WO2017185854A1 - Gène spl et son application dans l'amélioration de la tolérance des végétaux à la chaleur - Google Patents

Gène spl et son application dans l'amélioration de la tolérance des végétaux à la chaleur 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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Chinese (zh)
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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

La présente invention a trait à un gène SPL et son application dans l'amélioration de la tolérance des végétaux à la chaleur, et en particulier, une fonction et une utilisation du gène SPL. Le gène SPL, SPL1 ou SPL12, est un facteur clé nécessaire à l'adaptation aux températures élevées et à la tolérance d'une plante et/ou le maintien de la maturation et de la germination d'une semence sous température élevée. Arabidopsis thaliana transgénique surexprimant SPL1 ou SPL2 peut améliorer la tolérance des organes floraux aux températures extrêmes et à la température élevée.
PCT/CN2017/073473 2016-04-29 2017-02-14 Gène spl et son application dans l'amélioration de la tolérance des végétaux à la chaleur WO2017185854A1 (fr)

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