CN113388013A - Heat-resistance related protein SLG1 and application of coding gene thereof in regulation and control of rice heat resistance - Google Patents

Abstract

The invention discloses a heat-resistance related protein SLG1 and application of a coding gene thereof in regulation and control of rice heat resistance. The heat-resistant related protein SLG1 disclosed by the invention is A1), A2) or A3) as follows: A1) the amino acid sequence is the protein of sequence 3; 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 3 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 SLG1 and the coding gene thereof can regulate and control the heat resistance of plants: after the gene is introduced into a wild type plant, the survival rate of the obtained transgenic plant after heat treatment is obviously increased compared with the wild type plant; the survival rate of the transgenic plant with reduced gene expression after heat treatment is obviously reduced compared with the wild type. Therefore, the SLG1 and the coding gene thereof can regulate and control the heat resistance of plants.

Description

Heat-resistance related protein SLG1 and application of coding gene thereof in regulation and control of rice heat resistance

Technical Field

The invention relates to the field of biotechnology, and discloses application of a heat-resistant related protein SLG1 and a coding gene thereof in regulation and control of rice heat resistance.

Background

With the increase of population and the development of industrialization, global warming has become a non-negligible problem. Rice, as an important food crop, lives more than half of the global population, and the yield and quality of the rice are continuously threatened by global warming. Then, how should we live an ever-increasing population in such an environment? The analysis of the rice high-temperature response mechanism, the discovery of high-temperature resistant genes and the application of the high-temperature resistant genes to the improvement of rice varieties are urgent tasks of global breeders.

For decades, scientists have been screening heat-resistant germplasm resources to construct multiple mapping populations, such as chromosome fragment replacement lines (CSSL), Near Isogenic Lines (NIL), Recombinant Inbred Lines (RIL) and F₂And positioning a population and the like, and positioning a plurality of QTLs related to the heat resistance of the rice by fully utilizing various molecular markers, wherein the QTLs are respectively responsible for the heat resistance of the rice in a seedling stage, a booting stage, a flowering stage and a filling stage. However, due to the limitation of heat treatment conditions and the imperfect heat resistance evaluation method, the heat resistance QTL of the rice which is really finely positioned is very few, and the number of the cloned rice which is verified by genetic test is more than the number of the cloned rice which is obtained by TT1 cloned by the forest Hongkong research group of the famous academy of China. TT1 encodes a 26S proteasome α 2 subunit protein involved in ubiquitination protein degradation. Compared with the allele OsTT1 in oryza sativa, the ogTT1 from oryza africana is capable of more efficiently clearing toxic denatured proteins, maintaining the thermal response process and protecting cells from heat stress. The natural variation of the TT1 locus is selected by different climatic temperatures and plays an important role in the evolution process of rice adapting to local environment. In addition, the heat resistance of rice, arabidopsis thaliana and festuca arundinacea can be obviously improved by over-expressing OgTT 1; when high temperature is met in the flowering or filling period, the yield of the single plant containing the near isogenic line of OgTT1 is also obviously improved compared with that of a control plant. This finding is a global climate changeThe possibility of guaranteeing the safety of the grains is brought under the threat of warming. In addition, a study team of Homopsis from the institute of plant physiology and ecology of Shanghai academy of Chinese sciences discovers that the overexpression of the arabidopsis leucine receptor kinase ERECTA in model crops of arabidopsis, monocotyledonous crops of rice and dicotyledonous crops of tomato can obviously improve the high-temperature resistance of the arabidopsis leucine receptor kinase ERECTA. By means of an artificial incubator, after rice in a reproductive growth period is transferred to 42 ℃/35 ℃ (day/night) for treatment for 10 days and is recovered at 28 ℃ for 7 days, the leaves and tillers of most ERECTA overexpression transgenic (ER-OE) rice plants can keep green and survive, the transferred no-load control plants are withered and threatened to die, and the maturing rate (55-70%) of ER-OE transgenic rice in a later period is obviously higher than that of the control plants (about 35%). And field experiments in Shanghai, Wuhan and Changsha for years prove that the ER-OE transgenic rice can not only remarkably improve the high-temperature maturing rate, but also increase the biomass and the single-plant yield of the rice. The discovery is significant for artificially designing the paddy rice with different heat resistance by a genetic means so as to meet the requirements of different temperature areas. Furthermore, since ERECTA homologous proteins are present throughout higher plants, researchers can search for ERECTA alleles with high expression levels or high activity in superior crop germplasm resources and use them in crop heat tolerance improvement by combining molecular breeding. In addition, some genes are reported to be involved in the rice high temperature resistance process, and genetic evidence is obtained. Wang et al found that TOGR1 encodes a DEAD-Box RNA helicase, and that the growth and development of rice can be regulated by directly regulating the enzymatic activity at high temperature; qiao et al found that overexpression of rice calcium ion-binding membrane protein OsANN1 can enhance rice seedling heat resistance by regulating hydrogen peroxide production. Lin and the like find that HSA32 and HSP101 are strongly induced by high temperature, and the HSA32 and the HSP101 can form a positive feedback loop to positively regulate and control the heat resistance of rice in the seedling stage; liu et al found that OsHTAS encodes a ubiquitin ligase localized in nucleus and cytoplasm, and influences rice seedling stage heat resistance by regulating hydrogen peroxide-induced stomatal closure and ABA pathway. Zhang et al found that inhibition of expression of rice OsMADS7 by composition can stabilize the content of amylose in grains at high temperature, ensure the quality of rice, but bring about the negative effect of reduced maturing rate, butThe expression of the OsMADS7 is specifically reduced in endosperm, so that the normal setting rate is ensured, and the stability of the amylose content of the grains under the heat stress is improved, which brings possibility for improving the quality of the high-temperature sensitive rice. Chen et al found that AET1 encodes a histidine tRNA guanosine transferase which, in addition to affecting the maturation of the histidine tRNA, affects the overall translational state of the protein, can bind directly to ORF regions upstream of OsARF19 and OsARF23 mRNA and affect the growth of rice under normal and high temperature conditions by affecting auxin response.

However, many rice heat resistance related researches are basic theoretical researches, and because of the limitation of heat treatment conditions and the imperfect heat resistance evaluation method, the rice heat resistance genes which are truly cloned and verified by genetic tests are very few, and the rice heat resistance related researches can be applied to heat resistance improvement breeding of rice varieties.

Disclosure of Invention

The technical problem to be solved by the invention is how to improve the heat resistance of the plant.

In order to solve the technical problems, the invention firstly provides any one of the following applications of the heat-resistance related protein or the substance for regulating the activity or the content of the heat-resistance related protein:

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

D2) preparing a product for regulating and controlling the heat resistance of the plant;

D3) improving the heat resistance of the plant;

D4) preparing a product for improving the heat resistance of the plant;

D5) plant breeding;

the heat-resistant related protein is SLG1 and is A1), A2) or A3):

A1) the amino acid sequence is the protein of sequence 3;

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 3 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 in A1), the amino terminal or the carboxyl terminal of the protein consisting of the amino acid sequence shown in the sequence 3 in the sequence table is attached with the tags shown in the following table.

Table: sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The SLG1 protein in A2) above is a protein having 75% or more identity to the amino acid sequence of the protein shown in SEQ ID NO. 3 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 SLG1 protein in A2) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding SLG1 protein in A2) above can be obtained by deleting one or several amino acid residues from the DNA sequence shown in SEQ ID NO. 2, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching a coding sequence of the tag shown in the above table to the 5 'end and/or 3' end thereof. Wherein, the DNA molecule shown in the sequence 2 encodes SLG1 protein shown in the sequence 1.

The invention also provides any one of the following applications of the biological material related to SLG 1:

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

D2) preparing a product for regulating and controlling the heat resistance of the plant;

D3) improving the heat resistance of the plant;

D4) preparing a product for improving the heat resistance of the plant;

D5) plant breeding;

the biomaterial is any one of the following B1) to B22):

B1) a nucleic acid molecule encoding SLG 1;

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

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism containing the recombinant vector of B4);

B9) a transgenic plant cell line comprising the nucleic acid molecule of B1);

B10) a transgenic plant cell line comprising the expression cassette of B2);

B11) a transgenic plant cell line comprising the recombinant vector of B3);

B12) a transgenic plant cell line comprising the recombinant vector of B4);

B13) transgenic plant tissue comprising the nucleic acid molecule of B1);

B14) transgenic plant tissue comprising the expression cassette of B2);

B15) transgenic plant tissue containing the recombinant vector of B3);

B16) transgenic plant tissue containing the recombinant vector of B4);

B17) a transgenic plant organ containing the nucleic acid molecule of B1);

B18) a transgenic plant organ containing the expression cassette of B2);

B19) a transgenic plant organ containing the recombinant vector of B3);

B20) a transgenic plant organ containing the recombinant vector of B4);

B21) a nucleic acid molecule that reduces the expression level of SLG 1;

B22) 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 B21).

In the above application, the nucleic acid molecule of B1) may be any one of the following B1) -B6):

b1) the coding sequence is a cDNA molecule or DNA molecule at 37 th-1431 th site of the sequence 2 in the sequence table;

b2) a cDNA molecule or DNA molecule shown in 37 th-1431 th site of a sequence 2 in a sequence table;

b3) a cDNA molecule or a DNA molecule shown in a sequence 2 in a sequence table;

b4) DNA molecule shown in sequence 1 in the sequence table;

b5) a cDNA or DNA molecule having 75% or more identity to the nucleotide sequence defined in b1) or b2) or b3) or b4) and encoding SLG 1;

b6) a cDNA or DNA molecule which hybridizes with the nucleotide sequence defined by b1) or b2) or b3) or b4) or b5) under strict conditions and codes SLG 1;

B21) the nucleic acid molecule is shown as the 1174-1749 site of the sequence 2 in the sequence table.

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 SLG1 protein of the present invention can be easily 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 SLG1 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 SLG1 protein and have the function of the SLG1 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 3 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 ℃ in1 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 ℃; can also be used as a medicine: 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 SLG1 protein (SLG1 gene expression cassette) described in B2) refers to a DNA capable of expressing SLG1 protein in a host cell, and the DNA may include not only a promoter that initiates transcription of SLG1 gene but also a terminator that terminates transcription of SLG1 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) Plant Physiol 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, such as the millet seed-specific promoter pF128(CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin, and soybean beta conglycin (Beachy et al (1985) EMBO J.4: 3047-3053)). They can be used alone or in combination with other plant promoters. Incorporated herein by referenceAll 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) Nature 313: 810; rosenberg et al (1987) Gene,56: 125; guerineau et al (1991) mol.gen.genet,262: 141; proudfoot (1991) Cell,64: 671; sanfacon et al Genes Dev.,5: 141; mogen et al (1990) Plant Cell,2: 1261; munroe et al (1990) Gene,91: 151; ballad et al (1989) Nucleic Acids Res.17: 7891; joshi et al (1987) Nucleic Acid Res, 15: 9627).

The recombinant vector containing the SLG1 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 may be specifically pZH2B vector or pZH2Bi vector.

B3) The recombinant vector can be pZH2B-SLG 1. The pZH2B-SLG1 is a recombinant vector obtained by replacing a DNA fragment between XbaI and SacI recognition sequences of a vector pZH2B with an SLG1 gene shown in a sequence 1 in a sequence table, and the pZH2B-SLG1 can express an SLG1 protein shown in a sequence 3 in the sequence table.

B22) The recombinant vector can be a recombinant vector which contains the DNA fragment shown in the 1174-1749 site of the sequence 2 in the sequence table and a reverse fragment thereof and can inhibit the expression of the SLG1 gene. The recombinant vector can be pZH2Bi-SLG1-RNAi, the pZH Bi-SLG1-RNAi is a recombinant vector obtained by inserting a DNA fragment shown in the 1174-1749 th position of the sequence 2 in the sequence table into a vector pZH2Bi by using a restriction enzyme XbaI, and inserting a DNA fragment shown in the 1174-1749 th position of the sequence 2 in the sequence table into a vector pZH2Bi by using a restriction enzyme SpeI-HF, and the directions of two insertions of the DNA fragment shown in the 1174-1749 th position of the sequence 2 in the sequence table are opposite. pZH2Bi-SLG1-RNAi can be used for inhibiting the expression of SLG1 gene.

In the above application, the microorganism may be yeast, bacteria, algae or fungi. Wherein the bacteria can be Agrobacterium, such as Agrobacterium EHA 105.

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

The invention also provides a product for regulating and controlling the heat resistance of the plant, and the product contains SLG1 or the biological material.

The product can take SLG1 or the biological material as its active ingredient, and can also combine SLG1 or the biological material with substances with the same function as its active ingredient.

Above, the plant may be m1) or m2) or m 3):

m1) a monocotyledonous or dicotyledonous plant;

m2) a gramineous plant;

m3) rice.

The invention also provides a method for improving heat resistance of a plant, which comprises the following steps: increasing the activity and/or content of SLG1 in a recipient plant, or promoting the expression of a gene encoding SLG1, to obtain a target plant having improved heat resistance as compared with the recipient plant.

The invention also provides a method for cultivating plants with improved heat resistance, which comprises the following steps: increasing the activity and/or content of SLG1 in a recipient plant, or promoting the expression of a gene encoding SLG1, to obtain a target plant having improved heat resistance as compared with the recipient plant.

As described above, the target plant may be a transgenic plant having increased expression of the thermotolerant-associated protein as compared to the recipient plant, which is obtained by introducing a gene encoding SLG1 into the recipient plant.

The encoding gene of SLG1 may be B1).

Above, the recipient plant may be m1) or m2) or m 3):

m1) a monocotyledonous or dicotyledonous plant;

m2) a gramineous plant;

m3) rice.

In the above, the gene encoding SLG1 can be modified as follows and then introduced into a recipient plant to achieve better expression effect:

1) modifying and optimizing according to actual needs to enable the gene to be efficiently expressed; for example, the amino acid sequence of the gene encoding SLG1 of the present invention may be changed to conform to plant preferences while maintaining the amino acid sequence thereof, depending on the preferred codons of the recipient plant; 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 SLG1 encoding gene can be introduced into a recipient plant using a recombinant expression vector containing the SLG1 encoding gene. The recombinant expression vector can be specifically the pZH2B-SLG 1.

The recombinant expression vector can be introduced into Plant cells by conventional biotechnological methods using Ti plasmids, Plant viral vectors, direct DNA transformation, microinjection, electroporation, etc. (Weissbach,1998, Method for Plant Molecular Biology VIII, academic Press, New York, pp.411-463; Geiserson and Corey,1998, Plant Molecular Biology (2nd Edition)).

The plant of interest is understood to include not only the first generation plants in which the SLG1 protein or the gene encoding it has been altered, but also the progeny thereof. 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 present invention, the heat resistance may specifically be resistance of a plant to a temperature higher than that required for normal growth of the plant. In one embodiment of the invention, the heat resistance is resistance to a 45 ℃ environment.

In one embodiment of the invention, the plant is a seedling stage plant. The seedling stage plant is specifically a two-leaf one-heart stage plant.

Experiments prove that the SLG1 and the coding gene thereof can regulate and control the heat resistance of plants: after the gene is introduced into a wild type plant, the survival rate of the obtained transgenic plant after heat treatment is obviously increased compared with the wild type plant; the survival rate of the transgenic plant with reduced gene expression after heat treatment is obviously reduced compared with the wild type. The SLG1 gene and the heat-resistant related protein coded by the same can regulate and control the heat resistance of plants.

Biological material preservation instructions

Classification nomenclature of biological materials: rice (Oryza sativa)

Strain number of biological material: slg1

Deposit name of biological material: china general microbiological culture Collection center

The preservation unit of the biological material is abbreviated as: CGMCC (China general microbiological culture Collection center)

Deposit unit address of biological material: west road No.1, north west of the township, beijing, ministry of sciences, china, institute of microbiology, zip code: 100101

Preservation date of biological material: 12 month and 02 day 2019

Accession number to the collection of biological materials: CGMCC No.18785

Drawings

FIG. 1 shows the result of identification of transgenic plants. WT is wild type empty-bred KY131, slg1 represents slg1 mutant, slg1-C represents slg1-C transgenic positive plant.

FIG. 2 shows the phenotypes of wild-type empty-bred KY131(WT), SLG1 mutant, SLG1-C positive transgenic plant and SLG1-RNAi positive transgenic plant before and after heat treatment in examples 1 and 2. WT is wild type empty-bred KY131, SLG1 shows SLG1 mutant, SLG1-C shows SLG1-C transgenic positive plant, and SLG1-RNAi shows SLG1-RNAi positive transgenic plant.

FIG. 3 shows the comparison of SLG1 expression levels of wild-type and SLG1-RNAi transgenic positive plants (A) and the survival rate statistics of wild-type, SLG1 mutant, SLG1-C positive transgenic plant and SLG1-RNAi positive transgenic plant after heat treatment (B). WT is wild type empty-bred KY131, SLG1 shows SLG1 mutant, SLG1-C shows SLG1-C transgenic positive plant, and SLG1-RNAi shows SLG1-RNAi positive transgenic plant.

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.

Japonica rice (Oryza sativa subsp. japonica) slg1 is a mutant obtained by treating a northeast cultivated rice variety of air-cultivated rice 131(KY131) with sodium azide by an inventor, and is marked as slg1 mutant, and the air-cultivated rice 131(Oryza sativa l. ssp. japonica) is disclosed in the document "dundlong, shin-chang, dawn-chang, topaz-air-cultivated 131 different densities influence on yield relation, agriculture and technology, No. 5 in 2008", and the public can be obtained from institute of genetics and developmental biology of Chinese academy of sciences. slg1 mutant has been deposited in China general microbiological culture Collection center (CGMCC; address: West Lu No.1, Beijing, Chaoyang, Naja, Ministry of microbiology, China academy of sciences; zip code: 100101) in 2019, 12.02.D., with the deposit number of CGMCC NO. 18785.

Example 1 SLG1 Gene and its encoded protein can regulate Rice Heat resistance

The embodiment provides a gene which is derived from rice (Oryza sativa) air-cultured 131 and codes heat-resistant related protein, is named as SLG1 gene, the genome sequence of the gene in the rice air-cultured 131 is sequence 1 in a sequence table, the cDNA sequence of the gene is sequence 2 in the sequence table, and SLG1 protein shown as a coding sequence 3 at the 37 th-1431 th site of the sequence 2. The SLG1 gene and SLG1 protein coded by the gene can regulate and control the heat resistance of rice, and the specific detection method comprises the following steps:

1. constructing a recombinant vector:

5' -addition of the primer Com-XbaI-FtctagaGATGTATGCGCCAATTGCGTACAAC-3' and Com-SacI-R: 5-gagctcACCTGTAGTGTATCTGTGCATGCGATG-3' using genome DNA of wild type empty-breeding 131 as a template to perform PCR, connecting the obtained PCR product with correct sequence into an intermediate vector pEASY (Beijing hologold organism, cat # CB101-01), obtaining a recombinant plasmid, then using restriction enzymes XbaI (NEB, # R0145S) and SacI-HF (NEB, # R3156S) to perform enzyme digestion to obtain a DNA fragment with cohesive end, and connecting the DNA fragment with a vector pZH2B (Song, L., Wang, R., Zhang, L., Wang, Y., and Yao, S. (2016) (CRR 1 coding synthesis in overview expansion by afula cell cellular tissue coding in plant strain J.88, 620-4, using T4 to perform DNA ligation. The recombinant vectors with correct sequences obtained by connection are named as pZH2B-SLG1, pZH2B-SLG1, and are obtained by replacing DNA fragments between XbaI and SacI recognition sequences of the vector pZH2B with SLG1 genes shown in a sequence 1 in a sequence table, and pZH2B-SLG1 can express a sequence 3 in the sequence tableSLG1 protein shown.

2. Obtaining of transgenic plants:

introducing pZH2B-SLG1 obtained in the step 1 into agrobacterium EHA105 to obtain recombinant agrobacterium infection callus induced by SLG1 mutant embryo, and redifferentiating the infected callus on a hygromycin-containing culture medium to obtain a plant, namely a SLG1-C transgenic plant.

According to the method, pZH2B-SLG1 is replaced by a vector pZH2B, and other steps are not changed to obtain an empty vector control plant.

3. Identification of transgenic plants:

and (3) carrying out PCR amplification on the genome DNA of the transgenic plant obtained in the step (2) by utilizing SLG1-MS-F and SLG1-MS-R, carrying out enzyme digestion on an obtained PCR product by using a restriction enzyme PstI-HF (NEB, # R3140S), carrying out electrophoresis, and determining whether the transgenic plant is a positive transgenic plant or not according to the size of the obtained fragment. Wild-type air-bred KY131 and slg1 mutants were used as controls. The primer sequences are as follows:

SLG1-MS-F:5′-GCAACATCTCGATAGGCCTTCGAAC-3′；

SLG1-MS-R:5′-CAAGCTATGGCTGTCAGATATGGTATC-3′。

the results show that the band types of wild type empty-cultivated KY131(WT) and slg1 mutant and enzyme digestion products of slg1-C transgenic positive plants are different, the enzyme digestion product of WT is completely digested by PstI-HF and is represented by two bands of about 400bp and 200bp, the enzyme digestion product of slg1 mutant can not be digested by PstI-HF and is represented by one band of about 600bp, the slg1-C transgenic positive plant contains both WT and slg1 mutant DNA, and the enzyme digestion products are represented by three bands of about 600bp, 400bp and 200bp (figure 1). The obtained positive transgenic plant was subjected to PCR amplification of its genomic DNA using a primer set consisting of Com-SacI-F and Com-KpnI-R, and the result showed that the sequence of the obtained PCR product contained the correct genomic sequence of SLG1 gene shown in SEQ ID No. 1.

And marking the plants without the transferred target gene in the transgenic plants as transgenic negative plants.

4. Phenotypic identification of transgenic plants

Harvestingslg1-C Positive transgenic plant T₃Seeds, seeded on 30mg/L hygromycin and all homozygous T's that grow healthily on hygromycin are selected₃The strain is subjected to normal conditions (14h illumination, light intensity of 200 mu mol)^-2s^-2In 10H of darkness, the humidity is 65 percent and the temperature is 28 ℃), healthy two-leaf one-heart rice growing in the dark is transferred to an artificial incubator (SANYO, MLR-351H) for heat treatment under the condition of 14H of illumination and the illumination intensity is 200 mu molm^-2s^-2And treating for 44h in the dark for 10h at the humidity of 65% and the temperature of 45 ℃, then transferring to the normal condition to recover and culture for 10d, and counting the survival rate. Partial plant results are shown in FIG. 2 and B in FIG. 3 by using wild type empty-bred KY131(WT) and SLG1 mutant, transgenic negative plants as controls, and SLG1-RNAi represents SLG1-RNAi positive transgenic plants in example 2.

The results show that the survival rate of the slg1 mutant and the transgenic negative plant is not significantly different after heat treatment; compared with the slg1 mutant, the slg1-C positive transgenic plant has obviously better growth vigor after being subjected to high temperature treatment at 45 ℃, and the survival rate is obviously higher than that of the slg1 mutant. The survival rates of the wild type empty-bred KY131(WT) and slg1 mutant, transgenic negative plants and slg1-C positive transgenic plants are respectively 88 +/-5.1%, 6.7 +/-2.9%, 7.2 +/-3.1% and 55 +/-8.25%.

Example 2 SLG1 Gene can control Rice Heat resistance

1. Constructing a recombinant vector:

5' -Biopsis with primer SLG1-RNAi-XbaI-FtctagaCCGTTCAGTGAATCAGAACTCCAG-3' and SLG1-RNAi-SpeI-R: 5-actagtGTCTTGAGATCGGTGGAAACTTGCTG-3' using cDNA of wild type empty-bred KY131 as template to perform PCR, obtaining PCR products with correct sequence, connecting into intermediate vector pEASY (Beijing hologold organism, cat # CB101-01), obtaining recombinants, cleaving with restriction endonucleases XbaI (NEB, # R0145S) and SpeI-HF (NEB, # R3133S), inserting into pZH2Bi vector (Song, L., Wang, R., Zhang, L., Wang, Y., and Yao, S. (2016) CRR1 encoding restriction enzymes synthesis in extract by fermentation of vacuum cell paper in plant J.88,620-632.),the obtained recombinant vector with the correct sequence is named as pZH2Bi-SLG1-RNAi, the recombinant vector is obtained by inserting a DNA fragment shown in the 1174-1749 th position of the sequence 2 in the sequence table into a vector pZH2Bi by using a restriction enzyme XbaI, and inserting a DNA fragment shown in the 1174-1749 th position of the sequence 2 in the sequence table into a vector pZH2Bi by using a restriction enzyme SpeI-HF, and the directions of two insertions of the DNA fragment shown in the 1174-1749 th position of the sequence 2 in the sequence table are opposite in the vector pZH2 Bi. pZH2Bi-SLG1-RNAi can be used for inhibiting the expression of SLG1 gene.

2. Obtaining of transgenic plants:

introducing pZH 2-2 Bi-SLG1-RNAi obtained in the step 1 into agrobacterium EHA105, infecting callus induced by wild type empty-cultured KY131 embryo with the obtained recombinant agrobacterium, and redifferentiating the infected callus on a hygromycin-containing culture medium to obtain a plant, namely a transgenic plant of SLG 1-RNAi.

According to the method, pZH2Bi-SLG1-RNAi is replaced by a vector pZH2Bi, and other steps are not changed to obtain an RNAi no-load control plant.

3. Identification of transgenic plants:

extracting total RNA of each material, and detecting the expression level of SLG1 in each material by using a qRT-PCR method. Total RNA was extracted using trizol extraction: before sampling, burning tweezers, scissors and blades required by sampling by using alcohol lamp flame, taking down the material for RNA extraction, putting the material into tin foil paper, wrapping the material, quickly putting the wrapped material into liquid nitrogen for quick freezing, and transferring the sample without immediate RNA extraction into an ultra-low temperature refrigerator at minus 80 ℃ for later use. Grinding appropriate amount of tissue in mortar to powder, adding 1mL trizol extract, rapidly rotating mortar rod to cover trizol on plant tissue, melting, grinding the mixed solution with mortar rod to clear, transferring the grinding solution to 1.5mL RNase-free centrifuge tube, standing at room temperature for 5min, centrifuging at 4 deg.C and 12,000rpm for 5min, transferring supernatant to new centrifuge tube, adding one fifth volume of chloroform, shaking vigorously for 15s, standing at room temperature for 5min, centrifuging at 4 deg.C and 12,000rpm for 15min, transferring supernatant to new centrifuge tube, adding isovolumetric isopropanol, gently mixing, standing at room temperature for 10min, centrifuging at 4 ℃, 12,000rpm for 10min, removing supernatant, washing the precipitate with 75% ethanol, centrifuging at 4 ℃, 12,000rpm for 5min, removing ethanol, carefully sucking residual ethanol with a gun head, drying at room temperature for 3-5 min until RNA edges are transparent, and adding 30 mu L of DEPC water for reverse transcription. The quality of the RNA can be detected by agarose gel electrophoresis.

First strand cDNA Synthesis: the RNA concentration was measured by a Nanodrop apparatus, 1. mu.g of total RNA was digested with 1. mu.L of DNase I (Thermo Scientific) at 37 ℃ for 30min, heated at 70 ℃ for 10min to denature the DNase I, and then placed on ice. Adding a reagent required by reverse transcription according to the dosage of a Promega reverse transcription kit, incubating for 1h at 42 ℃, immediately placing a reaction system on ice after denaturation for 5min at 95 ℃, separating a first strand of cDNA from an RNA template, adding sterilized ultrapure water with four times volume, and storing at-20 ℃ for later use.

qRT-PCR detection analysis: adding Roche SYBR Green Master I enzyme premix and qRT primer to the first strand cDNA as template, and using Roche

The Nano instrument performs PCR. The reaction conditions of PCR were: 5min at 94 ℃; 5s at 94 ℃; the annealing temperature (55-60 ℃) of the corresponding primer is 15 s; 10s at 72 ℃; 45 cycles, and OsActin1 of rice is used as an internal reference gene.

The primer sequence of the reference gene is as follows:

ACTIN-Q-F：5′-TGCTATGTACGTCGCCATCCAG-3′；

ACTIN-Q-R：5′-AATGAGTAACCACGCTCCGTCA-3′。

qRT-PCR detection the RNA level of SLG1 was analyzed using the primers:

SLG1-Q-F：5′-CTGATGTCCAGTATGTTGATACACGGTG-3′；

SLG1-Q-R：5′-TCGAAGGCCTATCGAGATGTTGCTG-3′。

plants with more than 3 times lower expression level than the wild type were selected as SLG1-RNAi positive transgenic plants for further phenotypic identification (A in FIG. 3). Plants with expression levels not significantly different from wild type were selected as RNAi transgene negative plants as controls for further experiments.

4. Phenotypic identification of transgenic plants

Harvesting the seeds of the SLG1-RNAi positive transgenic plant, transferring healthy two-leaf one-heart-stage rice growing under normal conditions into an artificial culture box (SANYO, MLR-351H) for heat treatment under the condition of 14H illumination and the illumination intensity of 200 mu molm^-2s^-2And treating for 44h in the dark for 10h at the humidity of 65% and the temperature of 45 ℃, then transferring to the normal condition to recover and culture for 10d, and counting the survival rate. The results of the partial plants are shown in FIG. 2 and A in FIG. 3 using wild type KY131(WT), RNAi null control plant and slg1 mutant as controls.

The results show that after heat treatment, the survival rate of RNAi transgenic negative plants and WT have no significant difference; compared with WT, SLG1-RNAi positive transgenic plants have obviously poorer growth vigor after being subjected to high temperature treatment at 45 ℃, and the survival rate is obviously lower than that of WT. The survival rates of the SLG1-RNAi positive transgenic plant, the wild KY131(WT), the RNAi transgenic negative plant and the SLG1 mutant are respectively 15 +/-3.7%, 88 +/-5.1%, 86 +/-7.6% and 6.7 +/-2.9%.

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

<120> application of heat-resistance-related protein SLG1 and coding gene thereof in regulation and control of rice heat resistance

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 4871

<212> DNA

<213> Rice (Oryza sativa)

<400> 1

gatgtatgcg ccaattgcgt acaacgggat gaacattggg ctcgatctga acattgatac 60

atgattcaga agtctcagct cacaactgga caatgcagtt catgttaatc acaacgagtt 120

catactaacc atgcaatgaa ttcaacacaa attagccatt ctggaattac ttatatagtt 180

ccagttcgtc atcagttact gaaggatcat aggtaacacc ggtatcaaac tggaactggt 240

cctgaaactt gtgaagaact gaagacgagc aaggctaatt gtgcactact acacttgcat 300

catgggacgg ttcaatttgc ccccaaattt gcatgtgaca atttttttgt tgtcttcatg 360

aaagacatat acttttgaag agaatttgta aaattaagat actatatgtt tgctgtgaat 420

tttgtaagca atgcaaaatc tagtcgcttt tgattctttt tggaagcaaa tttttaagtc 480

ccattgtctc aatatctcat ttccagtttc cacctcgaag catgatctta agcacacgta 540

gaattattat tttatcaatt ttatttactt gttcatgttg gctcatgcgt ggagttgacc 600

tgatttaaat gaatttgtaa atagccaaca taccgaactg acaacaaggt tcacaatctc 660

gattggccag tgaatttcaa gccacaatga gacgtagatt ttttttttgt taattttgat 720

gaatttcgac aaattttgaa tgaaattgaa aaataatcaa acaaaaacat ttgagatcta 780

cattcgggcc agctcgagat gttcaaaatt cagagattgt gaaccctgag tacgtttttt 840

tcctcttttt tctaattata gccttataca tgattataat cttctaactt cttcttaact 900

ggctgttatt ttatcaaaat aaatcttatc aaattttggc aatgtcaaaa ttttggtaag 960

tgggtaatat tgccaaattt tgataggatt ccttatatat ttaccaaagt ttggtaaaaa 1020

aactaaatgt atatctttgg caattttttt aaaaaagaca tggtttgaaa tgacactaat 1080

ctaaacaacc cttatacttt taatatagtt ggtatcacta acttctaaaa ctcagtatgc 1140

aagtgtacaa catcgtcatc cactgttaag tattatctat agtattttaa atcatataca 1200

agtacgtcac atcatcactc actaacaatt attatatcga ttattttaaa tatcatgtta 1260

gcatgacata gtataattta tgattttgtt gtatttttaa aacttaatac gtaaacaatg 1320

tcaaatcgtc atctccacat tacttttaca ttatttaaag atgtaacata cattcatcca 1380

aatataacgt agcaaagtgc tagtactgta tcaatatgaa acactgaaac ctaacaccag 1440

ctggtgcgtt tcatttcaaa tattcaaagc aaaagggaaa aaaatgggcc taattcgagg 1500

ggacggccca ggtgaaagcc cattgggcct ggactgagac ggcccagcta ggcccaattc 1560

gaggagtagt atagaatccc aaagccctaa actaaaccta cgcgctcgcg tgcttacccg 1620

cctcgcgtac ccacaccatg gccgccgccg ccgcctcctc ctgcggcggc gccggctgcg 1680

ggccccactg ctcctcctcc gcctccgccg ccgcggtgga ggatgctgct gctgcggcgg 1740

cggagaaggt aggccgcctg tcactctccc gcgagtgcgg caagtgcggc ggcggcgccg 1800

ctgccgtcgc cgtcgccgga ggcctcggcc tgtgcggcga gtgcttccgg gccaacctgc 1860

tcgggaagtt caagctcgcc gtcacgagca acgccatggt tcgccccacc gactccgtcc 1920

tcctcgcctt ctccggcggc cccgcctcca ggttcgcttc cctcttcttt tttttttcct 1980

tcctaatttt catttctttt gtgctacata aagttattac tcttcctttg catataattc 2040

ttcacgtgtt tatgatcatt agatttgaag tgttattgta ttatacttta catagaattc 2100

ttcatgtgtt tatgatcatt agagtggaag tgttattgta ttatagtact ttgcatagaa 2160

tcttcatgtg tttatgatca ttagagtgga agtgttattg tattatagta ctttgcatag 2220

aatcttcatg tgtttatgat cattagagtg gaagtgtaat tgtactacta cgtgatgtaa 2280

ttatagggta gcgctgcaat tcatacatga gatgcggtgc aaggcgattg agagctggga 2340

cgtgagcaat tcacaagcct taccggtatt cggtgtcggg gtggcattcg tggatgaaag 2400

tgttctctgt tcgaagccga gagacgagat tgagatggca attgaagata tcaggtcgat 2460

cgtgtcgagt ttgtcaacag gcgttaaagc gatgcacatt gcgcgtcttg aggatgtgtt 2520

ctccaccgaa tcggaggatg gggaacgcag gttgagggag gcggtggata tgattggtga 2580

tgacactggg agggaggatt tccttcggtg cttacgcatg ctttcgttgc aaaaggtccg 2640

tcagtgtgct tttttgctct tgtattatgc tttttgatca gtggatgagt taatgtctat 2700

tgtttcatgt tgatgtgttg ttgatcagat tgctatggaa aacgggtacg ctaagatcat 2760

gctaggatca tgtgcttctg caatagcatg ccatgttctg tccgcgactg tgaaggtata 2820

ccatgtgctg ctttctctgt attctgttct ttgttaccaa ttcataattc ttgaaaagaa 2880

catgatggaa attagaggag cactatgaaa ttgatatctc tacccagggg caaggttact 2940

ctttacctgc tgatgtccag tatgttgata cacggtggga aatacctgtt gttctcccac 3000

tccgtgattg cctagcccaa gagcttactc tcctctgtga actagatagg taagagaagg 3060

gggaaaaacc ttatttgttt atttcttgaa atgccattga ttctaactga atgtgtttag 3120

tttaaaaaca cagcaacatc tcgataggcc ttcgaacggc atcaacagtt tagttgcatc 3180

ctttattaaa cgactacggg taagttcagt cctatcatgc caattactac atgtcaaatt 3240

acacttcacc tattcttagt tgaactacat ccttgctcag cagttatttt tgttaattgt 3300

ttggaggaca acacatcttt acagctatta ttaccatctg caggaagaga acccttctcg 3360

agagcatacc attgtaagaa ctgcgcaaaa gctcaagccc ttctctttta acaaattctc 3420

ggcagatggc tatcatgatt tcctaccatc gcgactacgt ccaaaattcc agaagtttga 3480

tagcgatgag tccacttttt ctgagattct ctgtctgatg tgtggaagtc cgttcagtga 3540

atcagaactc cagaatttgg aaagcacaaa acacaaggct cagaagaaaa ttgatcttta 3600

tactgctcat tgttgtcaaa gctgttattt ccagattcta ccagccggtg aaaacttgaa 3660

tgaacatttc ttttcactac taccaaaatt atggactgga aaaatggata ccatatctga 3720

cagccatagc ttgctaaggt gatagtggtt tctgtttctt tatggagtac ttcatttctt 3780

gttataaaca gtgagcgcta ttggtagttt agcaaatgaa aacattgaac ctttggatat 3840

tggagcttgt ttgtctgata atgtttgatt taacgtgacc atgtgtagta tgtgcagttg 3900

gcccattcat ctgttaaagt cctgccctat tcagtccttc agtgcactga tttgctaaat 3960

tgtttatggc attttttttc agagatcaaa tagaagagta cctgcttgaa gaaaatgatg 4020

atggaaactg aacttgatat ttcagtgaag cccgaatata ttgtaagaga gaatctttcg 4080

cttcatcctt ttattttgtg ctgtaaaaat cttctctgca tacggtaatg ctgtaaaagt 4140

cattttggaa tgtggtaatc atttatctta tagtttatgc tgagttgctg acatatattc 4200

ttcaaattgt tttgttgtag tgccagaaag acagcagaga ttcctgccta gttgggctcc 4260

tcacattggc ataatcacat aatccaaact tgcttcagat aatagtgttt ctatctagca 4320

tggcattggt ttattcagaa ttctgccaga tacttacaat ttcaacacat tatatccttg 4380

actcaattcc tgatgtttct gtatgtcctt cctactctta agagttgaaa cctgtatgaa 4440

atccgtatag tgatagcctg atggggcaga ccgtcacttg ccagcaagtt tccaccgatc 4500

tcaagaccac agacattgga cagaaacttc catgtgatta cagtattctg attggtcaca 4560

tctcctgtac caactattct gtctgcaaaa gattgtaacc tgaagaattt attcgattat 4620

tttggaacaa tcttctctat acaagatttg cacatactat ggtttcaatc aagtacaacg 4680

tcctgttggc tcttcccatt tatcatgcct tcaaggaaac ttttaaaatt aactttgtac 4740

atatatactg catatttgtt attgattgcc tggtaagatt gaatcattga aggatggatc 4800

attctattat ggttgcaaaa taagaatcaa aatcacaaac aatacatcgc atgcacagat 4860

acactacagg t 4871

<210> 2

<211> 1966

<212> DNA

<213> Rice (Oryza sativa)

<400> 2

gcgctcgcgt gcttacccgc ctcgcgtacc cacaccatgg ccgccgccgc cgcctcctcc 60

tgcggcggcg ccggctgcgg gccccactgc tcctcctccg cctccgccgc cgcggtggag 120

gatgctgctg ctgcggcggc ggagaaggta ggccgcctgt cactctcccg cgagtgcggc 180

aagtgcggcg gcggcgccgc tgccgtcgcc gtcgccggag gcctcggcct gtgcggcgag 240

tgcttccggg ccaacctgct cgggaagttc aagctcgccg tcacgagcaa cgccatggtt 300

cgccccaccg actccgtcct cctcgccttc tccggcggcc ccgcctccag ggtagcgctg 360

caattcatac atgagatgcg gtgcaaggcg attgagagct gggacgtgag caattcacaa 420

gccttaccgg tattcggtgt cggggtggca ttcgtggatg aaagtgttct ctgttcgaag 480

ccgagagacg agattgagat ggcaattgaa gatatcaggt cgatcgtgtc gagtttgtca 540

acaggcgtta aagcgatgca cattgcgcgt cttgaggatg tgttctccac cgaatcggag 600

gatggggaac gcaggttgag ggaggcggtg gatatgattg gtgatgacac tgggagggag 660

gatttccttc ggtgcttacg catgctttcg ttgcaaaaga ttgctatgga aaacgggtac 720

gctaagatca tgctaggatc atgtgcttct gcaatagcat gccatgttct gtccgcgact 780

gtgaaggggc aaggttactc tttacctgct gatgtccagt atgttgatac acggtgggaa 840

atacctgttg ttctcccact ccgtgattgc ctagcccaag agcttactct cctctgtgaa 900

ctagatagtt taaaaacaca gcaacatctc gataggcctt cgaacggcat caacagttta 960

gttgcatcct ttattaaacg actacgggaa gagaaccctt ctcgagagca taccattgta 1020

agaactgcgc aaaagctcaa gcccttctct tttaacaaat tctcggcaga tggctatcat 1080

gatttcctac catcgcgact acgtccaaaa ttccagaagt ttgatagcga tgagtccact 1140

ttttctgaga ttctctgtct gatgtgtgga agtccgttca gtgaatcaga actccagaat 1200

ttggaaagca caaaacacaa ggctcagaag aaaattgatc tttatactgc tcattgttgt 1260

caaagctgtt atttccagat tctaccagcc ggtgaaaact tgaatgaaca tttcttttca 1320

ctactaccaa aattatggac tggaaaaatg gataccatat ctgacagcca tagcttgcta 1380

agagatcaaa tagaagagta cctgcttgaa gaaaatgatg atggaaactg aacttgatat 1440

ttcagtgaag cccgaatata tttgccagaa agacagcaga gattcctgcc tagttgggct 1500

cctcacattg gcataatcac ataatccaaa cttgcttcag ataatagtgt ttctatctag 1560

catggcattg gtttattcag aattctgcca gatacttaca atttcaacac attatatcct 1620

tgactcaatt cctgatgttt ctgtatgtcc ttcctactct taagagttga aacctgtatg 1680

aaatccgtat agtgatagcc tgatggggca gaccgtcact tgccagcaag tttccaccga 1740

tctcaagacc acagacattg gacagaaact tccatgtgat tacagtattc tgattggtca 1800

catctcctgt accaactatt ctgtctgcaa aagattgtaa cctgaagaat ttattcgatt 1860

attttggaac aatcttctct atacaagatt tgcacatact atggtttcaa tcaagtacaa 1920

cgtcctgttg gctcttccca tttatcatgc cttcaaggaa actttt 1966

<210> 3

<211> 464

<212> PRT

<213> Rice (Oryza sativa)

<400> 3

Met Ala Ala Ala Ala Ala Ser Ser Cys Gly Gly Ala Gly Cys Gly Pro

1 5 10 15

His Cys Ser Ser Ser Ala Ser Ala Ala Ala Val Glu Asp Ala Ala Ala

20 25 30

Ala Ala Ala Glu Lys Val Gly Arg Leu Ser Leu Ser Arg Glu Cys Gly

35 40 45

Lys Cys Gly Gly Gly Ala Ala Ala Val Ala Val Ala Gly Gly Leu Gly

50 55 60

Leu Cys Gly Glu Cys Phe Arg Ala Asn Leu Leu Gly Lys Phe Lys Leu

65 70 75 80

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

85 90 95

Ala Phe Ser Gly Gly Pro Ala Ser Arg Val Ala Leu Gln Phe Ile His

100 105 110

Glu Met Arg Cys Lys Ala Ile Glu Ser Trp Asp Val Ser Asn Ser Gln

115 120 125

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

130 135 140

Leu Cys Ser Lys Pro Arg Asp Glu Ile Glu Met Ala Ile Glu Asp Ile

145 150 155 160

Arg Ser Ile Val Ser Ser Leu Ser Thr Gly Val Lys Ala Met His Ile

165 170 175

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

180 185 190

Arg Leu Arg Glu Ala Val Asp Met Ile Gly Asp Asp Thr Gly Arg Glu

195 200 205

Asp Phe Leu Arg Cys Leu Arg Met Leu Ser Leu Gln Lys Ile Ala Met

210 215 220

Glu Asn Gly Tyr Ala Lys Ile Met Leu Gly Ser Cys Ala Ser Ala Ile

225 230 235 240

Ala Cys His Val Leu Ser Ala Thr Val Lys Gly Gln Gly Tyr Ser Leu

245 250 255

Pro Ala Asp Val Gln Tyr Val Asp Thr Arg Trp Glu Ile Pro Val Val

260 265 270

Leu Pro Leu Arg Asp Cys Leu Ala Gln Glu Leu Thr Leu Leu Cys Glu

275 280 285

Leu Asp Ser Leu Lys Thr Gln Gln His Leu Asp Arg Pro Ser Asn Gly

290 295 300

Ile Asn Ser Leu Val Ala Ser Phe Ile Lys Arg Leu Arg Glu Glu Asn

305 310 315 320

Pro Ser Arg Glu His Thr Ile Val Arg Thr Ala Gln Lys Leu Lys Pro

325 330 335

Phe Ser Phe Asn Lys Phe Ser Ala Asp Gly Tyr His Asp Phe Leu Pro

340 345 350

Ser Arg Leu Arg Pro Lys Phe Gln Lys Phe Asp Ser Asp Glu Ser Thr

355 360 365

Phe Ser Glu Ile Leu Cys Leu Met Cys Gly Ser Pro Phe Ser Glu Ser

370 375 380

Glu Leu Gln Asn Leu Glu Ser Thr Lys His Lys Ala Gln Lys Lys Ile

385 390 395 400

Asp Leu Tyr Thr Ala His Cys Cys Gln Ser Cys Tyr Phe Gln Ile Leu

405 410 415

Pro Ala Gly Glu Asn Leu Asn Glu His Phe Phe Ser Leu Leu Pro Lys

420 425 430

Leu Trp Thr Gly Lys Met Asp Thr Ile Ser Asp Ser His Ser Leu Leu

435 440 445

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

450 455 460

Claims (10)

1. Any one of the following applications of the heat-resistant related protein or the substance for regulating the activity or the content of the heat-resistant related protein:

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

D2) preparing a product for regulating and controlling the heat resistance of the plant;

D3) improving the heat resistance of the plant;

D4) preparing a product for improving the heat resistance of the plant;

D5) plant breeding;

the heat-resistant related protein is A1), A2) or A3) as follows:

A1) the amino acid sequence is the protein of sequence 3;

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 3 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).

2. Use of a biomaterial related to a protein of heat-resistance related as claimed in claim 1 in any one of the following applications:

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

D2) preparing a product for regulating and controlling the heat resistance of the plant;

D3) improving the heat resistance of the plant;

D4) preparing a product for improving the heat resistance of the plant;

D5) plant breeding;

the biomaterial is any one of the following B1) to B22):

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

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

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism containing the recombinant vector of B4);

B9) a transgenic plant cell line comprising the nucleic acid molecule of B1);

B10) a transgenic plant cell line comprising the expression cassette of B2);

B11) a transgenic plant cell line comprising the recombinant vector of B3);

B12) a transgenic plant cell line comprising the recombinant vector of B4);

B13) transgenic plant tissue comprising the nucleic acid molecule of B1);

B14) transgenic plant tissue comprising the expression cassette of B2);

B15) transgenic plant tissue containing the recombinant vector of B3);

B16) transgenic plant tissue containing the recombinant vector of B4);

B17) a transgenic plant organ containing the nucleic acid molecule of B1);

B18) a transgenic plant organ containing the expression cassette of B2);

B19) a transgenic plant organ containing the recombinant vector of B3);

B20) a transgenic plant organ containing the recombinant vector of B4);

B21) a nucleic acid molecule that reduces the expression level of the thermostable protein of claim 1;

B22) 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 B21).

3. Use according to claim 2, characterized in that: B1) the nucleic acid molecule is any one of the following b1) -b 6):

b1) the coding sequence is a cDNA molecule or DNA molecule at 37 th-1431 th site of the sequence 2 in the sequence table;

b2) a cDNA molecule or DNA molecule shown in 37 th-1431 th site of a sequence 2 in a sequence table;

b3) a cDNA molecule or a DNA molecule shown in a sequence 2 in a sequence table;

b4) DNA molecule shown in sequence 1 in the sequence table;

b5) a cDNA molecule or DNA molecule having 75% or more identity with the nucleotide sequence defined in b1) or b2) or b3) or b4) and encoding the heat-resistant related protein of claim 1;

b6) a cDNA molecule or DNA molecule which hybridizes with the nucleotide sequence defined by b1) or b2) or b3) or b4) or b5) under stringent conditions and codes for the heat-resistant related protein as claimed in claim 1;

B21) the nucleic acid molecule is shown as the 1174-1749 site of the sequence 2 in the sequence table.

4. The product for regulating and controlling the heat resistance of the plants is characterized in that: the product contains the protein related to heat resistance in claim 1 or the biological material in claim 2 or 3.

5. Use according to any one of claims 1 to 3, or a product according to claim 4, wherein: the plant is m1) or m2) or m 3):

m1) a monocotyledonous or dicotyledonous plant;

m2) a gramineous plant;

m3) rice.

6. A method of increasing the heat tolerance of a plant comprising: increasing the activity and/or content of the protein related to heat tolerance in the receptor plant as defined in claim 1, or promoting the expression of the gene encoding the protein related to heat tolerance in claim 1, to obtain the target plant with improved heat tolerance compared with the receptor plant.

7. A method of growing a heat-tolerant enhanced plant comprising: increasing the activity and/or content of the protein related to heat tolerance in the receptor plant as defined in claim 1, or promoting the expression of the gene encoding the protein related to heat tolerance in claim 1, to obtain the target plant with improved heat tolerance compared with the receptor plant.

8. The method according to claim 6 or 7, characterized in that: the target plant is a transgenic plant having increased expression of the heat-resistant related protein as compared with the recipient plant, which is obtained by introducing the gene encoding the heat-resistant related protein of claim 1 into the recipient plant.

9. The method of claim 8, wherein: the gene encoding the protein related to thermotolerance according to claim 1, wherein the gene is the nucleic acid molecule according to B1) of claim 3.

10. The method according to any one of claims 6-9, wherein: the recipient plant is m1) or m2) or m 3):

m1) a monocotyledonous or dicotyledonous plant;

m2) a gramineous plant;

m3) rice.

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