WO2019063009A1 - 一种提高水稻产量的aba受体pyl家族基因组合敲除的方法及其应用 - Google Patents

一种提高水稻产量的aba受体pyl家族基因组合敲除的方法及其应用 Download PDF

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WO2019063009A1
WO2019063009A1 PCT/CN2018/109061 CN2018109061W WO2019063009A1 WO 2019063009 A1 WO2019063009 A1 WO 2019063009A1 CN 2018109061 W CN2018109061 W CN 2018109061W WO 2019063009 A1 WO2019063009 A1 WO 2019063009A1
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plant
pyl
gene
pyl1
wild type
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朱健康
苗春波
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中国科学院上海生命科学研究院
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008—Methods for regeneration to complete plants
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    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
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    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
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    • 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
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    • 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
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/8267—Seed dormancy, germination or sprouting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/8291—Hormone-influenced development
    • C12N15/8293—Abscisic acid [ABA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to the field of agriculture and biotechnology, and in particular to a method for knockdown of ABA receptor PYL family gene combination for improving rice yield and application thereof.
  • Abscisic acid is a hormone that regulates plant growth and enhances plant resistance to stress. Adversity stress, especially drought, can induce an increase in ABA content in plants. An increase in ABA levels will cause a broad and rapid physiological response of plants, thereby enhancing plant resistance. However, this increase in stress resistance is often accompanied by growth inhibition.
  • ABA is recognized by its receptor protein PYR/PYL/RCAR (pyrabactin resistance 1/PYR1-like/regulatory components of ABA receptors). The binding of ABA to PYL causes a conformational change of the protein, which causes and promotes the binding of PYL to the Clad A type 2C protein phosphatase protein.
  • SnRK2 serine nonfermenting 1-related protein kinase 2
  • the PYL protein is encoded by a gene family. There are 13 PYL genes in the rice nuclear genome. Although ABA plays a key role in plant stress tolerance and growth regulation, there are few studies on PYL in rice, and the specific gene function of its family members is still unclear. There are no reports of rice PYL mutants yet.
  • CRISPR/Cas9 technology is a gene editing technology that has emerged in recent years. After its first publication in 2013, the technology was quickly applied to genetic editing of plants and animals.
  • Cas9 nuclease cleaves a DNA sequence complementary to the sgRNA recognition region under the guidance of a short sgRNA (single guide RNA); multiple genes can be edited simultaneously by expressing multiple sgRNAs in one cell.
  • a first aspect of the invention provides a method of improving a plant comprising the steps of:
  • the desired trait characteristic is selected from the group consisting of increased plant height, increased yield, increased biomass, near-normal seed dormancy, no significant delay in heading, or a combination thereof.
  • a comprehensive evaluation of plant height, yield, biomass, seed dormancy, and heading date is performed to select plants having desired shape characteristics.
  • the desired trait characteristic is selected from the group consisting of increased biomass, increased yield, near-normal seed dormancy, no significant delay in heading, or a combination thereof.
  • the mutation comprises reducing the expression or activity of N members of the PYL gene family.
  • the "reduction" means that the expression or activity of N members of the PYL gene family is decreased to satisfy the following conditions:
  • the ratio of A1/A0 is ⁇ 80%, preferably ⁇ 60%, more preferably ⁇ 40%, most preferably 0-30%;
  • A1 is the expression or activity of N members of the PYL gene family
  • A0 is the expression or activity of N members of the same PYL gene family in the wild type of the same type of plant.
  • the decrease means that the expression level E1 of the member of the PYL family in the plant is 0-80% of the wild type compared to the expression level E0 of the member of the wild-type PYL family, preferably 0-60%, more preferably 0-40%.
  • the expression or activity of N members of the PYL gene family in the reduced plant is achieved by a method selected from the group consisting of gene mutation, gene knockout, gene disruption, RNA interference technology, Crispr technology, Or a combination thereof.
  • the plant having the desired trait characteristics is selected from the group consisting of pyl1/4/6 or pyl1/6.
  • the plant comprises a crop, a forestry plant, a flower; preferably comprises a Poaceae, a legume, and a crucifer, more preferably rice, corn, sorghum, wheat, or soybean.
  • the genetic engineering comprises genetic editing of members of the PYL gene family with a plurality of sgRNA-mediated Cas9 nucleases.
  • the gene editing comprises genetic editing of a gene of the PYL gene family selected from the group consisting of PYL1, PYL2, PYL3, PYL4, PYL5, PYL6, PYL12, or a combination thereof.
  • the gene editing further comprises gene editing of a PYL gene family selected from the group consisting of PYL7, PYL8, PYL9, PYL10, PYL11, PYL13, or a combination thereof.
  • the gene editing comprises gene editing of a gene selected from the group consisting of PYL gene family: PYL1, PYL2, PYL3, PYL4, PYL5, PYL6, PYL7, PYL8, PYL9, PYL10, PYL11, PYL12 , PYL13, or a combination thereof.
  • a second aspect of the invention provides a genetically engineered plant tissue or plant cell in which a mutation occurs in N members of the PYL gene family, wherein N >
  • the mutation comprises reducing the expression or activity of N members of the PYL gene family.
  • a third aspect of the invention provides a method of preparing a genetically engineered plant tissue or plant cell, comprising the steps of:
  • N The N members of the PYL gene family in plant tissues or plant cells are mutated to obtain genetically engineered plant tissues or plant cells, wherein N ⁇ 2.
  • a fourth aspect of the invention provides a method of preparing a transgenic plant, comprising the steps of:
  • the genetically engineered plant tissue or plant cell prepared by the method of the third aspect of the invention is regenerated into a plant body to obtain a transgenic plant.
  • a fifth aspect of the invention provides a transgenic plant prepared by the method of the fourth aspect of the invention.
  • a sixth aspect of the invention provides a method of producing food comprising the steps of:
  • the crop is selected from the group consisting of Poaceae, Leguminosae, and Cruciferous, and more preferably rice, corn, sorghum, wheat, or soybean.
  • Figure 1 shows a phylogenetic tree analysis of the rice PYL protein sequence.
  • Group I class I genes
  • group II class II genes.
  • Figure 2 shows that mutations in the class I PYL gene promote rice growth.
  • a rice PYL multi-gene knockout strategy.
  • b Comparison of seedling stage of wild type and class I gene mutants. Ruler, 10cm. c, wild type and class I mutants seedlings seedling length and fresh weight. Each column represents an independent line. "+” indicates a wild type gene and "-” indicates a mutant gene. "***”, the P value of the difference from the wild type is less than 0.001; "**”, the P value of the difference from the wild type is less than 0.01; “*”, the P value of the difference from the wild type is less than 0.05. P values were obtained using Student's t-test.
  • Figure 3 shows the phenotypic identification of class I gene mutants.
  • a wild type, pyl1/4/6 and pyl1/2/3/4/5/6 seedling stage comparison chart.
  • Ruler, 10cm. b Comparison of wild type and pyl1/2/3/4/5/6 at the initial stage of grouting.
  • Ruler, 10cm. c the height of the class I gene plant at maturity. Each column represents an independent line. "+” indicates a wild type gene and "-” indicates a mutant gene. "***”, the P value difference from the wild type is less than 0.001. P values were obtained using Student's t-test.
  • d wild type and pyl1/4/6 ear length and internode length comparison data.
  • the left histogram shows the length and ear length between each section; the right histogram shows the length of the ear and the ratio of the length of each section to the plant height.
  • e wild type and pyl1/4/6 ear length and internode length comparison.
  • Ruler 10cm.
  • Figure 4 shows the sensitivity of wild-type and class I gene mutants to high temperature weather.
  • a wild-type and class I gene mutants experienced phenotypic comparisons after the 2016 Shanghai high temperature climate.
  • b Comparison of the maturity of wild-type and class I gene mutants in Shanghai's high temperature climate in 2016.
  • Figure 5 shows the effect of rice PYL gene mutation on seed dormancy.
  • a class I gene mutant seed PHS map.
  • Ruler 10cm.
  • b the frequency of pre-harvest sprouting (PHS) of wild type and class I gene mutant seeds. All data were counted at the end of the pyl1/2/3/4/5/6/12 filling period and the statistical work was completed within one day.
  • Each column represents an independent line, and each strain system counts all seeds of 3 plants. "+” indicates a wild type gene and "-” indicates a mutant gene. "***", the P value difference from the wild type is less than 0.001.
  • P 0.104173
  • P values were obtained using Student's t-test. c, wild type and class II gene mutant seed PHS frequency. Seed harvest was delayed by approximately 25 days and the PHS frequency was counted. Each column represents an independent line, and each strain system counts all seeds of 3 plants. "***", the P value difference from the wild type is less than 0.001.
  • Figure 6 shows wild type and pyl1/4/6 yield trait analysis and testing.
  • a wild type and pyl1/4/6 main spike comparison chart.
  • Ruler 5cm.
  • b wild type and pyl1/4/6 main ear length data.
  • "***” the P value difference from the wild type is less than 0.001.
  • c wild-type and pyl1/4/6 seed 1000-grain weight data.
  • d wild type and pyl1/4/6 points statistic data.
  • ** the P value difference from the wild type is less than 0.01.
  • e wild-type and pyl1/4/6 main ear one branch data.
  • *** the P value difference from the wild type is less than 0.001.
  • Figure 7 shows the effect of the 2016 Shanghai pyl1/4/6 production test.
  • the present invention utilizes gene knockout technology (such as CRISPR/Cas9 polygene editing technology) for the first time to knock out the rice PYL gene, and found that by knocking out different PYL gene family members, rice growth can be promoted or Some of the agronomic traits required were improved.
  • gene knockout technology such as CRISPR/Cas9 polygene editing technology
  • PYL is an ABA receptor protein-encoding gene, and PYL exists in the form of a gene family. Previous studies have shown that the genes of the PYL family are highly conserved and critical for plant growth (especially stress resistance). Studies of the present invention suggest that modification of a single PYL gene (e.g., knockout or down-regulation) does not exhibit an improvement in plant traits.
  • N genes (N ⁇ 2) of the PYL gene of any plant species can be knocked out, and representative plants include, but are not limited to, forestry plants, agricultural plants, such as grasses, cruciferae , legumes, etc., such as rice, corn, sorghum, wheat, soybean, or a combination thereof.
  • the PYL gene includes all known PYL genes from the plant (or species), and PYL genes which may be found in the future, and homologous genes having homology to these PYL genes.
  • “having homology” means having > 70%, preferably > 80%, more preferably > 90%, and most preferably > 95% identity between the two sequences.
  • the common name of the homologous gene of the PYL gene is the PYL gene, and the Latin name abbreviation of the species may be added before the name of the PYL gene.
  • the wheat PYL gene is also called TaPYL; the corn PYL gene is also called ZmPYL; the soybean PYL gene is also called GmPYL.
  • PYL genes are known, namely PYL1, PYL2, PYL3, PYL4, PYL5, PYL6, PYL7, PYL8, PYL9, PYL10, PYL11, PYL12, PYL13.
  • two or more PYL genes may be expressed in combination, for example, "pyl1/4/6" means pyl1, pyl4 and pyl6.
  • the knockout type of the rice PYL gene is as follows: pyl1/4/6, pyl1/6.
  • the mutation comprises reducing the expression or activity of N members of the PYL gene family.
  • said "reducing” means reducing the expression or activity of N members of the PYL gene family to satisfy the following conditions:
  • the ratio of A1/A0 is ⁇ 80%, preferably ⁇ 60%, more preferably ⁇ 40%, most preferably 0-30%;
  • A1 is the expression or activity of N members of the PYL gene family
  • A0 is the expression or activity of N members of the same PYL gene family in the wild type of the same type of plant.
  • the decrease means that the expression level E1 of the member of the PYL family in the plant is 0-80% of the wild type compared to the expression level E0 of the member of the wild-type PYL family, preferably 0-60%, more preferably 0-40%.
  • the expression or activity of N members of the PYL gene family in the reduced plants is achieved by a method selected from the group consisting of gene mutation, gene knockout, gene disruption, RNA interference technology, Crispr technology, Or a combination thereof.
  • plants having poor traits are excluded based on the results of the trait test.
  • the method further comprises the step (iv) of further screening the plants having the desired shape characteristics selected in the step (iii), thereby screening for the ability to balance plant height, yield, biomass, seed dormancy, heading. For plants with traits such as water loss and water loss performance, the comprehensive traits of the plants are optimal.
  • the invention also provides a method of producing food comprising the steps of:
  • the present invention tests the knockout traits of members of the PYL gene family of different plants (such as rice), and selects plants having the desired trait characteristics (increased biomass, increased yield).
  • the present invention finds for the first time that simultaneous knockout of PYL1, PYL4 and PYL6 can exhibit the best growth state and agronomic traits, and can greatly increase rice yield.
  • Cas9 is mediated by the maize Ubiquitin promoter; four sgRNAs are mediated by the OsU3-1, OsU6-1, OsU3-2, and OsU6-2 promoters respectively (promoter sequences are shown in sequence 1 to sequence 4) ), four sgRNA expression cassettes are arranged in tandem on the vector.
  • a primer that specifically recognizes a target gene comprising a 20 bp target recognition sequence and a 4-5 bp tag sequence.
  • the primers anneal to form a double-stranded DNA with a sticky end (20 bp double-stranded region).
  • the double-stranded DNA is ligated seamlessly with the promoter and the downstream sequence under the action of T4 ligase to construct an sgRNA expression cassette.
  • the tandemly aligned sgRNA expression cassette was first constructed in the PUC19 intermediate vector and then subcloned into the PCAMBIA1300 backbone with the Cas9 expression cassette.
  • class I mutants continue to exhibit stronger plant types than wild type (Fig. 2d and Fig. 3b).
  • Fig. 2d and Fig. 3b To compare the differences between different mutants and wild type at maturity, we measured the plant height of various mutants. The plant height data at maturity showed a pattern similar to the seedling data, with pyl1/4/6 having the highest plant growth and the best growth among all class I gene mutants (Fig. 2d and Fig. 3c).
  • the heading date affects the geographical distribution of rice and its adaptability to the season.
  • the heading stage of the mutant lines was significantly delayed starting from the four mutants (Fig. 2h).
  • the heading stage of pyl1/2/3/4/5/6 and pyl1/2/3/4/5/6/12 was delayed by about 9 days
  • pyl1/2/3/4/6 It was delayed for about 7 days
  • pyl1/2/3/4 was delayed for about 5 days
  • pyl1/4/6 was delayed for about 1 day.
  • the statistic also shows a surprising phenomenon, the PHS frequency of pyl1/6 is lower than that of pyl1, and the PHS frequency of pyl1/4/6 is lower than that of pyl1 and pyl1/4, indicating that PYL6 and other class I genes are involved in seed dormancy.
  • There may be antagonism (Fig. 5b).
  • pyl1/4/6 exhibited the lowest PHS frequency, and its PHS frequency was almost similar to the wild type (the P values of the two independent lines compared with the wild type were 0.104173 and 0.02361) (Fig. 5b).
  • the biomass of pyl1/4/6 at maturity increased by about 55.3% compared to wild type, although its tiller number was significantly reduced compared to wild type (Fig. 6d and 6h).

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Abstract

一种提高水稻产量的ABA受体PYL家族基因组合敲除的方法及其应用。

Description

一种提高水稻产量的ABA受体PYL家族基因组合敲除的方法及其应用 技术领域
本发明涉及农业和生物技术领域，具体地，涉及一种提高水稻产量的ABA受体PYL家族基因组合敲除的方法及其应用。
背景技术
脱落酸(abscisic acid，ABA)是一种能够调节植物生长和增强植物对逆境抵抗力的激素。逆境胁迫，特别是干旱，能够诱导植物体内ABA含量的增加，ABA含量水平的增加将会引起植物广泛迅速的生理响应，从而增强植物的抗逆性。然而，这种抗逆性的增加往往伴随着生长抑制。在植物体内，ABA由其受体蛋白PYR/PYL/RCAR(pyrabactin resistance 1/PYR1-like/regulatory components of ABA receptors)识别。ABA与PYL的结合引起该蛋白的构象变化，从而引起和促进PYL与PP2C(clade A type 2C protein phosphatase)蛋白的结合。ABA-PYL-PP2C复合体的形成抑制了PP2C的活性，从而将SnRK2(sucrose nonfermenting 1-related protein kinase 2)蛋白的活性释放出来。激活的SnRK2将会磷酸化下游的众多蛋白因子，从而引起ABA响应基因的表达、气孔关闭和萌发抑制等生理响应。
PYL蛋白由基因家族编码。水稻核基因组中有13个PYL基因。尽管ABA在植物抗逆和生长调节上发挥着关键的作用，水稻中有关PYL的研究很少，对其家族成员的具体基因功能还不清楚。目前还未水稻PYL突变体的报道。
CRISPR/Cas9技术是最近几年兴起的一种基因编辑技术。2013年首次发表以后，该技术迅速被广泛应用于动植物的基因编辑。在该技术体系中，Cas9核酸酶在短的sgRNA(single guide RNA)引导下，去切割与sgRNA识别区互补的DNA序列；在一个细胞中表达多个sgRNA可同时对多个基因进行编辑。
发明内容
本发明的目的在于提供一种用于提高水稻产量的ABA受体PYL家族基因组合敲除的新方法。
本发明第一方面提供了一种改良植物的方法，包括步骤：
(i)对植物细胞或植物组织进行基因工程改造，从而使得PYL基因家族中的N个成员发生突变，其中N≥2；
(ii)将经基因工程改造的植物细胞或植物组织再生成植株，对再生的植株进行性状测试，所述性状选自下组：株高、抽穗期、种子休眠、产量、生物量、或其组合；
(iii)根据性状测试结果，选出具有所需性状特征的植株。
在另一优选例中，所述所需性状特征选自下组：株高增加、产量提高、生物量增加、种子休眠近乎正常、抽穗期未明显延迟、或其组合。
在另一优选例中，对株高、产量、生物量、种子休眠、抽穗期的综合评判，从而选出具有所需形状特征的植株。
在另一优选例中，所述所需性状特征选自下组：生物量增加、产量提高、种子休眠近乎正常、抽穗期未明显延迟、或其组合。
在另一优选例中，所述突变包括降低PYL基因家族中N个成员的表达或活性。
在另一优选例中，所述“降低”是指将PYL基因家族中N个成员的表达或活性降低满足以下条件：
A1/A0的比值≤80％，较佳地≤60％，更佳地≤40％，最佳地为0-30％；
其中，A1为PYL基因家族中N个成员的表达或活性；A0为野生型同种类型植物植株中相同PYL基因家族中N个成员的表达或活性。
在另一优选例中，所述的降低指与野生型PYL家族的成员的表达水平E0相比，所述植株中PYL家族的成员的表达水平E1为野生型的0-80％，较佳地0-60％，更佳地0-40％。
在另一优选例中，所述的降低植株中PYL基因家族中N个成员的表达或活性通过选自下组的方式实现：基因突变、基因敲除、基因中断、RNA干扰技术、Crispr技术、或其组合。
在另一优选例中，所述具有所需性状特征的植株选自下组：pyl1/4/6或pyl1/6。
在另一优选例中，所述的植物包括农作物、林业植物、花卉；优选地包括禾本科，豆科以及十字花科植物，更优选地包括水稻、玉米、高粱、小麦、或大豆。
在另一优选例中，所述基因工程改造包括用多个sgRNA介导的Cas9核酸酶对PYL基因家族的成员进行基因编辑。
在另一优选例中，所述基因编辑包括对选自下组的PYL基因家族的基因进行 基因编辑：PYL1、PYL2、PYL3、PYL4、PYL5、PYL6、PYL12、或其组合。
在另一优选例中，所述基因编辑还包括选自下组的PYL基因家族的基因进行基因编辑：PYL7、PYL8、PYL9、PYL10、PYL11、PYL13、或其组合。
在另一优选例中，所述基因编辑包括对选自下组的PYL基因家族的基因进行基因编辑：PYL1、PYL2、PYL3、PYL4、PYL5、PYL6、PYL7、PYL8、PYL9、PYL10、PYL11、PYL12、PYL13、或其组合。
本发明第二方面提供了一种基因工程的植物组织或植物细胞，所述植物组织或植物细胞中的PYL基因家族中的N个成员发生突变，其中N≥2。
在另一优选例中，所述突变包括降低PYL基因家族中N个成员的表达或活性。
本发明第三方面提供了一种制备基因工程的植物组织或植物细胞的方法，包括步骤：
将植物组织或植物细胞中的PYL基因家族中的N个成员发生突变，从而获得基因工程的植物组织或植物细胞，其中N≥2。
本发明第四方面提供了一种制备转基因植物的方法，包括步骤：
将本发明第三方面所述方法制备的基因工程的植物组织或植物细胞再生为植物体，从而获得转基因植物。
本发明第五方面提供了一种转基因植物，所述的植物是用本发明第四方面所述的方法制备的。
本发明第六方面提供了一种生产粮食的方法，包括步骤：
(i)种植农作物，所述农作物中PYL基因家族中的N个成员发生突变，其中N≥2；
(ii)收获所述农作物的粮食(谷物)。
在另一优选例中，所述农作物选自下组包括禾本科，豆科以及十字花科植物，更优选地包括水稻、玉米、高粱、小麦、或大豆。
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显示了水稻PYL蛋白序列的进化树分析。Group I，I类基因；group II， II类基因。
图2显示了I类PYL基因突变促进水稻生长。a，水稻PYL多基因敲除策略。b，野生型和I类基因突变体苗期对比图。标尺，10cm。c，野生型和I类基因突变体幼苗苗长和鲜重。每一个柱子代表一个独立的株系。“+”表示野生型基因，“-”表示突变体基因。“***”,与野生型差异的P值小于0.001；“**”,与野生型差异的P值小于0.01；“*”,与野生型差异的P值小于0.05。P值采用Student t-检验得出。d，成熟期野生型、pyl1/4/6和pyl1/2/3/4/5/6对比图。标尺，10cm。e，灌浆期野生型、pyl7/8/9/13和pyl7/8/9/10/11/13对比图。标尺，10cm。f，野生型和pyl1/4/6茎杆和穗子对比图。标尺，10cm。g，野生型、pyl1/4/6和pyl1/2/3/4/5/6倒二节间直径。P值采用Student t-检验得出，显示与野生型相比的显著性。h，野生型和I类基因突变体抽穗期对比图。标尺，10cm。
图3显示了I类基因突变体表型鉴定。a，野生型、pyl1/4/6和pyl1/2/3/4/5/6苗期对比图。标尺，10cm。b，灌浆初期野生型和pyl1/2/3/4/5/6对比图。标尺，10cm。c，成熟期I类基因植株高度。每一个柱子代表一个独立的株系。“+”表示野生型基因，“-”表示突变体基因。“***”,与野生型差异的P值小于0.001。P值采用Student t-检验得出。d，野生型和pyl1/4/6穗长和节间长度对比数据。左边柱状图表示每一节间的长度和穗长；右边柱状图表示穗长和每一节间长度占株高的比例。e，野生型和pyl1/4/6穗长和节间长度对比。标尺，10cm。
图4显示了野生型和I类基因突变体对高温天气的敏感性。a，野生型和I类基因突变体经历2016年上海高温气候后的表型对比。标尺，10cm。b，经历2016年上海高温气候的野生型和I类基因突变体成熟期对比图。标尺，10cm。
图5显示了水稻PYL基因突变对种子休眠的影响。a，I类基因突变体种子PHS图。标尺，10cm。b，野生型和I类基因突变体种子收获前萌发(pre-harvest sprouting，PHS)频率。在pyl1/2/3/4/5/6/12灌浆期末期统计所有数据，统计工作一天内完成。每一个柱子代表一个独立的株系，每个株系统计3个植株的所有种子。“+”表示野生型基因，“-”表示突变体基因。“***”,与野生型差异的P值小于0.001。“P＝0.104173”和“P＝0.02361”表示与野生型差异的P值分别为0.104173和0.02361。P值采用Student t-检验得出。c，野生型和II类基因突变体种子PHS频率。种子收获被延迟大约25天后统计PHS 频率。每一个柱子代表一个独立的株系，每个株系统计3个植株的所有种子。“***”,与野生型差异的P值小于0.001。
图6显示了野生型和pyl1/4/6产量性状分析和测试。a，野生型和pyl1/4/6主穗对比图。标尺，5cm。b，野生型和pyl1/4/6主穗长度数据。“***”，与野生型差异的P值小于0.001。c，野生型和pyl1/4/6种子千粒重数据。d，野生型和pyl1/4/6分蘖数统计数据。“**”，与野生型差异的P值小于0.01。e，野生型和pyl1/4/6主穗一次分枝数据。“***”，与野生型差异的P值小于0.001。f，野生型和pyl1/4/6主穗二次分枝数据。“***”，与野生型差异的P值小于0.001。g，野生型和pyl1/4/6主穗小花数。“***”，与野生型差异的P值小于0.001。h，野生型和pyl1/4/6成熟期生物量数据。“***”，与野生型差异的P值小于0.001。i，野生型和突变体小区产量试验数据。“**”，与野生型差异的P值小于0.01。
图7显示了2016年上海pyl1/4/6产量试验效果图。
具体实施方式
本发明人经过广泛而深入地研究，首次意外地发现，对PYL基因家族的N个成员(N≥2)进行敲除，居然可以显著改良植物的某些性状，例如提高植物(如水稻)的产量。具体地，本发明首次利用基因敲除技术(如CRISPR/Cas9多基因编辑技术)对水稻PYL基因进行了敲除研究，发现通过对不同水稻PYL基因家族成员的敲除，可以促进水稻的生长或改良某些所需的农艺性状，其中，PYL1、PYL4和PYL6的同时敲除表现出最好的生长状态和农艺性状，可大幅提高水稻产量。在此基础上，本发明人完成了本发明。
PYL基因
PYL是ABA受体蛋白编码基因，PYL以基因家族的形式存在。已有的研究表明，PYL家族的基因有高度的保守性，并且对于植物的生长而言(尤其是抗逆性)是至关重要的。本发明的研究提示，对单个PYL基因的改造(如敲除或下调)，不会表现出对植物性状的改善。
在本发明中，可对任何植物品种的PYL基因的N个基因(N≥2)进行敲除，代表性的植物包括，但并不限于：林业植物、农用植物，例如禾本科、十字花科、豆科等，如水稻、玉米、高粱、小麦、大豆、或其组合。
应理解，不同的植物中可能含有多个PYL基因(即来自PYL家族的多个基因)。在本发明中，所述的PYL基因包括来自所述植物(或物种)的全部已知的PYL基因，以及将来可能发现的PYL基因，以及与这些PYL基因具有同源性的同源基因。其中，所述的“具有同源性”指两个序列之间具有≥70％，较佳地≥80％，更佳地≥90％，最佳地≥95％相同性。
在其他植物中，一般地，PYL基因的同源基因的通用名称为PYL基因，也可在PYL基因名称前加物种拉丁名缩写。比如，小麦PYL基因也称为TaPYL；玉米PYL基因也称为ZmPYL；在大豆PYL基因也称为GmPYL。
以水稻为例，已知至少有13个PYL基因，即PYL1、PYL2、PYL3、PYL4、PYL5、PYL6、PYL7、PYL8、PYL9、PYL10、PYL11、PYL12、PYL13。在本发明中，两个或多个PYL基因可合并表述，例如“pyl1/4/6”表示pyl1、pyl4和pyl6。
在一优选实施方式中，所述水稻PYL基因的的敲除类型如下所示：pyl1/4/6、pyl1/6。
改良植物的方法
在本发明中，还提供了一种改良植物的方法，包括步骤：
(i)对植物细胞或植物组织进行基因工程改造，从而使得PYL基因家族中的N个成员发生突变，其中N≥2；
(ii)将经基因工程改造的植物细胞或植物组织再生成植株，对再生的植株进行性状测试，所述性状选自下组：株高、抽穗期、种子休眠、产量、或其组合；
(iii)根据性状测试结果，选出具有所需性状特征的植株。
在本发明中，所述突变包括降低PYL基因家族中N个成员的表达或活性。
在一优选实施方式中，所述“降低”是指将PYL基因家族中N个成员的表达或活性降低满足以下条件：
A1/A0的比值≤80％，较佳地≤60％，更佳地≤40％，最佳地为0-30％；
其中，A1为PYL基因家族中N个成员的表达或活性；A0为野生型同种类型植物植株中相同PYL基因家族中N个成员的表达或活性。
在一优选实施方式中，所述的降低指与野生型PYL家族的成员的表达水平E0相比，所述植株中PYL家族的成员的表达水平E1为野生型的0-80％，较佳地0-60％，更佳地0-40％。
在一优选实施方式中，所述的降低植株中PYL基因家族中N个成员的表达或 活性通过选自下组的方式实现：基因突变、基因敲除、基因中断、RNA干扰技术、Crispr技术、或其组合。
在本发明中，根据性状测试结果，排除性状不好的植株。
在本发明中，还进一步包括步骤(iv)，对步骤(iii)选出的具有所需形状特征的植株作进一步的筛选，从而筛选出能够平衡株高、产量、生物量、种子休眠、抽穗期、失水性能等性状的植株，所述植株的综合性状表现最优。
生产粮食的方法
本发明还提供了一种生产粮食的方法，包括步骤：
(i)种植农作物，所述农作物中PYL基因家族中的N个成员发生突变，其中N≥2；
(ii)收获所述农作物的粮食(谷物)。
本发明的主要优点包括：
(a)本发明首次发现通过对不同植物(如水稻)PYL基因家族成员的敲除，可以显著地促进植物生长,提高产量。
(b)本发明首次对不同植物(如水稻)PYL基因家族成员的敲除后的性状进行测试，挑出具有所需性状特征(生物量增加、产量增加)的植株。
(c)本发明首次发现PYL1、PYL4和PYL6的同时敲除可表现出最好的生长状态和农艺性状，可大幅提高水稻产量。
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，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)或植物分子生物学-实验手册(Plant Molecular Biology-A Laboratory Mannual,Melody S.Clark编,Springer-verlag Berlin Heidelberg,1997)中所述的条件，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。
实施例1.水稻PYL基因家族成员的多基因敲除
为敲除PYL基因家族，我们构建了一套CRISPR/Cas9多基因敲除体系。在该体系中，Cas9由玉米Ubiquitin启动子介导表达；四个sgRNA分别由OsU3-1、OsU6-1、OsU3-2、OsU6-2启动子介导表达(启动子序列见序列1到序列4)，四个sgRNA表达盒在载体上串联排列。为特异性地靶向水稻PYL基因，我们合成了特异识别靶基因的引物，其包含20bp的目标识别序列和4-5bp的标签序列。引物退火后形成带黏性末端的双链DNA(20bp双链区)。该双链DNA与启动子及下游序列在T4连接酶作用下无缝连接，从而构建sgRNA表达盒。串联排列的sgRNA表达盒先被构建于PUC19中间载体，然后被亚克隆于带Cas9表达盒的PCAMBIA1300骨架。
我们构建了两个多基因靶向的CRISPR/Cas9载体(靶位点序列见表1)，每个载体靶向水稻PYL进化树上的一簇基因，一个载体靶向I类(group I)基因(PYL1–PYL6和PYL12)，另一个载体靶向II类(group II)基因(PYL7–PYL11和PYL13)(图1和图2a)。为单独敲除每一个PYL基因，我们针对每一个靶基因设计了一个特异性靶位点(表2)，构建了13个单基因靶向的载体。
通过农杆菌介导的转化方法，我们将载体转化进了水稻品种日本晴。到T2代，获得了153个I类基因突变株系(A1–A108来自多基因敲除系，A109–A153来自单基因敲除系)和84个II类基因突变株系(B6–B51来自多基因敲除系，B52–B89来自单基因敲除系)(附件1)。在多基因编辑系中，我们未获得pyl7/8/9/10/11/13突变体。为获得pyl7/8/9/10/11/13，我们将pyl7/8/9/10/13与pyl11杂交，在F2代分离群体中鉴定出5个纯合的pyl7/8/9/10/11/13(B1–B5)。为确保多基因编辑系基因型的稳定性，我们对每一代的植株进行了基因型鉴定，挑选基因型稳定或不含Cas9的植株进行种子收获和表型鉴定。
表1.水稻PYL多基因编辑的靶位点序列
Figure PCTCN2018109061-appb-000001
Figure PCTCN2018109061-appb-000002
表2.水稻PYL单基因编辑的靶位点序列
Figure PCTCN2018109061-appb-000003
实施例2.水稻PYL亚家族基因突变促进水稻生长
本研究获得的纯合突变体材料于每年6月中旬和12月下旬被分别播种于 上海市和海南省陵水黎族自治县中科院植物生理生态研究所水稻转基因基地。大田表型观测发现所有基因的单突变系和II类基因突变系与野生型无明显形态差异(图2e)，而许多I类基因突变体表现出比野生型更好的生长状态(图2b和2d)。在幼苗期，从pyl1/6到pyl1/2/3/4/5/6/12，大多数突变体比野生型长势旺盛，生长势强，特别是pyl1/4/6三突变体生长状态最好(图2b和3a)。为对比不同突变类型和野生型的生长差异，我们统计了苗期的株高和鲜重(图2c)。统计结果显示pyl1/4/6具有最大的株高和鲜重，表明PYL1、PYL4和PYL6在水稻生长抑制上发挥着非常重要的作用。双突变体(pyl1/6、pyl1/4、pyl1/2和pyl1/12)中，唯有pyl1/6比野生型形体大，其他的与野生型无明显差异；三突变体(pyl1/4/6、pyl1/2/4、pyl1/4/5和pyl1/3/4)中，唯有pyl1/4/6比野生型形体大，其他的与野生型无明显差异；从统计数据中，我们还发现，从三突变体到七突变体，含pyl6的突变系比其他株系生长得好，表明PYL6在水稻生长抑制上发挥着关键的作用(图2c)。
成熟期，I类突变体继续表现出比野生型更强壮的株型(图2d和图3b)。为对比不同突变体与野生型在成熟期的差异，我们测量了各种突变体的株高。成熟期的株高数据呈现出与苗期数据相似的模式，pyl1/4/6在所有I类基因突变体中植株最高，生长最好(图2d和图3c)。
在2016年上海，从7月中旬开始，高温的气候(中午最高温度大约40℃)持续了至少两周。经历这种高温天气，我们发现从四突变体到七突变体，I类基因突变材料表现出不同程度的生长停滞，突变越多，生长停滞越严重；而这期间pyl1/4/6仍然比野生型高大(图4a)。抽穗期后，六突和六突以下的突变体恢复了较大的生长表型，七突变体呈现出与野生型相似的株高(图4b)。II类基因突变体在高温期间及之后与野生型无明显的表型差异。
在成熟期，我们还测量了突变体的茎杆直径。发现与野生型相比，pyl1/2/3/4/5/6和pyl1/4/6的茎杆较粗(图2f和2g)。我们还测量了野生型和pyl1/4/6的茎节长度。与野生型相比，pyl1/4/6的穗长和每一节间都增长了，特别是下部的节间增长幅度大(图3d和3e)。
以上表型鉴定结果表明I类PYL基因突变可促进水稻生长，其中PYL1、PYL4和PYL6同时突变对生长促进效果最好。
实施例3.水稻PYL基因对抽穗期的影响
抽穗期影响水稻的地理分布和对季节的适应性。在本研究中，我们发现在I类基因突变体中，从四突变体开始，突变株系的抽穗期被明显延迟(图2h)。与野生型相比，pyl1/2/3/4/5/6和pyl1/2/3/4/5/6/12的抽穗期被延迟了大约9天，pyl1/2/3/4/6被延迟了大约7天，pyl1/2/3/4被延迟了大约5天，pyl1/4/6被延迟了大约1天。I类基因的单突变体、双突变体及其他三突变体的抽穗期与野生型无明显差异。II类基因突变体抽穗期与野生型无差异。
实施例4.水稻PYL基因对种子休眠的影响
ABA促进种子休眠，因此我们调查了突变体种子休眠的情况。种子休眠的缺陷会导致种子在收获前萌发(pre-harvest sprouting，PHS)。在I类基因突变体中，我们观察到了明显的PHS(图5a)。为对比不同突变体种子休眠的缺陷，我们在2016年的田间统计了突变体和野生型的PHS。结果显示在所有的I类基因突变体中，pyl1/2/3/4/5/6/12的PHS频率最高；在所有单突变体中，只有pyl1和pyl12展现出比野生型明显高的PHS频率，表明PYL1和PYL12在种子休眠上发挥着关键的作用(图5b)。该统计数据还展示了一个令人吃惊的现象，pyl1/6的PHS频率比pyl1低，pyl1/4/6的PHS频率比pyl1和pyl1/4低，表明PYL6与其他I类基因在种子休眠方面可能存在拮抗作用(图5b)。实际上从I类双突变体到七突变体，pyl1/4/6展现出最低的PHS频率，其PHS频率几乎近似于野生型的水平(两个独立株系与野生型比较的P值分别为0.104173 and 0.02361)(图5b)。在其他年份和季节，我们在pyl1/4/6上未观察到明显的PHS。
在正常的收获时间，在II类基因的突变体上未观察到明显的PHS，但是当我们将收获时间延迟大约25天时，pyl7/8/9/10/13和pyl7/8/9/10/11/13展现出比野生型略微高的PHS频率(图5c)，表明水稻II类基因也在种子休眠上发挥着调节作用。
实施例5.pyl1/4/6突变提高水稻产量试验
以上研究结果显示在众多的pyl突变体中，pyl1/4/6表现出最好的生长状态、正常的种子休眠和抽穗期，这些结果表明pyl1/4/6可能具有潜在的应用价值。因此，我们仔细调查了pyl1/4/6的农艺性状，发现pyl1/4/6穗的着粒密度与野生型无明显差异，但是相对于野生型，pyl1/4/6具有更长的穗型(图 6a和6b)。pyl1/4/6主穗的一次分枝和二次分枝的数目比野生型明显多(图6e和6f)，因此主穗小花数目比野生型多(图6g)。成熟期pyl1/4/6的生物量比野生型增加约55.3％，尽管其分蘖数比野生型明显减少(图6d和6h)。pyl1/4/6种子的千粒重与野生型无明显差异(图6c，P＝0.230034)。
接下来，我们做了田间小区块测产试验(图7)。野生型和突变体被种植于交互排列的小区块中。在每一个小区块中，植株间距固定在15cm，共12行12列。每个材料设3个小区重复。2016年上海测产结果显示与野生型相比，pyl1/4/6将籽粒产量提高了25％(野生型小区块产量，1522.4±36.3g；pyl1/4/6小区块产量，1899.8±30.8g)。2017年海南测产结果pyl1/4/6的产量比野生型提高了31％(野生型小区块产量，1317.5±25.7g；pyl1/4/6小区块产量，1727.1±34.9g)。
以上研究结果表明，pyl1/4/6突变可以大幅度提高水稻产量。

Claims (10)

  1. 一种改良植物的方法，其特征在于，包括步骤：
    (i)对植物细胞或植物组织进行基因工程改造，从而使得PYL基因家族中的N个成员发生突变，其中N≥2；
    (ii)将经基因工程改造的植物细胞或植物组织再生成植株，对再生的植株进行性状测试，所述性状选自下组：株高、抽穗期、种子休眠、产量、生物量、或其组合；
    (iii)根据性状测试结果，选出具有所需性状特征的植株。
  2. 如权利要求1所述的方法，其特征在于，所述突变包括降低PYL基因家族中N个成员的表达或活性。
  3. 如权利要求1所述的方法，其特征在于，所述的植物包括农作物、林业植物、花卉；优选地包括禾本科，豆科以及十字花科植物，更优选地包括水稻、玉米、高粱、小麦、或大豆。
  4. 如权利要求1所述的方法，其特征在于，所述基因工程改造包括用多个sgRNA介导的Cas9核酸酶对PYL基因家族的成员进行基因编辑。
  5. 如权利要求4所述的方法，其特征在于，所述基因编辑包括对选自下组的PYL基因家族的基因进行基因编辑：PYL1、PYL2、PYL3、PYL4、PYL5、PYL6、PYL12、或其组合。
  6. 一种基因工程的植物组织或植物细胞，其特征在于，所述植物组织或植物细胞中的PYL基因家族中的N个成员发生突变，其中N≥2。
  7. 一种制备基因工程的植物组织或植物细胞的方法，其特征在于，包括步骤：
    将植物组织或植物细胞中的PYL基因家族中的N个成员发生突变，从而获得基因工程的植物组织或植物细胞，其中N≥2。
  8. 一种制备转基因植物的方法，其特征在于，包括步骤：
    将权利要求7所述方法制备的基因工程的植物组织或植物细胞再生为植物体，从而获得转基因植物。
  9. 一种转基因植物，其特征在于，所述的植物是用权利要求8所述的方法制备的。
  10. 一种生产粮食的方法，其特征在于，包括步骤：
    (i)种植农作物，所述农作物中PYL基因家族中的N个成员发生突变，其中N≥2；
    (ii)收获所述农作物的粮食(谷物)。
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