CN107325162B - SPL gene and application thereof in enhancing heat resistance of plants - Google Patents

Abstract

The invention provides an SPL gene and application thereof in enhancing the heat resistance of plants. Specifically, the invention provides a function and application of an SPL gene, the SPL gene (SPL1 or SPL12) is a key factor necessary for high temperature adaptation and tolerance of plants and maintenance of seed developmental maturity and germination under high temperature conditions, and transgenic Arabidopsis with over-expression of SPL1 or SPL12 can enhance the tolerance of floral organs to extreme and mild high temperature. The SPL gene has wide application value and provides a new way for cultivating high temperature stress resistant crops.

Description

SPL gene and application thereof in enhancing heat resistance of plants

Technical Field

The invention relates to the field of botany, in particular to an SPL gene and application thereof in enhancing heat resistance of plants.

Background

Temperature is one of the key physical factors on earth that affect vital activities. Ambient temperature affects the entire food chain and ecosystem. High temperature stress adversely affects almost all aspects of plant growth, development, reproduction and yield, including hindered germination of seeds, scorched leaves and shoots, sun damage of shoots and stems, senescence and abscission of leaves, growth inhibition of roots and stems, discoloration of fruits and reduction in yield, and even death of the whole plant. All plant tissues are vulnerable to heat stress, wherein the sensitivity of reproductive organs is highest, and the increase of several degrees in temperature during flowering period can lead to a sharp decrease in crop yield and even the extinction of the whole agricultural production. The reproductive growth phase of plants is more sensitive to high temperature than the vegetative growth phase, while the flowering phase of most crops is in the high temperature phase in summer, so that the research on the heat shock response mechanism of the reproductive growth phase of plants is urgent. With global warming, it is becoming increasingly important to study the mechanisms by which high temperatures affect plant growth and development as well as crop yield.

The plants form a signal path for sensing the change of the environmental temperature in the evolution process, and adjust the metabolism and the cell function of the plants to prevent the body damage caused by environmental stress. These different signaling pathways are tissue-and species-specific, and there is a large difference in heat shock response signals, especially during reproductive and vegetative growth of plants. At present, the research on the heat resistance of the plants in the reproductive growth period still stays at the morphological, physiological and biochemical levels, but the research on the molecular mechanism is less, and a referable regulation and control network is not available.

Therefore, there is an urgent need in the art to develop genes that regulate plant heat resistance and to study their functional applications accordingly.

Disclosure of Invention

The invention aims to provide an SPL gene for regulating and controlling heat resistance of plants and application thereof in enhancing the heat resistance of the plants.

In a first aspect of the present invention, there is provided a use of an SPL gene or a protein encoding the same, the SPL gene being selected from the group consisting of an SPL1 gene, an SPL12 gene, or a combination thereof, and the SPL gene or the protein encoding the same for a use selected from the group consisting of:

(a) for the preparation of agents or compositions for enhancing the heat resistance of plants;

(b) used for enhancing the heat resistance of plants.

In another preferred embodiment, the plant is selected from the group consisting of: gramineae, and cruciferae.

In another preferred embodiment, the plant is selected from the group consisting of: arabidopsis, tobacco, rice, and wheat.

In another preferred embodiment, the plant is Arabidopsis thaliana.

In another preferred embodiment, the SPL genes are SPL1 gene and SPL12 gene.

In another preferred embodiment, the "enhancing heat resistance of plants" includes one or more properties selected from the group consisting of:

(i) the heat resistance of the inflorescence is enhanced;

(i i) enhancing germination rate of seeds that are set in high temperature environment;

(ii) for enhancing the tolerance of the plant to a high temperature environment;

(iv) enhancing heat shock response of the plant in a high temperature environment;

(v) enhancing the oxidation resistance of the plant in a high-temperature environment;

(vi) enhancing heat resistance of plant roots, stems, and/or leaves;

(vii) and the maturing rate of the plants in the high-temperature environment is enhanced.

In another preferred embodiment, the high temperature environment is an environment with a temperature of 30-50 ℃, preferably an environment with a temperature of 32-45 ℃, more preferably an environment with a temperature of 35-42 ℃, such as 30 ℃,35 ℃, 37 ℃ and 42 ℃.

In another preferred embodiment, the heat-resistant property of the enhanced inflorescence comprises: enhance the survival rate and/or flowering rate of the inflorescences in the high temperature environment.

In another preferred embodiment, the enhancing the tolerance of the plant to the high-temperature environment comprises: improving the survival rate of the plants in the high-temperature environment.

In another preferred embodiment, the enhanced heat shock response comprises up-regulating gene expression selected from the group consisting of:

WRKY15, WRKY25, WRKY33, WRKY39, ERF020, ERF1, ERF2, RAP2.1, ERF054, CRJ1, ABRE1, RAP2.6, or a combination thereof.

In another preferred embodiment, the antioxidant property of the plant refers to the ability of the plant to scavenge ROS in vivo.

In another preferred embodiment, said enhancing the antioxidant ability of the plant means increasing the SOD expression and/or activity of the plant.

In another preferred example, the "enhancing the heat resistance of the plant" comprises enhancing the heat resistance of the plant in the growth period and the reproduction period.

In another preferred embodiment, the SPL gene includes a wild-type SPL gene and a mutant SPL gene.

In another preferred embodiment, the mutant form comprises a mutant form in which the function of the encoded protein is not altered after mutation (i.e., the function is the same or substantially the same as the wild-type encoded protein).

In another preferred embodiment, the mutant SPL gene encodes a polypeptide that is the same or substantially the same as the polypeptide encoded by the wild-type SPL gene.

In another preferred embodiment, the mutant SPL gene comprises a polynucleotide having a homology of 80% or more (preferably 90% or more, more preferably 95% or more) with respect to the wild-type SPL gene.

In another preferred embodiment, the mutant SPL gene comprises a polynucleotide that is truncated or added by 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides at the 5 'end and/or 3' end of the wild-type SPL gene.

In another preferred embodiment, the gene comprises genomic DNA, cDNA, and/or mRNA.

In another preferred embodiment, the CDS sequence of the SPL1 gene is shown in SEQ ID No. 1.

In another preferred embodiment, the protein encoded by the SPL1 gene is shown in SEQ ID No. 2.

In another preferred example, the genome sequence of the SPL1 gene is shown in SEQ ID No. 3.

In another preferred embodiment, the CDS sequence of the SPL12 gene is shown in SEQ ID No. 4.

In another preferred embodiment, the protein encoded by the SPL12 gene is shown in SEQ ID No. 5.

In another preferred example, the genome sequence of the SPL12 gene is shown in SEQ ID No. 6.

In another preferred embodiment, the SPL gene is derived from a plant, preferably from a gramineae, and a crucifer, more preferably from: arabidopsis, tobacco, rice, and wheat.

In a second aspect of the present invention, there is provided a method of modifying heat tolerance in a plant, comprising the steps of:

(a) introducing an exogenous construct into a plant cell, wherein the construct comprises an exogenous SPL gene sequence, an exogenous nucleotide sequence that promotes expression of the SPL gene, or an exogenous nucleotide sequence that inhibits expression of the SPL gene, thereby obtaining a plant cell into which the exogenous construct is introduced;

(b) regenerating the plant cell into which the exogenous construct is introduced, obtained in the previous step, into a plant: and

(c) optionally identifying said regenerated plants, thereby obtaining plants with altered heat tolerance;

wherein the SPL gene is selected from the group consisting of the SPL1 gene, the SPL12 gene, or a combination thereof.

In another preferred embodiment, said plant with altered heat tolerance is a plant with altered heat tolerance compared to the parent plant.

In another preferred embodiment, the exogenous SPL gene sequence further comprises a promoter and/or a terminator operably linked to the ORF sequence.

In another preferred embodiment, the promoter is selected from the group consisting of: constitutive promoters, tissue specific promoters, inducible promoters, and strong promoters.

In another preferred embodiment, the constitutive promoter comprises a 35S promoter.

In another preferred embodiment, the exogenous nucleotide sequence comprises a nucleotide sequence that interferes with the expression of the SPL gene.

In another preferred embodiment, the exogenous nucleotide sequence comprises an RNA interference sequence.

In a third aspect of the present invention, there is provided a method of enhancing the heat resistance of a plant, the method comprising the steps of: in the plant, promoting expression of an SPL gene or promoting activity of an SPL protein, wherein the SPL gene is selected from the group consisting of an SPL1 gene, an SPL12 gene, or a combination thereof.

In another preferred embodiment, the method comprises administering to the plant an enhancer of the SPL gene or a polypeptide encoded thereby.

In another preferred embodiment, the method comprises introducing an exogenous SPL gene into a plant.

In another preferred example, the method comprises the steps of:

(i) providing a plant or plant cell; and

(i i) introducing the SPL gene sequence into the plant or plant cell, thereby obtaining a transgenic plant or plant cell.

In another preferred example, the method comprises the steps of:

(a) providing agrobacterium carrying an expression vector of the SPL gene sequence;

(b) contacting a plant cell or tissue or organ with the agrobacterium of step (a) such that the gene sequence of the SPL is transferred into the plant cell and integrated into the chromosome of the plant cell;

(c) selecting plant cells or tissues or organs into which the SPL gene sequence has been transferred; and

(d) regenerating the plant cell or tissue or organ of step (c) into a plant.

In another preferred embodiment, the SPL gene is derived from a plant, preferably from a gramineae, and a crucifer, more preferably from: arabidopsis, tobacco, rice, and wheat.

In a fourth aspect of the invention, there is provided a use of a modulator of an SPL gene or a protein encoded thereby for modulating thermotolerance in a plant, or for the preparation of an agent or composition for modulating thermotolerance in a plant, wherein the SPL gene is selected from the group consisting of an SPL1 gene, an SPL12 gene, or a combination thereof.

In another preferred embodiment, the composition comprises an agricultural composition.

In another preferred embodiment, the regulating agent comprises an accelerating agent and an inhibiting agent.

In another preferred example, the regulating agent is an accelerant, and the regulation refers to enhancing the heat resistance of the plant.

In another preferred embodiment, the regulating agent is an inhibitor, and the regulation refers to weakening the heat resistance of the plant.

In another preferred embodiment, the modulator comprises a small molecule compound, or a nucleic acid.

In another preferred embodiment, the nucleic acid is selected from the group consisting of: miRNA, shRNA, siRNA, or a combination thereof.

In a fifth aspect of the invention, a transgenic plant is provided, wherein an SPL gene is introduced, wherein the SPL gene is selected from the group consisting of an SPL1 gene, an SPL12 gene, and a combination thereof.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the tissue expression characteristics of the SPL1 and SPL12 genes.

FIG. 1A shows the expression pattern of the GUS gene driven by the SPL1 promoter. pSPL1 GUS transgenic plants GUS staining: the upper layer is a seedling, a root mature area, a root tip and a stem which grow for 7d on 1/2MS culture medium from left to right; the lower layer is seedlings, inflorescences and fruit pods growing for 20d from left to right.

FIG. 1B shows the expression pattern of the GUS gene driven by the SPL12 promoter. pSPL12 GUS transgenic plants were stained for GUS in the same order as in FIG. 1A.

FIGS. 1C-1F show the staining results of pSPL1, GUS transgenic plants at different flowering stages (stages 1-16), respectively. Wherein FIG. 1C shows periods 1-12; FIGS. 1D and 1E show periods 13-14, with red arrows indicating pollen; FIG. 1F shows time periods 15-16.

FIG. 1G shows the results of Real-Time PCR to detect the expression levels of SPL1 and SPL12 in flowers, including the buds (stage12 and before) and the flowers that have bloomed (stage13 and after).

FIG. 2 shows that the down-regulation of SPL1, SPL12 expression, affects flower development and seed yield.

FIG. 2A shows the reproductive growth phase phenotypes of col-0, spl1, spl12 and spl1spl 12. Wherein spl1spl12 has abnormal flower development and is partially aborted;

FIG. 2B shows the col-0 current day flowering (stage13) phenotype.

FIG. 2C shows the spl1spl12 current day flower (stage13) phenotype, with flowers that do not unfold normally.

FIG. 2D shows the internal morphological structure after the calyx in D has been opened with sharp forceps; spl1spl12 calyx adhesion.

FIG. 2E shows the col-0 flower ripening and abscission process.

FIG. 2F shows the spl1sgpl12 flower maturation abscission process.

FIG. 2G shows the myc-gSPL1, RNAi-1 and RNAi-2 phenotypes in transgenic plants 35S.

FIG. 2H shows Realtime-PCR detection of the expression levels of SPL1 in transgenic plants 35S: gSPL1(line1 and line4),35S: myc-gSPL1(line4 and line12), pSPL1: SPL1(line3 and line4) and mutants SPL1 and SPL1SPL 12.

FIG. 2I shows the detection of the expression level of SPL12 in transgenic plants 35S: gSPL12(line2 and line4), RNAi-1, RNAi-2 and mutants SPL12 and SPL1SPL12 by Realtime-PCR. (RNAi-1 and RNAi-2 are two transgenic lines silencing SPL12 in the SPL1 background)

FIG. 2J shows the expression levels of SPL1 in Col-0, SPL1-1SPL12-1, ox-MS1d-1, ox-MS1d-4, ox-MS1d-7 and ox-MS1c-1 and the expression levels of SPL12 in Col-0, SPL1-1SPL12-1, ox-S12c-3 and ox-S12c-4, RNA1-1, RNAi-2.

FIG. 3 shows analysis of average daily floral patency status of inflorescences at the top of the stems.

FIG. 3A shows the average daily floral openness (NOF) of col-0, SPL1-1SPL12-1 and ox-SPL1 inflorescences at 22 ℃ and 30 ℃. Wherein n is 20-30, ± SD; p-value is less than or equal to 0.01, p-value is less than or equal to 0.05

FIG. 3B shows the relative change in NOF mean values in FIG. 3A. Absolute change values were divided by the mean NOF at 22 ℃ of the corresponding plants.

FIG. 4 shows statistical analysis and morphological features of the floral patency per day of the inflorescence at the top of the stem.

FIG. 4A shows statistics of daily floral patency for col-0, SPL1-1SPL12-1 and ox-SPL1 inflorescences at the apical end of the stems at 22 ℃ and 30 ℃. Statistics for 0, 1 and 4d are shown, with n being 20-30, showing one of three replicates.

FIG. 4B shows morphological features of the top inflorescence of col-0, SPL1-1SPL12-1 and ox-SPL1 stems at 22 ℃ and 30 ℃.

FIG. 5 shows the tolerance of Arabidopsis floral organs to extreme hyperthermia.

FIG. 5A shows the effect of treatment at 37 ℃ for 1d on the morphological characteristics of ox-SPL1, col-0 and SPL1-1SPL12-1 flower organs.

FIG. 5B shows Real-time PCR detection of the expression level of SPL1 in ox-SPL1, col-0 and SPL1-1SPL12-1 plant inflorescences.

FIG. 5C shows the effect of 1d treatment at 37 ℃ on the survival of ox-SPL1, col-0 and SPL1-1SPL12-1 floral organs. Wherein, the temperature of the seeds is recovered to 22 ℃ for 1d after being treated at 37 ℃ for 1d, the number of inflorescences which are completely withered and can not normally bloom is observed and counted, and the survival rate of the plants which are not treated is 100 percent by taking the plants which are not treated as a control; (n ═ 20-30,. + -. SD;. represents p-value ≦ 0.01; and. represents p-value ≦ 0.05)

FIG. 6 shows the effect of high temperature treatment on Arabidopsis inflorescence SOD activity, seed yield and germination rate.

FIG. 6A shows the effect of high temperature treatment on col-0 and spl1-1spl12-1 inflorescence SOD activity. Wherein CK represents 22 ℃, HS represents arabidopsis thaliana grown for 5 weeks and treated at 37 ℃ for 4 h. (3times, ± SD;) represents p-value ≦ 0.01);

FIG. 6B shows the effect of high temperature treatment on col-0 and spl1-1spl12-1 seed yield. Wherein CK represents 22 ℃ and HS represents 22 ℃ for restoring 22 ℃ growth after being treated for 1h at 42 ℃. (n ═ 18, ±, SD;, represents p-value ≦ 0.01, and ≦ 0.05);

FIG. 6C shows the effect of high temperature treatment on the germination rate of col-0 and spl1-1spl12-1 seeds. Wherein CK represents 22 ℃, HS represents the seed germination rate after 7 days of growth at 30 ℃. (n ═ 60-70, 3times, ±, SD;. represents p-value ≦ 0.01; and. represents p-value ≦ 0.05)

FIG. 7 shows Real-time PCR detection of heat shock response expression of candidate transcription factors.

FIG. 7A shows heat shock responsive expression of WRKY transcription factors.

FIGS. 7B and 7C show heat shock response expression of ERF class transcription factors. Wherein, the total RNA is taken from inflorescences treated at 37 ℃ for 1 hour and 4 hours respectively, and the untreated inflorescences at 22 ℃ are taken as a control (0 hour).

FIG. 8 shows that overexpression of SPL1 or SPL12 improves heat resistance in plants.

FIG. 8A shows the effect of treatment at 42 ℃ on growth of wild type, spl1-1spl12-1 and transgenic plants ox-MS1c-1, ox-MS1d-1 and ox-S12c-3 grown for 14 days. The plants grown for 14 days were treated at a high temperature of 42 ℃ for 2d, and photographed after 5 days of recovery.

FIG. 8B shows the effect of treatment at 42 ℃ on the survival rates of wild type, spl1-1spl12-1 and transgenic plants ox-MS1c-1, ox-MS1d-1 and ox-S12c-3 grown for 14 days. The survival rate was counted after 5 days of recovery by 2d of the high temperature treatment at 42 ℃ on plants grown for 14 days.

FIG. 8C shows the effect of treatment at 42 ℃ on the growth of wild type, spl1-1spl12-1 and transgenic plants ox-MS1d-1, ox-MS1d-4 and ox-MS1d-7 inflorescences for 5 weeks of growth. The plant state was obtained by subjecting the plants grown for 5 weeks to a high temperature treatment at 42 ℃ for 5 hours.

FIG. 8D shows the effect of treatment at 42 ℃ on the growth of wild type, spl1-1spl12-1 and transgenic plants ox-MS1D-1, ox-MS1D-4 and ox-MS1D-7 inflorescences for 5 weeks of growth. The damaged state of inflorescence is counted after the plants growing for 5 weeks are treated at the high temperature of 42 ℃ for 5 hours.

The experiments in fig. 8A-8D were repeated at least three times, with representative primary results shown.

FIG. 8E shows that transgenic plants ox-MS1c-1, ox-MS1d-1, ox-S12c-3 and ox-S12c-3 have higher seed yields under high temperature stress. Left: growing at 22 ℃ all the time; the method comprises the following steps: treating the plants growing for 5 weeks at 42 deg.C for 5h, and recovering to 22 deg.C; and (3) right: plants grown for 32 days were transferred to 30 ℃ incubator for maturation.

FIG. 9A shows the results of homology alignment of SPL1, SPL12, SPL2, SPL9 and SPL 11. Wherein, red underlines represent conserved SBP-box domains.

FIG. 9B shows a tree diagram of SPL1, SPL12, SPL2, SPL9, and SPL 11.

FIG. 10 shows the results of protein sequence alignment of homologous genes SPL1 and SPL12 in Arabidopsis (At), tobacco (Nt), rice (Os), and wheat (Ta). Wherein, red underlines represent the conserved SBP-box domain, green represents the Ankrin repeat (ANK) domain, and blue represents the Transmembrane (TM) domain.

Detailed Description

The present inventors have extensively and intensively studied and found for the first time an SPL gene capable of regulating and controlling heat resistance of plants. Experiments show that SPL1 and SPL12 are key factors necessary for high-temperature adaptation and tolerance of arabidopsis flower organs and maintaining development, maturity and germination of seeds under high-temperature conditions, transgenic arabidopsis with over-expression of SPL1 or SPL12 can enhance the tolerance of the flower organs to extreme and mild high temperatures, and further prove that SPL1 and SPL12 regulate the arabidopsis heat-resistant process through multiple signal pathways. The research result provides a new visual angle for understanding how the plant protects and maintains the normal reproductive growth and development under the condition of heat stress and further deeply researching the heat shock response mechanism of the plant in the reproductive growth period and cultivating the crops resistant to high-temperature stress. The present invention has been completed based on this finding.

Specifically, the present invention identifies SBP-box transcription factor genes SPL1 and SPL12 involved in regulating the heat shock response of Arabidopsis thaliana. The homology of the protein sequences of the SPL1 and the SPL12 reaches 72 percent, the protein sequences are widely expressed in arabidopsis tissues, and the protein sequences are highly expressed at the late development stage of flowers. Phenotypic analysis of the SPL1 and SPL12 function-deficient mutants shows that the SPL1 and the SPL12 function-deficient mutants participate in the late maturation process of flower development, and the SPL1-1SPL12-1 double-mutant flower organs show sensitive phenotypes to extreme and mild high temperatures, so that the seed yield under high-temperature stress is remarkably reduced compared with that of wild type, which indicates that SPL1 and SPL12 are key genes necessary for the arabidopsis flower organs to resist the high-temperature stress and have redundant functions.

To explore the mechanism of response to high temperature in the reproductive phase of Arabidopsis thaliana and to find heat shock response pathways and related genes regulated by SPL1 and SPL12, we performed whole genome transcriptional profiling on inflorescence material of 5-week-old wild-type and SPL1-1SPL12-1 double mutant plants under control temperature (22 ℃) and heat treatment (42 ℃, 1 h). Analysis of heat shock response differential expression genes in the wild type and the SPL1-1SPL12-1 double mutant proves that the deletion of SPL1 and SPL12 seriously influences the transcriptional regulation of arabidopsis thaliana on high-temperature stress.

More importantly, the invention obtains transgenic arabidopsis thaliana with high temperature heat resistance and over-expressed by a plurality of SPL1 and SPL12, and compared with wild type, the transgenic arabidopsis thaliana shows heat resistance phenotype in the reproductive growth phase and the vegetative growth phase, and higher yield is obtained under high temperature stress.

Term(s) for

As used herein, the term "functionally redundant" refers to two or more (n) genes having the same function, one or more (n-1) of which are deleted or reduced in expression, and the individual still exhibits a normal phenotype.

As used herein, the term "specifically expressed" refers to the expression of a gene of interest at a particular time and/or in a particular tissue in a plant.

As used herein, "exogenous" or "heterologous" refers to the relationship between two or more nucleic acid or protein sequences of different origin. For example, a promoter is foreign to a gene of interest if the combination of the promoter and the sequence of the gene of interest is not normally found in nature. A particular sequence is "foreign" to the cell or organism into which it is inserted.

SPL Gene

As used herein, the terms "SPL gene", "heat resistance-related gene", and "gene of the present invention" are used interchangeably and refer to a gene of the present invention that modulates heat resistance in plants.

In a preferred embodiment, the gene of the present invention refers to the SPL1 gene and/or the SPL12 gene. More preferably, the SPL gene of the present invention is derived from Arabidopsis thaliana.

The SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) gene encodes a plant-specific PROTEIN comprising a highly conserved SBP-box consisting of 76 amino acids. Arabidopsis contains 17 SPL genes, wherein SPL1 and SPL12 do not contain miR156 regulatory sequences, and no function report exists so far.

The invention discovers for the first time that SPL1 and SPL12 are key factors necessary for the adaptation and tolerance of the flower organ of Arabidopsis thaliana to high temperature and the maintenance of the developmental maturity and germination of seeds under the high temperature condition, transgenic Arabidopsis thaliana over-expressing SPL1 or SPL12 can enhance the tolerance of the flower organ to extreme and mild high temperature, and further proves that SPL1 and SPL12 regulate the heat-resisting process of Arabidopsis thaliana through a plurality of signal pathways. The research result provides a new visual angle for understanding how the plant protects and maintains the normal reproductive growth and development under the condition of heat stress and further deeply researching the heat shock response mechanism of the plant in the reproductive growth period and cultivating the crops resistant to high-temperature stress.

The SPL1 gene and/or the SPL12 gene of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The genomic DNA may be identical to the sequences shown in SEQ ID NO. 3, 6 or degenerate variants. The DNA of the present invention may be single-stranded or double-stranded, and the DNA may be a coding strand or a non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the sequences of the coding regions shown in SEQ ID NO. 1, 4 or may be a degenerate variant.

As used herein, "degenerate variant" refers in the present invention to nucleic acid sequences which encode proteins having the sequences of SEQ ID No.:2, 5, but differ from the coding region sequences shown in SEQ ID No.:1, 4 or the genomic sequences shown in SEQ ID No.:3, 6.

Polynucleotides encoding the mature polypeptides of SEQ ID No. 2, 5 include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.

The present invention also relates to variants of the above polynucleotides which encode polypeptides having the same amino acid sequence as the present invention or fragments, analogs and derivatives of the polypeptides. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the polypeptide encoded thereby.

The present invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the present invention. In the present invention, "stringent conditions" refer to (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 90% or more, preferably 95% or more. And, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID No. 2.

The invention also relates to nucleic acid fragments which hybridize to the sequences described above. As used herein, a "nucleic acid fragment" is at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides in length. The nucleic acid fragments can be used in nucleic acid amplification techniques (e.g., PCR) to determine and/or isolate polynucleotides encoding polypeptides associated with thermotolerant properties.

Polypeptide encoded by SPL gene

As used herein, the terms "heat resistance-related polypeptide", "polypeptide of the present invention", "polypeptide encoded by SPL gene", "protein encoded by SPL gene" and "SPL polypeptide" are used interchangeably and refer to a polypeptide of the present invention that modulates heat resistance in a plant.

In a preferred embodiment, the polypeptide of the invention refers to SPL1 and/or SPL 12. More preferably, the polypeptide of the present invention is derived from Arabidopsis thaliana.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. The polypeptides of the invention can be naturally purified products, or chemically synthesized products, or using recombinant technology from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated or may be non-glycosylated. The polypeptides of the invention may or may not also include an initial methionine residue.

The invention also includes fragments, derivatives, and analogs of the SPL polypeptides. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as a native SPL polypeptide of the present invention. A polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.

In a preferred embodiment, the polypeptide of the invention refers to a polypeptide having the sequence of SEQ ID No. 2 with heat resistance. Also included are variants of the sequence of SEQ ID No. 2 that have the same function as the SPL polypeptide. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of SPL polypeptides.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes to DNA of the SPL polypeptide under conditions of high or low stringency, and polypeptides or proteins obtained using antisera directed against the SPL polypeptide. The invention also provides other polypeptides, such as fusion proteins comprising an SPL polypeptide or fragment thereof. In addition to nearly full-length polypeptides, the invention also encompasses soluble fragments of SPL polypeptides. Typically, the fragment has at least about 10 contiguous amino acids, typically at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the SPL polypeptide sequence.

The invention also provides SPL polypeptides or analogs thereof. These analogs can differ from the native SPL polypeptide by amino acid sequence differences, by modifications that do not affect the sequence, or by both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, site-directed mutagenesis, or other known molecular biological techniques. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (generally without altering primary structure) forms include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or to optimize solubility.

In the present invention, the term "SPL polypeptide conservative variant polypeptide" refers to a polypeptide formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or similar properties, as compared with the amino acid sequence of SEQ ID No. 2. In such proteins, substitutions with amino acids of similar or analogous nature will not generally alter the function of the protein, nor will the addition of one or more amino acids at the C-terminus and/or \ terminus. These conservative variants are preferably produced by amino acid substitutions according to the following table.

Homology of SPL proteins

Arabidopsis contains a total of 17 SPL genes, and all SPL proteins contain a highly conserved SBP-box protein consisting of 76 amino acids. The homology of the arabidopsis thaliana SPL1 and SPL12 protein is 69%, the homology of SPL1 and SPL12 and other arabidopsis thaliana SPL proteins such as SPL2, SPL9 and SPL11 is less than 20%, and the homology comparison results are shown in the table and FIG. 9.

	SPL1	SPL12	SPL2	SPL9	SPL11
						SPL1
	100	69	10	19	10
						SPL12		100	8	15	10
SPL2			100	59	69
						SPL9				100	58
SPL11					100

Homology comparison of Arabidopsis, tobacco, rice, and wheat

Results of homology comparison of Arabidopsis, tobacco, rice, and wheat

The protein sequence alignment results of the homologous genes SPL1 and SPL12 in Arabidopsis (At), tobacco (Nt), rice (Os) and wheat (Ta) show that the homology of the SPL1 protein among the species is 50-62%, and the homology of the SPL12 protein among the species is about 55-61%.

Recombinant techniques and plant improvements

The full-length sequence of the heat resistance related gene or the fragment thereof can be obtained by a PCR amplification method, a recombination method or an artificial synthesis method. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to vectors comprising a polynucleotide of the invention, as well as genetically engineered host cells engineered with a vector or SPL polypeptide coding sequence of the invention, and methods for producing a polypeptide of the invention by recombinant techniques.

The polynucleotide sequences of the present invention may be used to express or produce recombinant SPL polypeptides by conventional recombinant DNA techniques (Science, 1984; 224: 1431). Generally, the following steps are performed:

(1) transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention, or with a recombinant expression vector comprising the polynucleotide;

(2) a host cell cultured in a suitable medium;

(3) separating and purifying protein from culture medium or cell.

The polynucleotide sequences of the present invention may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector well known in the art. In general, any plasmid or vector can be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing the polynucleotides of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells (e.g., cells of crops and forestry plants). Representative examples are: escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells, and the like.

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase transcription of a gene.

It will be clear to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The transformed plant may also be transformed by Agrobacterium transformation or gene gun transformation, such as leaf disk method. The transformed plant cells, tissues or organs can be regenerated into plants by conventional methods to obtain plants with altered heat resistance.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell disruption by osmosis, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

Recombinant SPL polypeptides have a variety of uses. For example for screening compounds, polypeptides or other ligands with modulated thermotolerance. Screening polypeptide libraries with expressed recombinant SPL polypeptides can be used to find valuable polypeptide molecules that inhibit, or promote, plant thermotolerance.

In another aspect, the invention also includes polyclonal and monoclonal antibodies, particularly monoclonal antibodies, specific for the SPL polypeptide. The present invention includes not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, or chimeric antibodies.

The antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art. For example, the purified SPL polypeptide gene product, or antigenic fragment thereof, can be administered to an animal to induce the production of polyclonal antibodies. The antibodies of the invention can be obtained by conventional immunization techniques using fragments or functional regions of heat resistance-related gene products. These fragments or functional regions can be prepared by recombinant methods or synthesized using a polypeptide synthesizer. Antibodies that bind to an unmodified form of a gene product associated with thermotolerance can be generated by immunizing an animal with a gene product produced in a prokaryotic cell (e.g., e.coli); antibodies that bind to post-translationally modified forms (e.g., glycosylated or phosphorylated proteins or polypeptides) can be obtained by immunizing an animal with a gene product produced in a eukaryotic cell (e.g., a yeast or insect cell). The antibody against the SPL polypeptide can be used for detecting the heat-resistant performance related polypeptide in a sample.

The invention also relates to a test method for quantitatively and positionally detecting the level of the heat-resistant performance-related polypeptide. These assays are well known in the art. The level of the polypeptide related to the heat resistance detected in the test can be used for explaining the function of regulating and controlling the heat resistance of the polypeptide related to the heat resistance.

A method for detecting whether heat-resistant performance-related polypeptide exists in a sample is to detect by using a specific antibody of SPL polypeptide, and comprises the following steps: contacting the sample with an antibody specific for the SPL polypeptide; observing whether an antibody complex is formed, wherein the formation of the antibody complex indicates that the polypeptide related to the heat-resistant property exists in the sample.

A part or all of the polynucleotide of the present invention can be used as a probe to be fixed on a microarray or a DNA chip (also called a "gene chip") for analyzing the differential expression analysis of genes in tissues. The transcription product of SPL polypeptides can also be detected by RNA-polymerase chain reaction (RT-PCR) in vitro amplification using primers specific for SPL polypeptides.

The main advantages of the invention include:

(a) provides a gene related to the heat resistance of plants and a polypeptide coded by the gene;

(b) the SPL gene can enhance the heat resistance of plants;

(c) provides a method for enhancing the heat resistance of plants in a targeted manner;

(d) the SPL genes and methods provided are suitable for genetic improvement in a variety of plants.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

General procedure

a. Arabidopsis thaliana genome DNA extraction

0.1g of plant tissue was taken, 0.3mL of lysis buffer was added, and homogenized. Add 0.3mL phenol/chloroform (1:1) and mix well. Centrifuge at 12,000rpm for 5min, transfer the supernatant to another centrifuge tube, add 30. mu.L of 3M NaAc (pH 5.2) and 500. mu.L of absolute ethanol. And (5) uniformly mixing. Centrifuge for 10min, wash the precipitate with 70% ethanol, dry in vacuo, and dissolve in 50. mu.L TE (pH 8.0).

b. Extraction of Total RNA from Arabidopsis thaliana

The material (about 100mg) was ground well in liquid nitrogen. The mixture was transferred to a 1.5mL centrifuge tube, and 1mL Trizol (Invitrogen, Cat.15596-018) was added thereto, mixed well, and left at room temperature for 5 min. Centrifuge at 12,000rpm for 10min and discard the pellet. Add 200. mu.L chloroform to the supernatant, mix well, centrifuge at 12,000rpm for 10 min. The supernatant was collected and 500. mu.L of isopropanol was added to precipitate RNA. Centrifuging at 12,000rpm for 10min, washing precipitate with 70% ethanol, vacuum drying, and dissolving in 20-50 μ L H₂O (RNase free). After the RNA was completely dissolved, RNase free DNase (0.5-1. mu.L) was added thereto, and the mixture was left at 37 ℃ for half an hour and inactivated at 65 ℃ for 20 min. The concentration of RNA was determined using NANODROP 2000c (thermo).

Reverse transcription of cDNA

PolyA mRNA first strand Reverse transcription was performed using M-MLV Reverse Transcriptase polymerase system (Invitrogen, Cat. C28025-021) in the following reaction scheme:

the system was mixed thoroughly, reacted at 65 ℃ for 5min, quickly placed on ice and left for 5 min. Then adding to the system:

mixing the systems, reacting at 37 deg.C for 2min, adding 1 μ L M-MLV Reverse Transcriptase, mixing, reacting at 37 deg.C for 50min, reacting at 70 deg.C for 15min to inactivate Reverse Transcriptase, heating at 95 deg.C for 5min, and placing on ice. The reverse transcription product can be directly used for qRT-PCR detection after being diluted by 3-10 times.

PCR reaction

Ex-Taq PCR reaction system (20. mu.L) was as follows:

the renaturation temperature and the extension time of the PCR reaction are determined by the length of the primers and the amplified fragment. The general reaction conditions are: denaturation at 94 deg.C for 5 min; denaturation at 94 ℃ for 30s, renaturation at 57 ℃ for 30s, extension at 72 ℃ for 30s, amplification for 30-35 cycles; keeping the temperature at 72 ℃ for 10 min. Keeping the temperature at 4 ℃.

The PCR primer sequence is shown in SEQ ID No. 7-16.

PrimeSTAR HS reaction system (50. mu.L) was as follows:

the general reaction conditions are: denaturation at 98 ℃ for 10 sec; renaturation at 55 ℃ for 5 or 15s, extension at 72 ℃ for 1min/kb, amplification for 30 to 35 cycles; keeping the temperature at 4 ℃.

real-time-PCR analysis

The Real-time RT-PCR detection adopts a chimeric fluorescence method (

Premix Ex Taq^TMII, TaKaRa, DRR 041A). The PCR primer sequence is shown in SEQ ID NO. 11-16. The reaction system is as follows:

the Arabidopsis thaliana S18(AT1G07210) gene was used as an internal standard reference. Data were analyzed using Realplex v2.0(Eppendorf, Hamburg, Germany). The experiment was repeated three times, and the mean and variance of each group of data were taken and plotted.

f. Vector construction

DNA agarose gel electrophoresis, cleavage of fragments, purification and ligation are described in molecular cloning (Sambrook and Russell,2001) and instructions from the manufacturer of the relevant reagents and enzymes. The promoter and gene coding sequence of the constructed vector are obtained by high fidelity PCR enzyme amplification, and TA is cloned and sequenced to ensure the correct sequence.

Example 1

Cloning and vector construction of SPL1 and SPL12 genes

The 35S promoter and NOS terminator of pBI121 were ligated to the multiple cloning site of pCAMBIA1300 by EcoRI/SacI and PstI/HindIII, respectively, by high fidelity enzymatic amplification, and then the 35S promoter of the resistance gene was replaced with the NOS promoter (from pBI121) to give pCAMBIA 1300S. The 6 Xmyc fragment is amplified by a pBSK-tag template and high fidelity enzyme, and the product is cloned into a pCAMBIA1300S vector by KpnI/SmaI enzyme digestion to obtain pCAMBIA 1300S-myc. The genome sequence of SPL1 is amplified by PrimeSTAR HS DNA polymerase, and the product is cut by BamH1 enzyme and cloned to pCAMBIA1300S-myc vector to obtain 35S myc-gSPL 1. The genome sequence of SPL12 was amplified with PrimeSTAR HS DNA polymerase, and the SPL12 product was ligated to JW819(pCAMBIA3300 engineered) vector by TA cloning to obtain p35S: gSPL 12.

Example 2

Tissue-specific analysis of SPL1 and SPL12 Gene expression

The isolated SPL1 and SPL12 genes encode proteins with 881 and 921 amino acids, respectively, with 72% sequence homology. The nucleotide sequences of the SPL1 and SPL12 genes are shown in SEQ ID NO. 1 and SEQ ID NO. 4, and the sequences of the coded amino acids are shown in SEQ ID NO. 2 and SEQ ID NO. 5.

To investigate the tissue expression specificity of SPL1, SPL12 in Arabidopsis plants, vectors pSPL1: GUS and pSPL12: GUS, in which the promoter of SPL1, SPL12 (about 3.1kb upstream of ATG) driven GUS gene expression, were transformed into wild type Arabidopsis. The Arabidopsis transformation adopts a soaking method, a single-insertion independent strain with the resistance ratio of 3:1 is selected from plants of T2 generation, and a homozygous strain is selected from T3 generation for subsequent analysis. The plants were cultivated at 22 ℃ in a 16h light/8 h dark phytotron.

GUS staining was performed on multiple independent transgenic lines (at least 8 each). Staining experiments with reporter genes showed that pSPL1: GUS and pSPL12: GUS is widely expressed in various tissues of Arabidopsis thaliana (FIG. 1A, FIG. 1B), including 1) cotyledon, hypocotyl vascular tissue; 2) main and lateral roots (except for the root cap); 3) rosette leaves (containing epidermal hair), the expression level of which in old leaves is higher than that of young leaves; 4) stem epidermis (containing epidermal hair); 5) inflorescence and inflorescence stalks; 6) the top of the pod, where the pod joins the stalk and the mature pod, but is not expressed in the seed. The reporter gene in the pSPL12: GUS transgenic line stained slightly less than pSPL1: GUS, and the expression pattern in the remaining tissues was similar to that of pSPL1: GUS except that it was not expressed in the hypocotyls and shoot coat, suggesting that SPL1 and SPL12 may be functionally redundant and present partial tissue specificity.

SPL1 and SPL12 began expression at the apical end of the buds at late stages of flower development (stages 10-16), extending throughout the calyx, petals, stigma, stamens, and pollen grains to late stages (FIGS. 1C-1F). The Real-Time PCR experiment also shows that the expression level of SPL1 and SPL12 in bloomed flowers (stage13 and later) is obviously higher than that of unraveled buds (FIG. 1G), suggesting that the expression level may play an important role in the late development of floral organs.

Example 3

Downregulation of SPL1, SPL12 expression affects floral development and seed set

To reveal the biological functions of SPL1 and SPL12, their T-DNA insertion mutants were analyzed as SPL1-1(SALK _134584) and SPL12-1(SALK _142295), respectively (FIG. 2A). RT-PCR detection shows that the expression of SPL1 in the SPL1-1 mutant and the expression of SPL12 in the SPL12-1 mutant are almost zero (FIG. 2B), which indicates that SPL1-1 and SPL12-1 are all functional complete deletion mutants. The SPL1-1SPL12-1 double mutant, i.e., the SPL1 and SPL12 fully function-deficient mutant, was obtained by hybridizing the SPL1-1 and SPL12-1 single mutants (FIG. 2B).

Under normal lighting and temperature conditions, the spl1-1 and spl12-1 single mutants did not have any visible abnormal phenotype, whereas the spl1-1spl12-1 double mutant plants showed partial failure of normal flower development by the reproductive growth period (FIG. 2C, FIG. 2E, FIG. 2F), calyx edge adhesion (FIG. 2G), spatially blocked the fertilization process due to insufficient flower organ development on the day of flowering, resulting in reduced seed set (FIG. 2C, FIG. 2D). The wild type flowers shed naturally soon after flowering, whereas the double mutant flowers matured and shed slower, and the calyx could not shed normally (fig. 2H, fig. 2I).

Transgenic plants transformed with the spl1-1spl12-1 double mutant with 35S: myc-gSPL1 and 35: gSPL 12-vectors both restored wild type phenotype (FIG. 2D). In addition, RNAi (silencing SPL12 in the SPL1-1 background) transgenic line RNAi-1 plants (FIG. 2J) also showed phenotypes similar to the SPL1-1SPL12-1 double mutant (FIG. 2D).

The above results indicate that the SPL1-1SPL12-1 double mutant floral dysplasia phenotype is indeed caused by deletion of the SPL1 and SPL12 genes, and that there is a functional redundancy between SPL1 and SPL12 (FIG. 2).

Example 4

Expression levels of SPL1, SPL12 affect the response of Arabidopsis floral organs to mild hyperthermia

Tracking statistics of flower openness of wild type Col-0, spl1-1spl12-1 double mutants and transgenic plant ox-MS1c-1(35S: myc-gSPL1, Col-0 background) after mild high temperature (30 ℃) treatment were performed for 4 consecutive days. The condition that the calyx of the flower blossoming on the current day on the inflorescence at the top end of the stem is completely unfolded, the petal is naturally unfolded is regarded as 'open', the condition that the calyx or the petal cannot be naturally unfolded is regarded as 'failure to bloom', and the Number of blossoms on the current day on each inflorescence at the top end of the stem is counted, namely the daily flower openness (NOF for short).

Arabidopsis grown for five weeks, with average NOF of inflorescences at the top of the shoot for four consecutive days as shown in FIG. 3: at 22 ℃, the wild-type arabidopsis NOF was 2.5, while the spl1-1spl12-1 double mutant NOF was 1.4. And four consecutive days at 30 ℃ with wild type arabidopsis NOF equal to 1.7 and spl1-1spl12-1 double mutant NOF equal to 0.4. There was no significant difference between ox-SPL1 and wild type, with NOFs at 22 ℃ and 30 ℃ of 2.9 and 1.9, respectively. Demonstrating that accumulated mild hyperthermia affects arabidopsis daily floral patency, the spl1-1spl12-1 double mutant is more sensitive to hyperthermia.

FIG. 4 shows morphological features of Arabidopsis thaliana shoot apices at 22 ℃ and 30 ℃ together with statistics of daily floral openness, showing 0d, 1d and 4d, respectively. As can be seen, the number of flowers opened daily on the apical inflorescence of the wild type Arabidopsis thaliana stem under the normal growth condition of 22 ℃ (0d-4d) is 2-3, accounting for 80-90%. The ratio of 0, 1, 4 and 5 to 10-20% of flowers opened per day is added. The ratio of the number of flowers opened per day by the tip inflorescence of the spl1-1spl12-1 double mutant stem is only 45 percent, the rest is 55 percent, wherein the percentage is mainly 0 and 1, and the percentage of 4 and 5 is only 1 percent. It was demonstrated that the absence of SPL1 and SPL12 reduced the daily flower openness at normal temperature in Arabidopsis (FIG. 4A, FIG. 4B).

On the first day (1d) after 30 ℃, the daily flower openness of wild type arabidopsis thaliana is not obviously changed, while the daily flower openness of the spl1-1spl12-1 double mutant is obviously reduced, the proportion of the number of the opened flowers 2 and 3 is reduced from 45% to 12%, and the proportion of 0 and 1 is increased from 51% to 88%. By the fourth day after the treatment, the flower openness of wild type Arabidopsis thaliana was also reduced to some extent, with the ratio of flower openness numbers 2 and 3 being reduced from 88% to 29%, and the ratio of 0 and 1 being increased from 9% to 70%. The spl1-1spl12-1 double mutant has larger change range of daily flower openness, and the proportion of 0 flower openness accounts for 95%, 1 is 5% and the rest is 0%. The phenotype of ox-SPL1 was not significantly different from wild type (FIG. 4A, FIG. 4B). The above results demonstrate that accumulated mild hyperthermia alters the growth and morphological development of arabidopsis inflorescences to some extent, whereas the absence of SPL1 and SPL12 affects the response of arabidopsis flowers to mild hyperthermia.

By combining the results and morphological anatomical analysis of the double mutant flowers, it is speculated that SPL1 and SPL12 not only participate in regulating and controlling normal opening and developmental maturation of Arabidopsis flowers, but also play an important role in the response process of plants to mild high temperature.

Example 5

Expression levels of SPL1 and SPL12 affect tolerance of Arabidopsis floral organs to extreme hyperthermia

Further analyzing the tolerance of arabidopsis thaliana floral organs to extreme high temperature, the young stem at the top of the wild plant and the floral organs can be rapidly wilted by treating the plant which grows at 22 ℃ for five weeks at 37 ℃ for 1 minute, but the plant gradually returns to normal after half an hour, and ox-MS1c-1 is similar to the wild type but the wilting degree is not as good as that of the wild type. The wilting degree of the double mutant spl1-1spl12-1 is severe, and part of inflorescences can not be recovered to a normal state after half an hour, even some parts of inflorescences are rapidly burnt. The inflorescence (FIG. 5A) was restored to 22 ℃ for 1 day after treatment at 37 ℃ for 1 day, and the number of inflorescences which were completely scorched and failed to flower normally was observed and counted, and the survival rates of the flowers were ox-MS 1C-164.5%, wild type 43.2%, spl1-1spl12-1 double mutant 16.7%, respectively (FIG. 5C), and the survival rate was 100% with respect to the untreated plants as a control. It is demonstrated that the deletion of SPL1 and SPL12 reduces the tolerance of Arabidopsis floral organs to extreme high temperature, while the overexpression of SPL1 enhances the tolerance, and the expression level thereof is positively correlated with the high temperature tolerance (FIG. 5B, FIG. 5C).

Superoxide Dismutase (SOD) is the first substance in plants to scavenge free radicals. The level of SOD in the organism is the visual index of aging and death. The activity of SOD indirectly reflects the ability of organism to eliminate ROS. High temperature stress can cause ROS accumulation in plants, and a large amount of accumulated active oxygen can cause damage to cells and can cause cell death in severe cases. SOD activity of inflorescences of the wild type grown at 22 ℃ for 5 weeks and the spl1-1spl12-1 double mutant after treatment at 22 ℃ and 37 ℃ for 4h was examined. The wild type Arabidopsis inflorescence SOD activity is reduced after high temperature stress, and the wild type Arabidopsis inflorescence SOD activity is reduced more seriously in SPL1-1SPL12-1 (figure 6A), which shows that SPL1 and SPL12 play an important role in maintaining a certain antioxidant capacity of the Arabidopsis inflorescence under high temperature stress. The above results suggest that SPL1 and SPL12 are key genes necessary for maintaining tolerance to high temperature in Arabidopsis reproductive organs.

Example 6

Influence of reduced expression of SPL1 and SPL12 on seed yield and germination rate of Arabidopsis at high temperature

Wild type and double mutant plants grown for 6 weeks were restored to 22 ℃ culture after 1 day of treatment at 42 ℃ and finally the total dry weight of seed per plant was counted, with the yield of wild type seed cultured at 22 ℃ being 100% and the seed yields of mutant and differently treated plants were counted. The results showed that under normal conditions spl1-1spl12-1 double mutant seed yield was 69% of wild type. High temperature treatment resulted in a reduction in wild type seed yield to 85% and double mutant seed yield to 25%, indicating that the absence of SPL1, SPL12 not only affected seed yield, but that this effect was more severe at high temperatures (fig. 6B).

The seed germination rate under high-temperature stress is detected, and the seed germination rates of the wild type and the double mutant under the condition of 22 ℃ are both more than 90%, the wild type germination rate after the wild type grows for 7 days at 30 ℃ is 80%, and the germination rate of the spl1-1spl12-1 double mutant is 47% (fig. 6C), which indicates that the influence of the high temperature at 30 ℃ on the germination rate of the wild type seeds is weak, and the germination rate of the spl1-1spl12-1 double mutant seeds is sensitive to the high temperature at 30 ℃. And the germination rates of the wild type and spl1-1spl12-1 double mutant seeds are both 0 under the condition of extreme high temperature such as 37 ℃, and the germination of the arabidopsis thaliana seeds can be completely inhibited at 37 ℃. The above results indicate that SPL1 and SPL12 have an important role in maintaining seed yield and germination rate of Arabidopsis thaliana at high temperature.

Example 7

QRT-PCR validation of SPL1, SPL12 regulated Heat shock responsive transcription factors

Transcription factors play an important role in the processes of plant growth and development and interaction with the environment. Through analysis of transcriptome and differential transcription factor expression genes, more than ten reported transcription factor genes related to drought stress, ABA signal regulation and control pathway and heat resistance pathway are screened, and heat shock response expression conditions of the transcription factor genes in inflorescences are analyzed. The results of Real-time PCR experiments show that the genes are expressed in the wild type by high temperature induction, but the induction in the spl1-1spl12-1 double mutant is blocked, which is consistent with the results of RNA-seq (FIG. 7A, FIG. 7B and FIG. 7C). Suggesting that the heat shock response induction of these genes requires the participation of SPL1 and SPL 12. It is further confirmed that these genes may be heat shock response transcription factors regulated by SPL1 and SPL12, and play an important role in maintaining heat resistance of Arabidopsis inflorescences.

Example 8

Overexpression of SPL1 or SPL12 to improve heat resistance in plants

To further explore the role of SPL1 and SPL12 in the heat resistance of Arabidopsis thaliana, 6 Xmyc-SPL 1(ox-MS1) or SPL12(ox-S12) vectors driven by the 35S promoter were transformed in wild type and double mutants, respectively. 6 transgenic lines with the highest expression level of the target gene are selected, wherein ox-MS1c-1, ox-S12c-3 and ox-S12c-4 are wild type backgrounds, and ox-MS1d-1, ox-MS1d-4 and ox-MS1d-7 are spl1-1spl12-1 double mutant backgrounds (figure 2J). The wild type growing for 14 days, spl1-1spl12-1 and transgenic plants ox-MS1c-1, ox-MS1d-1 and ox-S12c-3 are subjected to 2d high-temperature treatment at 42 ℃, the survival rate is counted after 5 days of recovery, and the results show that the survival rates of ox-MS1c-1, ox-MS1d-1 and ox-S12c-3 are all obviously higher than those of the wild type and the double mutant (fig. 8A and fig. 8B). ox-MS1D-1, ox-MS1D-4 and ox-MS1D-7 inflorescences showed higher survival rates after high temperature treatment at 42 ℃ (FIG. 8C, FIG. 8D). In addition, higher seed yields were obtained at 42 ℃ ox-MS1c-1, ox-MS1d-1, ox-S12c-3 (FIG. 8E), and at 30 ℃ ox-MS1c-1, ox-MS1d-1, ox-S12 c-4. The above results indicate that overexpression of SPL1 or SPL12 can improve the heat resistance in the vegetative and reproductive growth phases of Arabidopsis.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (21)

1. A kind ofSPLUse of a gene or its encoded protein, characterized in thatSPLThe gene is selected fromSPL1Gene, gene,SPL12Genes or combinations thereof, andSPLuse of a gene or its encoded protein for a protein selected from the group consisting of:

(a) for the preparation of agents or compositions for enhancing the heat resistance of plants;

(b) is used for enhancing the heat-resistant performance of plants,

wherein, theSPL1The CDS sequence of the gene is shown as SEQ ID NO:1SPL12The CDS sequence of the gene is shown in SEQ ID NO. 4.

2. Use according to claim 1, wherein the plant is selected from the group consisting of gramineae and cruciferae.

3. The use according to claim 1, wherein the plant is selected from arabidopsis, tobacco, rice or wheat.

4. The use according to claim 1, wherein the plant is Arabidopsis thaliana.

5. The use according to claim 1, wherein saidSPLThe gene isSPL1Genes andSPL12a gene.

6. The use according to claim 1, wherein said "enhanced plant heat tolerance properties" comprise one or more properties selected from the group consisting of:

(i) the heat resistance of the inflorescence is enhanced;

(ii) enhancing the germination rate of the seeds which are fruited in the high-temperature environment;

(iii) Enhancing heat shock response of the plant in a high temperature environment;

(iv) Enhancing the oxidation resistance of the plant in a high-temperature environment;

(v) Enhancing the heat resistance of the plant roots, stems and/or leaves;

(vi) and the maturing rate of the plants in the high-temperature environment is enhanced.

7. Use according to claim 6, wherein said heat-resistant properties of the inflorescence are enhanced by: the survival rate and/or the flowering rate of the inflorescences in the high-temperature environment are enhanced.

8. The use according to claim 1, wherein said "enhancing the heat resistance of plants" comprises enhancing the tolerance of plants to high temperature environments.

9. The use of claim 8, wherein said enhancing the tolerance of a plant to a high temperature environment comprises: improving the survival rate of the plants in the high-temperature environment.

10. The use of claim 6, wherein said enhanced heat shock response comprises up-regulating gene expression selected from the group consisting of:

WRKY15、WRKY25、WRKY33、WRKY39、ERF020、ERF1、ERF2、RAP2.1、ERF054、ABRE1、RAP2.6or a combination thereof.

11. The use of claim 6, wherein said antioxidant properties of a plant are the ability of a plant to scavenge ROS in vivo.

12. The use as claimed in claim 6, wherein the enhancing of antioxidant properties of plants is increasing of SOD expression and/or activity of plants.

13. The use according to claim 1, wherein said "enhancing the heat tolerance of plants" comprises enhancing the heat tolerance of plants during the growth and reproduction periods.

14. The use according to claim 1, wherein saidSPLThe gene is wild typeSPLA gene.

15. The use according to claim 1, wherein saidSPLThe gene is derived from a plant.

16. The use according to claim 15, wherein saidSPLThe gene is derived from gramineae or cruciferae.

17. The use according to claim 16, wherein saidSPLThe genes are derived from: arabidopsis, tobacco, rice or wheat.

18. A method of modifying the heat tolerance of a plant comprising the steps of:

(a) introducing an exogenous construct into a plant cell, wherein said construct comprises an exogenous constructSPLGene sequence, promotionSPLExogenous nucleotide sequences or inhibition of gene expressionSPLOutside of gene expressionA source nucleotide sequence, thereby obtaining a plant cell into which the exogenous construct is introduced;

(b) regenerating the plant cell introduced with the exogenous construct obtained in the previous step into a plant; and

(c) optionally identifying said regenerated plants, thereby obtaining plants with altered heat tolerance;

wherein, theSPLThe gene is selected fromSPL1Gene, gene,SPL12A gene or a combination thereof;

wherein, theSPL1The CDS sequence of the gene is shown as SEQ ID NO:1SPL12The CDS sequence of the gene is shown in SEQ ID NO. 4.

19. The method of claim 18, wherein the exogenous source isSPLThe gene sequence further comprises a promoter and/or a terminator operably linked to the ORF sequence.

20. A method of enhancing the heat tolerance of a plant, said method comprising the steps of: in the plant, promotingSPLExpression or promotion of genesSPLActivity of a protein, whereinSPLThe gene is selected fromSPL1Gene, gene,SPL12A gene or a combination thereof;

wherein, theSPL1The CDS sequence of the gene is shown as SEQ ID NO:1SPL12The CDS sequence of the gene is shown in SEQ ID NO. 4.

21. A kind ofSPLUse of a gene or its encoded protein as a modulator for modulating the thermotolerance of a plant or for the preparation of an agent or composition for modulating thermotolerance of a plant, wherein the gene or protein encoded therebySPLThe gene is selected fromSPL1Gene, gene,SPL12A gene or a combination thereof;

wherein, theSPL1The CDS sequence of the gene is shown as SEQ ID NO:1SPL12The CDS sequence of the gene is shown in SEQ ID NO. 4.

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