-75 th positions of nucleotide sequence, so that the encoded protein is terminated in advance at 65 th amino acid, and the OsEIL1 gene in eil1-2 has 8bp insertion after 418 th position, so that the encoded protein is terminated in advance at 146 th amino acid.

Example 2 application of OsEIL1 gene in regulation of drought tolerance of rice

Construction of OsEIL1 gene expression vector pCANBIA2300-OsEIL1

The primer sequences are as follows:

EIL-LP:5’-CGGGATCCATGGGAGGTGGTCTGGTGAT-3’(BamHⅠ)；

EIL-RP:5’-GCTCTAGATCAGTAGTACCAATTCGAGCCGTCA-3’(XbaⅠ)。

the cDNA reverse transcription of the total RNA of Nipponbare of a rice variety is used as a template, and primers EIL-LP and EIL-RP are used for PCR amplification to obtain a PCR product of about 1940bp, namely OsEIL1 full-length cDNA (1-1923 th nucleotides from the 5' end of a sequence 1) containing BamHI and XbaI recognition sequences.

And carrying out double enzyme digestion on the PCR product by using BamHI and XbaI, recovering the enzyme digestion product, and connecting the enzyme digestion product with a vector pCAMBIA2300 subjected to the same enzyme digestion to obtain the recombinant vector.

After sequencing, the recombinant vector was obtained by replacing the DNA fragment between BamHI and XbaI recognition sequences of the vector pCAMBIA2300 with OsEIL1 gene shown at 1-1923 from the 5' end of the sequence 1, and was named pCAMBIA2300-OsEIL1 (FIG. 1). The pCAMBIA2300-OsEIL1 can express OsEIL1 protein shown in a sequence 2 in a sequence table, and the expression of OsEIL1 gene in the pCAMBIA2300-OsEIL1 is driven by a 35S promoter.

Second, obtaining of OsEIL1 transgenic rice plant

1. Obtaining of recombinant Agrobacterium

And (3) introducing the pCAMBIA2300-OsEIL1 obtained in the step one into Agrobacterium AGL1 to obtain a recombinant bacterium, and marking the recombinant bacterium as AGL1/pCAMBIA2300-OsEIL 1.

2. Agrobacterium-mediated transformation of rice

a. Induction of immature embryogenic callus

Taking Japanese sunny seeds of the rice variety, sterilizing the Japanese sunny seeds for 1 minute by 70 percent ethanol, and washing the Japanese sunny seeds for 2 to 3 times by using sterile water; then the embryo is sterilized by sodium hypochlorite solution with 2 percent of available chlorine for more than 60 minutes by shaking, the embryo is washed by sterile water for 4-5 times, then the embryo is separated under the aseptic condition and is inoculated on N6D2 culture medium (N6 salt and vitamin, 500g/L hydrolyzed casein, 30g/L cane sugar, 2mg/L2,4-D,2.5g/L firming agent (gelrite), pH5.8) to be cultured for 4 days at 28 ℃ in the dark.

b. Cultivation of Agrobacterium

AGL1/pCAMBIA2300-OsEIL1 was inoculated into 20ml LB liquid medium containing kanamycin to a final concentration of 50. mu.g/ml and rifampicin to 25. mu.g/ml, and shake-cultured at 28 ℃ until late logarithmic growth; then 0.5ml of the resulting culture was transferred to 50ml of LB medium containing 50. mu.g/ml kanamycin and 25. mu.g/ml rifampicin at the same final concentration, and cultured under the same conditions until OD₆₀₀About 0.5, and obtaining the agrobacterium culture solution.

c. Co-culture and transformation, screening, differentiation

Centrifuging 4000g of the agrobacterium culture solution obtained in the step b for 10 minutes, and then re-suspending the thallus precipitate by using an AAM-AS culture medium (the AAM-AS culture medium consists of AA salt, amino acid, MS vitamin, acetosyringone and water, wherein the AA salt and the amino acid have the same composition and concentration AS those of the AA culture medium, the MS vitamin has the same composition and concentration AS those of the MS culture medium, the acetosyringone has the concentration of 100mM, and the pH of the AAM-AS culture medium is 5.2) with the same volume to obtain an AAM-AS bacterial solution; after the callus derived from young rice Nipponbare embryos which are pre-cultured for 4 days is immersed in the AAM-AS bacterial liquid for 20 minutes, the water of the callus is sucked off by sterile filter paper and then transferred to an N6D2C culture medium (the culture medium is a culture medium which is obtained by adding glucose and acetosyringone into an N6D2 culture medium and has the concentrations of the glucose and the acetosyringone of 10g/L and 100mM respectively, and the pH value is 5.2) (the surface of the culture medium is paved with the sterile filter paper), and the callus is co-cultured for 3 days at the temperature of 25 ℃ in the dark.

After the co-culture is finished, washing the callus with sterile water containing 300mg/L of cef mycin (cef) for 4-5 times, sucking the callus through sterile filter paper, transferring the callus to an N6D2S1 culture medium (the culture medium is a culture medium which is obtained by adding hygromycin and cef mycin into an N6D2 culture medium, the concentrations of the hygromycin and the cef mycin are respectively 25mg/L and 600mg/L, and the pH value is 5.8), and culturing to screen a first generation; after two weeks, the cells were transferred to N6D2S2 medium (the medium was a medium obtained by adding hygromycin and cefamycin to N6D2 medium to obtain hygromycin and cefamycin concentrations of 50mg/L and 300mg/L, respectively, and pH5.8) and screened for the second generation (2 weeks/generation).

Taking out the resistant callus which grows vigorously after twice screening, transferring to a differentiation culture medium (the culture medium consists of MS salt and vitamins, hydrolyzed casein, 150mg/L G418, 6-BA, KT, ZT, a curing agent (gelrite) and water, wherein the composition and the concentration of the MS salt and the vitamins in the differentiation culture medium are the same as those of the MS culture medium, the concentrations of the hydrolyzed casein, G418, 6-BA, KT, ZT and the curing agent (gelrite) are respectively 300G/L, 150mg/L, 3G/L, 2.5mg/L, 0.2mg/L and 2.5G/L, and the pH of the differentiation culture medium is 5.8), and culturing in a differentiation culture box (12 hours of light/12 hours of darkness, 28 ℃ in the daytime and 25 ℃) for 7 days; then transferring the seedlings to a differentiation culture medium, and culturing the seedlings in a differentiation culture box until regenerated seedlings are generated; the regeneration seedlings are placed on a rooting and seedling strengthening culture medium (the culture medium consists of MS salt, MS vitamins, paclobutrazol, NAA, agar powder and water, in the rooting and seedling strengthening culture medium, the composition of the MS salt is the same as that of the MS culture medium, the concentration of each component is 1/4 of the MS culture medium, the composition and the concentration of the MS vitamins are the same as that of the MS culture medium, the concentrations of the paclobutrazol, the NAA and the agar powder are respectively 1mg/L, 0.5mg/L and 6.5g/L, and the pH of the rooting and seedling strengthening culture medium is 5.8) to take roots and strengthen seedlings; opening the sealing film of the container when the young seedling grows to about 10cm, hardening the seedling for 2-3 days to obtain the transgenic seedling, and transferring the transgenic seedling to a phytotron for cultivation. 32T strains were obtained in total₀Transgenic OsEIL1 gene rice, and the screening antibiotic used in seedling is G418, and the concentration is 150 mg/L.

T₁Generation represents T₀Produced by selfingSeeds and plants grown from the seeds.

The empty vector pCAMBIA2300 is transferred into Nipponbare of a rice variety according to the method to obtain 9T₀Breeding the rice to obtain T₃No-load control rice was used.

3. Identification of OsEIL1 transgenic rice plant

Extracting Nipponbare plants (WT) and T of rice variety₀Generation no-load control rice plant (OX-0), T₀Performing Real-time PCR identification analysis on total RNA of a rice plant with a transgenic OsEIL1 gene, wherein primers used by the Real-time PCR are EIL-LP and EIL-RP, an internal reference is Ubiquitin, and the primer of the internal reference is YQ19: TCACCAGGCTCAGGAAGGAG; YQ20: CAGATCAGAGCAAAGCGAGC. The results showed that 32T strains₀In the transgenic OsEIL1 gene rice plants, the expression level of 21 OsEIL1 genes is remarkably higher than that of the rice variety Nipponbare T₀No-load control rice generation (OX-0). The positive T is the rice with OsEIL1 gene expression level higher than that of Nipponbare plant of rice variety₀Transgenic OsEIL1 gene rice is represented by OsEIL 1-OX.

Three lines with different expression levels (OX2-3, OX3-5 and OX4-4) were selected from OsEIL1-OX lines for further phenotypic identification (FIG. 2). Warp beam pair T₂Selection of the plant generations, T₃Performing generation identification to obtain pure line T of the three strains₃Transgenic OsEIL1 gene rice lines OX2-3, OX3-5 and OX 4-4.

Identification of T by the same method₃The expression of the OsEIL1 gene in the generation no-load control rice has no obvious difference with the expression of the gene in wild type Nipponbare.

Drought tolerance experiments on OX2-3, OX3-5 and OX4-4 showed that the mutant has similar phenotype under drought stress, and the injury is larger than that of the control OX-0 and the mutant. OX4-4 was therefore selected for repeated experiments.

FIG. 3 shows the results of measurement of the expression level of OsEIL1 gene in mutants eil1-1 and eil1-2, indicating that it is difficult to detect the expression of OsEIL1 gene in both mutants, indicating that mutants eil1-1 and eil1-2 are deletion mutants. The expression level of the OsEIL1 gene in OX4-4 is obviously higher than that of a control (WT).

Third, drought tolerance test of control and OsEIL1 transgenic rice and mutant

The plants to be tested are: t is₃No-load contrast rice OX-0, pure line T₃Transgenic OsEIL1 gene rice line OX4-4, mutant eil1-1 and mutant eil1-2, Nipponbare.

And (3) stopping watering for 9 days at the room temperature of 25 ℃ for 2-week seedlings (namely 14-day seedlings) of the plants to be detected, rehydrating for 6 days, observing and photographing, and counting the survival rate. The experiment was repeated three times, and each plant to be tested was 30 plants per repetition.

The results are shown in FIG. 4, where the watering was stopped for 9 days and 6 days after rehydration, the control (Nipponbare), OX4-4 and mutants eil1-1, eil1-2 grew significantly differently, the control and OX4-4 wilted severely and the mutant seedlings began to rejuvenate. Survival statistics showed that the control and mutant survivors were approximately 35% and 58%, respectively (average survival of eil1-1 and eil 1-2), and that the survival of OX4-4 was approximately 40%. T is₃No significant difference exists between the phenotype and the survival rate of the generation no-load control rice OX-0 and Nipponbare. The result shows that compared with a control, the drought tolerance of a transgenic plant is reduced by overexpression of the OsEIL1 gene in OX4-4, the drought tolerance of the two mutants eil1-1 and eil1-2 of the OsEIL1 is obviously higher than that of the control and reaches a very significant level, and meanwhile, the improvement of the drought tolerance is unrelated to the mutation site of the OsEIL1 gene.

The results show that OsEIL1 and the coding gene thereof negatively regulate the drought tolerance of plants, the high expression of OsEIL1 in the plants reduces the drought tolerance of the plants, and the reduced expression of OsEIL1 improves the drought tolerance of the plants.

<110> institute of genetics and developmental biology of Chinese academy of sciences

<120> protein for regulating and controlling plant drought resistance, coding gene and application thereof

<160>2

<170>PatentIn version 3.5

<210>1

<211>1923

<212>DNA

<213> Rice (Oryza sativa)

<400>1

atgggaggtg gtctggtgat ggaccagggc atgatgttcc ccggcgtgca caacttcgtg 60

gatctcctgc agcagaacgg cggcgacaag aacctcggct tcggcgcgct cgtgccgcag 120

acgtcgtcgg gggagcagtg cgtgatgggg gagggcgacc tcgtggaccc gccgccggag 180

agcttcccgg acgccggtga ggacgacagc gacgacgacg tggaggacat cgaggagctg 240

gagcgccgca tgtggcgcga ccgcatgaag ctgaagcggc tcaaggagct gcagctgagc 300

cggggcaagg accccgcggg cggcgtcgtg ggcgacccgt ccaagccgcg gcagtcgcag 360

gagcaggcgc ggcggaagaa gatgtcgcgc gcgcaggacg gcatcctcaa gtacatgctc 420

aagatgatgg aggtgtgccg cgcgcagggg ttcgtgtacg ggatcatccc ggagaagggc 480

aagccggtga gcggcgcctc cgacaacctc cgcggctggt ggaaggagaa ggtccgcttc 540

gaccgcaacg gccccgccgc catcgccaag taccaggccg acaacgccgt cccgggcttc 600

gagagcgagc tcgcctccgg caccgggagc ccgcactcgc tgcaggagct gcaggacacc 660

accctcgggt cgctgctctc ggcgctcatg cagcactgcg accctccgca gcggcggtac 720

ccgctcgaga agggcgtccc tccgccgtgg tggcccaccg gcgacgagga gtggtggccg 780

gagctcggca tccccaagga ccagggcccg cctccgtaca agaagcccca tgacctcaag 840

aaggcctgga aggtcagcgt gctcaccgct gtcatcaagc acatgtcgcc ggacatcgag 900

aagatccgcc ggctggtccg gcagtccaag tgcctccagg acaagatgac cgccaaggag 960

atctccacct ggctggccgt cgtcaagcag gaagaggagc tgtacctgaa gctgaacccc 1020

ggtgcccgcc ctccggcacc taccggcggc atcaccagcg ccatatcgtt caacgccagc 1080

tcaagtgagt acgacgtcga cgtcgtcgac gactgcaagg gcgacgaggc cggcaaccag 1140

aaggctgttg ttgtcgccga cccgaccgcg ttcaacctcg gcgcggctat gctgaacgac 1200

aagttcctca tgccggcgtc catgaaggag gaggccaccg atgtcgagtt catccagaag 1260

aggagcgcgt ctggcgcgga gcctgagctg atgctgaaca accgtgtcta cacctgccac 1320

aatgtccagt gcccgcatag cgactatgga tacgggttcc ttgaccggaa cgcgcgcaac 1380

agccaccaat acacttgcaa gtacaatgat ccactccagc agagcacgga gaacaagcca 1440

tcgccaccgg ccatcttccc ggcaacctac aacacgccga accaggctct gaacaatctg 1500

gatttcggcc tgcccatgga tggccagagg tcaattacag agctgatgaa catgtacgac 1560

aacaacttcg tggccaacaa gaaccttagc aacgacaatg ccacgatcat ggagaggcct 1620

aatgcagtca acccaaggat acagattgaa gaaggctttt ttggacaggg aagtggcatc 1680

ggcggcagca acggaggtgt gttcgaagat gtcaatggca tgatgcagca accgcagcag 1740

accaccccgg cacagcagca gttcttcatc cgcgacgata ctccattcgg taaccagatg 1800

ggcgacatca atggcgcatc ggagttcagg ttcggctctg gtttcaacat gtcaggtgcc 1860

gtcgaatacc ccggcgcaat gcagggccag cagaagaatg acggctcgaa ttggtactac 1920

tga 1923

<210>2

<211>640

<212>PRT

<213> Rice (Oryza sativa)

<400>2

Met Gly Gly Gly Leu Val Met Asp Gln Gly Met Met Phe Pro Gly Val

1 5 10 15

His Asn Phe Val Asp Leu Leu Gln Gln Asn Gly Gly Asp Lys Asn Leu

20 25 30

Gly Phe Gly Ala Leu Val Pro Gln Thr Ser Ser Gly Glu Gln Cys Val

35 40 45

Met Gly Glu Gly Asp Leu Val Asp Pro Pro Pro Glu Ser Phe Pro Asp

50 55 60

Ala Gly Glu Asp Asp Ser Asp Asp Asp Val Glu Asp Ile Glu Glu Leu

65 70 75 80

Glu Arg Arg Met Trp Arg Asp Arg Met Lys Leu Lys Arg Leu Lys Glu

85 90 95

Leu Gln Leu Ser Arg Gly Lys Asp Pro Ala Gly Gly Val Val Gly Asp

100 105 110

Pro Ser Lys Pro Arg Gln Ser Gln Glu Gln Ala Arg Arg Lys Lys Met

115 120 125

Ser Arg Ala Gln Asp Gly Ile Leu Lys Tyr Met Leu Lys Met Met Glu

130 135 140

Val Cys Arg Ala Gln Gly Phe Val Tyr Gly Ile Ile Pro Glu Lys Gly

145 150 155 160

Lys Pro Val Ser Gly Ala Ser Asp Asn Leu Arg Gly Trp Trp Lys Glu

165 170 175

Lys Val Arg Phe Asp Arg Asn Gly Pro Ala Ala Ile Ala Lys Tyr Gln

180 185 190

Ala Asp Asn Ala Val Pro Gly Phe Glu Ser Glu Leu Ala Ser Gly Thr

195 200 205

Gly Ser Pro His Ser Leu Gln Glu Leu Gln Asp Thr Thr Leu Gly Ser

210 215 220

Leu Leu Ser Ala Leu Met Gln His Cys Asp Pro Pro Gln Arg Arg Tyr

225 230 235 240

Pro Leu Glu Lys Gly Val Pro Pro Pro Trp Trp Pro Thr Gly Asp Glu

245 250 255

Glu Trp Trp Pro Glu Leu Gly Ile Pro Lys Asp Gln Gly Pro Pro Pro

260 265 270

Tyr Lys Lys Pro His Asp Leu Lys Lys Ala Trp Lys Val Ser Val Leu

275 280 285

Thr Ala Val Ile Lys His Met Ser Pro Asp Ile Glu Lys Ile Arg Arg

290 295 300

Leu Val Arg Gln Ser Lys Cys Leu Gln Asp Lys Met Thr Ala Lys Glu

305 310 315 320

Ile Ser Thr Trp Leu Ala Val Val Lys Gln Glu Glu Glu Leu Tyr Leu

325 330 335

Lys Leu Asn Pro Gly Ala Arg Pro Pro Ala Pro Thr Gly Gly Ile Thr

340 345 350

Ser Ala Ile Ser Phe Asn Ala Ser Ser Ser Glu Tyr Asp Val Asp Val

355 360 365

Val Asp Asp Cys Lys Gly Asp Glu Ala Gly Asn Gln Lys Ala Val Val

370 375 380

Val Ala Asp Pro Thr Ala Phe Asn Leu Gly Ala Ala Met Leu Asn Asp

385 390 395 400

Lys Phe Leu Met Pro Ala Ser Met Lys Glu Glu Ala Thr Asp Val Glu

405 410 415

Phe Ile Gln Lys Arg Ser Ala Ser Gly Ala Glu Pro Glu Leu Met Leu

420 425 430

Asn Asn Arg Val Tyr Thr Cys His Asn Val Gln Cys Pro His Ser Asp

435 440 445

Tyr Gly Tyr Gly Phe Leu Asp Arg Asn Ala Arg Asn Ser His Gln Tyr

450 455 460

Thr Cys Lys Tyr Asn Asp Pro Leu Gln Gln Ser Thr Glu Asn Lys Pro

465 470 475 480

Ser Pro Pro Ala Ile Phe Pro Ala Thr Tyr Asn Thr Pro Asn Gln Ala

485 490 495

Leu Asn Asn Leu Asp Phe Gly Leu Pro Met Asp Gly Gln Arg Ser Ile

500 505 510

Thr Glu Leu Met Asn Met Tyr Asp Asn Asn Phe Val Ala Asn Lys Asn

515 520 525

Leu Ser Asn Asp Asn Ala Thr Ile Met Glu Arg Pro Asn Ala Val Asn

530 535 540

Pro Arg Ile Gln Ile Glu Glu Gly Phe Phe Gly Gln Gly Ser Gly Ile

545 550 555 560

Gly Gly Ser Asn Gly Gly Val Phe Glu Asp Val Asn Gly Met Met Gln

565 570 575

Gln Pro Gln Gln Thr Thr Pro Ala Gln Gln Gln Phe Phe Ile Arg Asp

580 585 590

Asp Thr Pro Phe Gly Asn Gln Met Gly Asp Ile Asn Gly Ala Ser Glu

595 600 605

Phe Arg Phe Gly Ser Gly Phe Asn Met Ser Gly Ala Val Glu Tyr Pro

610 615 620

Gly Ala Met Gln Gly Gln Gln Lys Asn Asp Gly Ser Asn Trp Tyr Tyr

625 630 635 640

Claims

1. The use of a protein or a substance which modulates the activity or content of said protein, as defined in any one of the following:

D1) regulating and controlling the drought resistance of the plant;

D3) cultivating drought-resistant enhanced plants;

D4) preparing and cultivating drought resistance enhancing plant products;

D5) cultivating drought-resistance-reduced plants;

D6) preparing and cultivating plant products with reduced drought resistance;

D7) plant breeding;

the protein is A1), A2) or A3) as follows:

A1) the amino acid sequence is the protein of sequence 2;

2. Use of a biological material related to a protein according to claim 1, wherein the biological material is selected from the group consisting of:

D1) regulating and controlling the drought resistance of the plant;

D3) cultivating drought-resistant enhanced plants;

D4) preparing and cultivating drought resistance enhancing plant products;

D5) cultivating drought-resistance-reduced plants;

D6) preparing and cultivating plant products with reduced drought resistance;

D7) plant breeding;

the biomaterial is any one of the following B1) to B9):

B1) a nucleic acid molecule encoding the protein of claim 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B8) a nucleic acid molecule that reduces the expression level of the protein of claim 1;

3. Use according to claim 2, characterized in that: B1) the nucleic acid molecule is b11) or b12) or b13) or b14) as follows:

b12) a cDNA molecule or a DNA molecule of a sequence 1 in a sequence table;

b13) a cDNA or DNA molecule having 75% or more identity to the nucleotide sequence defined in b11) or b12) and encoding the protein of claim 1;

b14) a cDNA molecule or a DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined in b11) or b12) or b13) and encodes the protein of claim 1.

4. Use according to any one of claims 1 to 3, characterized in that: the plant is M1) or M2) or M3):

m1) monocotyledonous or dicotyledonous plants;

m2) gramineous plants;

m3) rice, wheat, corn, turf grass, cucumber, tomato or alfalfa.

5. Any one of the following methods:

x1) a method for cultivating plants with reduced drought resistance, which comprises allowing the receptor plant to express the protein of claim 1, or increasing the content of the protein of claim 1 in the receptor plant, or increasing the activity of the protein of claim 1 in the receptor plant, to obtain a target plant with reduced drought resistance;

x2) a method for reducing drought resistance of plants, which comprises the steps of enabling a receptor plant to express the protein in the claim 1, or increasing the content of the protein in the claim 1 in the receptor plant, or increasing the activity of the protein in the claim 1 in the receptor plant, obtaining a target plant with reduced drought resistance, and realizing the reduction of the drought resistance of the plant;

x3) a method for cultivating drought-resistant-enhanced plants, comprising reducing the content of the protein of claim 1 in a recipient plant, or reducing the activity of the protein of claim 1 in a recipient plant, or inhibiting the expression of the gene encoding the protein of claim 1, or knocking out the gene encoding the protein of claim 1 in a recipient plant, to obtain a target plant with enhanced drought resistance;

x4), comprising reducing the content of the protein in the claim 1 in the receptor plant, or reducing the activity of the protein in the claim 1 in the receptor plant, or inhibiting the expression of the coding gene of the protein in the claim 1, or knocking out the coding gene of the protein in the claim 1 in the receptor plant, so as to obtain the target plant with enhanced drought resistance, thereby realizing the enhancement of the drought resistance of the plant.

6. The method of claim 5, wherein: x1) and X2) is increased in the content of the protein of claim 1 in a recipient plant by introducing the gene encoding the protein of claim 1 into the recipient plant and expressing the gene.

7. The method of claim 6, wherein: the coding gene is the nucleic acid molecule of B1) in claim 2 or 3.

8. The method according to any one of claims 5-7, wherein: the recipient plant is M1) or M2) or M3):

m1) monocotyledonous or dicotyledonous plants;

m2) gramineous plants;

m3) rice, wheat, corn, turf grass, cucumber, tomato or alfalfa.

9. A product having a function of D1) or D2) as follows, containing the protein of claim 1 or the biomaterial of claim 2 or 3:

D1) regulating and controlling the drought resistance of the plant;

D2) reducing the drought resistance of the plants.

10. The product of claim 9, wherein: the plant is M1) or M2) or M3):

m1) monocotyledonous or dicotyledonous plants;

m2) gramineous plants;

m3) rice, wheat, corn, turf grass, cucumber, tomato or alfalfa.