WO2017185854A1 - Spl基因及其在增强植物耐热性能中的应用 - Google Patents

Spl基因及其在增强植物耐热性能中的应用 Download PDF

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WO2017185854A1
WO2017185854A1 PCT/CN2017/073473 CN2017073473W WO2017185854A1 WO 2017185854 A1 WO2017185854 A1 WO 2017185854A1 CN 2017073473 W CN2017073473 W CN 2017073473W WO 2017185854 A1 WO2017185854 A1 WO 2017185854A1
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gene
plant
spl1
spl
spl12
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French (fr)
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陈晓亚
朝鲁门
刘尧倩
曹俊峰
毛颖波
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中国科学院上海生命科学研究院
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)

Definitions

  • the present invention relates to the field of botany, and more particularly to the use of the SPL gene and its enhancement of plant heat tolerance.
  • Temperature is one of the key physical factors affecting life activities on Earth. 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 germination of seeds, scorch of leaves and shoots, sunburn of shoots and stems, senescence and shedding of leaves, and growth inhibition of roots and stems. The discoloration of the fruit and the decrease in yield even caused the death of the entire plant. All plant tissues are susceptible to heat stress, with the highest sensitivity of the reproductive organs, and a few degrees of temperature increase during flowering can lead to a sharp decline in crop yields and even the entire agricultural production. Compared with the vegetative growth period, the plant reproductive growth period is more sensitive to high temperature. However, the flowering period of most crops is in the high temperature period in summer, so the research on the heat shock response mechanism of plant reproductive growth is imminent. As the global climate warms, the mechanisms by which high temperatures affect plant growth and crop yields become more important.
  • plants form a signal pathway that senses the change of environmental temperature, and adjusts their metabolism and cell function to prevent damage caused by environmental stress.
  • These different signaling pathways are tissue-specific and species-specific, especially in the plant reproductive growth phase and vegetative growth phase.
  • the research on the heat resistance of plant reproductive growth period remains at the level of morphological, physiological and biochemical, but there are few studies on its molecular mechanism, and there is no regulatory network that can be referenced.
  • an SPL gene selected from the SPL1 gene, the SPL12 gene, or a combination thereof, and the SPL gene or a protein encoded thereby for use in the selection thereof Uses from the following groups:
  • the plant is selected from the group consisting of a gramineous plant, and a cruciferous plant.
  • the plant is selected from the group consisting of Arabidopsis thaliana, tobacco, rice, and wheat.
  • the plant is Arabidopsis thaliana.
  • the SPL gene is the SPL1 gene and the SPL12 gene.
  • the "enhancing plant heat resistance” includes one or more properties selected from the group consisting of:
  • the high temperature environment refers to an environment having a temperature of 30-50 ° C, preferably an environment of 32-45 ° C, more preferably an environment of 35-42 ° C, such as 30 ° C, 35. °C, 37 ° C, 42 ° C.
  • the heat resistance of the reinforcing inflorescence comprises: enhancing the survival rate of the inflorescence in a high temperature environment, and/or the flowering rate.
  • the enhancing the tolerance of the plant to a high temperature environment comprises: increasing the survival rate of the plant in a high temperature environment.
  • the enhancing heat shock response comprises upregulating gene expression selected from the group consisting of:
  • the antioxidant properties of the plant refers to the ability of the plant to scavenge ROS in the body.
  • said enhancing the antioxidant properties of the plant means increasing the SOD expression and/or activity of the plant.
  • the "enhancing plant heat resistance” includes enhancing the heat resistance of the plant during the growth and reproductive stages.
  • the SPL gene comprises a wild-type SPL gene and a mutant SPL gene.
  • the mutant comprises a mutant form in which the function of the encoded protein is not altered (i.e., the function is identical or substantially identical to the wild-type encoded protein).
  • polypeptide encoded by the mutant SPL gene is identical or substantially identical to the polypeptide encoded by the wild-type SPL gene.
  • the mutant SPL gene comprises a polynucleotide having a homology of > 80% (preferably > 90%, more preferably > 95%) compared to the wild type SPL gene.
  • the mutant SPL gene comprises truncating or adding 1-60 (preferably 1-30, more preferably 1) at the 5' end and/or the 3' end of the wild type SPL gene. -10) nucleotide polynucleotides.
  • the gene comprises genomic DNA, cDNA, and/or mRNA.
  • CDS sequence of the SPL1 gene is set forth in SEQ ID NO.: 1.
  • the encoded protein of the SPL1 gene is set forth in SEQ ID NO.: 2.
  • genomic sequence of the SPL1 gene is set forth in SEQ ID NO.: 3.
  • CDS sequence of the SPL12 gene is set forth in SEQ ID NO.: 4.
  • the encoded protein of the SPL12 gene is set forth in SEQ ID NO.: 5.
  • genomic sequence of the SPL12 gene is set forth in SEQ ID NO.: 6.
  • the SPL gene is derived from a plant, preferably from a gramineous plant, and a cruciferous plant, more preferably from Arabidopsis thaliana, tobacco, rice, and wheat.
  • introducing a foreign construct into a plant cell wherein the construct contains an exogenous SPL gene sequence, an exogenous nucleotide sequence that promotes expression of the SPL gene, or an exogenous nucleotide that inhibits expression of the SPL gene. a sequence to obtain a plant cell into which the exogenous construct is introduced;
  • the SPL gene is selected from the group consisting of the SPL1 gene, the SPL12 gene, or a combination thereof.
  • the plant having a change in heat resistance means that the heat resistance is changed as compared with the parent plant.
  • the exogenous SPL gene sequence further comprises a promoter and/or terminator operably linked to the ORF sequence.
  • the promoter is selected from the group consisting of a constitutive promoter, a tissue-specific promoter, an inducible promoter, and a strong promoter.
  • the constitutive promoter comprises a 35S promoter.
  • the exogenous nucleotide sequence comprises a nucleotide sequence that interferes with expression of the SPL gene.
  • the exogenous nucleotide sequence comprises an RNA interference sequence.
  • a method for enhancing heat resistance of a plant comprising the steps of: promoting expression of an SPL gene or promoting activity of an SPL protein in the plant, wherein the SPL The gene is selected from the SPL1 gene, the SPL12 gene, or a combination thereof.
  • the method comprises administering a promoter of a plant SPL gene or a polypeptide encoded thereby.
  • the method comprises introducing an exogenous SPL gene into the plant.
  • the method comprises the steps of:
  • the method comprises the steps of:
  • step (b) contacting the plant cell or tissue or organ with the Agrobacterium in step (a), thereby transferring the SPL gene sequence into the plant cell and integrating it into the chromosome of the plant cell;
  • step (d) regenerating the plant cell or tissue or organ in step (c) into a plant.
  • the SPL gene is derived from a plant, preferably from a gramineous plant, and a cruciferous plant, more preferably from Arabidopsis thaliana, tobacco, rice, and wheat.
  • a modulator of an SPL gene or a protein encoded thereby A reagent or composition for regulating the heat resistance of a plant, or for preparing a heat-resistant property of a plant, wherein the SPL gene is selected from the group consisting of the SPL1 gene, the SPL12 gene, or a combination thereof.
  • the composition comprises an agricultural composition.
  • the modulator comprises an accelerator, an inhibitor.
  • the modulator is an accelerator, and the regulation refers to enhancing the heat resistance of the plant.
  • the modulator is an inhibitor, and the regulation refers to attenuating the heat resistance of the plant.
  • the modulator comprises a small molecule compound, or a nucleic acid substance.
  • the nucleic acid species is selected from the group consisting of miRNA, shRNA, siRNA, or a combination thereof.
  • a transgenic plant into which an SPL gene is introduced, the SPL gene being selected from the group consisting of the SPL1 gene, the SPL12 gene, or a combination thereof.
  • Figure 1 shows the tissue expression characteristics of the SPL1 and SPL12 genes, in which A shows the GUS gene expression pattern driven by the SPL1 promoter, and the GUS staining of the pSPL1:GUS transgenic plants: the upper layer grows from left to right in 1/2 MS medium. 7d seedlings, root mature areas, root tips and stems; lower layer from left to right for 20d seedlings, inflorescences and pods; B shows the SPL12 promoter-driven GUS gene expression pattern.
  • pSPL12 GUS transgenic plants were stained with GUS in the same order as A; CF showed the staining results of different stages of flower development of pSPL1:GUS transgenic plants (stage1-16), respectively, where C shows period 1-12; D and E show Periods 13-14, red arrows indicate pollen; F shows periods 15-16; G shows the results of Real-Time PCR detection of SPL1 and SPL12 expression levels in flowers, including flower buds (stage12 and before) and flowering flowers ( Stage13 and later).
  • Figure 2 shows that down-regulation of SPL1 and SPL12 affects flower development and seed yield, where A is the SPL1 and SPL12 gene structure and mutant T-DNA insertion site; B is RT-PCR for spl1-1, spl12-1 and spl1-1spl12 Expression of SPL1 and SPL12 in the -1 mutant; C shows the reproductive growth phenotype of Col-0, spl1-1, spl12-1 and spl1-1spl12-1; D shows the transgenic plant ox-MS1c-1, RNAi- 1 and RNAi-2 phenotype, the internal morphological structure after picking up the flower buds in D; the spl1spl12 flower bud adhesion; E showing the col-0 flowering (stage13) phenotype; F showing the spl1-1spl12-1 part of the day Flowering (stage13) phenotype, flowers can not be unfolded normally; G shows the internal morph
  • Figure 3 shows the average daily flower openness analysis of stem tip inflorescences, where A shows col-0, spl1-1spl12-1 and ox-SPL1 stem apical inflorescences at 22 °C and 30 °C (NOF) )average value.
  • n 20-30, ⁇ SD; ** represents p-value ⁇ 0.01, * represents p-value ⁇ 0.05;
  • B shows the relative change value of the NOF average value in FIG. 3A. The absolute change value was obtained by dividing the average NOF of the corresponding plant at 22 °C.
  • Figure 4 shows the statistical analysis and morphological characteristics of the daily flower opening of the stem apical inflorescence, where A shows the daily flower opening of col-0, spl1-1spl12-1 and ox-SPL1 stem apical inflorescence at 22 °C and 30 °C. statistics.
  • Figure 5 shows the tolerance of Arabidopsis flower organs to extreme high temperatures, where A shows the effect of 1d treatment at 37 °C on floral organ morphological characteristics of ox-SPL1, col-0 and spl1-1spl12-1; B shows Real-time PCR was used to detect the expression level of SPL1 in oxa-SPL1, col-0 and spl1-1spl12-1 plant inflorescences; C showed that flower organs survived at 37 °C for 1d on ox-SPL1, col-0 and spl1-1spl12-1 The impact of the rate.
  • Figure 6 shows the effect of high temperature treatment on the SOD activity, seed yield and germination rate of Arabidopsis inflorescence
  • A shows the effect of high temperature treatment on the SOD activity of col-0 and spl1-1spl12-1 inflorescence.
  • CK stands for 22 ° C
  • HS stands for 37 ° C for 4 h
  • B shows the effect of high temperature treatment on the seed yield of col-0 and spl1-1spl12-1.
  • CK represents 22 ° C
  • HS represents 42 ° C treatment for 1 h and then resumes 22 ° C growth.
  • C shows the effect of high temperature treatment on the germination rate of col-0 and spl1-1spl12-1 seeds.
  • CK represents 22 ° C
  • Figure 7 shows the expression of heat shock response of candidate transcription factors by Real-time PCR, in which A shows the expression of heat shock response of WRKY transcription factors; B and 7C show the expression of heat shock response of ERF transcription factors, among which The RNAs were taken from the inflorescences treated at 37 ° C for 1 and 4 h, respectively, and the inflorescences under untreated 22 ° C conditions were used as controls (0 h).
  • Figure 8 shows that overexpression of SPL1 or SPL12 enhances plant heat tolerance, where A shows 42 °C treatment for wild type 14 days of growth, spl1-1spl12-1 and transgenic plants ox-MS1c-1, ox-MS1d-1 and ox -S12c-3 growth effect, planting for 14 days of growth for 2 days at 42 °C high temperature treatment, photographing after 5 days of recovery; B showed 42 °C treatment for 14 days of growth of wild type, spl1-1spl12-1 and transgenic plants ox - MS1c-1, ox-MS1d-1 and ox-S12c-3 survival rate, for 14 days of growth of plants for 2 days of 42 ° C high temperature treatment, recovery after 5 days of statistical survival; C showed 42 ° C treatment for growth 5 weeks of wild type, spl1-1spl12-1 and transgenic plants ox-MS1d-1, ox-MS1d-4 and The effects of ox-MS1d-7
  • the plants in the 5th week of growth were subjected to high temperature treatment at 42 °C for 5 hours, and the damaged inflorescences were counted.
  • E showed that the transgenic plants ox-MS1c-1, ox-MS1d-1, ox-S12c-3 and ox-S12c-3 were Higher seed yield under high temperature stress.
  • FIG. 9 shows the results of the SPL1, SPL12, SPL2, SPL9 and SPL11 homology alignments. Among them, the red underline represents the conservative SBP-box domain.
  • FIG. 9 B shows the evolution tree diagrams of SPL1, SPL12, SPL2, SPL9, and SPL11.
  • Figure 10 shows the results of protein sequence alignment of SPL1 and SPL12 homologous genes in Arabidopsis thaliana (At), tobacco (Nt), rice (Os), and wheat (Ta).
  • the underlined line represents the conserved SBP-box domain
  • the double-crossed line represents the Ankrin repeat (ANK) domain
  • ANK Ankrin repeat
  • TM transmembrane
  • Figure 11 shows that the transfer of Arabidopsis thaliana genes AtSPL1 and AtSPL12 to N. benthamiana did not affect the normal growth and development of tobacco, and A showed that transgenic tobacco grown at 22 °C for three weeks was no different from wild type.
  • the scale is 5 cm.
  • Nb represents wild-type Nicotiana benthamiana
  • 1H represents transgenic tobacco transferred to 35S::6MYC-AtSPL1
  • 12B represents transgenic tobacco transferred to 35S::AtSPL12, the same below
  • B shows transgenic tobacco grown at 22 °C for forty days and The wild type is the same, the scale is 5 cm
  • C and D show the transgenic tobacco grown at 22 °C, the development of the inflorescence apical meristem and the pod is the same as that of the wild type N. benthamiana, the scale is 5 cm
  • E shows Semi-quantitative PCR identified AtSPL12 expression in transgenic tobacco
  • F showed Western blot analysis of MYC-AtSPL1 protein expressed in transgenic tobacco.
  • Figure 12 shows that transgenic tobacco (vegetative growth phase) showing growth for three weeks showed an increase in resistance to heat, with A showing the phenotype of the three-week old wild-type N. benthamiana and transgenic tobacco after treatment at 42 ° C for 16 hours.
  • the scale is 5 cm;
  • B shows the statistical proportion of each of the normally viable tobacco and the withered (injured) tobacco in Figure 12 (continued) A.
  • Fisher exact test statistical significance difference, n 15, * represents p-value ⁇ 0.05, **** represents p-value ⁇ 0.001.
  • Figure 13 shows the survival rate of flowers that continue to develop after heating of the apical meristem of transgenic tobacco flowers to improve flowering heat resistance. It is not susceptible to heat damage.
  • A shows that 35 days of tobacco is opening flowers. After 42 °C and 3 hours of high temperature stress, the flower of transgenic tobacco is lighter than wild type, and the scale is 5 cm.
  • B shows protein.
  • Western Blot confirmed that the MYC-AtSPL1 protein in the transgenic tobacco did not degrade after heating, and the heat resistance was enhanced by the overexpression of the protein;
  • C showed that the newly formed apical segmentation was about 35 days after growth.
  • the tissue tobacco was cultured at 45 ° C for 6 hours and then recovered at 22 ° C for three weeks.
  • the survival rate of the fertile flowers in the first 7 flowers was counted, and the transgenic tobacco was significantly better than the wild type.
  • One-Way ANOVA test statistically significant difference, n 10, ** represents p-value ⁇ 0.01.
  • SPL1 and SPL12 are the key factors for the adaptation and tolerance of Arabidopsis flower organs to high temperature, and the maintenance of seed development and germination under high temperature conditions.
  • Transgenic Arabidopsis overexpressing SPL1 or SPL12 can enhance flower organs. Tolerance to extreme and mild hyperthermia, and further demonstrated that SPL1 and SPL12 regulate the heat-resistance process of Arabidopsis through multiple signaling pathways.
  • the results of this study provide an insight into how plants can protect and maintain their normal reproductive growth under heat stress conditions and provide a new perspective for further research into the thermogenic response mechanisms of plant reproductive growth and the cultivation of crops resistant to high temperature stress.
  • the present invention has been completed on this basis.
  • the present invention identifies SBP-box transcription factor genes SPL1 and SPL12 involved in the regulation of the heat shock response of Arabidopsis.
  • the SPL1 and SPL12 protein sequences have a homology of 72%, are widely expressed in Arabidopsis tissues, and are highly expressed in late flower development. Phenotypic analysis of SPL1 and SPL12 loss-of-function mutants indicated that both of them were involved in the late maturation process of flower development.
  • the spl1-1spl12-1 double mutant flower organ showed a sensitive phenotype for extreme and mild high temperature, resulting in high temperature stress.
  • the seed yield under the seed was significantly lower than that of the wild type, indicating that SPL1 and SPL12 are the key genes necessary for the tolerance of high temperature stress in Arabidopsis flower organs, and they are functionally redundant.
  • the present invention obtains a number of SPL1 and SPL12 overexpressing transgenic Arabidopsis having high temperature heat resistance, which exhibits a thermotolerant phenotype during reproductive growth and vegetative growth compared to wild type. Higher yields are obtained under high temperature stress.
  • AtSPL1 and AtSPL12 from Arabidopsis thaliana to N. benthamiana does not affect the normal growth and development of tobacco, including seedlings, inflorescences and fruit pods.
  • the transferred protein did not degrade under high temperature stress.
  • Transgenic tobacco can significantly improve the survival rate under high temperature stress during vegetative growth.
  • the apical meristem of the inflorescence returned to growth after being heated, and it was found that the survival rate of the fertile flowers in the flowers immediately developed in the transgenic tobacco was significantly higher than that in the wild type, that is, the transgenic tobacco flowers were more heat-resistant at high temperatures.
  • the present invention is applicable not only to Arabidopsis, but also to heterologous transfer into tobacco, and can also improve the heat tolerance of tobacco vegetative growth period and reproductive growth period, and does not affect the normal growth and development of tobacco at normal growth temperature. High application value.
  • the term "functional redundancy” refers to a table in which two or more (n) genes have the same function, deleting or reducing the expression level of one or more (n-1), and the individual still behaves normally. type.
  • telomere As used herein, the term "specific expression” refers to a specific time and/or characteristic of a gene of interest in a plant. The expression of the organization.
  • exogenous or “heterologous” refers to the relationship between two or more nucleic acid or protein sequences from different sources. For example, if the combination of a promoter and a gene sequence of interest is generally not naturally occurring, the promoter is foreign to the gene of interest. A particular sequence is “exogenous” 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 having a plant heat-resistant property.
  • the gene of the present invention refers to the SPL1 gene and/or the SPL12 gene. More preferably, the SPL gene of the invention is derived from Arabidopsis thaliana.
  • SPL SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE
  • the present invention finds for the first time that SPL1 and SPL12 are essential factors for the adaptation and tolerance of Arabidopsis flower organs to high temperature, maintaining seed development and germination under high temperature conditions, and transgenic Arabidopsis overexpressing SPL1 or SPL12 can enhance flowers.
  • the tolerance of organs to extreme and mild hyperthermia and further demonstrated that SPL1 and SPL12 regulate the heat-resistance process of Arabidopsis through multiple signaling pathways.
  • the results of this study provide an insight into how plants can protect and maintain their normal reproductive growth under heat stress conditions and provide a new perspective for further research into the thermogenic response mechanisms of plant reproductive growth and the cultivation of crops resistant to high temperature stress.
  • the SPL1 gene and/or SPL12 gene of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • the genomic DNA may be the same as or identical to the sequence shown in SEQ ID NO.: 3, 6.
  • 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 coding region sequence encoding the mature polypeptide may be identical to the coding region sequence shown in SEQ ID NO.: 1, 4 or a degenerate variant.
  • degenerate variant in the present invention refers to a protein encoding SEQ ID NO.: 2, 5, but to the coding region sequence shown in SEQ ID NO.: 1, 4 or SEQ ID NO .: The nucleic acid sequences differing in the genomic sequence shown in 3 and 6.
  • Polynucleotides encoding the mature polypeptides of SEQ ID NOS.: 2, 5 include: coding sequences encoding only mature polypeptides; coding sequences for mature polypeptides and various additional coding sequences; coding sequences for mature polypeptides (and optional additional coding) Sequence) and non-coding sequences.
  • polynucleotide encoding a polypeptide can be a polynucleotide comprising the polypeptide, or a polynucleotide further comprising additional coding and/or non-coding sequences.
  • the invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of polypeptides or polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby. .
  • the invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the invention particularly relates to polynucleotides that hybridize to the polynucleotides of the invention under stringent conditions.
  • stringent conditions means: (1) hybridization and elution at a lower ionic strength and higher temperature, such as 0.2 x SSC, 0.1% SDS, 60 ° C; or (2) hybridization a denaturant such as 50% (v/v) formamide, 0.1% calf serum / 0.1% Ficol l, 42 ° C, etc.; or (3) at least 90% identity between the two sequences, More preferably, hybridization occurs more than 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO.: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” is at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more.
  • Nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR) to identify and/or isolate polynucleotides encoding heat resistant related polypeptides.
  • thermoresistant property-related polypeptide As used herein, the terms "heat-resistant property-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 the present invention.
  • polypeptide of the invention refers to SPL1 and/or SPL12. More preferably, the polypeptide of the 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 may be naturally purified products, either chemically synthesized or produced recombinantly from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects, and mammalian cells).
  • the polypeptide of the invention may be glycosylated or may be non-glycosylated, depending on the host used in the recombinant production protocol. Polypeptides of the invention may also or may not include an initial methionine residue.
  • the invention also includes fragments, derivatives and analogs of SPL polypeptides.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of a native SPL polypeptide of the invention.
  • the polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It 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 mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, for example Polyethylene glycol) a polypeptide formed by fusion, or (iv) a polypeptide formed by fused an additional amino acid sequence to the polypeptide sequence (such as a leader or secretion sequence or a sequence or proprotein sequence used to purify the polypeptide, or fusion) protein).
  • the polypeptide of the invention refers to a polypeptide of the sequence of SEQ ID NO.: 2 having thermostable properties.
  • variant forms 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, one or more (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acid deletions , Insertion and/or Substitution, and the addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus.
  • the function of the protein is generally not altered.
  • the addition of one or several amino acids at the C-terminus and/or N-terminus will generally not alter the function of the protein.
  • the term also encompasses active fragments and active derivatives of SPL polypeptides.
  • Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA encoded by DNA that hybridizes to the DNA of the SPL polypeptide under high or low stringency conditions.
  • the invention also provides other polypeptides, such as fusion proteins comprising SPL polypeptides or fragments thereof. In addition to nearly full length polypeptides, the invention also includes soluble fragments of SPL polypeptides.
  • the fragment has at least about 10 contiguous amino acids of the SPL polypeptide sequence, 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.
  • a contiguous amino acid typically at least about 10 contiguous amino acids of the SPL polypeptide sequence, 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.
  • the invention also provides SPL polypeptides or analogs thereof.
  • the difference between these analogs and the native SPL polypeptide may be a difference in amino acid sequence, or a difference in the modification form that does not affect the sequence, or both.
  • These polypeptides include natural or induced genetic variants. Induced variants can be obtained by a variety of techniques, such as random mutagenesis by irradiation or exposure to a mutagen, or by site-directed mutagenesis or other techniques known to molecular biology.
  • Analogs also include analogs having residues other than the native L-amino acid (such as D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (such as beta, gamma-amino acids). It is to be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • Modifications include chemically derived 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 such as phosphotyrosine, phosphoserine, phosphothreonine. Also included are polypeptides modified to increase their resistance to proteolytic properties or to optimize solubility properties.
  • SPL polypeptide conservative variant polypeptide means up to 10, preferably up to 8, more preferably up to 5, most preferably up to the amino acid sequence of SEQ ID NO.: 2.
  • the three amino acids are replaced by amino acids of similar or similar nature to form a polypeptide.
  • the function of the protein is usually not changed, and the addition of one or several amino acids at the C-terminus and/or the end does not usually change the function of the protein.
  • These conservative variant polypeptides are preferably produced by amino acid substitutions according to the following table.
  • substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln;His;Lys;Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu;Val;Met;Ala;Phe Leu
  • Arabidopsis contains a total of 17 SPL genes, all of which contain a highly conserved SBP-box protein consisting of 76 amino acids.
  • the homology of SPL1 and SPL12 in Arabidopsis thaliana is 69%.
  • the homology between SPL1 and SPL12 and other Arabidopsis SPL proteins, such as SPL2, SPL9 and SPL11, is less than 20%.
  • the results of homology comparison are shown in the following table and graph. 9 is shown.
  • 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
  • SPL1 and SPL12 homologous genes in Arabidopsis thaliana (At), tobacco (Nt), rice (Os), and wheat (Ta) showed that the SPL1 protein homology between the species was 50-62%. Between the SPL12 species, the homology between the species is about 55-61%.
  • the full-length sequence of the heat-resistant property-related gene of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art.
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.
  • DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the invention also relates to vectors comprising the polynucleotides of the invention, as well as host cells genetically engineered using the vector or SPL polypeptide coding sequences of the invention, and methods of producing the polypeptides of the invention by recombinant techniques.
  • polynucleotide sequences of the present invention can be used to express or produce recombinant SPL polypeptides by conventional recombinant DNA techniques (Science, 1984; 224: 1431). Generally there are the following steps:
  • the polynucleotide sequence of the present invention can be inserted into a recombinant expression vector.
  • 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.
  • any plasmid and vector can be used as long as it can replicate and stabilize in the host.
  • An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control element.
  • expression vectors containing the polynucleotides of the invention and suitable 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 can be operably linked to an appropriate 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.
  • the expression vector preferably comprises one or more selectable marker genes to provide for selection Phenotypic traits of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell (such as a cell of a crop or a forestry plant).
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a plant cell (such as a cell of a crop or a forestry plant).
  • Representative examples are: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells, and the like.
  • an enhancer sequence is inserted into the vector.
  • An enhancer is a cis-acting factor of DNA, usually about 10 to 300 base pairs, acting on a promoter to enhance transcription of the gene.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2 .
  • Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • Transformed plants can also be subjected to methods such as Agrobacterium transformation or gene gun transformation, such as the leaf disc method.
  • Agrobacterium transformation or gene gun transformation such as the leaf disc method.
  • the plants can be regenerated by a conventional method to obtain plants having a change in 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 cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
  • 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 (salting method), centrifugation, osmotic sterilizing, ultrafiltration treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption Chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • Recombinant SPL polypeptides have a variety of uses. For example, for screening compounds, polypeptides or other ligands having regulatory thermostable properties. A library of screening polypeptides using expressed recombinant SPL polypeptides can be used to find valuable polypeptide molecules that inhibit or promote the heat resistance of plants.
  • the invention also encompasses polyclonal and monoclonal antibodies, particularly monoclonal antibodies, that are specific for SPL polypeptides.
  • the present invention encompasses not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, or chimeric antibodies.
  • Antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art. For example, a purified SPL polypeptide gene product or a fragment thereof having antigenicity can be administered to an animal to induce multiple grams. Production of antibodies.
  • the antibodies of the present invention can be obtained by conventional immunological techniques using fragments or functional regions of gene products related to heat resistance. These fragments or functional regions can be prepared by recombinant methods or synthesized using a polypeptide synthesizer.
  • An antibody that binds to an unmodified form of a heat-resistant performance-related gene product can be produced by immunizing an animal with a gene product produced in a prokaryotic cell (for example, E.
  • the protein or polypeptide can be obtained by immunizing an animal with a gene product produced in a eukaryotic cell such as yeast or insect cells.
  • Antibodies against SPL polypeptides can be used to detect heat resistance related polypeptides in a sample.
  • the invention also relates to a test method for quantifying and locating the level of a polypeptide associated with the detection of heat resistance. These tests are well known in the art. The level of heat-resistance-related polypeptides detected in the test can be used to explain the function of heat-resistant properties-related peptides to regulate heat resistance.
  • a method for detecting the presence or absence of a heat-resistant polypeptide in a sample is carried out by using a specific antibody of the SPL polypeptide, which comprises: contacting the sample with an SPL polypeptide-specific antibody; observing whether an antibody complex is formed, forming an antibody complex This indicates the presence of a thermostable-related polypeptide in the sample.
  • a part or all of the polynucleotide of the present invention can be immobilized as a probe on a microarray or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis of genes in tissues. Transcription products of SPL polypeptides can also be detected by RNA-polymerase chain reaction (RT-PCR) in vitro amplification using SPL polypeptide-specific primers.
  • RT-PCR RNA-polymerase chain reaction
  • SPL gene of the present invention can enhance the heat resistance of plants
  • 0.1 g of plant tissue was taken, 0.3 mL of lysis buffer was added, and homogenized. Add 0.3 mL of phenol/chloroform (1:1) and mix. After centrifugation at 12,000 rpm for 5 min, the supernatant was transferred to another centrifuge tube, and 30 ⁇ L of 3 M NaAc (pH 5.2) and 500 ⁇ L of absolute ethanol were added. Mix well. After centrifugation for 10 min, the precipitate was washed with 70% alcohol, dried in vacuo, and dissolved in 50 ⁇ L of TE (pH 8.0).
  • the material (about 100 mg) was thoroughly ground in liquid nitrogen. Transfer to a 1.5 mL centrifuge tube, add 1 mL Trizol (Invitrogen, Cat. 15596-018), mix and let stand for 5 min at room temperature. Centrifuge at 12,000 rpm for 10 min and discard the precipitate. 200 ⁇ L of chloroform was added to the supernatant, mixed, and centrifuged at 12,000 rpm for 10 min. The supernatant was taken, and 500 ⁇ L of isopropanol was added to precipitate RNA. After centrifugation at 12,000 rpm for 10 min, the precipitate was washed with 70% ethanol, dried under vacuum, and dissolved in 20-50 ⁇ L of H 2 O (RNase free).
  • Trizol Invitrogen, Cat. 15596-018
  • the first strand reverse transcription of PolyA mRNA was carried out using the M-MLV Reverse Transcriptase system (Invitrogen, Cat. C28025-021).
  • the reaction system was as follows:
  • the system was thoroughly mixed, reacted at 37 ° C for 2 min, finally added 1 ⁇ L of M-MLV Reverse Transcriptase, mixed, reacted at 37 ° C for 50 min, reacted at 70 ° C for 15 min to inactivate reverse transcriptase, heated at 95 ° C for 5 min, placed on ice.
  • the reverse transcription product can be directly used for qRT-PCR detection after dilution by 3-10 fold.
  • the Ex-Taq PCR reaction system (20 ⁇ L) is as follows:
  • the renaturation temperature and extension time of the PCR reaction are determined by the length of the primer and the amplified fragment.
  • the general reaction conditions were: denaturation at 94 ° C for 5 min; denaturation at 94 ° C for 30 s, renaturation at 57 ° C for 30 s, extension at 72 ° C for 30 s, amplification for 30 to 35 cycles, and incubation at 72 ° C for 10 min. 4 ° C insulation.
  • the PCR primer sequences are shown in SEQ ID NO.: 7-16.
  • the PrimeSTAR HS reaction system (50 ⁇ L) is as follows:
  • the general reaction conditions are: denaturation at 98 ° C for 10 sec; renaturation at 55 ° C for 5 or 15 s, extension at 72 ° C for 1 min / kb, amplification for 30 to 35 cycles; incubation at 4 ° C.
  • the Arabidopsis thaliana S18 (AT1G07210) gene was used as an internal standard reference. Data analysis was performed using Realplex v2.0 (Eppendorf, Hamburg, Germany). The experiment was repeated three times, and the average and variance of each group of data were taken to draw a chart.
  • DNA agarose gel electrophoresis, fragment digestion, purification and ligation are described in "Molecular Cloning” (Sambrook and Russell, 2001) and related reagents and enzyme manufacturer's instructions.
  • the promoter and gene coding sequence of the constructed vector were amplified by high-purity PCR, and the TA was cloned and sequenced to ensure the sequence was correct.
  • Protein extraction SDS-PAGE electrophoresis, transfer to cellulose acetate membrane, blocking, binding of primary antibody to target protein, enzyme-linked secondary immunoreactivity and color development are referred to the Guide to Fine Molecular Biology (Frederick M. Ausubel, 1995) and related reagent manufacturer's operating instructions.
  • the 35S promoter and NOS terminator of pBI121 were amplified by high-fidelity enzyme, ligated into the multiple cloning site of pCAMBIA1300 via EcoRI/SacI and PstI/HindIII, respectively, and then the 35S promoter of the resistance gene was NOS promoter (from pBI121) instead, get pCAMBIA1300S.
  • the 6 ⁇ myc fragment was amplified by high-fidelity enzyme using pBSK-tag template, and the product was cloned into pCAMBIA1300S vector by KpnI/SmaI to obtain pCAMBIA1300S-myc.
  • the SPL1 genomic sequence was amplified with PrimeSTAR HS DNA polymerase, and the product was digested with BamH1 into the pCAMBIA1300S-myc vector to obtain 35S:myc-gSPL1.
  • the genomic sequence of SPL12 was amplified with PrimeSTAR HS DNA polymerase, and the SPL12 product was ligated to JW819 (pCAMBIA3300 modified) vector to obtain p35S: gSPL12.
  • the isolated SPL1 and SPL12 genes encode proteins containing 881 and 921 amino acids, respectively.
  • the source is 72%.
  • 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 amino acids encoded are shown in SEQ ID NO.: 2 and SEQ ID NO.: 5.
  • the promoters of SPL1 and SPL12 (about 3.1 kb upstream of ATG) drive the expression of GUS gene pSPL1:GUS and pSPL12:GUS to wild-type south Mustard.
  • Arabidopsis thaliana transformation was performed by soaking method. Single-insertion independent lines with a resistance ratio of 3:1 were selected in T2 plants, and homozygous lines were screened for subsequent analysis by T3 generation. The plants were cultured in an artificial climate chamber at 22 ° C, 16 h light / 8 h dark.
  • GUS staining was performed on a number of independent transgenic lines (at least 8 each). Reporting gene staining experiments showed that pSPL1:GUS and pSPL12:GUS were widely expressed in Arabidopsis tissues (A in Figure 1, B in Figure 1), including 1) cotyledon, hypocotyl vascular tissue; 2) primary root and Lateral root (except the root crown); 3) rosette leaves (including epidermal hair), the expression level of the old leaves is higher than that of the young leaves; 4) the stem epidermis (including epidermal hair); 5) the inflorescence and the inflorescence stalk; 6) the top of the pod The fruit pod is combined with the fruit stem and the mature fruit pod, but is not expressed in the seed.
  • the staining degree of the reporter gene in pSPL12:GUS transgenic lines was slightly weaker than that of pSPL1:GUS, except that it was not expressed in hypocotyls and stem epidermis, and the expression pattern in other tissues was similar to that of pSPL1:GUS, suggesting that SPL1 and SPL12 may function. Redundant and partially tissue specific.
  • SPL1 and SPL12 are expressed at the apical end of the flower bud at the late flowering stage (periods 10-16) and extend throughout the flower bud, petal, stigma, stamen and pollen grains (C-F in Figure 1).
  • Real-Time PCR results also showed that SPL1 and SPL12 were significantly higher in open flowers (stage13 and later) than in unopened calyx (G in Figure 1), suggesting that it may be important in the late development of floral organs. The role.
  • spl1-1 and spl12-1 single mutants did not show any visible abnormal phenotype, while spl1-1spl12-1 double mutant plants showed that some flowers could not develop normally during the reproductive growth period (Fig. 2) C, E, F), the edge of the calyx adhesion (G in Fig. 2), due to the inability of the floral organ to fully expand on the day of flowering, spatially prevented the fertilization process, resulting in a decrease in seed set rate (Fig. 2C, D).
  • the wild-type flower naturally fell off after flowering, while the double mutant flower matured and fell off slowly, and the flower bud could not fall off normally (Fig. 2H, I).
  • transgenic plants obtained by transforming the spl1-1spl12-1 double mutant with the 35S:myc-gSPL1 and 35:gSPL12-vectors were able to restore the wild-type phenotype (D in Figure 2).
  • RNAi stressing SPL12 in spl1-1 background
  • transgenic line RNAi-1 plants J in Figure 2 also showed a phenotype similar to the spl1-1spl12-1 double mutant (D in Figure 2).
  • SPL1 and SPL12 affect the response of Arabidopsis flower organs to mild high temperature
  • the flower openness of the wild-type Col-0, spl1-1spl12-1 double mutant and the transgenic plant ox-MS1c-1 (35S: myc-gSPL1, Col-0 background) treated at mild high temperature (30 ° C) was carried out. Tracking statistics for 4 consecutive days. The flower of the flower that blooms on the top of the stem is completely unfolded, and the natural extension of the flower is regarded as “open”. The condition that the flower bud or the flower petals cannot be naturally unfolded is regarded as “disappearing”, and the flower on the top of each stem is counted on the same day. The number is referred to as Number of open flowers per inflorescence per day (NOF).
  • NOF Number of open flowers per inflorescence per day
  • Figure 4 shows the morphological characteristics of the stem inflorescence and the daily flower opening statistics of Arabidopsis thaliana at 22 °C and 30 °C, showing 0d, 1d and 4d, respectively. It can be seen that under normal growth conditions of 22 °C (0d-4d), the number of flowers open daily in the apical inflorescence of wild-type Arabidopsis thaliana stems is 2-3, accounting for 80-90%. The proportion of flowers open daily, 0, 1, 4, and 5 is 10-20%.
  • the spl1-1spl12-1 double mutant stem apical inflorescence has a daily flowering number of 2-3, only ⁇ 45%, and the rest ⁇ 55%, of which mainly 0 and 1, while 4 and 5 are only ⁇ 1%. This indicates that the deletion of SPL1 and SPL12 reduces the daily flower opening at normal temperature in Arabidopsis (A, B in Figure 4).
  • the spl1-1spl12-1 double mutant had a greater variation in flower opening per day, and the ratio of flower opening number to 0 was 95%, 1 was 5%, and the rest was 0%.
  • the phenotype of ox-SPL1 was not significantly different from that of wild type (A, B in Figure 4). The above results indicated that the accumulation of mild high temperature changed the growth and morphological development of Arabidopsis to some extent, while the deletion of SPL1 and SPL12 affected the response of Arabidopsis flowers to mild high temperature.
  • SPL1 and SPL12 not only participate in the regulation of the normal opening and development of Arabidopsis flowers, but also play an important role in the response of plants to mild high temperature.
  • SPL1 and SPL12 affect the tolerance of Arabidopsis flower organs to extreme high temperatures
  • Super Oxide Dismutase is the primary substance for scavenging free radicals in plants.
  • the level of SOD in the body is a visual indicator of aging and death.
  • the level of SOD activity indirectly reflects the body's ability to scavenge ROS.
  • High temperature stress can cause accumulation of ROS in plants, and a large amount of accumulated reactive oxygen species can cause damage to cells, which can lead to cell death in severe cases.
  • the SOD activity of the inflorescences of wild type and spl1-1spl12-1 double mutants grown at 22 °C for 5 weeks after treatment at 22 ° C and 37 ° C for 4 h was examined.
  • the seed germination rate under high temperature stress was detected. It was found that the seed germination rate of wild type and double mutant was more than 90% at 22 °C, and the wild type germination rate was 80% after 7 days of growth at 30 °C. spl1-1spl12-1 double The germination rate of the mutant was 47% (C in Figure 6), indicating that the effect of 30 °C high temperature on the germination rate of wild-type seeds was weak, while the seed germination rate of spl1-1spl12-1 double mutant was sensitive to the high temperature of 30 °C.
  • the seed germination rate of wild type and spl1-1spl12-1 double mutant was 0 at extreme high temperature such as 37 °C, and 37 °C could completely inhibit the seed germination of Arabidopsis thaliana.
  • extreme high temperature such as 37 °C
  • 37 °C could completely inhibit the seed germination of Arabidopsis thaliana.
  • SPL1 and SPL12 play an important role in maintaining seed yield and germination rate in Arabidopsis at high temperatures.
  • transcriptome and differential transcription factor expression genes More than ten reported transcription factor genes involved in drought stress, ABA signaling pathway and heat-resistant pathway were screened, and their expression of heat shock response in inflorescence was analyzed.
  • Real-time PCR test results show that these genes are in the wild type Expression was induced at high temperature, and induction was blocked in the spl1-1spl12-1 double mutant, consistent with the RNA-seq results (A, B, C in Figure 7). It is indicated that the induction of heat shock response of these genes requires the participation of SPL1 and SPL12. It was further confirmed that these genes may be heat shock response transcription factors regulated by SPL1 and SPL12, and play an important role in the process of maintaining heat resistance of Arabidopsis inflorescences.
  • the 35S promoter-driven 6x myc-SPL1 (ox-MS1) or SPL12 (ox-S12) vector was transformed in wild-type and double mutants, respectively.
  • Six transgenic lines with the highest target gene expression were selected, among which ox-MS1c-1, ox-S12c-3, and ox-S12c-4 were wild-type background, ox-MS1d-1, ox-MS1d-4 and ox-MS1d-7 is the spl1-1spl12-1 double mutant background (J in Figure 2).
  • the survival rates of ox-MS1c-1, ox-MS1d-1 and ox-S12c-3 were significantly higher than those of wild-type and double mutants (A, B in Figure 8).
  • the ox-MS1d-1, ox-MS1d-4 and ox-MS1d-7 inflorescences showed higher survival rates after treatment at 42 °C (C, D in Figure 8).
  • the 35S::6-MYC-AtSPL1 vector and the 35S::AtSPL12 vector were transferred from the leaf disc method to N. benthamiana, and the homozygous plants were expanded and identified.
  • Two transgenic lines were identified by semi-quantitative PCR and Western Blot. (EF in Figure 11) 1H-2, 1H-9, 12B-1 and 12B-4, where 1H represents transgenic tobacco transferred to 35S::6MYC-AtSPL1, and 12B represents transgenic tobacco transferred to 35S::AtSPL12, with.
  • transgenic lines and wild-type tobacco that were grown at normal temperature for about 20 days in the vegetative growth period were found to be transgenic tobacco 1H-2, 1H-9, 12B-1 and 12B-4 after treatment at 42 ° C for 16 hours.
  • the proportion of withered damage was significantly lower than that of the wild type, while the proportion of normal growth was significantly higher than that of the wild type, showing significant heat resistance (Fig. 12).
  • transgenic tobacco 1H-2, 1H-9, 12B-1 and 12B-4 were significantly higher than those of wild type (C in Figure 13), ie transgenic tobacco flowers were more heat resistant.
  • AtSPL1 and AtSPL12 can improve the heat tolerance of N. benthamiana vegetative growth and reproductive growth.

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Abstract

提供了SPL基因及其在增强植物耐热性能中的应用。具体地，提供了一种SPL基因的功能与用途，SPL基因，SPL1或SPL12，是植物对高温的适应和耐受、高温条件下维持种子的发育成熟和萌发所必须的关键因子，过表达SPL1或SPL12的转基因拟南芥可以增强花器官对极端及温和高温的耐受性。

Description

SPL基因及其在增强植物耐热性能中的应用 技术领域
本发明涉及植物学领域，更具体地涉及SPL基因及其增强植物耐热性能中的应用。
背景技术
温度是地球上影响生命活动的关键物理因子之一。环境温度影响整个食物链与生态***。高温胁迫对植物生长、发育、繁殖和产量等几乎所有方面产生不利影响，包括种子发芽受阻、叶片及嫩芽的焦枯、枝条及茎的晒伤、叶片的衰老和脱落、根和茎的生长抑制，果实的变色和产量的降低，甚至造成整个植物的死亡。所有的植物组织都容易受到热胁迫伤害，其中生殖器官的敏感性最高，在开花时期温度升高几度便可导致作物产量急剧下降，甚至整个农业生产的绝收。相比营养生长期，植物生殖生长期对高温更为敏感，然而大多数农作物的花期正在夏季高温时期，因此植物生殖生长期热激响应机制的研究迫在眉睫。随着全球气候的变暖，研究高温对植物生长发育以及作物产量影响的机理变得愈发重要。
植物在进化过程中都形成感知环境温度变化的信号通路，并调整自身的代谢和细胞功能，防止环境胁迫造成的机体损坏。这些不同的信号途径具有组织特异性和物种特异性，尤其在植物生殖生长期和营养生长期热激响应信号存在很大的差异。目前对植物生殖生长期抗热性的研究仍停留在形态、生理和生化水平，而对其分子机制研究尚少，更没有可参考的调控网络。
因此，本领域迫切需要开发调控植物耐热性的基因，并对其功能应用进行相应的研究。
发明内容
本发明的目的在于提供一种调控植物耐热性能的SPL基因及其在增强植物耐热性能中的应用。
在本发明的第一方面，提供了一种SPL基因或其编码蛋白的用途，所述SPL基因选自SPL1基因、SPL12基因、或其组合，并且所述的SPL基因或其编码蛋白用于选自下组的用途：
(a)用于制备增强植物耐热性能的试剂或组合物；
(b)用于增强植物的耐热性能。
在另一优选例中，所述的植物选自下组：禾本科植物、和十字花科植物。
在另一优选例中，所述的植物选自下组：拟南芥、烟草、水稻、和小麦。
在另一优选例中，所述的植物为拟南芥。
在另一优选例中，所述的SPL基因为SPL1基因和SPL12基因。
在另一优选例中，所述的“增强植物耐热性能”包括选自下组的一种或多种性能：
(i)增强花序的耐热性能；
(ii)增强高温环境下结实的种子的萌发率；
(iii)用于增强植物对高温环境的耐受性；
(iv)增强高温环境下植物的热激响应；
(v)增强高温环境下植物的抗氧化性能；
(vi)增强植物根、茎、和/或叶的耐热性能；
(vii)增强高温环境下植物的结实率。
在另一优选例中，所述的高温环境是指温度为30-50℃的环境，较佳地为32-45℃的环境，更佳地为35-42℃的环境，如30℃、35℃、37℃、42℃。
在另一优选例中，所述的增强花序的耐热性能包括：增强高温环境下花序的存活率、和/或开花率。
在另一优选例中，所述的增强植物对高温环境的耐受性包括：提高植物在高温环境下的存活率。
在另一优选例中，所述的增强热激响应包括上调选自下组的基因表达：
WRKY15、WRKY25、WRKY33、WRKY39、ERF020、ERF1、ERF2、RAP2.1、ERF054、CRJ1、ABRE1、RAP2.6、或其组合。
在另一优选例中，所述的植物的抗氧化性能是指植物清除体内ROS的能力。
在另一优选例中，所述的增强植物的抗氧化性能是指提高植物的SOD表达和/或活性。
在另一优选例中，所述的“增强植物耐热性能”包括增强植物生长期和生殖期的耐热性能。
在另一优选例中，所述的SPL基因包括野生型SPL基因和突变型SPL基因。
在另一优选例中，所述的突变型包括突变后编码蛋白的功能未发生改变的突变形式(即功能与野生型编码蛋白相同或基本相同)。
在另一优选例中，所述的突变型SPL基因编码的多肽与野生型SPL基因所编码的多肽相同或基本相同。
在另一优选例中，所述的突变型SPL基因包括与野生型SPL基因相比，同源性≥80％(较佳地≥90％，更佳地≥95％)的多核苷酸。
在另一优选例中，所述的突变型SPL基因包括在野生型SPL基因的5＇端和/或3＇端截短或添加1-60个(较佳地1-30，更佳地1-10个)核苷酸的多核苷酸。
在另一优选例中，所述的基因包括基因组DNA、cDNA、和/或mRNA。
在另一优选例中，所述的SPL1基因的CDS序列如SEQ ID NO.:1所示。
在另一优选例中，所述的SPL1基因的编码蛋白如SEQ ID NO.:2所示。
在另一优选例中，所述的SPL1基因的基因组序列如SEQ ID NO.:3所示。
在另一优选例中，所述的SPL12基因的CDS序列如SEQ ID NO.:4所示。
在另一优选例中，所述的SPL12基因的编码蛋白如SEQ ID NO.:5所示。
在另一优选例中，所述的SPL12基因的基因组序列如SEQ ID NO.:6所示。
在另一优选例中，所述的SPL基因来源于植物，较佳地来源于禾本科植物、和十字花科植物，更佳地来源于：拟南芥、烟草、水稻、和小麦。
在本发明的第二方面，提供了一种改变植物耐热性能的方法，包括步骤：
(a)将外源的构建物导入植物细胞，其中所述的构建物含有外源的SPL基因序列、促进SPL基因表达的外源核苷酸序列、或抑制SPL基因表达的外源核苷酸序列，从而获得导入外源构建物的植物细胞；
(b)将上一步骤获得的所述导入外源构建物的植物细胞，再生成植株：和
(c)任选地对所述再生的植株进行鉴定，从而获得耐热性能改变的植物；
其中，所述SPL基因选自SPL1基因、SPL12基因、或其组合。
在另一优选例中，所述的耐热性能改变的植物是指与亲本植物相比，耐热性能改变。
在另一优选例中，所述外源的SPL基因序列还包含与ORF序列操作性连接的启动子和/或终止子。
在另一优选例中，所述的启动子选自下组：组成型启动子、组织特异性启动子、诱导型启动子、和强启动子。
在另一优选例中，所述的组成性启动子包括35S启动子。
在另一优选例中，所述的外源核苷酸序列包括干扰所述SPL基因表达的核苷酸序列。
在另一优选例中，所述的外源核苷酸序列包括RNA干扰序列。
在本发明的第三方面，提供了一种增强植物耐热性能的方法，所述方法包括以下步骤：在所述植物中，促进SPL基因的表达或促进SPL蛋白的活性，其中，所述SPL基因选自SPL1基因、SPL12基因、或其组合。
在另一优选例中，所述方法包括给予植物SPL基因或其编码的多肽的促进剂。
在另一优选例中，所述方法包括向植物中导入外源的SPL基因。
在另一优选例中，所述方法包括步骤：
(i)提供一植物或植物细胞；和
(ii)将SPL基因序列导入所述植物或植物细胞，从而获得转基因的植物或植物细胞。
在另一优选例中，所述方法包括步骤：
(a)提供携带SPL基因序列的表达载体的农杆菌；
(b)将植物细胞或组织或器官与步骤(a)中的农杆菌接触，从而使SPL的基因序列转入植物细胞，并且整合到植物细胞的染色体上；
(c)选择已转入SPL基因序列的植物细胞或组织或器官；和
(d)将步骤(c)中的植物细胞或组织或器官再生为植株。
在另一优选例中，所述的SPL基因来源于植物，较佳地来源于禾本科植物、和十字花科植物，更佳地来源于：拟南芥、烟草、水稻、和小麦。
在本发明的第四方面，提供了一种SPL基因或其编码蛋白的调控剂的用途， 用于调控植物的耐热性能，或用于制备调控植物耐热性能的试剂或组合物，其中，所述SPL基因选自SPL1基因、SPL12基因、或其组合。
在另一优选例中，所述的组合物包括农用组合物。
在另一优选例中，所述的调控剂包括促进剂、抑制剂。
在另一优选例中，所述的调控剂为促进剂，并且所述的调控是指增强植物耐热性能。
在另一优选例中，所述的调控剂为抑制剂，并且所述的调控是指减弱植物耐热性能。
在另一优选例中，所述的调控剂包括小分子化合物、或核酸类物质。
在另一优选例中，所述的核酸类物质选自下组：miRNA、shRNA、siRNA、或其组合。
在本发明的第五方面，提供了一种转基因植株，所述转基因植株中导入SPL基因，所述SPL基因选自SPL1基因、SPL12基因、或其组合。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了SPL1和SPL12基因的组织表达特征，其中A显示了SPL1启动子驱动的GUS基因表达模式，pSPL1:GUS转基因植株GUS染色：上层从左到右依次为在1/2MS培养基上生长7d的幼苗、根成熟区、根尖和茎；下层从左到右为生长20d的苗、花序和果荚；B显示了SPL12启动子驱动的GUS基因表达模式。pSPL12:GUS转基因植株GUS染色，顺序同A；C-F分别显示了pSPL1:GUS转基因植株花发育期不同阶段(stage1-16)的染色结果，其中，C显示了时期1-12；D和E显示了时期13-14，红色箭头指示花粉；F显示了时期15-16；G显示了Real-Time PCR检测SPL1和SPL12在花中的表达水平的结果，包括花苞(stage12及之前)和已开花花朵(stage13及之后)。
图2显示SPL1，SPL12表达下调影响花发育与种子产量，其中A为SPL1和SPL12基因结构与突变体T-DNA***位点；B为RT-PCR检测spl1-1,spl12-1和spl1-1spl12-1突变体中SPL1和SPL12的表达；C显示了Col-0,spl1-1,spl12-1和spl1-1spl12-1生殖生长期表型；D显示了转基因植物ox-MS1c-1,RNAi-1和RNAi-2表型，用尖镊挑开D中花萼之后的内部形态结构；spl1spl12花萼粘连；E显示了col-0当日开花(stage13)表型；F显示了spl1-1spl12-1部分当日开花(stage13)表型，花不能正常展开；G显示了为用尖镊挑开F中花萼之后的内部形态结构；spl1-1spl12-1花萼粘连；H显示了Col-0花成熟脱落过程；I显示了spl1-1spl12-1花成熟脱落过程；J显示了Realtime-PCR检测Col-0,spl1-1spl12-1以及转基因植物ox-MS1d-1,ox-MS1d-4,ox-MS1d-7和ox-MS1c-1中SPL1的相对表达量(左)；Realtime-PCR检测Col-0, spl1-1spl12-1,ox-S12c-3和ox-S12c-4,RNA1-1,RNAi-2中SPL12表达量。
图3显示了茎顶端花序平均每日花开放度情况分析，其中A显示了22℃和30℃条件下col-0,spl1-1spl12-1和ox-SPL1茎顶端花序每日花开放度(NOF)平均值。其中，n＝20-30，±SD；**代表p-value≤0.01，*代表p-value≤0.05；B显示了图3A中NOF平均值相对变化值。绝对变化值除以相应植物22℃的平均NOF获得。
图4显示了茎顶端花序每日花开放度统计分析与形态特征，其中A显示了22℃和30℃条件下col-0,spl1-1spl12-1和ox-SPL1茎顶端花序每日花开放度统计。图中显示了0，1和4d的统计数值，n＝20-30，展示三次重复结果之一；B显示了22℃和30℃条件下col-0,spl1-1spl12-1和ox-SPL1茎顶端花序形态特征。
图5显示了拟南芥花器官对极端高温的耐受性情况，其中A显示了37℃处理1d对ox-SPL1,col-0和spl1-1spl12-1花器官形态特征的影响；B显示了Real-time PCR检测ox-SPL1,col-0和spl1-1spl12-1植物花序中SPL1的表达水平；C显示了37℃处理1d对ox-SPL1,col-0和spl1-1spl12-1花器官存活率的影响。其中，将37℃处理1d之后重新恢复到22℃生长1d，观察并统计完全焦枯而不能正常开花的花序数目，以没有做处理的植物作为对照，其存活率为100％；(n＝20-30，±SD；**代表p-value≤0.01，*代表p-value≤0.05)
图6显示了高温处理对拟南芥花序SOD活性、种子产率和萌发率的影响，其中A显示了高温处理对col-0和spl1-1spl12-1花序SOD活性的影响。其中，CK代表22℃，HS代表37℃处理4h，生长5周的拟南芥为材料。(3 times，±SD；**代表p-value≤0.01)；B显示了高温处理对col-0和spl1-1spl12-1种子产率的影响。其中，CK代表22℃，HS代表42℃处理1h后恢复22℃生长。(n＝18，±SD；**代表p-value≤0.01，*代表p-value≤0.05)；C显示了高温处理对col-0和spl1-1spl12-1种子萌发率的影响。其中，CK代表22℃，HS代表30℃生长7天后统计种子萌发率。(n＝60-70，3times，±SD；**代表p-value≤0.01，*代表p-value≤0.05)
图7显示了Real-time PCR检测候选转录因子的热激响应表达，其中A显示了WRKY转录因子的热激响应表达情况；B和7C显示了ERF类转录因子的热激响应表达，其中，总RNA分别取自37℃处理1和4h的花序，以未处理的22℃条件下的花序为对照(0h)。
图8显示过表达SPL1或SPL12提高植物抗热性，其中A显示了42℃处理对生长14天的野生型，spl1-1spl12-1和转基因植物ox-MS1c-1，ox-MS1d-1和ox-S12c-3生长的影响，对生长14天的植物进行2d的42℃高温处理，恢复5天后拍照；B显示了42℃处理对生长14天的野生型，spl1-1spl12-1和转基因植物ox-MS1c-1，ox-MS1d-1和ox-S12c-3存活率的影响，对生长14天的植物进行2d的42℃高温处理，恢复5天后统计存活率；C显示了42℃处理对生长5周的野生型，spl1-1spl12-1和转基因植物ox-MS1d-1，ox-MS1d-4和 ox-MS1d-7花序生长的影响，对生长5周的植物进行42℃高温处理5h后的植物状态；D显示了42℃处理对生长5周的野生型，spl1-1spl12-1和转基因植物ox-MS1d-1，ox-MS1d-4和ox-MS1d-7花序生长的影响。对生长5周的植物进行42℃高温处理5h后对花序受损状态进行统计；E显示了转基因植物ox-MS1c-1，ox-MS1d-1，ox-S12c-3和ox-S12c-3在高温胁迫下具有更高的种子产量。左：一直在22℃生长；中：将生长5周的植物用42℃处理5h后恢复至22℃生长；右：将生长32天的植物移至30℃培养箱生长至成熟；其中A-D中的实验至少重复三次，图中显示代表性的一次结果。
图9中A显示了SPL1、SPL12、SPL2、SPL9和SPL11同源性比对结果。其中，红色下划线代表保守的SBP-box结构域。
图9中B显示了SPL1、SPL12、SPL2、SPL9和SPL11的进化树图。
图10显示了拟南芥(At)、烟草(Nt)、水稻(Os)、和小麦(Ta)中SPL1和SPL12同源基因的蛋白序列比对结果。其中，下划横线代表保守的SBP-box结构域，双横线代表Ankrin repeat(ANK)结构域，……代表跨膜(TM)结构域。
图11显示了将拟南芥基因AtSPL1，AtSPL12分别转入本氏烟草后并不影响烟草正常的生长发育，其中A显示了22℃生长三周的转基因烟草与野生型无异。比例尺为5厘米。Nb表示野生型本氏烟草，1H表示转入35S::6MYC-AtSPL1的转基因烟草，12B表示转入35S::AtSPL12的转基因烟草，下同；B显示了22℃生长四十天的转基因烟草与野生型无异，比例尺为5厘米；C和D显示了22℃生长的转基因烟草，其花序顶端分生组织和果荚的发育与野生型本氏烟草无异，比例尺为5厘米；E显示了半定量PCR鉴定AtSPL12在转基因烟草中有所表达；F显示了蛋白免疫印记杂交实验(Western Blot)鉴定MYC-AtSPL1蛋白在转基因烟草中有所表达。
图12显示了生长三周的转基因烟草(营养生长期)表现出对抗热性的增强，其中A显示了三周大的野生型本氏烟草与转基因烟草在42℃处理16小时后的表型，比例尺为5厘米；B显示了图12(续)A中正常存活的烟草与枯萎(损伤)的烟草各自的统计比例。Fisher精确检验统计显著性差异，n＝15，*代表p-value≤0.05，****代表p-value≤0.001.
图13显示了转基因烟草花朵顶端分生组织受热后继续发育的花朵的存活率，来提高花期抗热性。不易受热损伤，其中A显示了35天的烟草正在开放的花朵，在经历42℃，3小时的高温胁迫后，转基因烟草的花朵受损程度较野生型轻，比例尺为5厘米；B显示了蛋白免疫印记杂交实验(Western Blot)验证受热后转基因烟草内MYC-AtSPL1蛋白并未发生降解，抗热性的增强是由该蛋白过表达引起；C显示了将生长35天左右的刚形成顶端分生组织的烟草，经过45℃，6小时的高温处理后在22℃恢复生长三周后，统计发育出的前7朵花中可育花朵的存活比例，转基因烟草明显优于野生型。One-Way ANOVA检验统计学显著性差异，n＝10,**代表p-value≤0.01。
具体实施方式
本发明人经过广泛而深入地研究，首次发现了一种能够调控植物耐热性能的SPL基因。实验表明，SPL1和SPL12是拟南芥花器官对高温的适应和耐受、高温条件下维持种子的发育成熟和萌发所必须的关键因子，过表达SPL1或SPL12的转基因拟南芥可以增强花器官对极端及温和高温的耐受性，并进一步证明了SPL1和SPL12经过多条信号途径调控拟南芥耐热过程。这一研究结果为理解植物如何在热胁迫条件下保护并维持其正常的生殖生长发育，并为进一步深入研究植物生殖生长期热激响应机制和培育抗高温胁迫的农作物提供了一个新视角。在此基础上完成了本发明。
具体地，本发明鉴定了参与调控拟南芥热激响应的SBP-box转录因子基因SPL1和SPL12。SPL1和SPL12蛋白序列同源性达72％,在拟南芥组织中广泛表达，且花发育晚期高表达。对SPL1和SPL12功能缺失突变体的表型分析表明两者共同参与花发育晚期成熟过程，spl1-1spl12-1双突变体花器官对极端及温和高温均表现出敏感的表型，导致其高温胁迫下的种子产量相比野生型显著降低，说明SPL1和SPL12是拟南芥花器官耐受高温胁迫所必需的关键基因，且功能冗余。
为了探索拟南芥生殖生长期对高温的响应机制，并寻找SPL1和SPL12调控的热激响应途径及相关的基因，我们以生长5周的野生型和spl1-1spl12-1双突变体植物在对照温度(22℃)和热处理(42℃，1h)条件下的花序材料进行全基因组转录谱测序。通过对野生型和spl1-1spl12-1双突变体中热激响应差异表达基因的分析，证明了SPL1和SPL12的缺失严重影响了拟南芥对高温胁迫的转录调控。
更重要的是，本发明获得了具有高温耐热性的若干SPL1和SPL12过表达的转基因拟南芥，相比野生型它们在生殖生长期和营养生长期均表现出耐热的表型，在高温胁迫下获得更高的产量。
同时，将拟南芥的AtSPL1和AtSPL12转入本氏烟草后，并不影响烟草正常的生长发育，包括小苗，花序和果荚。转入的蛋白在高温胁迫下并未发生降解。转基因烟草可以显著提高营养生长期高温胁迫下的存活率。花序顶端分生组织受热后再恢复生长，可以发现，转基因烟草中随即发育的花朵中，可育花朵的存活率显著高于野生型，即转基因烟草花朵在高温下更具有耐热性。因此本发明不仅适用于拟南芥，异源转入烟草中，也可以提高烟草营养生长期与生殖生长期的抗热性，并且在正常生长温度下不会影响烟草正常的生长发育，具有较高的应用价值。
术语
如本文所用，术语“功能冗余”是指是指两个或多个(n)基因具有相同功能,缺失或降低其中一个或多个(n-1)的表达量，个体仍表现正常的表型。
如本文所用，术语“特异性表达”是指目的基因在植物中特定的时间和/或特 定的组织的表达。
如本文所用，“外源的”或“异源的”是指不同来源的两条或多条核酸或蛋白质序列之间的关系。例如，如果启动子与目的基因序列的组合通常不是天然存在的，则启动子对于该目的基因来说是外源的。特定序列对于其所***的细胞或生物体来说是“外源的”。
SPL基因
如本文所用，术语“SPL基因”、“耐热性能相关基因”、“本发明基因”可以互换使用，都是指本发明的具有调控植物耐热性能的基因。
在一优选例中，本发明基因指SPL1基因和/或SPL12基因。更佳地，本发明SPL基因来源于拟南芥。
SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE(SPL)基因编码一类植物特有的包含由76个氨基酸组成的高度保守的SBP-box的蛋白。拟南芥含有17个SPL基因，其中，SPL1和SPL12不含有miR156调控序列，至今尚无功能报道。
本发明首次发现SPL1和SPL12是拟南芥花器官对高温的适应和耐受、高温条件下维持种子的发育成熟和萌发所必须的关键因子，过表达SPL1或SPL12的转基因拟南芥可以增强花器官对极端及温和高温的耐受性，并进一步证明了SPL1和SPL12经过多条信号途径调控拟南芥耐热过程。这一研究结果为理解植物如何在热胁迫条件下保护并维持其正常的生殖生长发育，并为进一步深入研究植物生殖生长期热激响应机制和培育抗高温胁迫的农作物提供了一个新视角。
本发明的SPL1基因和/或SPL12基因可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。基因组DNA可以是与SEQ ID NO.:3、6所示的序列相同或者是简并的变异体。本发明的DNA可以是单链的或是双链的，DNA可以是编码链或非编码链。编码成熟多肽的编码区序列可以与SEQ ID NO.:1、4所示的编码区序列相同或者是简并的变异体。
如本文所用，“简并的变异体”在本发明中是指编码具有SEQ ID NO.:2、5的蛋白质，但与SEQ ID NO.:1、4所示的编码区序列或SEQ ID NO.:3、6所示的基因组序列有差别的核酸序列。
编码SEQ ID NO.:2、5的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多肽或多肽的片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和***变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或***，但不会从实质上改变其编码的多肽的功能。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指:(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC,0.1％SDS,60℃；或(2)杂交时加有变性剂,如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficol l，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上,更好是95％以上时才发生杂交。并且,可杂交的多核苷酸编码的多肽与SEQ ID NO.:2所示的成熟多肽有相同的生物学功能和活性。
本发明还涉及与上述的序列杂交的核酸片段。如本文所用，“核酸片段”的长度至少含15个核苷酸，较好是至少30个核苷酸，更好是至少50个核苷酸，最好是至少100个核苷酸以上。核酸片段可用于核酸的扩增技术(如PCR)以确定和/或分离编码耐热性能相关多肽的多聚核苷酸。
SPL基因编码的多肽
如本文所用，术语“耐热性能相关多肽”、“本发明多肽”、“SPL基因编码的多肽”、“SPL基因编码的蛋白”、“SPL多肽”可以互换使用，都是指本发明的具有调控植物耐热性能的多肽。
在一优选例中，本发明多肽指SPL1和/或SPL12。更佳地，本发明多肽来源于拟南芥。
本发明的多肽可以是重组多肽、天然多肽、合成多肽，优选重组多肽。本发明的多肽可以是天然纯化的产物，或是化学合成的产物，或使用重组技术从原核或真核宿主(例如，细菌、酵母、高等植物、昆虫和哺乳动物细胞)中产生。根据重组生产方案所用的宿主，本发明的多肽可以是糖基化的，或可以是非糖基化的。本发明的多肽还可包括或不包括起始的甲硫氨酸残基。
本发明还包括SPL多肽的片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明的天然SPL多肽相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
在优选例中，本发明多肽指具有耐热性能的SEQ ID NO.:2序列的多肽。还包括具有与SPL多肽相同功能的、SEQ ID NO.:2序列的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、***和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。 例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括SPL多肽的活性片段和活性衍生物。
该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与SPL多肽的DNA杂交的DNA所编码的蛋白、以及利用抗SPL多肽的抗血清获得的多肽或蛋白。本发明还提供了其他多肽，如包含SPL多肽或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了SPL多肽的可溶性片段。通常，该片段具有SPL多肽序列的至少约10个连续氨基酸，通常至少约30个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
本发明还提供SPL多肽或其类似物。这些类似物与天然SPL多肽的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。这些多肽包括天然或诱导的遗传变异体。诱导变异体可以通过各种技术得到，如通过辐射或暴露于诱变剂而产生随机诱变，还可通过定点诱变法或其他已知分子生物学的技术。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的多肽并不限于上述例举的代表性的多肽。
修饰(通常不改变一级结构)形式包括：体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
在本发明中，“SPL多肽保守性变异多肽”指与SEQ ID NO.:2的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。在所述蛋白中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能，在C末端和/或\末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。这些保守性变异多肽最好根据下表进行氨基酸替换而产生。
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
SPL蛋白的同源性
拟南芥共含有17个SPL基因，所有的SPL蛋白都包含由76个氨基酸组成的高度保守的SBP-box的蛋白。拟南芥SPL1与SPL12蛋白同源性为69％，SPL1和SPL12与其他拟南芥SPL蛋白，如SPL2、SPL9及SPL11等同源性均小于20％，同源性比对结果如下表和图9所示。
  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
拟南芥、烟草、水稻、和小麦的同源性比较
拟南芥、烟草、水稻、和小麦的同源性比较结果如下。
Figure PCTCN2017073473-appb-000001
拟南芥(At)、烟草(Nt)、水稻(Os)、和小麦(Ta)中SPL1和SPL12同源基因的蛋白序列比对结果表明，各物种间SPL1蛋白同源性在50-62％之间，SPL12各物种间的同源性在55-61％左右。
重组技术和植物改良
本发明耐热性能相关基因的全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明所公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或SPL多肽编码序列经基因工程产生的宿主细胞，以及经重组技术产生本发明所述多肽的方法。
通过常规的重组DNA技术(Science，1984；224：1431)，可利用本发明的多聚核苷酸序列可用来表达或生产重组的SPL多肽。一般来说有以下步骤：
(1)用本发明的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2)在合适的培养基中培养的宿主细胞；
(3)从培养基或细胞中分离、纯化蛋白质。
本发明的多核苷酸序列可***到重组表达载体中。术语“重组表达载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒或其他载体。总之，只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含本发明多核苷酸和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择 转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如植物细胞(如农作物和林业植物的细胞)。代表性例子有：大肠杆菌，链霉菌属、农杆菌；真菌细胞如酵母；植物细胞等。
本发明的多核苷酸在高等真核细胞中表达时，如果在载体中***增强子序列时将会使转录得到增强。增强子是DNA的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。
本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
转化植物也可使用农杆菌转化或基因枪转化等方法，例如叶盘法。对于转化的植物细胞、组织或器官可以用常规方法再生成植株，从而获得耐热性能变化的植物。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超滤处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
重组的SPL多肽有多方面的用途。例如用于筛选具有调控耐热性能的化合物、多肽或其它配体。用表达的重组SPL多肽的筛选多肽库可用于寻找有价值的能抑制、或促进植物耐热性能的多肽分子。
另一方面，本发明还包括对SPL多肽具有特异性的多克隆抗体和单克隆抗体，尤其是单克隆抗体。本发明不仅包括完整的单克隆或多克隆抗体，而且还包括具有免疫活性的抗体片段、或嵌合抗体。
本发明的抗体可以通过本领域内技术人员已知的各种技术进行制备。例如，纯化的SPL多肽基因产物或者其具有抗原性的片段，可被施用于动物以诱导多克 隆抗体的产生。本发明的各类抗体可以利用耐热性能相关基因产物的片段或功能区，通过常规免疫技术获得。这些片段或功能区可以利用重组方法制备或利用多肽合成仪合成。与耐热性能相关基因产物的未修饰形式结合的抗体可以用原核细胞(例如E.Coli)中生产的基因产物来免疫动物而产生；与翻译后修饰形式结合的抗体(如糖基化或磷酸化的蛋白或多肽)，可以用真核细胞(例如酵母或昆虫细胞)中产生的基因产物来免疫动物而获得。抗SPL多肽的抗体可用于检测样品中的耐热性能相关多肽。
本发明还涉及定量和定位检测耐热性能相关多肽水平的测试方法。这些试验是本领域所熟知的。试验中所检测的耐热性能相关多肽水平，可用于解释耐热性能相关多肽调控耐热性能的功能。
一种检测样品中是否存在耐热性能相关多肽的方法是利用SPL多肽的特异性抗体进行检测，它包括：将样品与SPL多肽特异性抗体接触；观察是否形成抗体复合物，形成了抗体复合物就表示样品中存在耐热性能相关多肽。
本发明的多核苷酸的一部分或全部可作为探针固定在微阵列(microarray)或DNA芯片(又称为“基因芯片”)上，用于分析组织中基因的差异表达分析。用SPL多肽特异的引物进行RNA-聚合酶链反应(RT-PCR)体外扩增也可检测SPL多肽的转录产物。
本发明的主要优点包括：
(a)提供了植物耐热性能相关的基因及其所编码的多肽；
(b)本发明的SPL基因可以增强植物的耐热性能；
(c)提供了一种有针对性的增强植物的耐热性能的方法；
(d)提供的SPL基因和方法适用于多种植物的遗传改良。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
通用方法
a.拟南芥基因组DNA提取
取植物组织0.1g，加裂解缓冲液0.3mL，匀浆。加0.3mL酚/氯仿(1:1)，混匀。12,000rpm离心5min，上清转入另一离心管，加入30μL 3M NaAc(pH 5.2)和500μL无水乙醇。混匀。离心10min，沉淀经70％酒精洗涤，真空干燥，溶于50μL TE(pH 8.0)中。
b.拟南芥总RNA的提取
取材料(约100mg)于液氮中充分研磨。转移至1.5mL离心管中，加入1mL Trizol(Invitrogen，Cat.15596-018)，混匀，室温放置5min。12,000rpm离心10min，弃取沉淀。上清中加入200μL三氯甲烷，混匀，12,000rpm离心10min。取上清，加入500μL异丙醇沉淀RNA。12,000rpm离心10min，沉淀用70％乙醇洗涤，真空干燥，溶于20-50μL H2O(RNase free)。待RNA 全部溶解之后，加入RNase free的DNase(0.5-1μL)，37℃放置半小时，65℃失活20min。用NANODROP 2000c(Thermo)测定RNA的浓度。
c.cDNA反转录
PolyA mRNA第一链反转录采用M-MLV Reverse Transcriptase体系(Invitrogen，Cat.C28025-021)，反应体系如下：
Figure PCTCN2017073473-appb-000002
体系充分混合，65℃反应5min，迅速置于冰上，放置5min。然后向体系中加入：
Figure PCTCN2017073473-appb-000003
体系充分混合，37℃反应2min，最后加入1μL的M-MLV Reverse Transcriptase，混匀，37℃反应50min，70℃反应15min使逆转录酶失活，95℃加热5min，置于冰上。反转录产物稀释3-10倍之后可直接用于qRT-PCR检测。
d.PCR反应
Ex-Taq PCR反应体系(20μL)如下：
Figure PCTCN2017073473-appb-000004
PCR反应的复性温度和延伸时间由引物和扩增片段长度决定。一般反应条件为：94℃变性5min；94℃变性30s，57℃复性30s，72℃延伸30s，扩增30至35个循环；72℃保温10min。4℃保温。
PCR引物序列如SEQ ID NO.:7-16所示。
PrimeSTAR HS反应体系(50μL)如下：
Figure PCTCN2017073473-appb-000005
Figure PCTCN2017073473-appb-000006
一般反应条件为：98℃变性10sec；55℃复性5或15s，72℃延伸1min/kb，扩增30至35个循环；4℃保温。
e.Real-time-PCR分析
Real-time RT-PCR检测采用嵌合荧光法(
Figure PCTCN2017073473-appb-000007
Premix Ex TaqTM II，TaKaRa，DRR041A)。PCR引物序列如SEQ ID NO.:11-16所示。反应体系如下：
Figure PCTCN2017073473-appb-000008
以拟南芥S18(AT1G07210)基因作为内标参考。数据分析采用Realplex v2.0(Eppendorf，Hamburg，Germany)。实验重复三次，取各组数据的平均值和方差，绘制图表。
f.载体构建
DNA琼脂糖凝胶电泳、片段的酶切、纯化和连接参考《分子克隆》(Sambrook and Russell,2001)以及相关试剂和酶生产商的操作说明。构建载体的启动子和基因编码序列均采用高保真PCR酶扩增得到，TA克隆并测序以保证序列正确。
g.Western Blot实验
蛋白质的提取、SDS-PAGE电泳、转移至醋酸纤维膜，封闭，第一抗体与靶蛋白的结合，酶联二级免疫反应及显色等步骤参考《精编分子生物学指南》(Frederick M.Ausubel,1995)及相关试剂生产商的操作说明。
实施例1
SPL1，SPL12基因克隆和载体构建
将pBI121的35S启动子和NOS终止子通过高保真酶扩增，分别经EcoRI/SacI和PstI/HindIII连入pCAMBIA1300的多克隆位点，然后将抗性基因的35S启动子用NOS启动子(来自pBI121)替代，得到pCAMBIA1300S。以pBSK-tag模板，高保真酶扩增6×myc片段，产物经KpnI/SmaI酶切克隆到pCAMBIA1300S载体中，得到pCAMBIA1300S-myc。用PrimeSTAR HS DNA聚合酶扩增SPL1基因组序列，产物经BamH1酶切克隆到pCAMBIA1300S-myc载体，获得35S:myc-gSPL1。用PrimeSTAR HS DNA聚合酶扩增SPL12的基因组序列，SPL12产物经TA克隆连接到JW819(pCAMBIA3300经过改造得到)载体，获得p35S:gSPL12。
实施例2
SPL1和SPL12基因表达的组织特异性分析
分离的SPL1和SPL12基因分别编码含有881和921个氨基酸的蛋白，序列同 源性为72％。SPL1和SPL12基因的核苷酸序列如SEQ ID NO.:1和SEQ ID NO.:4所示，其所编码的氨基酸的序列如SEQ ID NO.:2和SEQ ID NO.:5所示。
为研究SPL1，SPL12在拟南芥植株中的组织表达特异性，将SPL1，SPL12的启动子(ATG上游约3.1kb)驱动GUS基因表达的载体pSPL1:GUS和pSPL12:GUS转化到野生型拟南芥中。拟南芥转化采用浸泡法，在T2代植物中挑选抗性比为3:1的单***独立株系，T3代筛选纯合的株系用于后续分析。植物在22℃，16h光照/8h黑暗的人工气候室中培养。
对多个独立的转基因株系(各至少8个)进行GUS染色观察。报告基因的染色实验显示pSPL1:GUS和pSPL12:GUS在拟南芥各个组织中广泛表达(图1中A，图1中B)，包括1)子叶、下胚轴维管组织；2)主根和侧根(除根冠)；3)莲座叶(含表皮毛)，其老叶中的表达量高于嫩叶；4)茎表皮(含表皮毛)；5)花序及花序柄；6)果荚顶端，果荚与果柄结合处及成熟果荚，但在种子中不表达。pSPL12:GUS转基因株系中报告基因的染色程度略弱于pSPL1:GUS，除了不在下胚轴和茎表皮毛中表达，其余组织中的表达模式与pSPL1:GUS相似，暗示了SPL1和SPL12可能功能冗余，并存在部分组织特异性。
SPL1和SPL12在花发育晚期(时期10-16)在花苞顶端开始表达，到后期延伸到整个花萼、花瓣、柱头、雄蕊及花粉粒(图1中C-F)。Real-Time PCR实验结果也显示，SPL1和SPL12在已开的花(stage13及之后)中表达量明显高于未开的花苞(图1中G)，暗示可能在花器官晚期发育过程中有重要的作用。
实施例3
SPL1，SPL12表达下调影响花发育和结实
为了揭示SPL1和SPL12的生物学功能，分析了其T-DNA***突变体，分别为spl1-1(SALK_134584)以及spl12-1(SALK_142295)(图2中A)。RT-PCR检测显示spl1-1突变体中SPL1的表达和spl12-1突变体中SPL12的表达几乎为零(图2中B)，说明spl1-1和spl12-1均为功能完全缺失型突变体。通过杂交spl1-1和spl12-1单突变体获得spl1-1spl12-1双突变体，即SPL1和SPL12功能完全缺失型突变体(图2中B)。
在正常光照和温度条件下，spl1-1和spl12-1单突变体没有任何可见异常表型，而spl1-1spl12-1双突变体植株到了生殖生长期表现出部分花不能正常展开(图2中C、E、F)，花萼边缘粘连(图2中G)，由于开花当日花器官不能充分展开，空间上阻止了受精过程，从而导致结实率降低(图2C、D)。野生型花在开花后很快自然脱落，而双突变体花成熟和脱落过程较慢，花萼不能正常脱落(图2H、I)。
用35S:myc-gSPL1和35:gSPL12-载体转化spl1-1spl12-1双突变体获得的转基因植物均能恢复到野生型表型(图2中D)。另外，RNAi(在spl1-1背景下，沉默SPL12)转基因株系RNAi-1植物(图2中J)也表现出类似spl1-1spl12-1双突变体的表型(图2中D)。
以上结果说明spl1-1spl12-1双突变体花发育异常表型确实是由SPL1和SPL12基因的缺失引起的，且SPL1和SPL12间存在功能冗余(图2)。
实施例4
SPL1，SPL12表达水平影响拟南芥花器官对温和高温的响应
对野生型Col-0、spl1-1spl12-1双突变体和转基因植物ox-MS1c-1(35S:myc-gSPL1，Col-0背景)在温和高温(30℃)处理后的花开放度进行了连续4天的跟踪统计。将茎顶端花序上当日开的花朵其花萼完全展开，花瓣自然伸展的情况视为“开放”，花萼或花瓣不能自然展开的情况视为“败开”，统计每个茎顶端花序上当日开花的数目，简称为每日花开放度(Number of open flowers per inflorescence per day，简称NOF)。
生长五周的拟南芥，连续四天时间内茎顶端花序平均NOF情况如图3所示：22℃情况下，野生型拟南芥NOF＝2.5，而spl1-1spl12-1双突变体NOF＝1.4。而30℃连续处理四天，野生型拟南芥NOF＝1.7，spl1-1spl12-1双突变体NOF＝0.4。ox-SPL1与野生型间没有显著差异，其22℃和30℃的NOF分别为2.9和1.9。说明累积的温和高温影响拟南芥每日花开放度，spl1-1spl12-1双突变体对高温更加敏感。
图4显示了22℃和30℃下拟南芥茎顶端花序形态特征与每日花开放度统计结果，分别显示了0d、1d和4d的情况。可以看到，正常22℃生长条件下(0d-4d)野生型拟南芥茎顶端花序上每日开放的花数为2-3，所占比例为80-90％。而每日开放的花数为0、1、4、5所占比例加起来为10-20％。而spl1-1spl12-1双突变体茎顶端花序每日开放的花数为2-3的比例只有～45％，其余占～55％，其中主要为0和1，而4和5所占比例仅～1％。说明SPL1和SPL12的缺失降低了拟南芥正常温度下的每日花开放度(图4中A、B)。
30℃处理后第一天(1d)，野生型拟南芥每日花开放度并没有发生明显的变化，而spl1-1spl12-1双突变体每日花开放度显著降低，开放花数为2和3的所占比例从45％降到12％，而0和1的所占比例从51％上升到88％。到处理后第四天，野生型拟南芥花开放度也有一定程度降低，花开放数为2和3的比例从88％降到29％，0和1的比例从9％升到70％。spl1-1spl12-1双突变体每日花开放度变化幅度更大，花开放数为0的比例占到95％、1为5％、其余为0％。ox-SPL1的表型与野生型没有明显的差异(图4中A、B)。以上结果说明了累积的温和高温在一定程度上改变了拟南芥花序的生长与形态发育，而SPL1和SPL12的缺失影响了拟南芥花对温和高温的响应。
综合以上结果和对双突变体花的形态解剖分析，推测SPL1和SPL12不仅参与调控拟南芥花的正常开放与发育成熟，同时在植物对温和高温的响应过程中发挥重要作用。
实施例5
SPL1，SPL12表达水平影响拟南芥花器官对极端高温的耐受性
进一步分析拟南芥花器官对极端高温的耐受性，将在22℃生长五周的植物用37℃处理1分钟便可以看到野生型植物顶端幼嫩茎及花器官快速萎蔫，但半 个小时之后又逐渐恢复正常，ox-MS1c-1与野生型类似，但萎蔫程度不如野生型。而spl1-1spl12-1双突变体的萎蔫度较严重，半个小时之后部分花序不能恢复正常状态，甚至有些快速焦枯。将37℃处理1天之后花序(图5中A)重新恢复到22℃生长1天，观察并统计完全焦枯而不能正常开花的花序数目，结果显示花存活率分别是ox-MS1c-1 64.5％、野生型43.2％、spl1-1spl12-1双突变体16.7％(图5中C)，以没有做处理的植物作为对照，其存活率为100％。说明SPL1和SPL12的缺失降低了拟南芥花器官对极端高温的耐受性，而SPL1的过量表达增强其耐受性，其表达量与高温耐受性正相关(图5中B、C)。
超氧化物歧化酶(Super Oxide Dismutase，SOD)是植物体内清除自由基的首要物质。SOD在生物体内的水平高低是衰老与死亡的直观指标。SOD活力的高低间接反应了机体清除ROS的能力。高温胁迫可以造成植物体内ROS的积累，大量积累的活性氧对细胞造成伤害，严重时会导致细胞的死亡。检测了22℃生长5周的野生型和spl1-1spl12-1双突变体在22℃和37℃处理4h后花序的SOD活性。高温胁迫后野生型拟南芥花序SOD活性降低，在spl1-1spl12-1中降低更为严重(图6中A)，说明SPL1和SPL12对拟南芥花序在高温胁迫下维持一定抗氧化能力起着重要作用。以上结果暗示着SPL1和SPL12是拟南芥生殖器官维持高温耐受性所必需的关键基因。
实施例6
SPL1，SPL12表达下降对拟南芥高温下种子产量和萌发率的影响
将生长6周的野生型和双突变体植物在42℃处理1天后重新恢复到22℃培养，最后统计每棵植物的种子总干重，以22℃培养的野生型种子产量为100％，统计突变体和不同处理植物的种子产率。结果显示，正常条件下spl1-1spl12-1双突变体种子产量为野生型的69％。高温处理导致野生型种子产率降低至85％，而双突变体种子产率降低至25％，说明SPL1，SPL12的缺失不仅影响了种子产量，且这种影响在高温下更为严重(图6中B)。
检测了高温胁迫下种子萌发率，发现22℃条件下野生型和双突变体的种子萌发率均为90％以上，30℃生长7天后的野生型萌发率为80％，spl1-1spl12-1双突变体萌发率为47％(图6中C)，说明30℃高温对野生型种子萌发率的影响较弱，而spl1-1spl12-1双突变体种子萌发率对30℃高温敏感。而极端高温如37℃情况下野生型和spl1-1spl12-1双突变体种子萌发率均为0，37℃可以完全抑制拟南芥种子萌发。以上结果表明SPL1和SPL12对于拟南芥在高温下维持种子产量和萌发率方面具有重要作用。
实施例7
QRT-PCR验证SPL1，SPL12调控的热激响应转录因子
转录因子在植物生长发育和与环境互作过程中起着十分重要的角色。通过转录组和差异转录因子表达基因的分析，筛选了十余个已被报道的参与干旱胁迫、ABA信号调控途径、抗热途径相关的转录因子基因，分析它们在花序中的热激响应表达情况。Real-time PCR实验检测结果显示这些基因在野生型中被 高温诱导表达，而在spl1-1spl12-1双突变体中诱导受阻，与RNA-seq结果一致(图7中A、B、C)。说明这些基因的热激响应诱导需要SPL1、SPL12的参与。进一步证实这些基因可能是SPL1、SPL12调控的热激响应转录因子，在拟南芥花序维持抗热性过程中起着重要作用。
实施例8
过表达SPL1或SPL12提高植物抗热性
为了进一步探索SPL1和SPL12在拟南芥抗热性的作用，在野生型和双突变体中分别转化35S启动子驱动的6×myc-SPL1(ox-MS1)或SPL12(ox-S12)载体。选择了目标基因表达量最高的6个转基因株系，其中ox-MS1c-1,ox-S12c-3,和ox-S12c-4为野生型背景，ox-MS1d-1，ox-MS1d-4和ox-MS1d-7为spl1-1spl12-1双突变体背景(图2中J)。对生长14天的野生型，spl1-1spl12-1和转基因植物ox-MS1c-1，ox-MS1d-1和ox-S12c-3进行2d的42℃高温处理，恢复5天后统计存活率，结果显示ox-MS1c-1，ox-MS1d-1和ox-S12c-3的存活率均显著高于野生型和双突变体(图8中A、B)。ox-MS1d-1，ox-MS1d-4和ox-MS1d-7花序在42℃高温处理后表现出更高的存活率(图8中C、D)。此外，在42℃，ox-MS1c-1，ox-MS1d-1，ox-S12c-3获得更高的种子产量(图8中E)，在30℃，ox-MS1c-1，ox-MS1d-1，ox-S12c-4获得更高的种子产量。以上结果说明过表达SPL1或SPL12能够提高拟南芥营养生长期和生殖生长期的抗热性。
将35S::6-MYC-AtSPL1载体和35S::AtSPL12载体由叶盘法转入本氏烟草，扩繁并获得纯合植株后，各通过半定量PCR和Western Blot鉴定了两个转基因株系(图11中E-F)1H-2，1H-9,12B-1和12B-4，其中1H表示转入35S::6MYC-AtSPL1的转基因烟草，12B表示转入35S::AtSPL12的转基因烟草，下同。观察到22℃生长的条件下，生长三周大的营养生长期烟草以及生长40天左右的生殖生长期烟草，其花序顶端分生组织和果荚的发育在转基因烟草中均未观察到发育异常(图11中A-D)。说明拟南芥基因AtSPL1和AtSPL12异源转入本氏烟草并不影响本氏烟草正常的生长发育。
将正常温度生长20天左右处于营养生长期的转基因株系与野生型烟草，经42℃、16小时的高温处理后，发现转基因烟草1H-2，1H-9，12B-1和12B-4中枯萎受损的比例明显低于野生型，而正常生长的比例明显高于野生型，表现出了显著的抗热性(图12)。
将正常温度生长35天左右处于生殖生长期的转基因株系与野生型烟草，经42℃，3小时的短时高温处理后，观察正在开放的花朵在热胁迫下的受损情况，发现野生型花朵相较于转基因烟草1H-9和12B-4其花朵更易受损伤(图13中A)。为了证明转入的异源蛋白在热胁迫下仍稳定表达继而发挥其功能，我们选择了一直在22℃生长和高温处理后的1H-2和1H-9的花朵，以及1H-9叶片中，检测其中MYC-AtSPL1蛋白的表达量。Western Blot实验证明其异源蛋白在上述实验条件的组织中稳定表达，从而贡献于烟草抗热性的增强。为了观察SPL1和SPL12对烟草花序顶端分生组织的作用，我们对上述四个经鉴定的转基因株 系，在正常生长至35天大时，即花序顶端分生组织已经形成时，与同时期的野生型烟草，经45℃，6小时的短时高温处理后，恢复正常生长条件，三周后统计已经发育成熟的前7朵花中，可育花朵的数量作为花朵存活的标准，死去的花朵为不可育。转基因烟草1H-2，1H-9，12B-1和12B-4的花朵存活率明显高于野生型(图13中C)，即转基因烟草花朵更具有耐热性。以上结果说明AtSPL1和AtSPL12可提高本氏烟草营养生长期和生殖生长期抗热性。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种SPL基因或其编码蛋白的用途，其特征在于，所述SPL基因选自SPL1基因、SPL12基因、或其组合，并且所述的SPL基因或其编码蛋白用于选自下组的用途：
    (a)用于制备增强植物耐热性能的试剂或组合物；
    (b)用于增强植物的耐热性能。
  2. 如权利要求1所述的用途，其特征在于，所述的植物为禾本科植物或十字花科植物，较佳地，所述植物为拟南芥、烟草、水稻、或小麦。
  3. 如权利要求1所述的用途，其特征在于，所述的SPL基因为SPL1基因和SPL12基因。
  4. 如权利要求1所述的用途，其特征在于，所述的“增强植物耐热性能”包括选自下组的一种或多种性能：
    (i)增强花序的耐热性能；
    (ii)增强高温环境下结实的种子的萌发率；
    (iii)用于增强植物对高温环境的耐受性；
    (iv)增强高温环境下植物的热激响应；
    (v)增强高温环境下植物的抗氧化性能；
    (vi)增强植物根、茎、和/或叶的耐热性能；
    (vii)增强高温环境下植物的结实率。
  5. 如权利要求1所述的用途，其特征在于，所述的“增强植物耐热性能”包括增强植物生长期和生殖期的耐热性能。
  6. 如权利要求1所述的用途，其特征在于，所述的SPL基因包括野生型SPL基因和突变型SPL基因。
  7. 一种改变植物耐热性能的方法，其特征在于，包括步骤：
    (a)将外源的构建物导入植物细胞，其中所述的构建物含有外源的SPL基因序列、促进SPL基因表达的外源核苷酸序列、或抑制SPL基因表达的外源核苷酸序列，从而获得导入外源构建物的植物细胞；
    (b)将上一步骤获得的所述导入外源构建物的植物细胞，再生成植株：和
    (c)任选地对所述再生的植株进行鉴定，从而获得耐热性能改变的植物；
    其中，所述SPL基因选自SPL1基因、SPL12基因、或其组合。
  8. 如权利要求7所述的方法，其特征在于，所述外源的SPL基因序列还包含与ORF序列操作性连接的启动子和/或终止子。
  9. 一种增强植物耐热性能的方法，其特征在于，所述方法包括以下步骤：在所述植物中，促进SPL基因的表达或促进SPL蛋白的活性，其中，所述SPL基因选自SPL1基因、SPL12基因、或其组合。
  10. 一种SPL基因或其编码蛋白的调控剂的用途，其特征在于，用于调控植物的耐热性能，或用于制备调控植物耐热性能的试剂或组合物，其中，所述SPL基因选自SPL1基因、SPL12基因、或其组合。
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