WO2024031987A1 - 植物耐盐碱基因及其用途 - Google Patents

植物耐盐碱基因及其用途 Download PDF

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WO2024031987A1
WO2024031987A1 PCT/CN2023/081663 CN2023081663W WO2024031987A1 WO 2024031987 A1 WO2024031987 A1 WO 2024031987A1 CN 2023081663 W CN2023081663 W CN 2023081663W WO 2024031987 A1 WO2024031987 A1 WO 2024031987A1
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plant
seq
amino acid
similar
acid sequence
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PCT/CN2023/081663
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English (en)
French (fr)
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谢旗
于菲菲
张会丽
欧阳亦聃
张启发
张华伟
孙文静
孙生远
李旭
朱瑶瑶
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中国科学院遗传与发育生物学研究所
华中农业大学
北京大学现代农业研究院
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Priority claimed from CN202210968618.1A external-priority patent/CN116063425A/zh
Priority claimed from CN202211198322.2A external-priority patent/CN116162142B/zh
Application filed by 中国科学院遗传与发育生物学研究所, 华中农业大学, 北京大学现代农业研究院 filed Critical 中国科学院遗传与发育生物学研究所
Publication of WO2024031987A1 publication Critical patent/WO2024031987A1/zh

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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
    • 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
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • 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)

Definitions

  • the present invention relates to the plant salt-alkali tolerance related gene AT1 and its homologous genes and their use in cultivating salt-alkali tolerance plants.
  • the present invention also relates to a method for cultivating salt-alkali-tolerant plants, the obtained salt-alkali-tolerant plants and plant materials thereof.
  • Soil salinization and secondary salinization are global ecological and resource problems and one of the important abiotic stress factors causing crop yield reduction (Yamaguchi, T. and Blumwald, E. Developing salt-tolerant crop plants: challenges and opportunities.(2005).Trends in Plant Science 10:615-620).
  • Salinized soil generally refers to a type of soil affected by salinity, including saline soil and alkaline soil.
  • Saline soil refers to soil with a content of soluble salt substances in the soil exceeding 2 ⁇
  • alkaline soil refers to soil with a proportion of exchangeable sodium ions to soluble cations (ESP) greater than 20% and a pH value greater than 8.0 (Yang Jinsong. (2008). Saline in China The development history and prospects of soil research. Journal of Soil Science 45:837-845).
  • crops are harmed by varying degrees of saline-alkali stress throughout the growth period. Especially in spring, the surface will "return to salt".
  • alkaline saline soils have a high pH that reduces the rate of plant uptake of essential nutrients and the excretion of sodium ions (Na + ).
  • Alkaline saline soils have more negative effects on plant growth than salinity alone by inducing high cellular oxidative stress (M.Javid, R.Ford, MENicolas, Tolerance responses of Brassica juncea to salinity,alkalinity and alkaline salinity. Funct. Plant Biol. 39, 699-707(2012). doi:10.1071/FP12109).
  • the purpose of the present invention is to study the alkali/saline-alkali tolerance mechanism of plants at the genetic level, develop methods for cultivating salt-alkali-tolerant plants, and cultivate plants with higher saline-alkali tolerance.
  • AT1 an atypical G protein gamma subunit (G ⁇ subunit)
  • G ⁇ subunit an atypical G protein gamma subunit
  • the N-terminal domain of AT1 and its homologs plays a negative regulatory role in alkaline stress tolerance.
  • Crops containing C-terminal truncated AT1 protein are highly sensitive to alkali stress. This may be due to the inhibitory effect of the C-terminal domain, which is necessary for protein degradation in its rice homolog (S. Sun, L. Wang, H. Mao, L. Shao, X.
  • the present invention provides the use of AT1 or its homologous gene in regulating the salt-alkali tolerance of plants or cultivating plants with salt-alkali tolerance or salt-alkali sensitivity.
  • the AT1 or homologous gene thereof comprises a GGL domain or a GGL-like domain comprising at least 40% identity to SEQ ID NO: 15, preferably at least About 75% identical, more preferably at least about 80% identical, or more preferably at least about 90% or about 95% identical, or at least about 60% similar, preferably at least about 70%, about 80% or about 85% identical. % similarity, more preferably at least about 90% or about 95% similarity in amino acid sequences.
  • the GGL domain or GGL-like domain comprises SEQ ID NO: 15 and 86- Any amino acid sequence among 96.
  • increasing the expression level (e.g., overexpression) of AT1 or its homologous gene in a plant, or expressing or overexpressing a C-terminal truncated protein encoded by AT1 or its homologous gene variant can improve plant salt-alkali sensitivity.
  • the expression levels of all alleles of AT1 or its homologous genes are reduced in plants (e.g., by gene editing, targeted mutagenesis, chemical induction, radiation induction, natural mutation, RNAi, or addition Substances that inhibit the expression of target genes) or prevent the expression of all alleles can improve the salt-alkali tolerance of plants.
  • the growth of the salt-alkali tolerant plants of the present invention is better than that of wild-type plants under saline-alkaline conditions, wherein the saline-alkaline conditions include growth conditions of pH>7.5 and Na + concentration>75mM or pH>8.0 and Na + Growth conditions at concentrations >50mM.
  • knocking out the N-terminal GGL domain or GGL-like domain of all alleles of AT1 or its homologous genes in plants, or knocking out the N-terminal first exon can improve the tolerance of plants. Salinity.
  • the N-terminal GGL domain or GGL-like domain of AT1 or its homologues is conserved.
  • the invention provides a nucleic acid molecule encoding a nucleic acid molecule that is at least 40% identical to SEQ ID NO: 15, preferably at least about 75% identical, more preferably at least about 80% identical, or more preferably at least Amino acids that are about 90% or about 95% identical, or have at least about 60% similarity, preferably at least about 70%, about 80% or about 85% similarity, more preferably at least about 90% or about 95% similarity.
  • the protein sequence is used to regulate the salt-alkali tolerance of plants, or to breed plants with salt-alkali tolerance or salt-alkali sensitivity.
  • the nucleic acid molecule encodes a protein comprising the amino acid sequence of any one of SEQ ID NO: 15 and 86-96.
  • the nucleic acid molecule encodes an amino acid sequence selected from:
  • the present invention also provides a mutant protein, which is encoded by a variant nucleic acid molecule after a frameshift mutation occurs in a nucleic acid molecule encoding the following amino acid sequence:
  • the activity of the mutated protein is reduced or inactive compared with the protein encoded before the frameshift mutation occurs in the nucleic acid molecule.
  • the frameshift mutation includes an insertion or deletion of one or more (other than 3 or a multiple of 3) nucleotides.
  • the frameshift mutation can occur anywhere in the coding sequence of interest.
  • the invention also provides nucleic acid molecules encoding the mutant proteins.
  • the invention provides an expression cassette comprising a nucleic acid molecule of the invention.
  • the invention provides a recombinant vector comprising a nucleic acid molecule or expression cassette of the invention.
  • the invention provides a cell comprising the nucleic acid molecule of the invention, expressing cassette or recombinant vector.
  • the cells are selected from prokaryotic cells or eukaryotic cells, such as bacterial cells or fungal cells, such as, but not limited to, E. coli cells, yeast cells, or Agrobacterium cells; the eukaryotic cells Nuclear cells such as plant cells.
  • prokaryotic cells or eukaryotic cells such as bacterial cells or fungal cells, such as, but not limited to, E. coli cells, yeast cells, or Agrobacterium cells
  • the eukaryotic cells Nuclear cells such as plant cells.
  • the present invention provides a method for cultivating salt-alkali tolerant plants, the method comprising:
  • the encoding in the reduced plant includes at least 40% identity, preferably at least about 75% identity, more preferably at least about 80% identity, or more preferably at least about 90% or about 95% identity, or to expression levels of all alleles of a protein having an amino acid sequence that is less than about 60% similar, preferably at least about 70%, about 80% or about 85% similar, more preferably at least about 90% or about 95% similar, or rendering all said alleles inexpressible,
  • salt-alkali tolerant plants grow better than wild-type plants under saline-alkali conditions
  • the saline-alkaline conditions include growth conditions of pH>7.5 and Na + concentration>75mM or growth conditions of pH>8.0 and Na + concentration>50mM.
  • the expression levels of all alleles in the plant are reduced or reduced by gene editing methods, targeted mutagenesis, chemical induction, radiation induction, natural mutation, RNAi, or adding substances that inhibit the expression of the gene of interest. None of the alleles are expressed.
  • all of the alleles in the plant are knocked out or mutated, e.g., by knocking out the GGL domain or GGL-like domain of the gene by homologous recombination, or by editing the gene by CRISPR technology. GGL domain or GGL-like domain.
  • the N-terminal first exon portion of all alleles is knocked out or mutated, resulting in reduced or no activity of the encoded protein.
  • the GGL domain or GGL-like domain, or the N-terminal first exon portion of all alleles is knocked out or mutated, resulting in reduced or no activity of the encoded protein.
  • the expression level of the gene is reduced by at least 51%, preferably by 60%, 70% or 80%, more preferably by 85%, 90% or 95%, or even not compared to wild-type control plants. Express.
  • the plant is a monocotyledonous or dicotyledonous plant, e.g., a grass plant, such as, but not limited to, Sorghum (e.g., sorghum), Oryza (e.g., rice), millet, corn, wheat or soybeans.
  • a grass plant such as, but not limited to, Sorghum (e.g., sorghum), Oryza (e.g., rice), millet, corn, wheat or soybeans.
  • the method further includes identifying a parent plant that contains a knockout or mutation of all of the alleles, or a non-functional allele, and selfing or mating with another knockout that contains the gene. or mutation or One or more generations of progeny plants resulting from a cross between parent plants containing non-functional alleles, wherein knockout or mutation of all alleles results in reduced or no activity of proteins encoded by all alleles.
  • the GGL domain or GGL-like domain, or the N-terminal first exon portion is knocked out or mutated in all alleles of a gene encoding an amino acid sequence selected from:
  • the salt-alkali tolerant plants cultivated by the method of the present invention have improved survival rate, increased yield, and increased plant height under saline-alkaline growth conditions compared with the corresponding wild-type control. Or fresh weight.
  • the saline-alkaline growth conditions include culture conditions with pH>7 and Na + concentration>50mM.
  • the invention provides a plant or plant material, wherein the code comprises at least 40% identity, preferably at least about 75% identity, more preferably at least about 80% identity or more preferably with SEQ ID NO:15 At least about 90% or about 95% identical, or at least about 60% similar, preferably at least about 70%, about 80% or about 85% similar, more preferably at least about 90% or about 95% similar. All alleles of the protein of the amino acid sequence are knocked out or mutated, preferably, the GGL domain or GGL-like domain, or the N-terminal first exon part of all alleles of the gene is knocked out or mutation,
  • the knockout or mutation of all alleles makes the activity of the protein encoded by all alleles reduced or inactive.
  • the plant or plant material comprises a non-functional allele of the gene.
  • the plant is a monocotyledonous or dicotyledonous plant, e.g., a grass plant, such as, but not limited to, Sorghum (e.g., sorghum), Oryza (e.g., rice), millet, corn, wheat or soybeans.
  • a grass plant such as, but not limited to, Sorghum (e.g., sorghum), Oryza (e.g., rice), millet, corn, wheat or soybeans.
  • the plant material is a plant part, plant organ, plant tissue, seed, plant protoplast or plant cell, e.g., embryo, pollen, ovule, seed, leaf, flower, branch, fruit, stem, Roots, root tips, anthers, plant cell cultures or plant callus.
  • the present invention provides a method for preparing hybrid plant seeds, the method comprising:
  • the code comprises at least 40% identical to SEQ ID NO: 15, preferably at least about 75% Identity, more preferably at least about 80% identity, or more preferably at least about 90% or about 95% identity, or at least about 60% similarity, preferably at least about 70%, about 80% or about 85% similarity. , more preferably all alleles of the protein having at least about 90% or about 95% similarity in amino acid sequence are knocked out or mutated; and
  • the first parent plant and/or the second parent plant comprise a non-functional version of the gene, etc. bit gene.
  • the first parent plant and/or the second parent plant are inbred plants.
  • the present invention also provides a method for preparing conventional planting seeds, which method includes:
  • Propagating a parent seed to harvest its progeny seed wherein in the parent seed, the encoding includes at least 40% identity, preferably at least about 75% identity, more preferably at least about 80% identity with SEQ ID NO: 15, or More preferably at least about 90% or about 95% identical, or at least about 60% similar, preferably at least about 70%, about 80% or about 85% similar, more preferably at least about 90% or about 95% similar. All alleles of a protein with a specific amino acid sequence have been knocked out or mutated such that the protein is not expressed or has a reduced expression level compared to wild-type plants.
  • all alleles encoding alleles comprising the following amino acid sequence have been knocked out or mutated:
  • the invention provides a plant or plant material thereof grown from the seed of the fourth aspect.
  • the present invention provides a method for cultivating salt-alkali sensitive plants, the method comprising:
  • genes in the plant encoding a protein that is at least 40% identical to SEQ ID NO: 15, preferably at least about 75% identical, more preferably at least about 80% identical, or more preferably at least about 90% or about 95% identical, or to Expression levels of genes of proteins having amino acid sequences that are less than about 60% similar, preferably at least about 70%, about 80% or about 85% similar, more preferably at least about 90% or about 95% similar, or in plants Express or overexpress the C-terminal truncated protein encoded by the gene mutant.
  • the exogenous code comprising at least 40% identity to SEQ ID NO: 15, preferably at least about 75% identity, more preferably at least about 80% identity, or more preferably at least about 90% identity to SEQ ID NO: 15. % or about 95% identity, or to an amino acid sequence having at least about 60% similarity, preferably at least about 70%, about 80% or about 85% similarity, more preferably at least about 90% or about 95% similarity.
  • Protein nucleic acid molecules increase the expression level of the target gene.
  • genetic material carrying the nucleic acid molecule is introduced into the cells or tissues of the plant, and the genetic material is present in the plant in the form of free or integrated into the chromosome of the plant, and then The cells or tissues introduced with the genetic material are cultured into complete plants to obtain the salt-alkali sensitive plants.
  • the saline-alkaline conditions include growth conditions of pH>7.5 and Na + concentration>75mM or growth conditions of pH>8.0 and Na + concentration>50mM.
  • Figure 1 Evaluation conditions and phenotypic variation for salt-alkali tolerance identification of different varieties of sorghum populations.
  • A Analysis of the survival rates of 16 sorghum varieties under different concentrations of salt-alkali stress during the germination stage;
  • B shows the relative survival rate (RSR) analysis of 16 sorghum varieties under alkali stress for 5 consecutive weeks;
  • C treated with alkali stress 3 Phenotypic observations of representative sorghum varieties treated with or without alkali stress treatment (i.e., CK, control) for weeks.
  • RSR relative survival rate
  • the scale bar in the figure represents 5cm;
  • D The number of sorghum accessions from the sorghum association panel (SAP) with different relative survival rates on day 21 after treatment with 75mM mixed alkali solution ;
  • E Relative survival rate of 352 grain sorghum natural population materials under control (CK) and alkali treatment conditions (75mM mixed alkali treatment). Varieties with low germination rate ( ⁇ 80%) in the control were removed during counting.
  • F QQ-plot analysis of salt-alkali tolerance in SAP population.
  • FIG. 1 Phenotypic changes in sorghum treated with different saline-alkali levels.
  • A Statistical analysis of the seedling growth rate of 16 test materials under mixed alkali stress conditions of different concentrations
  • B Relative survival rate analysis of 16 test materials under 75mM mixed alkali stress and control conditions for different days.
  • CK represents the survival rate under control conditions
  • T represents the survival rate under 75mM mixed alkali stress.
  • Viability, T_CK represents the relative survival rate.
  • FIG. 3 Natural variation of SbAT1 gene related to salt-alkali tolerance in sorghum.
  • A Manhattan plot of a genome-wide association study (GWAS) of alkali tolerance in sorghum natural populations. Sex versus survival rates were obtained from sorghum plants sown and grown for 21 days with (alkali stress) and without (control) the addition of 75mM mixed alkali solution. The arrow in the figure marks the main locus of the SbAT1 gene.
  • B Scatter plot of the approximately 10 Mb genomic region before and after the SbAT1 locus on chromosome 1.
  • n the number, and the statistical P value was determined by a two-tailed unpaired t test.
  • F Relative expression levels of SbAT1 in alkali-tolerant (T) and alkali-sensitive (S) sorghum plants under 5- and 8-day stress of 75 mM mixed alkali treatment and under corresponding control conditions (CK). Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test. NS indicates no significance.
  • Figure 4 Schematic diagram of the protein structure of sorghum AT1 and its mutant at1.
  • Phenotypes of sorghum NIL parental lines and SbAT1 transgenic plants under alkali stress and/or salt stress treatment Phenotypes of NIL parental lines SN010 (NIL-AT1) and M-81E (NIL-at1) under alkali stress. SN010 (NIL-AT1) and M-81E (NIL-at1) seeds were sown in soil without or with 75mM mixed alkali and photographed 17 days later. The scale bar indicates 5cm.
  • B and C Relative survival rate (B) and relative plant height
  • C statistics of SN010 (NIL-AT1) and M-81E (NIL-at1) under alkali treatment compared with no alkali treatment (CK) analyze.
  • E and F Statistical analysis of relative survival rate (B) and relative plant height (C) of SN010 (NIL-AT1) and M-81E (NIL-at1) under the salt treatment of (D).
  • FIG. 6 Function of SbAT1 in alkaline tolerance of sorghum.
  • A Schematic representation of the proteins of SbAT1 and its truncated form Sbat1 in sorghum NIL-AT1 and NIL-at1 plants.
  • B Phenotypic analysis of sorghum NIL seedlings under alkali stress. Photographs were taken on the 14th day after seed sowing (CK, no alkali stress; 75mM mixed alkali), and the scale bar indicates 5cm.
  • C Statistical analysis of relative survival rate of seedlings in (B).
  • Figure 7 Field performance of near-isogenic sorghum lines sown in the saline-alkali soil of Ningxia, northwest China.
  • FIG. 8 Soil physical and chemical properties and corresponding seedling establishment rate statistics of near-isogenic line materials on saline-alkali soils in different areas of Ningxia, China.
  • (A) and (B) are the soil physical and chemical properties of the saline-alkali land in Huiwei Village and Dongfeng Village, Ningxia, respectively.
  • C and D are the statistical analysis data of the survival rate of near-isogenic line materials in the saline-alkali land in Huiwei Village and Dongfeng Village, Ningxia, respectively.
  • Figure 9 Construction and gene editing identification results of sorghum overexpression transgenes and mutants.
  • FIG. 1 Construction of millet mutants and identification results of gene editing materials.
  • FIG. 11 Concentration screening for phenotypic identification of transgenic sorghum and millet.
  • A The growth of transgenic sorghum and millet genetic materials on the 14th day after control (CK) and different mixed alkali stress treatments
  • B Statistical analysis of the survival rate of transgenic sorghum and millet genetic materials under different mixed alkali stress conditions, The upper picture shows the statistical analysis of the survival rate of transgenic sorghum, and the lower picture shows the statistical analysis of the survival rate of transgenic millet.
  • FIG. 12 AT1 homolog G ⁇ -like subunit has conserved functions in alkali tolerance in millet, rice and maize.
  • A Schematic representation of millet SiAT1 and its truncated or non-functional versions in SiAT1 genetic plants. SiWT represents wild-type millet Ci846.
  • B Photographs of representative SiAT1 genetic plants on day 14 after sowing without or with 75mM mixed alkali stress. The scale bar indicates 5 cm.
  • C Statistical analysis of relative survival rate of millet in (B).
  • D Schematic representation of rice OsGS3 and its truncated or non-functional versions in OsGS3 genetic plants.
  • OsWT is wild-type rice ZH11.
  • H Photos of ZmWT and ZmGS3 ko maize on day 14 after sowing without or with 75mM mixed alkali stress, scale bar indicates 5cm.
  • FIG. 13 Gene editing information of AT1 in millet and maize and the phenotype of transgenic plants in response to alkali stress.
  • A Western blot analysis of Myc-SiAT1 124 expression in T0 generation of different SiAT1 124 overexpression (SiAT1 124 -OE) lines.
  • B Target sequence location and gene editing information of AT1 in millet SiAT1 102 plants. A base insertion occurs in SiAT1 102 , resulting in a frameshift mutation and translational extraction termination. A C-terminally truncated protein with predicted amino acids 1-102 was retained in SiAT1 102 .
  • C Phenotypic analysis and relative plant height statistical analysis of SiWT, SiAT1 124 -OE, SiAT1 102 and SiAT1 ko millet plants under alkali stress. Millet seeds were sown in soil without or with 75mM mixed alkali and photos were taken on the 14th day after sowing. The scale bar indicates 5cm.
  • D Growth morphology of rice OsWT, OsGS3-1OE, OsGS3-4OE, OsGS3 ko and OsGS3Ri without or with 75mM mixed alkali stress treatment. Photographs were taken on the 29th day after sowing, and the scale bar indicates 5cm.
  • E and F Statistical analysis of relative plant height (E) and relative chloroplast content (F) of the rice lines in (D). Data are the mean ⁇ SEM of 4 representative plants per line. Statistical significance was determined by one-way ANOVA with Tukey's multiple comparison test.
  • G Target sequence locations and gene editing information for ZmGS3 and ZmGS3 ko maize plants. A 34 bp deletion and 1 base mutation occurred in ZmGS3 ko maize plants, resulting in frameshift mutations and premature translation termination.
  • H Phenotypic analysis and relative plant height statistical analysis of maize ZmWT and ZmGS3 ko plants under alkali stress treatment.
  • Corn seeds were sown in soil without (CK) or with 75mM mixed alkali and photographed on the 14th day after sowing.
  • the scale bar indicates 5cm.
  • the right picture shows the statistical analysis of plant height of maize ZmWT and ZmGS3 ko plants under 75mM mixed alkali stress. Statistical differences were determined by two-tailed unpaired t-test.
  • Figure 14 Construction results of transgenic plants overexpressing rice GS3-1 or GS3-4 and inhibiting the expression of GS3.
  • A Schematic diagram of overexpression vector
  • B Schematic diagram of inhibitory expression vector
  • C Expression level detection results of overexpression and inhibition of expression transgenic plants.
  • FIG. 15 Schematic diagram of the construction process of the rice GS3 CRISPR knockout vector pYL-Cas9-gRNA-OsGS3.
  • A Schematic diagram of the structure of the rice GS3 gene and the location of the CRISPR target site;
  • B The two target sites T1 and T2 were generated by PCR. The process of inserting into pYL-OsU3-gRNA and pYL-OsU6a-gRNA to obtain pYL-OsU3-T1-gRNA and pYL-OsU6a-T2-gRNA;
  • C Schematic structural diagram of the expression vector pYL-Cas9-gRNA-OsGS3.
  • FIG. Schematic diagram of the construction process of the maize ZmGS3 CRISPR knockout vector pYL-Cas9-gRNA-ZmGS3.
  • A Structure of the maize ZmGS3 gene and schematic diagram of the CRISPR target;
  • B Maize ZmGS3 CRISPR knockout Except for the detection results of mutation sites in the T 1 generation of transgenic plants.
  • FIG. 18 Schematic diagram of the TaGS gene structure and target setting using CRISPR/Cas9 technology according to Embodiment 4.1 of the present invention.
  • FIG. 19 Schematic diagram of the sequencing results of the TaGS gene T2 mutant of the transgenic wheat plant E5 according to Embodiment 4.2 of the present invention; wherein, WT represents the wild-type gene sequence, "-" represents the sequence with deletion mutation, and the "-" following Numbers indicate the number of deleted or inserted nucleotides.
  • FIG 20 Salt-alkali tolerance phenotype of wheat under salt-alkali stress according to Embodiment 4.3 of the present invention.
  • A is the treatment with clean water
  • B is the treatment with 75mM mixed alkali salt solution (NaHCO 3 : Na 2 CO 3 at a molar ratio of 5:1)
  • the three plants on the left in A and B are wild-type Fielder wheat (i.e., the target wheat).
  • Annotated as WT the three strains on the right are wheat TaGS gene triple mutant E5, annotated as E5.
  • FIG. 21 Gene editing information of TaAT1 (also known as TaGS) in wheat and the corresponding alkali stress phenotype of transgenic wheat plants.
  • A Schematic representation of the T-DNA structure in the CRISPR/Cas9 construct.
  • B CRISPR/Cas9-induced mutagenesis of target genes. Target and PAM sequences are shown in blue and red, respectively. Mutation sites are shown with dashed lines.
  • C Phenotypic analysis of wild-type wheat TaWT and TaAT1 knockout (TaAT1 ko ) plants under alkali stress. Wheat seeds were sown in soil without or with 125mM mixed alkali. Photographs were taken on the 21st day after sowing. The scale bar indicates 5cm.
  • H The amount of H 2 O 2 detected by ROS detection probe (H 2 DCFDA) in the root tip of TaAT1-related genetic plants.
  • FIG. 22 OsGS3 non-functional allele contributes to alkali tolerance in rice.
  • A Phenotypes of KY NIL (GS3) and KY NIL (gs3 - ) seedlings treated with or without 75mM mixed alkali on day 21 after sowing. The scale bar indicates 5cm. The picture on the right shows the statistical analysis of the relative plant height of rice under alkali treatment compared with no alkali treatment.
  • B Phenotypes of rice KY NIL (GS3) and KY NIL (gs3 ⁇ ) grown in soil. The scale bar indicates 5cm. The right panel shows statistical analysis of offset survival rates. Data are the mean ⁇ SEM of four replicates for each treatment, with 40 plants tested for each replicate.
  • (C) Relative NIL of rice grown in naturally alkaline soil (pH9.45) and near-neutral soil (pH7.74) in a greenhouse in Jilinzhou, China Survival and grain yield. The number of panicles represents the number of rice panicles per plant, and the data are the mean ⁇ SEM (n 3 small experimental plots).
  • the scale bar indicates 2cm.
  • FIG 23 AT1/GS3 knockout and natural non-functional alleles improve crop yield in saline-alkali soil.
  • A Phenotype and grain yield of rice KY NIL (GS3) and KY NIL (gs3 - ) grown in alkaline soil (pH9.17) in Jilinzhou, China in 2021.
  • the first graph shows the phenotype of rice plants during the reproductive stage (3 months after planting in the field).
  • the number of ears indicates the number of rice ears per rice plant.
  • (B) Phenotype, survival rate, grain yield and total biomass of sorghum SbWT and SbAT1 ko grown in alkaline soil (pH9.10) in Ningxia Autonomous Region, China in 2021. Data are means ⁇ SEM (n 3 plots).
  • (D) Phenotype, survival rate and grain yield of millet lines grown in alkaline soil (pH9.10) in Ningxia Autonomous Region, China in 2021. Data are means ⁇ SEM (n 3 plots).
  • FIG. 24 Putative model of the G ⁇ subunit AT1-mediated alkali stress response in plants.
  • PIP2s functions as an H 2 O 2 export protein.
  • G ⁇ subunit AT1 may pair with G ⁇ to negatively regulate the phosphorylation of PIP2s, thereby reducing the H 2 O 2 export capacity of PIP2s, leading to excessive accumulation of H 2 O 2 and causing plant sensitivity to alkali stress.
  • the truncated form of AT1, i.e. at1 further inhibits H2O2 export activity and leads to high sensitivity of plants to alkali stress.
  • the natural non-functional form of AT1 or the knockout AT1 homolog releases the inhibitory effect on PIP2s and effectively improves the alkali stress tolerance of crops.
  • Figure 25 Amino acid sequence alignment of AT1 homologous genes of sorghum (Sb), rice (Os), millet (Si), cultivated soybean (Gm), and wild soybean (Gs). The part enclosed by the red box is the predicted conserved GGL domain.
  • plant is broadly understood to include references to whole plants, plant organs, plant tissues, seeds and plant cells, and their progeny.
  • Plant cells include, but are not limited to, cells from seeds, suspension cultures, germs, meristems, callus, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores.
  • Progeny includes any subsequent generations of the plant.
  • Root or corn means any rice or corn plant and includes all plant varieties with which rice or corn can be bred, including whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated Intact plant cells in plants, plant callus, plants or plant parts such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, etc.
  • saline-alkali soil As used herein, the terms "saline-alkali soil", “sodium-containing soil” or “alkaline soil” refer to soil types where salts accumulate, meaning that the salts contained in the soil affect the normal growth of plants (eg, crops).
  • the formation of alkaline soil and alkalized soil is mostly related to the accumulation of carbonates in the soil. Therefore, the degree of alkalinization is generally high, and plants in severe saline-alkali soil areas can hardly survive.
  • saline-alkali land is divided into light saline-alkali land, moderate saline-alkali land and heavy saline-alkali land.
  • light saline-alkali land refers to the seedling emergence rate when planting crops is 70%-80%, and the salt content is less than 3 thousandths; heavy saline-alkali land refers to the salt content exceeding 6 thousandths, and the seedling emergence rate is less than 50%;
  • the moderate saline-alkali land is expressed in terms of pH value: the pH value of the mild saline-alkali land is: 7.1-8.5, the pH value of the moderate saline-alkali land is: 8.5-9.5, and the pH value of the severe saline-alkali land is: above 9.5 .
  • Mixed sodium salts e.g., sodium carbonate and sodium bicarbonate
  • nucleic acids are written from left to right in 5' to 3' orientation; amino acid sequences are written from left to right in amino to carboxyl orientation.
  • Amino acids may be referred to herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Committee on Biochemical Nomenclature.
  • nucleotides may be represented by commonly accepted single-letter codes.
  • a numerical range includes the numbers that qualify the range.
  • nucleic acid includes reference to deoxyribonucleotides or ribonucleotide polymers in single- or double-stranded form and, unless otherwise limited, includes known analogs having the essential properties of natural nucleotides ( For example, peptide nucleic acids), the analogs hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides.
  • the term "encoding” or “encoded” when used in the context of a particular nucleic acid means that the nucleic acid contains instructions This nucleotide sequence translates into the necessary information for a specific protein. Codons are used to represent information encoding proteins.
  • a "full-length sequence" referring to a particular polynucleotide or the protein it encodes refers to the entire nucleic acid sequence or the entire amino acid sequence with natural (non-synthetic) endogenous sequences.
  • a full-length polynucleotide encodes the full-length, catalytically active form of that particular protein.
  • polypeptide polypeptide
  • polypeptide protein
  • proteins proteins are used interchangeably herein to refer to a polymer of amino acid residues. This term is used for amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids. The term is also used for naturally occurring amino acid polymers.
  • amino acids or “amino acid residue” or “amino acid” are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively, a "protein”).
  • the amino acids may be naturally occurring amino acids and, unless otherwise limited, may include known analogs of natural amino acids that may function in a manner similar to naturally occurring amino acids.
  • the nucleotide sequences of the present application can be altered to make conservative amino acid substitutions. Principles and examples of conservative amino acid substitutions are described further below.
  • the nucleotide sequence of the present application can be substituted in accordance with the disclosed monocot codon preference without changing the amino acid sequence. For example, codons encoding the same amino acid sequence can be replaced with monocot-preferred codons. , without changing the amino acid sequence encoded by the nucleotide sequence.
  • part of the nucleotide sequence in the present application is replaced with different codons encoding the same amino acid sequence, thereby changing the nucleotide sequence without changing the amino acid sequence it encodes.
  • Conservative variants include those sequences that encode the amino acid sequence of one of the proteins of the embodiments due to the degeneracy of the genetic code.
  • portions of the nucleotide sequences in this application are replaced based on monocot preferred codons.
  • amino acid additions and/or substitutions are generally based on the relative similarity of the amino acid side chain substituents, e.g., hydrophobicity, charge, size, etc. of the substituents.
  • amino acid substituents having various properties contemplated above include arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.
  • Guidance on appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.) (incorporated herein by reference) found in the model. Conservative substitutions such as exchanging one amino acid for another with similar properties can be made.
  • Constant amino acid substitutions are those substitutions of amino acids for different amino acids in which the substitution is predicted to least interfere with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially preserve the structure and function of the reference polypeptide. Table B below provides a list of exemplary conservative amino acid substitutions considered herein.
  • deletion refers to a change in the amino acid sequence that results in the absence of one or more amino acid residues.
  • Deletions may remove at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 200 or more amino acid residues.
  • Deletions may include internal and/or terminal deletions (eg, N-terminal truncation, C-terminal truncation, or both) of the reference polypeptide.
  • a "variant,” “mutant,” or “derivative" of a reference polypeptide sequence may include deletions relative to the reference polypeptide sequence.
  • a "fragment” is a portion of an amino acid sequence that is identical in sequence to a reference sequence but is shorter in length than the reference sequence. Fragments may contain up to the entire length of the reference sequence, minus at least one amino acid residue. For example, the fragments may each comprise 5 to 1000 contiguous amino acid residues of the reference polypeptide. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous amino acid residues of a reference polypeptide. Fragments may be preferentially selected from certain regions of the molecule. The term "at least one fragment" includes full-length polypeptides.
  • Fragments may include N-terminal truncations, C-terminal truncations, or both relative to the full-length protein.
  • a "variant,””mutant,” or “derivative" of a reference polypeptide sequence may include fragments of the reference polypeptide sequence.
  • Insertion and addition refer to changes in the amino acid sequence that result in the addition of one or more amino acid residues. Insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more amino acid residues.
  • "Variants,” “mutants,” or “derivatives” of a reference polypeptide sequence may include insertions or additions relative to the reference polypeptide sequence. Variants of the protein may have N-terminal insertions, C-terminal insertions, internal insertions, or any combination of N-terminal insertions, C-terminal insertions and internal insertions.
  • percent identity refers to the percentage of residue matches between at least two amino acid sequences aligned using a standardized algorithm. Methods for amino acid sequence alignment are well known. Some alignment methods take conservative amino acid substitutions into account. Such conservative substitutions, explained in more detail below, generally preserve the charge and hydrophobicity at the substitution site, thereby preserving the structure (and therefore function) of the polypeptide. Percent identity of amino acid sequences can be determined as understood in the art (see, eg, U.S. Patent No. 7,396,664, which is incorporated herein by reference in its entirety).
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Alignment Search Tool
  • the BLAST software suite includes various sequence analysis programs, including "blastp", which is used to align known amino acid sequences with other amino acid sequences from various databases.
  • percent identity may be measured over the entire length of a defined polypeptide sequence (e.g., as determined by a specific SEQ ID number), or may be measured over a shorter length, e.g., when taken from a larger.
  • the length of a fragment of a determined polypeptide sequence eg, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 contiguous residues is measured.
  • Such lengths are exemplary only, and it will be understood that any fragment length supported by the sequences shown herein in tables, figures, or sequence listings may be used to describe the lengths over which percent identity can be measured.
  • similarity refers to the proportion of identical bases or amino acids in the entire sequence between the detected sequence and the reference sequence (a relatively macroscopic description). In amino acid sequence alignment, similarity also includes, in addition to identical residues, whether the two residues at corresponding positions have similar characteristics, such as the size, charge, hydrophilicity, and hydrophobicity of the side chain groups. In other words, for proteins, "identity” requires that the amino acids at the compared positions are exactly the same, while “similarity” does not require that the amino acids at the compared positions are exactly the same. If the amino acids at the compared positions are conserved For substituted residues, the amino acids at that position are considered to be similar.
  • Protein-tag refers to a polypeptide or protein that is fused and expressed together with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and/or purification of the target protein.
  • Protein tags include but Not limited to Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag and/or SUMO tag, etc.
  • nucleic acid sequence identity refers to the sequence similarity between two polynucleotide sequences. When a position in two compared sequences is occupied by the same base, for example if each position in two DNA molecules is occupied by adenine, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ⁇ 100.
  • Determination of nucleic acid sequence identity includes hybridization techniques. For example, all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in cloned genomic DNA fragments from the selected organism. or a population of cDNA fragments (i.e., genomic library or cDNA library).
  • the hybridization probe may be a genomic DNA fragment, a cDNA fragment, an RNA fragment or other oligonucleotide, and may be labeled with a detectable group such as 32P or other detectable marker.
  • hybridization probes can be prepared by labeling synthetic oligonucleotides based on the sequences of embodiments.
  • Hybridization of the sequences can be performed under stringent conditions.
  • stringent conditions or “stringent hybridization conditions” means conditions under which a probe will hybridize to a detectably greater extent (e.g., at least 2 times background) relative to other sequences. , 5 times or 10 times) hybridizes to its target sequence.
  • Stringent conditions are sequence dependent and vary in different environments. By controlling hybridization stringency and/or controlling washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probe method). Alternatively, stringent conditions can be adjusted to allow for some sequence mismatches in order to detect lower similarities (heterologous probe approach).
  • probes are less than about 1000 or 500 nucleotides in length.
  • stringent conditions are conditions in which the salt concentration is less than about 1.5 M Na ions, typically about 0.01 M to 1.0 M Na ions (or other salts) at pH 7.0 to 8.3, and the temperature The conditions are: at least about 30°C when used with short probes (eg, 10 to 50 nucleotides); and at least about 60°C when used with long probes (eg, greater than 50 nucleotides).
  • Stringent conditions can also be achieved by adding destabilizing agents such as formamide.
  • Exemplary moderately stringent conditions include hybridization in 40% to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C and washes in 0.5 ⁇ to 1 ⁇ SSC at 55°C to 60°C.
  • Exemplary high stringency conditions include hybridization in 50% formamide, 1M NaCl, 1% SDS at 37°C and a final wash in 0.1 ⁇ SSC at 60°C to 65°C for at least about 20 minutes.
  • the wash buffer may contain about 0.1% to about 1% SDS.
  • the duration of hybridization is generally less than about 24 hours, and typically ranges from about 4 hours to about 12 hours. Specificity usually relies on post-hybridization clearance The key factors for cleaning are the ionic strength and temperature of the final cleaning solution.
  • the Tm (thermodynamic melting point) of the DNA-DNA hybrid can be approximated from the formula of Meinkoth and Wahl (1984) Anal. Biochem.
  • Tm 81.5°C+16.6(logM)+0.41(%GC)-0.61( % formamide)-500/L; where M is the molar concentration of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in DNA, "formamide %" is the percentage of formamide in the hybridization solution, and L is the base pair length of the hybrid.
  • Tm is the temperature (at a defined ionic strength and pH) at which 50% of the complementary target sequence hybridizes to a perfectly matched probe. Washing is typically performed at least until equilibrium is reached and low hybridization background levels are achieved, such as for 2 hours, 1 hour, or 30 minutes.
  • each 1% of mismatches should lower the Tm by approximately 1°C; thus, the Tm, hybridization and/or wash conditions can be adjusted to hybridize to sequences of desired identity. For example, if a sequence with ⁇ 90% identity is required, the Tm can be lowered by 10°C.
  • stringent conditions are chosen to be about 5°C lower than the Tm of the specific sequence and its complement at defined ionic strength and pH.
  • hybridization and/or washing can be performed at 4°C lower than the Tm; under moderately stringent conditions, hybridization and/or washing can be performed at 6°C lower than the Tm; Under low stringency conditions, hybridization and/or washing can be performed at 11°C below the stated Tm.
  • frameshift mutation refers to the insertion or loss of one or several (not 3 or multiples of 3) base pairs at a certain site in a DNA fragment, resulting in a series of coding sequence misalignments after the insertion or loss site. A mutation. It can cause abnormalities in subsequent genetic information at this site. Genes with frameshift mutations can change the amino acid sequence of the polypeptide chain when expressed, thus seriously affecting the structure and function of the protein or enzyme.
  • Change in some embodiments ⁇ 10% change compared to the specified amount, in some embodiments ⁇ 5% change compared to the specified amount, in some embodiments ⁇ 1% change compared to the specified amount, in some embodiments, a change of ⁇ 0.5% from the stated amount, and in some embodiments a change of ⁇ 0.1% from the stated amount, as such changes are suitable for performing the disclosed methods and/or using the disclosed compositions, nucleic acids, peptides, etc. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and the appended claims are approximations that may vary depending upon the desired characteristics sought to be obtained by the subject matter disclosed herein.
  • 352 grain sorghum and 38 sweet sorghum natural population materials were collected from the USDA-ARS (United States Department of Agriculture-Agricultural Research Service) germplasm resource system. After the freshly harvested seeds are sun-dried and naturally air-dried, they are placed in a 55°C oven for 5-7 days to break the dormancy of the seeds.
  • USDA-ARS United States Department of Agriculture-Agricultural Research Service
  • the data are the average of three replicates for each treatment, and 9 plants were tested for each replicate.
  • the seedling growth environment is as follows: light/dark time is 16h/8h, day and night temperature range is 28/26°C, and relative humidity is 60% to 70%. On the 23rd day of culture, the number of seedlings was counted and the relative survival rate was calculated. The experimental results showed that the salt-alkali tolerance during the germination period differed significantly between different varieties ( Figure 1B-F).
  • the seed germination rate, germination index and alkali tolerance index of 16 grain sorghum varieties under saline-alkali stress during the germination period were dynamically monitored through random selection.
  • the index of relative seedling rate was selected as a representative index for the evaluation of alkali resistance during the germination period to evaluate the degree of salt-alkali tolerance. Subsequently, it was found that the seedlings of sorghum materials under alkali treatment grew better in the first two weeks, but there was a phenomenon of seedling burning in the later stage of alkali treatment. The 23rd day was the "watershed" for this phenomenon. Therefore, the 23rd day was selected as the GWAS identification. time node ( Figure 2B).
  • S1_5577933 and S1_55779336 directly The interior of the Sobic.001G341700 gene was located and named SbAT1 (Alkali Tolerance 1, or AT1 for short) ( Figure 3B). It was initially determined that SbAT1 is a major gene that controls the relative seedling rate of sorghum under saline-alkali stress.
  • the genomic gene sequence of sorghum AT1 is shown in SEQ ID NO:1.
  • the AT1 gene contains 5 exons, of which the first exon is located at positions 1-111 of SEQ ID NO:1; the second exon is located in SEQ ID NO:1 ID NO:1 at positions 2447-2499; the third exon is located at positions 2846-2890 of SEQ ID NO:1; the fourth exon is located at positions 2972-3025 of SEQ ID NO:1; the fifth exon is located at positions 2972-3025 of SEQ ID NO:1 The exon is located at positions 4084-4417 of SEQ ID NO:1.
  • the sequence of the AT1 cDNA gene is shown in SEQ ID NO:2, and its coding sequence (CDS) is shown in SEQ ID NO:3.
  • AT1 encodes a highly conserved protein, consisting of 198 amino acids (SEQ ID NO: 4).
  • SEQ ID NO: 4 A sequence comparison of the protein product found that the total amino acid length of this gene is close to that of the homologous AT1 protein in millet (Setaria italica), and The N-terminal conserved sequence similarity is very high, so it is believed that sorghum AT1 is the homologous gene of millet SiAT1.
  • the genomic nucleotide sequence of millet SiAT1 gene is shown in SEQ ID NO:5, the amino acid sequence of millet SiAT1 protein is shown in SEQ ID NO:8, and the coding cDNA sequence of SiAT1 protein is shown in SEQ ID NO:6, which encodes The sequence (CDS) is shown in SEQ ID NO:7.
  • AT1 gene affects the alkali tolerance of sorghum
  • haplotype I Haplotype 1
  • haplotype II haplotype corresponding to the AT1 gene in the at1 mutant
  • P value 2.43 ⁇ 10 -10
  • haplotype I and haplotype II are alkali-resistant (AT1) and alkali-sensitive (at1) alleles, respectively (Fig.
  • the Sbat1 gene also referred to as at1 gene
  • Sbat1 gene which is the insertion of five bases (GTGGC) in the fifth exon of the wild-type AT1 gene (that is, in SEQ Five nucleotides of GTGGC were inserted between positions 3271-3272 of ID NO:1), resulting in a frameshift mutation at the 3' end of the AT1 gene, and protein translation was terminated prematurely, forming 137 amino acids ("at1-a in Figure 4 ” shown).
  • the nucleotide sequence of the cDNA of the at1 gene is shown in SEQ ID NO:9
  • amino acid sequence of the mutated at1 protein corresponding to the AT1 gene is shown in SEQ ID NO:10.
  • NIL-AT1 and NIL-at1 which only have a 58 kb interval on chromosome 1. The genotypes are different, and the remaining background region marker genes are consistent.
  • SEQ ID NO:4 The amino acid sequence of the AT1 gene of the NIL-AT1 material is shown in SEQ ID NO:4; the amino acid sequence of the at1 gene of the NIL-at1 material is shown in SEQ ID NO:10.
  • NIL was derived from a cross between two sorghum accessions, SN010 and M-81E.
  • SN010 is haplotype Hap1 (containing wild-type AT1)
  • M-81E is haplotype Hap2 (containing at1)
  • Figure 5A and Figure 6A-C survival rate and plant height data
  • SN010(NIL-AT1) show higher alkali resistance.
  • the method used in this embodiment is as follows:
  • the plant expression vector pCAMBIA2300-Ubi-Myc is preserved by our laboratory (see Liu Y, Sun J, Wu Y. Arabidopsis ATAF1 enhances the tolerance to salt stress and ABA in transgenic rice. J Plant Res. 2016 Sep; 129(5):955 -962.doi:10.1007/s10265-016-0833-0.Epub 2016 May 23.PMID:27216423, where the vectors described in lines 1-3 of Constructs and transformation in the right column of Page 2, Materials and methods, are The Ubi promoter and OCS terminator are added to the pCAMBIA2300 commercial vector, and a recombinant vector with a Myc tag sequence is also available to the public from the applicant to repeat the invention).
  • the restriction endonucleases Spe I and BamHI both purchased from New England Biolabs
  • the primers carrying the adapter to amplify the CDS coding region of the sorghum AT1 gene with the stop codon (the nucleotide sequence is shown in SEQ ID NO: 3) to obtain the target fragment with the adapter, and use the Seamless Cloning Kit pEASY-Uni Seamless Cloning and Assembly Kit (CU101-01) connects the obtained target fragment (gene fragment) with the adapter and the obtained linearized vector.
  • Seamless cloning kit ligation reaction system (10 ⁇ L): 5 ⁇ L of 2 ⁇ Assembly Mix, mix the linearized vector and the target fragment with the adapter at a molar ratio of 1:2, then add ddH2O to make up to 10 ⁇ L. After gentle mixing, react at 50°C for 15 minutes. After the reaction, place the centrifuge tube on ice to cool for a few seconds. Then the recombinant product can be transformed into E. coli XL1-Blue, the plasmid is extracted, and the positive clone recombinant plasmid pCAMBIA2300-Ubi-Myc-AT1 is confirmed by enzyme digestion and sent to Ruibo Company for further sequencing. verify.
  • the recombinant Agrobacterium EHA105/pCAMBIA2300-Ubi-Myc-AT1 contains the CDS nucleotide sequence of the sorghum AT1 gene (SEQ ID NO:3)
  • the recombinant Agrobacterium EHA105/pCAMBIA2300-Ubi-Myc-AT1 was transferred into the sorghum recipient material Wheatland through Agrobacterium-mediated method.
  • the method of genetic transformation of sorghum is as follows:
  • Plant Wheatland sorghum plants in the greenhouse After about 15 days of growth to the flowering stage, cut off the ears and squeeze out the young embryos from the filled seeds as much as possible on a sterile ultra-clean platform and place them evenly until the callus is ready. Callus culture was carried out on culture medium.
  • the recombinant Agrobacterium stored at -80°C was streak cultured in YEP solid medium supplemented with Kan.
  • the single colony that grew out was inoculated into 10 mL YEP liquid medium (containing Kan) for primary activation (28°C, shaking at 220 rpm overnight).
  • YEP liquid medium including Kan
  • T0 generation sorghum transformed seedlings with a plant height of about 5-7cm and good root growth into the soil, cultivate them in the greenhouse, and maintain a certain humidity to ensure the survival rate of the regenerated seedlings. After cultivating to maturity, harvest The mature seeds are T0 generation transgenic seeds.
  • CRISPR-Cas9 includes the gRNA expression cassette pYLsgRNA-OsU6a/LacZ
  • the vector and CRISPR-Cas9 vector pYLCRISPR/Cas9Pubi-B were kindly donated by Professor Liu Yaoguang of South China Agricultural University (Ma and Dicot Plants.Mol Plant.2015 Aug;8(8):1274-84.doi:10.1016/j.molp.2015.04.007.Epub 2015 Apr 24.PMID:25917172.).
  • the target adapter is prepared and the sgRNA vector is digested and then connected to the sgRNA expression cassette.
  • the nucleotide sequence of the sgRNA target is 5’-ACGCCTGAAAAGTTGACAGCTG-3’, targeting positions 2317-2338 of SEQ ID NO:1 of the AT1 gene.
  • the specific operation method is as follows:
  • Target adapters Dissolve the designed and synthesized adapter primers AT1-Target-F: 5'-GCCGCAAGTCGCCGCCTGCCTCGC-3' and AT1-Target-R: 5'-AAACGCGAGGCAGGCGGCGACTTG-3' with ddH2O into 100 ⁇ M mother solution, and take left and right of each Primers were mixed and diluted to a final concentration of 1 ⁇ M. Heat on a PCR machine (about 90°C for 30 seconds) and cool at room temperature to complete annealing to form a double-stranded target linker.
  • Enzyme digestion of sgRNA vector Take 1 ⁇ g of pYLsgRNA-OsU6a/LacZ plasmid and digest it with 10U Bsa I for 20 minutes in a 25 ⁇ L enzyme digestion reaction system to obtain the digested plasmid, and freeze it for storage.
  • sgRNA expression cassette ligation reaction Ligation reaction between the digested plasmid and the corresponding double-stranded target adapter (10 ⁇ L system): 1 ⁇ L 10 ⁇ T4 DNA ligase buffer, 10 ng of digested plasmid, 0.05 ⁇ M double-stranded target Adapter, 18 U T4 DNA ligase, make up to 10 ⁇ L with ultrapure water; ligate at room temperature for 20 minutes.
  • Second round of amplification After diluting the first round PCR product 10 times, take 1 ⁇ L as the template for the second round of PCR. Also use an appropriate amount of KOD Fx Neo enzyme and forward primer B1':5'- TTCAGAggtctcTctcgCACTGGAATCGGCAGCAAAGG-3' and reverse primer BL: 5'-AGCGTGggtctcGaccgGGTCCATCCACTCCAAGCTC-3'. Amplify for 30 cycles: 95°C for 10 s, 58°C for 15 s, and 68°C for 20 s. Detect the size of the target band (831bp) by running gel, and perform ethanol precipitation and purification. The obtained PCR purified product is sgRNA with OsU6a promoter: OsU6a-sgRNA.
  • pYLCRISPR/Cas9Pubi-B plasmid Take about 2 ⁇ g of pYLCRISPR/Cas9Pubi-B plasmid and digest it in an enzyme digestion system (30U Bsa I) for 2 hours. Use DNA gel electrophoresis to recover the digested plasmid fragments. Take 100ng of the pYLCRISPR/Cas9Pubi-B plasmid digested with BsaI, and add the OsU6a-sgRNA (20ng), the second round PCR purification product of step 6) that has been digested with BsaI, and add 35U of T4 to the 10 ⁇ L ligation reaction. Ligase, ligated through temperature cycle: 10°C 5min, 20°C 5min; 10-15 cycles) for 2-3h.
  • the plating medium is LB (25 ⁇ g/mL Kan, 0.5mM IPTG and appropriate amount of X-gal). Pick a single clone colony and use the colony PCR method, and use the vector primers SP1: 5'-CCCGACATAGATGCAATAACTTC-3' and SP2: 5'-GCGCGGTGTCATCTATGTTACT-3' to PCR amplify and sequence the specific target.
  • the amplified product band size is approximately About 1kb, the amplified product contains OsU6a-sgRNA after sequencing.
  • the positive clone obtained is the successfully recombined plasmid pYLCRISPR/Cas9Pubi-B-AT1-sgRNA.
  • the obtained positive Agrobacterium was used to infect the calli of sorghum (Wheatland) and millet (Ci846).
  • the genetic transformation steps of sorghum are the same as step 4.2.2.
  • the millet receptor material Ci846 was donated by Professor Sui Yi of the Institute of Crop Science, Chinese Academy of Agricultural Sciences (Related literature: Cheng Z, Sun Y, Yang S, Zhi H, Yin T, Ma X, Zhang H,Diao X,Guo Y,Li .).
  • Genetic transformation of millet was carried out with reference to the genetic transformation method of millet in the following patent documents: Method for Obtaining Embryogenic Calli of Millet for Genetic Transformation and Genetic Transformation, Publication No. CN108588002A.
  • Disinfection of mature embryos of millet seeds Disinfect the mature embryos of millet seeds in 10% sodium hypochlorite solution for 10-15 minutes, then rinse them repeatedly with sterile water 3-5 times, and finally use absorbent paper to absorb the water for later use.
  • Agrobacterium-mediated genetic transformation of millet embryonic cells activate the recombinant Agrobacterium EHA105/pYLCRISPR/Cas9Pubi-B-AT1-sgRNA in advance, and combine the prepared calli and Agrobacterium on the pre-medium Co-culture, and transfer the co-cultured callus to a screening medium containing corresponding resistance for subculture. After three subcultures, transfer the resistant callus to a differentiation and regeneration medium until seedlings grow.
  • the targeting effect is tested, that is, with the target as the center, primers are synthesized at approximately 200-300 bp on each side for PCR amplification. Design internal primers about 150-250 bp upstream of the target as sequencing primers to directly sequence the PCR products. If there are no overlapping peaks in the sequencing peak chart, it can be determined as wild type or homozygous mutation. If a plant with double peaks appears near the target point, it is a positive AT1 gene mutant plant.
  • sorghum transgenic T0 generation plants and Wheatland (wild-type sorghum) as templates, PCR amplified the genomic DNA fragments including the shear sites, and sequenced the amplified products to detect the knockout of the AT1 gene.
  • the previously predicted amino acids 22 to 88 of the N-terminus of AT1 protein in sorghum are highly conserved domains.
  • CRISPR-Cas9 transgenic plants were PCR amplified with primers F:5'-GTTGACAGCTGAACACATGGCT-3' and R:5'-ATACATCGTTAGGAATGGATCCG-3', and the sequencing results showed that the AT1 gene was edited, resulting in the loss of AT1 gene function. plant.
  • the transgenic plants with edited AT1 genes were named AT1 KO ( Figure 9C).
  • the gene-edited material is a material in which the conserved domain of the AT1 protein is completely deleted ( Figure 10).
  • the coding sequence of AT1 expressed by SiAT1 KO plants is shown in SEQ ID NO:13, and the amino acid sequence of the protein is shown in SEQ ID NO:14.
  • the nucleotide sequence of the sgRNA target of the millet SiAT1 gene is 5'-TATAATGGCTGCTGCGCCGG-3', targeting positions 393-412 of the SiAT1 gene, that is, SEQ ID NO:5; the adapter used in the preparation of the target adapter
  • the primers were SiAT1-Target-F: 5'-GGCGTATAATGGCTGCTGCGCCGG-3' and SiAT1-Target-R: 5'-AAACCCGGCGCAGCAGCCATTATA-3'.
  • T1 generation plants After selecting positive transgenic plants from the T0 generation of sorghum, the plants were selfed to obtain T1 generation plants.
  • primers F and R were used again to identify the target.
  • the target was confirmed. Edited and sequenced single peaks were retained as homozygous plants.
  • the plant is then PCR amplified using the final primers SP1: 5'-CCCGACATAGATGCAATAACTTC-3' and SP2: 5'-GCGCGGTGTCATCTATGTTACT-3'. If the amplification product cannot be obtained, it means that the Cas9 homozygous plant has been screened out. Use this material for subsequent experiments.
  • the screened T1 generation homozygous plants were selfed to obtain T2 generation homozygous plants.
  • the homozygous lines SbAT1 KO and SiAT1 KO isolated by the CRISPR-Cas9 vector and causing premature termination mutations at the target site were identified and screened in the T2 generation of transgenic sorghum and millet.
  • a total of 54 seedlings were obtained from the T0 generation of AT1-MYC overexpressing transgenic sorghum, and 12 T0 generation transgenic positive strains were identified by PCR, and 10 of them were identified by qPCR (named AT1-MYC-3 ⁇
  • the AT1 gene expression level of AT1-MYC-12) was much higher than that of wild-type Wheatland, with folds between 92 and 303 ( Figure 9B), using SbEIF as the internal reference gene.
  • water the treatment liquid water or alkali solution
  • water the treatment liquid water or alkali solution
  • water it with clean water as the control.
  • the growth environment is as follows: the day and night temperature is controlled at 28°C/22°C, the light/dark time is 16h/8h, and the relative humidity is 60% to 70%). It is filled with clean water in the later period.
  • Each group of treatments is repeated three times. The seed is considered to have germinated when its germ emerges from the soil surface.
  • the data statistics method is as follows: First, use Excel 2016 for data sorting and chart production, use DPS 7.5 software for data statistics, and use the least significant difference (LSD) and one-factor analysis of variance (ANOVA) to conduct significant variance analysis on the results. If P ⁇ 0.05, it is marked with lowercase letters, and the difference is significant; if P ⁇ 0.01, it is marked with uppercase letters, and the difference is extremely significant.
  • the preparation method of 50mM mixed alkali solution (NaHCO3:Na2CO3 with a molar ratio of 5:1) is to weigh 12.32g NaHCO3 and 3.11g Na2CO3 and dissolve it in 2 liters of water.
  • the solution pH is 10.03. After the soil fully absorbs the solution, the soil pH The value is 9.19; the preparation method of 75mM mixed alkali solution (NaHCO 3 : Na 2 CO 3 with a molar ratio of 5:1) is to weigh 18.48g NaHCO 3 and 4.66g Na 2 CO 3 and dissolve them in 2 liters of water.
  • the solution pH is 10.04.
  • the soil pH value is 9.32; the preparation method of 100mM mixed alkali solution (NaHCO 3 : Na 2 CO 3 at a molar ratio of 5:1) is to weigh 24.64g NaHCO 3 and 6.22g Na 2 CO 3 and dissolve them in 2 liters of water. , the solution pH is 10.03, and the soil pH value is 10.10 after the soil fully absorbs the solution.
  • Phenotypic observation results show that under normal growth conditions of the control (CK), there is no significant difference in survival rates between wild-type sorghum Wheatland, sorghum AT1 gene overexpression line AT1-OE, and sorghum AT1 gene deletion mutant line AT1 KO ( Figure 5H, Figure 6E), there is no significant difference in survival rate between wild-type millet Ci846 (SiWT in Figure 12B), millet SiAT1 gene overexpression SiAT1-OE and deletion mutant line SiAT1 KO ( Figure 12B); 75mM mixed base
  • the sorghum AT1-OE strain under the treatment conditions is extremely sensitive to alkali, while the sorghum mutant AT1 KO and the millet mutant SiAT1 KO with loss of gene function are better than the wild-type sorghum Wheatland and millet Ci846.
  • Example 1 shows that compared to the wild-type AT1 protein (SEQ ID NO: 1) with 198 amino acids, the C-terminal truncation only retains 136 amino acids in the NIL-at1 mutant.
  • the at1 (SEQ ID NO:10) is the protein corresponding to the natural variant allele of AT1.
  • Analysis of near-isogenic line materials found that compared with wild-type AT1, at1, which lacks the C-terminal but retains the complete N-terminal GGL domain, has greatly reduced alkali resistance. Therefore, all the above results in Example 1 and Example 2 show that the AT1 protein in the grass crops sorghum and millet is a key factor that negatively regulates the degree of alkali tolerance.
  • Gene editing of the AT1 gene and knocking out AT1 can improve the salt tolerance of crops. alkaline.
  • transgenic plants in which a stop codon was generated via gene editing at the same position as in sorghum to mimic the potential C-terminally truncated SiAT1 102 protein in millet.
  • millet for testing in this example is based on the following considerations: (1) Compared with sorghum, millet plant transformation is easier and faster, (2) Millet and sorghum are closely related taxonomically and have high genomic similarity. sex and similar environmental physiological performance, and (3) the single copy of millet SiAT1 and sorghum SbAT1 are 75.24% identical at the protein level.
  • AT1 homologous genes in alkali tolerance in other major monocotyledonous crops, rice and maize.
  • the ortholog of AT1 in rice has been identified as OsGS3, which is a major QTL regulating grain size (H.Mao, S.Sun, J.Yao, C.Wang, S.Yu, C.Xu et al., Linking differential domain functions of the GS3 protein to natural variation of grain size in rice.Proc.Natl.Acad.Sci.USA 107,19579-19584(2010).doi:10.1073/pnas.1014419107; C.Fan,Y.Xing, H.Mao,T.Lu,B.Han,C.Xu et al., GS3, a major QTL for grain length and weight and minor QTL for grain width and thickness in rice, encodes a putative transmembrane protein.Theor.Appl.Genet.112,1164-1171(2006).doi:
  • the complete rice GS (GS3-1) protein sequence is shown in SEQ ID NO:16
  • the rice GS3 C-terminal truncated protein (GS3-4) sequence is shown in SEQ ID NO:17
  • the maize GS3 protein sequence is shown in SEQ ID NO:18 shown.
  • the maize homologous gene of AT1 was previously identified as ZmGS3 (Q. Li, X. Yang, G. Bai, ML Warburton, G. Mahuku, M. Gore et al., Cloning and characterization of a putative GS3 ortholog involved in maize kernel development .Theor.Appl.Genet.120,753-763(2010).doi:10.1007/s00122-009-1196-x). Therefore, we named the gene AT1/GS3, with the prefix indicating the species; depending on the context, AT1 and GS3 may also be used alone or interchangeably.
  • ZmWT maize ZmGS3 knockout (ZmGS3 ko ) line was obtained.
  • Maize ZmGS3 ko has a 34bp deletion, and a base mutation occurs in the first exon of ZmGS3. These mutations resulted in frameshift mutations and premature translation termination of the predicted protein (Fig. 13G). After alkaline treatment, compared with wild-type corn, the growth performance of knockout corn plants on the 14th day of culture showed that they had stronger alkali tolerance ( Figure 12G-H, Figure 13H). After 50 days of alkali stress treatment, the phenotypic difference between the two was more significant; almost all wild-type maize seedlings died, while ZmGS3 ko survived and continued to grow ( Figure 12I, Figure 13I). This result supports that ZmGS3 ko can improve alkali tolerance in maize, similar to what we observed in sorghum, millet, and rice.
  • Design primers based on rice genome annotation information (GS3 genome sequence is shown in SEQ ID NO: 19), and use water
  • the full-length GS3 cDNA of the rice variety Guanglu Ai (Osigcea013f09t3) was used as a template to amplify the two allelic types of GS3, GS3-1 and GS3-4, respectively, with sizes of 696bp (as shown in SEQ ID NO: 20) and 450bp (as shown in SEQ ID NO:21).
  • the present invention designs the following primers:
  • Reverse primer of GS3-1OER (SEQ ID NO:37): 5'- agatct CAAGCAGGGGGGGCAGCAACG-3' (the underlined sequence is the BglII recognition site);
  • Reverse primer of GS3-4OER (SEQ ID NO:38): 5'- agatct ACGCCGCCCCACATGAGGA-3' (the underlined sequence is the BglII recognition site);
  • the primer combinations GS3OEF, GS3-1OER and GS3OEF, GS3-4OER were used to perform PCR amplification respectively, and the target fragments of GS3-1 and GS3-4 were obtained.
  • the total volume of the PCR reaction is 50 ⁇ l, including 2 ⁇ l cDNA template, 25 ⁇ l 2 ⁇ GC I buffer, 5 ⁇ l 10mM dNTP, 1 ⁇ l each of 10mM primers GS3OEF and GS3OER, 1 ⁇ l ExTaq enzyme, and add deionized water to 50 ⁇ l (2 ⁇ GC I used Buffer, dNTP, rTaq enzyme, etc. were purchased from Bao Bioengineering Dalian Co., Ltd.).
  • PCR reaction conditions are as follows: 194°C 4min, 294°C 30s, 358°C 30s, 472°C 1min, 5cycle from 2-4 33 times, 672°C 7min, 7save at 25°C.
  • the PCR product was detected by electrophoresis on a 1% (mass/volume) TBE agarose gel, and a GS3-1 length of 696 bp was recovered (a target DNA segment of 681 bp plus two restriction enzyme sites of 15 bp attached to the primer). ) and a DNA fragment of 462 bp GS3-4 (447 bp target DNA segment plus two 15 bp additional restriction enzyme sites on the primer).
  • pCAMBIA1301U This vector was modified by our laboratory: its basic skeleton is from the Australian CAMBIA laboratory (http://www.cambia.org/daisy/cambia/materials/ overview.html) pCAMBIA1301, by adding Ubi promoter to achieve expression control of transformed genes], transformed rice Zhonghua 11 (ZH11) to obtain over-expression plants GS3-1OE and GS3-4OE.
  • the carrier structure is shown in Figure 14A.
  • RNAi vector of GS3 was constructed in two steps. First, the plasmid containing the full-length cDNA of GS3 (osigcea013f09t3) was double-digested with BamHI and KpnI, and connected to the modified dsRNAi1301. Obtain the vector containing the first chain (forward) GS3; at the same time, connect the BamHI and KpnI digestion products to the intermediate vector GZ-1 (provided by Dr.
  • the method of using RNA interference to knock down the expression of GS3 gene or protein in plants is preferably to insert the coding sequence of the plant GS3 gene into pDS1301 in the forward and reverse directions respectively, and transform the plants with the resulting recombinant vector, thereby reducing the expression of the GS3 gene or protein. of plants.
  • the method of inserting the GS3 gene into the pDS1301 vector is preferably accomplished by enzyme digestion. For forward insertion, KpnI and BamHI double enzyme digestion and ligation were used. When inserting in the reverse direction, SacI and SpeI double enzyme digestion and ligation are used.
  • the nucleotide sequence is shown in SEQ ID NO:34.
  • the nucleotide sequence is shown in SEQ ID NO:35.
  • the pDS1301 vector was modified by our laboratory (see existing technology: Yuan B, Shen X, Li X, Xu C, Wang S (2007) Mitogen-activated protein kinase OsMPK6 negatively regulates rice disease resistance to bacterial pathogens.Planta226: 953–960); its basic skeleton is pCAMBIA1301 from the Australian CAMBIA laboratory (http://www.cambia.org/daisy/cambia/materials/overview.html), which controls the expression of transformed genes by adding the 35S promoter.
  • RNA from GS3-1OE, GS3-4OE, GS3-1RNAi and the 1cm long spikelets of wild-type plants at the spikelet differentiation stage The reagents for RNA extraction are purchased using the Trizol extraction kit produced by Invitrogen ( Please follow the instructions provided with the kit for specific operating steps).
  • the GS3 gene detection primers are GS3QRT-F and GS3QRT-R, and the Ubiquitin gene (LOC_Os03g13170) is used as the internal reference (the primer combination is UbiQRT-F and UbiQRT-R).
  • the sequence is as follows:
  • the above-prepared transgenic plants overexpressing the GS3 gene and plants with reduced GS3 expression were subjected to alkali treatment, and the differences in main agronomic traits and non-transgenic recipients were compared.
  • the details are as follows: An alkali treatment experiment was conducted using seeds grown under the same conditions, harvested at the same time, and stored under the same conditions.
  • the data statistics method is as follows: First, use Excel 2016 for data sorting and chart production, use DPS 7.5 software for data statistics, and use the least significant difference method (LSD) and one-factor analysis of variance (ANOVA) to perform significant variance analysis on the results. If P ⁇ 0.05, it is marked with lowercase letters, and the difference is significant; if P ⁇ 0.01, it is marked with uppercase letters, and the difference is extremely significant.
  • LSD least significant difference method
  • ANOVA one-factor analysis of variance
  • Phenotypic observation results show that: under normal water treatment, there is no significant difference in the survival rate between wild-type ZH11, GS3 gene overexpression lines GS3-1OE, GS3-4OE and GS3 inhibition expression plants GS3RNAi, while in 75mM mixed alkaline solution Under treatment, the survival rates of GS3-1OE and GS3-4OE transgenic plants decreased by 12.5% and 26.4% respectively compared with ZH11. On the contrary, the survival rate of GS3RNAi increased by 7.4% ( Figure 12F). The above results all show that GS3 in rice is a negative factor. Important genes that regulate alkali tolerance.
  • pYL-U6a-gRNA plasmid was used as the template, and primers B1' and T1R were used to amplify the OsU6a promoter and the 20bp T1 target sequence of the GS3 gene; similarly, pYL-U3-gRNA was used as the template, and primers T1F and B2 were used.
  • the second round of PCR uses the first round PCR product as a template and uses primers B1' and B2 to amplify the OsU6a-T1-gRNA-polyT fragment; the same method is used to obtain OsU6a- Fragment of T2-gRNA-polyT.
  • the first round of PCR uses pYL-U6a-gRNA plasmid as the template, and uses primers B2' and T2R to amplify the OsU6b promoter and GS3 gene 20bp T2 target sequence; also uses pYL-U6b-gRNA as the template, and uses primers T2F and BL to amplify GS3 gene T2 target sequence and gRNA-polyT; the second round of PCR uses the first round PCR product as a template and uses primers B2' and BL to amplify the OsU6a-T2-gRNA-polyT fragment (Error! No citation source found. 15B ).
  • the primer sequences used in step 1) are as follows:
  • B2 AGCGTG ggtctcGtcag GGTCCATCCACTCCAAGCTC-3 (underlined is the BsaI restriction site, SEQ ID NO: 44)
  • T1F AACGGATTCAGCCGGTCTCG GTTTTAGAGCTAGAAATAGCA (underlined is target T1, SEQ ID NO: 47)
  • T1R CGAGACCGGCTGAATCCGTT TGCCACGGATCATCTGCACA (underlined is target T1, SEQ ID NO: 48)
  • T2F GGGACTTGAACGGATTCAGC GTTTTAGAGCTAGAAATAGCA (underlined is target T2, SEQ ID NO: 49)
  • T2R GCTGAATCCGTTCAAGTCCC CGGCAGCCAAGCCAGCACCCG (underlined is target T2, SEQ ID NO: 50).
  • the total volume of the PCR reaction is 50 ⁇ l, including 2 ⁇ l cDNA template, 25 ⁇ l 2 ⁇ GC I buffer, 5 ⁇ l 10mM dNTP, 1 ⁇ l each of 10mM primers GS3OEF and GS3OER, 1 ⁇ l ExTaq enzyme, and add deionized water to 50 ⁇ l (the 2 ⁇ GC I used Buffer, dNTP, ExTaq enzyme, etc. were purchased from Bao Bioengineering Dalian Co., Ltd.); PCR reaction The conditions are as follows: 194°C 4min, 294°C 30s, 358°C 30s, 472°C 1min, 5cycle from 2-4 33 times, 672°C 7min, 7save at 25°C.
  • the PCR fragments of OsU6a-T1-gRNA-polyT, OsU6b-T2-gRNA-polyT and the pYLCRISPR/Cas9-MT vector were cut and ligated with BsaI, and the OsU6-T1-gRNA-polyT and OsU6-T2-gRNA were -polyT was connected to the pYLCRISPR/Cas9-MT vector ( Figure 15C) to obtain the pYL-Cas9-gRNA-OsGS3 vector, and transformed into Zhonghua 11 (Zhonghua 11) to obtain the transgenic plant OsGS3 ko .
  • Design primers at 111 bp upstream and 72 bp downstream of the target site perform PCR amplification of the DNA of the transgenic plant OsGS3 ko obtained in Example 2, sequence the amplified fragment, and determine the mutation status of the target site.
  • GS3CRJCF/GS3CRJCR primers were used for PCR amplification.
  • the primer sequence for identifying the effect of GS3 mutation is as follows.
  • the fragment size is 273bp.
  • GS3CRJCF TACATAGCTGCTGCACCGTC(SEQ ID NO:51);
  • GS3CRJCR GAAGCAAGATCGAAGGAGTATG (SEQ ID NO: 52).
  • the total volume of the PCR reaction is 20 ⁇ l, including 2 ⁇ l of DNA template, 20 ⁇ l of 2 ⁇ GC I buffer, 2 ⁇ l of 2mM dNTP, 0.2 ⁇ l of 10mM primers GS3OEF and GS3OER, 0.2 ⁇ l of rTaq enzyme, and add deionized water to 20 ⁇ l (the 2 ⁇ used GC I buffer, dNTP, rTaq enzyme, etc.
  • OsGS3 ko has a 2bp insertion (see Figure 16), and the sequence is shown in SEQ ID NO:30.
  • a similar method was used to inhibit the GS3 protein (SEQ ID NO: 18) in the corn inbred line KN5585 (variety rights application number 20191002444).
  • the maize GS3 genome sequence is shown in SEQ ID NO:22, and the cDNA sequence is shown in SEQ ID NO:23.
  • the target is also designed at the first exon, the sequence is shown in SEQ ID NO:26, and the synthetic sgRNA sequence is shown in SEQ ID NO:29.
  • primers are designed at 118 bp upstream and 113 bp downstream of the target site, and PCR amplification and sequencing are performed to determine the mutation status of the target site.
  • the primer sequence for identifying the effect of ZmGS3 mutation is as follows, and the fragment size is 255bp.
  • ZmGS36F ACTATAACAATCGACGACGTG (SEQ ID NO: 53);
  • ZmGS36R AGCAGTGCAGCGTAATCGAT (SEQ ID NO: 54).
  • the total volume of the PCR reaction is 20 ⁇ l, including 2 ⁇ l of DNA template, 20 ⁇ l of 2 ⁇ GC I buffer, 2 ⁇ l of 2mM dNTP, 0.2 ⁇ l of each of 10 mM primers ZmGS36F and ZmGS36R, 0.2 ⁇ l of rTaq enzyme, and add deionized water to 20 ⁇ l (the 2 ⁇ used GC I buffer, dNTP, rTaq enzyme, etc.
  • PCR reaction conditions are as follows: 1 94°C for 4 min, 2 94°C for 30 s, 3 58°C for 30 s, 4 72°C for 1 min, 5 33 cycles from 2 to 4 , 672°C 7min, 7Save at 25°C.
  • this experiment obtained a pure and knockout mutant, designated as ZmGS3 ko , with a 34bp deletion at the target site ( Figure 17).
  • the mutant gene sequence of ZmGS3 ko is shown in SEQ ID NO: 31, and the deduced amino acid sequence of the mutant gene sequence is shown in SEQ ID NO: 32.
  • mutant genes or mutant proteins can be bred into other rice or corn or other hybridizable materials through conventional cross-pollination, thereby cultivating new alkali-tolerant strains.
  • TaGS Triticum aestivum
  • TaGS-7A gene The homologous gene of AT1 in wheat.
  • TaGS-7D gene The genome sequences are shown in SEQ ID NO:59, SEQ ID NO:60 and SEQ ID NO:61 respectively.
  • TaGS proteins include any one or more of TaGS-4A1 protein, TaGS-4A2 protein, TaGS-7A protein or TaGS-7D protein.
  • TaGS-4A1 protein and TaGS-4A2 protein are two transcripts of the wheat TaGS-4A gene, and their amino acid sequences are SEQ ID NO: 55 and SEQ ID NO: 56 respectively.
  • the TaGS-7A gene expresses the TaGS-7A protein shown in SEQ ID NO:57.
  • the TaGS-7D gene expresses the TaGS-7D protein shown in SEQ ID NO:58.
  • the TaGS gene in wheat has three homologous genes, TaGS-4A, TaGS-7A and TaGS-7D, and the corresponding gene ID numbers are: TraesCS4A02G474000, TraesCS7A02G017700 and TraesCS7D02G015000. Find a suitable target site, use a conserved target sequence to target the three homologous genes, and construct the knockout vector to select a target site located in the first exon (Figure 18).
  • the selected target sequence of the TaGS gene is as follows, and the underlined base is PAM.
  • TaGS gene target sequence AAGTCCCCGCTCGACCCCTG CGG (SEQ ID NO:70).
  • the Cas9 protein cuts in the target sequence region, forming a DNA double-strand break, triggering the self-damage repair mechanism in the body, and mutations will be introduced during the process of cells spontaneously repairing the gap ( "Mutation” here refers to mutations in a broad sense, including insertions, deletions, mutations in a narrow sense, and other forms. The vast majority of these mutations are gene function-inactivating mutations).
  • TaGS-1F GGCG AAGTCCCCGCTCGACCCCTG (SEQ ID NO:71).
  • TaGS-1R AAAC CAGGGGTCGAGCGGGGACTT (SEQ ID NO:72).
  • TaGS-1 Anneal TaGS-1F and TaGS-1R to form a double-stranded DNA with sticky ends, named TaGS-1, and connect it to the gel recovery product BUE411 in step 1 to obtain the recombinant plasmid pBUE411-TaGS-1.
  • the structure of the recombinant plasmid pBUE411-TaGS-1 is described as follows: the recognition of the two restriction endonucleases BsaI of the pBUE411 plasmid The recombinant plasmid obtained by replacing the small fragments between the specific sequences with the DNA fragments shown in positions 1-20 of SEQ ID NO:70.
  • Agrobacterium EHA105 carrying the recombinant plasmid pBUE411-TaGS-1 constructed in Example 1 was used to genetically transform wheat immature embryo callus. After transformation, complete regenerated plants (i.e., T0 generation) were obtained through tissue culture.
  • the TaGS-4A mutation of the E5 plant is a 13bp deletion of the CCGCTCGACCCC (SEQ ID NO:73) base at positions 6-18; the first TaGS-7A mutation of the E5 plant is a 1bp deletion of the C base at position 18, and the second The first mutation is a 2bp deletion of the CC base at positions 17-18; the first mutation of TaGS-7D in the E5 plant is a 1bp deletion of the G base at position 20, and the second mutation is a TG base deletion at positions 19-20 2bp is missing.
  • the above-mentioned embodiments of the present invention have achieved the following technical effects: by gene editing the nucleic acid molecules used for transcribing and translating the TaGS protein in wheat, the expression amount of the TaGS protein in wheat is reduced, and wheat With the improvement of salt-alkali tolerance, new wheat plants with improved salt-alkali tolerance were obtained.
  • TaAT1 null mutants i.e., TaGS gene triple mutants
  • TaAT1 also known as TaGS
  • Phenotypic analysis showed that under 125mM (104.2mM NaHCO 3 and 20.8mM Na 2 CO 3 , pH 9.7-9.8) mixed alkali treatment, the survival rates of TaAT1 ko -1 and TaAT1 ko -2 were 122% and 164% higher than TaWT, respectively.
  • % Figure 21C, D, E and F. It can be seen that knocking out all copies of TaAT1 can greatly enhance the salt-alkali tolerance of wheat.
  • TaAT1 adopts a conserved mechanism to resist salt-alkali stress like sorghum and rice
  • DAB 3,3-diaminobenzidine
  • H2DCFDA staining methods were used to determine ROS accumulation in the TaAT1 ko line.
  • the intracellular H 2 O 2 level of the TaAT1 ko strain was significantly lower than that of TaWT, which is consistent with the AT1 ko phenotype of other species.
  • the results show that, like the AT1 gene in sorghum, corn and rice, the TaAT1 gene is an important gene that negatively regulates the salt-alkali tolerance of wheat. Gene modification of the TaAT1 gene can also effectively improve the growth performance of wheat in saline-alkali soil. And the mechanism is conservative.
  • AT1/GS3 genes in crop production, we conducted field tests in trona-containing high-sodium soils on sorghum, rice, corn, and millet, which have different natural et al. gene and genetically modified AT1/GS3 genes.
  • the fields are located in two areas of China's saline-alkali region: the Da'an area of Jilin province (northern China) and the Pinglou area of Ningxia (northwestern China). These two areas are China's main crop producing areas, but crop yields are limited due to the presence of large areas of saline-alkali land.
  • KY NIL (GS3) is an excellent rice variety Kongyu131 carrying OsGS3-2 (an in-frame 3-bp insertion at the C terminus relative to OsGS3-1).
  • the function of OsGS3-2 is equivalent to that of OsGS3-1.
  • KY NIL (gs3 - ) is an introgression of OsGS3-3 in the Kongyu131 background, a completely loss-of-function allele.
  • an alkali resistance test was conducted on KY NIL (GS3) and KY NIL (gs3 - ). The experiment was carried out in the greenhouse at the seedling stage with a mixed alkali concentration of 75mM. As expected, KY NIL (gs3 ⁇ ) showed higher alkaline resistance than Kongyu131 ( Figure 22A and B).
  • ZKF5 rice Zhongkefa5
  • OsGS3-3 non-functional allele of GS3
  • KY NIL gs3 -
  • ZKF5 field performance was then tested in summer 2021 in relatively high-sodium soil (pH 8.5-8.7) and low-sodium soil (pH 7.4-7.6).
  • the local farmers association obtained field production data from more than 30 hectares of land.
  • the yield in the sodium-containing field high sodium
  • was only reduced by 7.8% compared to the neutral field (low sodium) Figure 22K.
  • These large-area field production data also indicate that the use of the GS3-3 allele (i.e., the non-functional allele) in rice production on sodic lands can breed rice with increased crop yields.
  • the survival rate of the SbAT1 ko line was over 60%, whereas the survival rate of the Wheatland wild type was only 33% (Figure 23B, panels 1 and 2).
  • Leaf burn a symptom that commonly occurs in monocotyledonous crops affected by high salt or high sodium stress, was observed in most Wheatland wild-type plants but not in plants of the SbAT1 ko line (Fig. 23B, first panel).
  • the tiller number and panicle number of the SbAT1 ko line were lower than those of the control, but the yield was 20.1% higher than the control (Figure 23B, third panel).
  • Millet SiAT1 ko and its wild-type control Ci846 were also grown together with sorghum in the same area.
  • the survival rate of the SiAT1 ko strain at the seedling stage is close to 100%, while the survival rate of wild-type Ci846 is only about 75% ( Figure 23D, left panel).
  • the panicle size of the knockout line SiAT1 ko was also larger than that of the control (Fig. 23D, middle panel), and the yield of SiAT1 ko was approximately 19.5% higher than that of the control (Fig. 23D, right panel).
  • non-functional mutations in the AT1 homologous gene can improve crop biomass or yield when grown in sodic soils (saline-alkali soils). Field performance in terms of yield.
  • Examples 1-5 we have confirmed that the AT1/GS3 gene plays a conserved and important role in the salt-alkali stress response of five monocotyledonous cereals (sorghum, millet, rice, corn and wheat).
  • genetic modification of the AT1/GS3 gene can also regulate the salt-alkali tolerance of dicotyledonous plants (such as soybeans) requires further study, considering that the AT1 homologous gene in soybean has a certain identity with the sorghum AT1 gene, especially is the identity of the GGL domain higher (approximately 40% to 50%), our preliminary experimental results (data not shown) support that the AT1 homologous gene in soybean will play a similar role in regulating salt-alkali tolerance as the homologous genes in sorghum, millet, rice, corn, and wheat.
  • the conservative effect of sex for example, reducing the expression of all copies of AT1 homologous genes in soybeans, especially knocking out the GGL domain, or using all copies of non-functional alleles, can improve the salt-alkali tolerance of soybeans; On the contrary, if the expression of the AT1 homologous gene in soybean is increased (for example, overexpression), or the C-terminal truncation mutant of the AT1 homologous gene is expressed, it is also possible to obtain salt-alkali sensitive soybeans.
  • the AT1 homologous gene sequence in soybean is shown in Table 4.

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Abstract

涉及植物耐盐碱相关基因AT1及其同源基因以及它们在培育耐盐碱性植物中的用途。还涉及培育耐盐碱性植物的方法、得到的耐盐碱性植物及其植物材料。当植物中编码包含与SEQ ID NO:15具有至少40％同一性、优选至少约75％同一性、更优选至少约80％同一性或更优选至少约90％或约95％同一性、或至具有少约60％相似性、优选至少约70％、约80％或约85％相似性、更优选至少约90％或约95％相似性的氨基酸序列的蛋白的所有等位基因的表达水平减少或不表达时，该植物的耐盐碱性提高，当植物中编码包含与SEQ ID NO:15具有至少40％同一性、优选至少约75％同一性、更优选至少约80％同一性或更优选至少约90％或约95％同一性、或至具有少约60％相似性、优选至少约70％、约80％或约85％相似性、更优选至少约90％或约95％相似性的氨基酸序列的蛋白的基因的表达水平增加时，该植物的盐碱敏感性提高。

Description

植物耐盐碱基因及其用途
优先权信息
本申请要求2022年8月12日提交的中国专利申请号202210968618.1和2022年9月29日提交的中国专利申请号202211198322.2的优先权，两份在先申请的全部内容通过引用结合在本文中。
技术领域
本发明涉及植物耐盐碱相关基因AT1及其同源基因以及它们在培育耐盐碱性植物中的用途。本发明还涉及培育耐盐碱性植物的方法、得到的耐盐碱性植物及其植物材料。
背景技术
粮食安全受到全球人口增长和气候变化对农业生产的潜在负面影响的影响。气候干旱、地下水位提高、地势低洼没有排水出路，这些都是土地盐碱化的原因。同时由于大量施用化肥可能会造成在不远的未来使50％的良田变为盐碱地，严重威胁粮食生产(A.Kumar,S.Singh,A.K.Gaurav,S.Srivastava,J.P.Verma,Plant growth-promoting bacteria:Biological tools for the mitigation of salinity stress in plants.Front.Microbiol.11,1216(2020).doi:10.3389/fmicb.2020.01216)。土壤盐渍化和次生盐碱化是全球性的生态与资源问题，是造成作物减产的重要非生物胁迫因子之一(Yamaguchi,T.and Blumwald,E.Developing salt-tolerant crop plants:challenges and opportunities.(2005).Trends in Plant Science 10:615-620)。
据联合国粮农组织FAO 2015年的调查数据显示，目前，全球大约20％的耕地和50％的灌溉土地受到不同程度盐渍化的影响，有超过10亿公顷的土地受到盐渍的影响，大约60％的估计面积被归类为碱性土地(即，由于以NaHCO3和Na2CO3为主的高碱含量而具有高pH值的土地)。全球约25-33％的灌溉土地受到次生盐度的影响(B.P.Singh,A.L.Cowie,K.Y.and Chan,Soil health and climate change.New York:Springer-Verlag Berlin Heidelberg 29,(2011).doi:10.1007/978-3-642-20256-8)。由于普通作物很难在盐碱地上成活，因此盐碱地难以成为作物种植的耕地，除非经过非常复杂繁琐的 去盐碱化处理。
盐渍化土壤泛指受盐碱影响的一类土壤，包括盐土和碱土。盐土指土壤中可溶性盐类物质含量超过2‰的土壤，而碱土指交换性钠离子占可溶性阳离子的比例(ESP)大于20％、pH值大于8.0的土壤(杨劲松.(2008).中国盐渍土研究的发展历程与展望.土壤学报45:837-845)。在盐碱地分布的地区，作物在整个生育期都受到不同程度的盐碱胁迫的伤害。特别是在春季时地表会“返盐”，这是由于地表水分蒸发强烈，地下水中的盐分随毛管水的上升而聚集在土壤表层，而春季是农作物播种的时节，盐碱胁迫对农作物萌发期的伤害极大。为解决盐碱地如何变良田的问题，已有多种技术被应用于改善土壤盐碱化，如：化学和物理方法来转换盐碱化的程度，或是通过耕作措施的手段来改良盐碱地，这两者虽然都能改善土壤理化性质和土壤质地，但工期长且成本高。因此，通过解析植物应答盐碱胁迫的分子机制，通过分子生物学的手段培育耐盐碱的品种，才能从根本上解决盐碱地的有效利用问题(Glenn,E.P.,Brown,J.J.and Blumwald E.(1999).Salt tolerance and crop potential of halophytes.Critical Reviews in Plant Sciences 18:227-255；钱前，漆小泉，林荣呈，杨淑华，董爱武，左建儒，陈凡，萧浪涛，顾红雅，陈之端，白永飞，王台.(2019).2018年中国植物科学若干领域重要研究进展.植物学报54:405-440)。
另外，通过分析Web of Science(http://www.webofscience.com/)上近20年发表的论文，与耐盐性相关的发表论文多达22614篇，而与耐碱度相关的发表论文只有457篇。由于人们对碱胁迫认识不足，限制了对耐碱性/耐盐碱作物的培育以及盐碱地作物增产技术的发展。实际上，植物耐盐和耐碱是两个不同的性状，根据FAO在2015年的统计，在调查的大于100万公顷盐碱地中，有60％实际可定义为由Na2CO3或者NaHCO3引起的土地碱化。与中性盐度(pH值约为7)仅具有离子毒性不同，碱性盐渍土具有高pH值，降低了植物对必需养分的吸收速率和钠离子(Na+)排出。与单独的盐渍性相比，碱性盐渍土通过诱导高细胞氧化应激对植物生长产生更多的负面影响(M.Javid,R.Ford,M.E.Nicolas,Tolerance responses of Brassica juncea to salinity,alkalinity and alkaline salinity.Funct.Plant Biol.39,699-707(2012).doi:10.1071/FP12109)。
因此，土壤盐碱化可能成为影响植物生长和作物生产的全球性问题。利用这些盐碱地进行作物生产有助于满足未来的粮食需求。因此，研究作物耐碱/耐盐碱的机制将 有助于碱地/盐碱地的治理，培育耐碱/耐盐碱的作物新品种也可以扩展耕地面积，是提升作物总产量的有效措施，研究作物耐盐碱的机制以及培育更耐盐碱的作物可能是未来农业的优先考虑事项。
发明内容
本发明的目的在于从基因水平上研究植物耐碱/耐盐碱的机制，研发培育耐盐碱植物的方法以及培育具有更高耐盐碱性的植物。
在研究中，本发明人已经鉴定并证明了AT1(一种非典型G蛋白γ亚基(Gγ亚基))的天然等位基因有助于五种不同单子叶作物(高粱、谷子、水稻、玉米和小麦)的耐碱性。AT1及其同系物的N端结构域在耐碱性胁迫中起负调控作用。含有C端截短AT1蛋白的作物对碱胁迫高度敏感。这可能是由于C端结构域的抑制作用，在其水稻同系物中，C端结构域对于蛋白质降解是必要的(S.Sun,L.Wang,H.Mao,L.Shao,X.Li,J.Xiao et al.,A G-protein pathway determines grain size in 45rice.Nat.Commun.9,851(2018).doi:10.1038/s41467-018-03141-y,W.Yang,K.Wu,B.Wang,H.Liu,S.Guo,X.Guo et al.,The RING E3 ligase CLG1 targets GS3 for degradation via the endosome pathway to determine grain size in rice.Mol.Plant 14,1699-1713(2021).doi:10.1016/j.molp.2021.06.027)。因此，整个AT1蛋白的过表达产生更高量的该蛋白，导致对碱胁迫的敏感性更高，而C端截短蛋白的过表达导致对碱胁迫的敏感性更高。相比之下，所有五种作物中由于该基因的敲除/具有无功能等位基因的自然变异而导致缺乏N端GGL结构域，均观察到对碱度/盐碱度的高耐受性。在此基础上，本发明人完成了本发明。
在第一方面，本发明提供AT1或其同源基因在调控植物的耐盐碱性或培育具有耐盐碱性或盐碱敏感性的植物中的用途。
在一些实施方案中，所述AT1或其同源基因包含GGL结构域或GGL样结构域，所述GGL结构域或GGL样结构域包含与SEQ ID NO:15具有至少40％同一性、优选至少约75％同一性、更优选至少约80％同一性或更优选至少约90％或约95％同一性、或至具有少约60％相似性、优选至少约70％、约80％或约85％相似性、更优选至少约90％或约95％相似性的氨基酸序列。
在一些实施方案中，所述GGL结构域或GGL样结构域包含SEQ ID NO:15和86- 96中任一氨基酸序列。
在一些实施方案中，在植物中增加AT1或其同源基因的表达水平(例如，过表达)、或表达或过表达AT1或其同源基因变体编码的C端截短蛋白，能够提高植物的盐碱敏感性。
在一些实施方案中，在植物中降低AT1或其同源基因的所有等位基因的表达水平(例如，通过基因编辑、靶向诱变法、化学诱导、射线诱导、自然突变、RNAi、或添加抑制目的基因表达的物质)或使所述所有等位基因不表达，能够提高植物的耐盐碱性。
本发明所述的耐盐碱性植物在盐碱性条件下的生长优于野生型植物，其中所述盐碱性条件包括pH>7.5、Na+浓度>75mM的生长条件或pH>8.0、Na+浓度>50mM的生长条件。
在一些实施方案中，在植物中敲除AT1或其同源基因所有等位基因的N端GGL结构域或GGL样结构域、或敲除N端第一个外显子，能够提高植物的耐盐碱性。
在一些实施方案中，AT1或其同源基因的N端GGL结构域或GGL样结构域是保守的。
在一些实施方案中，本发明提供一种核酸分子，其编码包含与SEQ ID NO:15具有至少40％同一性、优选至少约75％同一性、更优选至少约80％同一性或更优选至少约90％或约95％同一性、或至具有少约60％相似性、优选至少约70％、约80％或约85％相似性、更优选至少约90％或约95％相似性的氨基酸序列的蛋白，其用于调控植物的耐盐碱性，或用于培育具有耐盐碱性或盐碱敏感性的植物。
在一些实施方案中，所述核酸分子编码包含SEQ ID NO:15和86-96中任一氨基酸序列的蛋白。
在一些实施方案中，所述核酸分子编码选自下述的氨基酸序列：
(i)与SEQ ID NO:4具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
(ii)与SEQ ID NO:8具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
(iii)与SEQ ID NO:16-17中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
(iv)与SEQ ID NO:18具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
(v)与SEQ ID NO:55-58中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；或
(vi)与SEQ ID NO:75、77、79、81、83或85中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列。
本发明还提供一种突变蛋白质，其由编码下述氨基酸序列的核酸分子发生移码突变后的变体核酸分子编码：
(i)与SEQ ID NO:4具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
(ii)与SEQ ID NO:8具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
(iii)与SEQ ID NO:16-17中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
(iv)与SEQ ID NO:18具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
(v)与SEQ ID NO:55-58中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；或
(vi)与SEQ ID NO:75、77、79、81、83或85中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
其中与核酸分子发生移码突变前所编码的蛋白质相比，所述突变蛋白质的活性降低或没有活性。
在一些实施方案中，所述移码突变包括一个或多个(非3或3的倍数)核苷酸的插入或缺失。所述移码突变可以发生在目的编码序列中的任意位置。
本发明还提供编码所述突变蛋白质的核酸分子。
在一些实施方案中，本发明提供一种表达盒，其包含本发明所述的核酸分子。
在一些实施方案中，本发明提供一种重组载体，其包含本发明所述的核酸分子或表达盒。
在一些实施方案中，本发明提供一种细胞，其包含本发明所述的核酸分子、表达 盒或重组载体。
在一些实施方案中，所述细胞选自原核细胞或真核细胞，所述原核细胞例如细菌细胞或真菌细胞，例如，但不限于，大肠杆菌细胞、酵母菌细胞或农杆菌细胞；所述真核细胞例如植物细胞。
在第二方面，本发明提供一种培育耐盐碱性植物的方法，所述方法包括：
减少植物中编码包含与SEQ ID NO:15具有至少40％同一性、优选至少约75％同一性、更优选至少约80％同一性或更优选至少约90％或约95％同一性、或至具有少约60％相似性、优选至少约70％、约80％或约85％相似性、更优选至少约90％或约95％相似性的氨基酸序列的蛋白的所有等位基因的表达水平或使所述所有等位基因不表达，
其中所述耐盐碱性植物在盐碱性条件下的生长优于野生型植物，
其中所述盐碱性条件包括pH>7.5、Na+浓度>75mM的生长条件或pH>8.0、Na+浓度>50mM的生长条件。
在一些实施方案中，通过基因编辑方法、靶向诱变法、化学诱导、射线诱导、自然突变、RNAi或添加抑制目的基因表达的物质来减少植物中所述所有等位基因的表达水平或使所述所有等位基因不表达。
在一些实施方案中，植物中所述所有等位基因被敲除或突变，例如，通过同源重组敲除所述基因的GGL结构域或GGL样结构域，或通过CRISPR技术编辑所述基因的GGL结构域或GGL样结构域。
在一些实施方案中，所述所有等位基因的N端第一个外显子部分被敲除或突变，从而使得所编码的蛋白活性降低或没有活性。
在一些实施方案中，所述所有等位基因的GGL结构域或GGL样结构域、或N端第一个外显子部分被敲除或突变，从而使得所编码的蛋白活性降低或没有活性。
在一些实施方案中，相较于野生型对照植物，所述基因的表达水平至少减少51％，优选减少60％、70％或80％，更优选85％、90％或95％，或甚至不表达。
在一些实施方案中，所述植物是单子叶植物或双子叶植物，例如，禾本科植物，例如，但不限于，高粱属植物(例如高粱)、稻属植物(例如水稻)、谷子、玉米、小麦或大豆。
在一些实施方案中，所述方法还包括鉴定包含所述所有等位基因的敲除或突变、或包含无功能等位基因的亲本植物，进行自交或与另一包含所述基因的敲除或突变或 包含无功能等位基因的亲本植物杂交得到的一代或多代后代植物，其中所述所有等位基因的敲除或突变使得所述所有等位基因编码的蛋白活性降低或没有活性。
在一些实施方案中，编码选自下述的氨基酸序列的基因的所有等位基因的GGL结构域或GGL样结构域、或N端第一个外显子部分被敲除或突变：
(i)与SEQ ID NO:4具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
(ii)与SEQ ID NO:8具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
(iii)与SEQ ID NO:16-17中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
(iv)与SEQ ID NO:18具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
(v)与SEQ ID NO:55-58中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；或
(vi)与SEQ ID NO:75、77、79、81、83或85中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列。
在一些实施方案中，通过本发明的方法培育的耐盐碱性植物，与相应的野生型对照相比，在盐碱性生长条件下，具有提高的存活率、提高的产量、提高的株高或鲜重。
在一些实施方案中，所述盐碱性生长条件包括pH>7、Na+浓度>50mM的培养条件。
在第三方面，本发明提供一种植物或植物材料，其中编码包含与SEQ ID NO:15具有至少40％同一性、优选至少约75％同一性、更优选至少约80％同一性或更优选至少约90％或约95％同一性、或至具有少约60％相似性、优选至少约70％、约80％或约85％相似性、更优选至少约90％或约95％相似性的氨基酸序列的蛋白的所有等位基因被敲除或突变，优选地，其中所述基因的所有等位基因的GGL结构域或GGL样结构域、或N端第一个外显子部分被敲除或突变，
其中所述所有等位基因的敲除或突变使得所述所有等位基因编码的蛋白活性降低或没有活性。
在一些实施方案中，所述植物或植物材料包含所述基因的无功能等位基因。
在一些实施方案中，所述植物是单子叶植物或双子叶植物，例如，禾本科植物，例如，但不限于，高粱属植物(例如高粱)、稻属植物(例如水稻)、谷子、玉米、小麦或大豆。
在一些实施方案中，所述植物材料为植物部分、植物器官、植物组织、种子、植物原生质体或植物细胞，例如，胚、花粉、胚珠、种子、叶、花、枝、果实、茎杆、根、根尖、花药、植物细胞培养物或植物愈伤组织。
在一些实施方案中，所述植物或植物材料中，编码选自下述的氨基酸序列的基因的所有等位基因的GGL结构域或GGL样结构域、或N端第一个外显子部分被敲除或突变：
(i)与SEQ ID NO:4具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
(ii)与SEQ ID NO:8具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
(iii)与SEQ ID NO:16-17中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
(iv)与SEQ ID NO:18具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
(v)与SEQ ID NO:55-58中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；或
(vi)与SEQ ID NO:75、77、79、81、83或85中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列。
在第四方面，本发明提供一种制备杂交种植物种子的方法，所述方法包括：
(i)将第一亲本植株与第二亲本植株杂交，其中在所述第一亲本植株和第二亲本植株中，编码包含与SEQ ID NO:15具有至少40％同一性、优选至少约75％同一性、更优选至少约80％同一性或更优选至少约90％或约95％同一性、或至具有少约60％相似性、优选至少约70％、约80％或约85％相似性、更优选至少约90％或约95％相似性的氨基酸序列的蛋白的所有等位基因被敲除或突变；以及
(ii)收获杂交植株或其后代的种子。
在一些实施方案中，所述第一亲本植株和/或第二亲本植株包含所述基因的无功能等 位基因。
在一些实施方案中，所述第一亲本植株和/或第二亲本植株是近交系植株。
本发明还提供一种制备常规种植物种子的方法，所述方法包括：
扩繁亲本种子以收获其后代种子，其中在所述亲本种子中，编码包含与SEQ ID NO:15具有至少40％同一性、优选至少约75％同一性、更优选至少约80％同一性或更优选至少约90％或约95％同一性、或至具有少约60％相似性、优选至少约70％、约80％或约85％相似性、更优选至少约90％或约95％相似性的氨基酸序列的蛋白的所有等位基因已被敲除或突变，使得所述蛋白不被表达或与野生型植物相比表达水平减少。
在一些实施方案中，在所述亲本种子中，编码包含下述氨基酸序列的所有等位基因的已被敲除或突变：
(i)与SEQ ID NO:4具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
(ii)与SEQ ID NO:8具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
(iii)与SEQ ID NO:16-17中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
(iv)与SEQ ID NO:18具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
(v)与SEQ ID NO:55-58中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；或
(vi)与SEQ ID NO:75、77、79、81、83或85中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列。
在第五方面，本发明提供由第四方面的种子长成的植物或其植物材料。
在第六方面，本发明提供一种培育盐碱敏感性的植物的方法，所述方法包括：
增加植物中编码包含与SEQ ID NO:15具有至少40％同一性、优选至少约75％同一性、更优选至少约80％同一性或更优选至少约90％或约95％同一性、或至具有少约60％相似性、优选至少约70％、约80％或约85％相似性、更优选至少约90％或约95％相似性的氨基酸序列的蛋白的基因的表达水平，或在植物表达或过表达所述基因突变体编码的C端截短蛋白。
在一些实施方案中，通过向植物中导入外源编码包含与SEQ ID NO:15具有至少40％同一性、优选至少约75％同一性、更优选至少约80％同一性或更优选至少约90％或约95％同一性、或至具有少约60％相似性、优选至少约70％、约80％或约85％相似性、更优选至少约90％或约95％相似性的氨基酸序列的蛋白的核酸分子而增加目的基因的表达水平。
在一些实施方案中，向所述植物的细胞或组织中导入携带所述核酸分子的遗传物质，所述遗传物质在所述植物内，以游离或整合至所述植物的染色体的形式存在，再将导入所述遗传物质后的细胞或组织培养成完整植株，获得所述盐碱敏感植物。
在本发明中，所述盐碱性条件包括pH>7.5、Na+浓度>75mM的生长条件或pH>8.0、Na+浓度>50mM的生长条件。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是，本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1.不同品种高粱群体耐盐碱性鉴定的评价条件和表型变异。(A)16个高粱品种种子在萌芽期时处于不同浓度盐碱胁迫状态下的存活率分析；(B-F)采用75mM混合碱溶液(即，摩尔比5：1的NaHCO3：Na2CO3，pH＝9.2-9.4)处理不同品种高粱种子，其中(B)显示在连续5周的碱胁迫下16个高粱品种的相对存活率(relative survival rate,RSR)分析；(C)用碱胁迫处理3周或不用碱胁迫处理(即，CK，对照)的代表性高粱品种的表型观察。图中的刻度条表示5cm；(D)在75mM混合碱溶液处理后第21天具有不同相对存活率的来自高粱联合群体(sorghum association panel，SAP)的高粱自交系材料(sorghum accessions)的数量；(E)352份籽粒高粱自然群体材料在对照(CK)和碱处理条件(75mM混合碱处理)下的相对存活率。计数时去除对照中低萌芽率(<80％)的品种。(F)SAP群体中耐盐碱性的QQ-plot分析。
图2.高粱在不同盐碱水平处理下的表型变化。(A)16份供试材料在不同浓度混合碱胁迫条件下的成苗率统计分析；(B)16份供试材料在75mM混合碱胁迫和对照条件下不同天数的相对存活率分析。CK代表对照条件下的存活率，T代表75mM混合碱胁迫的存 活率，T_CK代表相对存活率情况。
图3.高粱中与耐盐碱性相关的SbAT1基因的自然变异。(A)高粱自然群体中耐碱性全基因组关联分析(Genome wide association study,GWAS)的曼哈顿图(Manhattan plot)。性对存活率从在有(碱胁迫)和无(对照)75mM混合碱溶液添加的条件下播种并生长21天的高粱植株获得。图中箭头标示SbAT1基因的主要基因座。(B)1号染色体上SbAT1基因座前后约10Mb的基因组区域的散点图。(C)在检测到的29个SbAT1基因区序列变异与37个测序的高粱材料的耐碱性之间的基于SbAT1的关联性作图。29个因果位点(causal sites)之间的LD分析表明连锁关联性信号。五个主要变异位点(红点)显示具有强LD的强关联信号并用黑色线条突出显示。(D)基于五个主要变异位点检测到SbAT1的两个典型单倍体型(Hap1和Hap2)。Hap2中的移码突变(由“G”突变为“GGTGGC”)以红色突出显示。(E)Hap1中20个高粱材料和Hap2中17个高粱材料在进行碱胁迫(75mM混合碱处理)的相对存活率。n表示数量，统计学P值通过双尾不配对t检验确定。(F)耐碱性(T)和碱敏感性(S)高粱植株在75mM混合碱处理的5天和8天胁迫下以及在对应的对照条件(CK)下的SbAT1相对表达水平。统计学显著性通过附带Tukey’s多重比较检验的单向ANOVA确定。NS表示没有显著性。
图4.高粱AT1和其突变体at1的蛋白结构示意图。
图5.高粱NIL亲本系和SbAT1转基因植株在碱胁迫和/或盐胁迫处理下的表型。(A)NIL亲本系SN010(NIL-AT1)和M-81E(NIL-at1)在碱胁迫下的表型。SN010(NIL-AT1)和M-81E(NIL-at1)种子在无或有75mM混合碱的土壤中播种，17天后拍照。刻度条表示5cm。(B和C)与没有碱处理(CK)相比，SN010(NIL-AT1)和M-81E(NIL-at1)在碱处理下的相对存活率(B)和相对株高(C)统计学分析。(D)高粱NIL亲本系SN010(NIL-AT1)和M-81E(NIL-at1)在盐胁迫下的表型。SN010(NIL-AT1)和M-81E(NIL-at1)种子在包含75、100、150和200mM的NaCl的中性pH值土壤中播种，第14天后拍照。刻度条表示5cm。(E和F)SN010(NIL-AT1)和M-81E(NIL-at1)在(D)的盐处理下的相对存活率(B)和相对株高(C)统计学分析。(G)在SbAT1过表达(SbAT1-OE)植株T0代中SbAT1表达的qRT-PCR测定。(H)在对照(CK)和75mM混合碱胁迫下，SbWT、SbAT1-OE和SbAT1ko的表型分析。在播种后第14天拍照，刻度条表示5cm。(K)SbAT1和Sbat1的蛋白质印迹分析。上图是SbAT1及其截短形式Sbat1与GFP在C端融合的示意图。核糖体-1,5-二磷酸羧化酶/加氧酶(ribulose-1,5-bisphosphate carboxylase/oxygenase，RbcL)的大亚基用作上样对照。
图6.SbAT1在高粱耐碱性中的功能。(A)在高粱NIL-AT1和NIL-at1植株中SbAT1及其截短形式Sbat1的蛋白示意图。(B)高粱NIL幼苗在碱胁迫下的表型分析。在种子播种(CK，无碱胁迫；75mM混合碱)后第14天拍照，刻度条表示5cm。(C)(B)中幼苗的相对存活率统计分析。(D)SbWT、SbAT1过表达(SbAT1-OE)和SbAT1基因敲除(SbAT1ko)的植株中SbAT1及其无功能性版本的示意图。(E)bWT、SbAT1过表达(SbAT1-OE)和SbAT1基因敲除(SbAT1ko)植株在无碱胁迫(CK)和75mM混合碱胁迫处理下第14天的代表性幼苗照片。刻度条表示5cm。(F)(E)中幼苗的相对存活率统计学分析。
图7.高粱近等基因系材料播种在中国的西北地区宁夏盐碱地的田间表现情况。
图8.近等基因系材料在中国宁夏不同地区盐碱地上的土壤理化性质及对应的成苗率统计数据。(A)和(B)分别为宁夏惠威村和东风村盐碱地的土壤理化性质，C和D分别为近等基因系材料在宁夏惠威村和东风村盐碱地上的存活率统计分析数据。
图9.高粱过表达转基因及突变体的构建和基因编辑鉴定结果。
图10.谷子突变体的构建和基因编辑材料鉴定结果。
图11.转基因高粱和谷子表型鉴定浓度筛选。(A)转基因高粱和谷子遗传材料在对照(CK)和不同混合碱胁迫处理后第14天的生长情况，(B)转基因高粱和谷子遗传材料在不同混合碱胁迫条件下的存活率统计分析，上图为转基因高粱的存活率统计分析，下图为转基因谷子的存活率统计分析。
图12.AT1同源物Gγ样亚基在谷子(millet)、水稻和玉米耐碱性中具有保守性功能。(A)谷子SiAT1及其在SiAT1遗传植株中截短或无功能版本的示意图。SiWT表示野生型谷子Ci846。(B)代表性SiAT1遗传植株在无或有75mM混合碱胁迫下播种后第14天的照片，刻度条表示5cm。(C)(B)中谷子的相对存活率统计学分析。(D)水稻OsGS3及其在OsGS3遗传植株中截短或无功能版本的示意图。OsWT是野生型水稻ZH11。(E)代表性OsGS3遗传植株在无或有75mM混合碱胁迫下播种后第21天的照片，刻度条表示5cm。(F)(E)中水稻的相对存活率统计学分析。(G)野生型玉米ZmWT中野生型ZmGS3和ZmGS3ko玉米中无功能版本的示意图。ZmWT是野生型玉米KN5585。(H)ZmWT和ZmGS3ko玉米在无或有75mM混合碱胁迫下播种后第14天的照片，刻度条表示5cm。(I)在播种后第50天(H)中玉米植株的相对存活率统计学分析。
图13.AT1在谷子和玉米中的基因编辑信息和转基因植株响应碱胁迫的表型。(A)不同SiAT1124过表达(SiAT1124-OE)株系T0代的Myc-SiAT1124表达的蛋白质印迹分析。(B)谷子SiAT1102植株中AT1的靶序列位置和基因编辑信息。在SiAT1102发生一个碱基插入，导致移码突变和翻译提取终止。在SiAT1102中保留具有预测的1-102位氨基酸的C端截短的蛋白。(C)SiWT、SiAT1124-OE、SiAT1102和SiAT1ko谷子植株在碱胁迫下的表型分析和相对株高统计学分析。谷子种子播种在无或有75mM混合碱的土壤中在播种后第14天拍照，刻度条表示5cm。(D)水稻OsWT、OsGS3-1OE、OsGS3-4OE、OsGS3ko和OsGS3Ri在无或有75mM混合碱胁迫处理下的生长形态。在播种后第29天拍照，刻度条表示5cm。(E和F)(D)中水稻株系的相对株高(E)和相对叶绿体含量(F)的统计学分析。数据为每个株系4个代表性植株的平均值±SEM。统计学显著性通过附带Tukey’s多重比较检验的单向ANOVA确定。(G)ZmGS3和ZmGS3ko玉米植株的靶序列位置和基因编辑信息。在ZmGS3ko玉米植株中发生34bp的缺失和1个碱基突变，导致移码突变和翻译过早终止。(H)玉米ZmWT和ZmGS3ko植株在碱胁迫处理下的表型分析和相对株高统计学分析。玉米种子在无(CK)或有75mM混合碱的土壤中播种，并在播种后第14天拍照。刻度条表示5cm。右图显示玉米ZmWT和ZmGS3ko植株在75mM混合碱胁迫下的株高统计学分析。统计学差异通过双尾不配对t检验确定。(I)玉米ZmWT和ZmGS3ko植株在75mM混合碱胁迫处理下的表型。在播种后第50天拍照。刻度条表示5cm。
图14.超量表达水稻GS3-1或者GS3-4以及GS3抑制表达转基因植株的构建结果。(A)过表达载体示意图；(B)抑制表达载体示意图；(C)过表达以及抑制表达转基因植株表达量检测结果。
图15.水稻GS3 CRISPR敲除载体pYL-Cas9-gRNA-OsGS3构建过程示意图，(A)水稻GS3基因的结构以及CRISPR靶点位置示意图；(B)两个靶点T1以及T2分别通过PCR的方法插入到pYL-OsU3-gRNA以及pYL-OsU6a-gRNA，获得pYL-OsU3-T1-gRNA以及pYL-OsU6a-T2-gRNA的过程；(C)表达载体pYL-Cas9-gRNA-OsGS3结构示意图。
图16.水稻GS3 CRISPR敲除转基因植株T1代突变位点检测结果。
图17.玉米ZmGS3 CRISPR敲除载体pYL-Cas9-gRNA-ZmGS3构建过程示意图。(A)玉米ZmGS3基因的结构以及CRISPR靶点示意图；(B)玉米ZmGS3 CRISPR敲 除转基因植株T1代突变位点检测结果。
图18.根据本发明实施例4.1的TaGS基因结构及利用CRISPR/Cas9技术的靶点的设定示意图。
图19.根据本发明实施例4.2的转基因小麦植株E5的TaGS基因T2代突变体测序结果示意图；其中，WT表示野生型基因序列，“-”表示发生了删除突变的序列，“-”后边的数字表示删除或插入的核苷酸的数量。
图20.根据本发明实施例4.3的盐碱胁迫下小麦耐盐碱表型。其中，A为清水处理；B为75mM混合碱盐溶液(摩尔比5：1的NaHCO3：Na2CO3)处理；A和B中的左边三株为野生型Fielder小麦(即目的小麦)，标注为WT；右边三株为小麦TaGS基因三突突变体E5，标注为E5。
图21.小麦中TaAT1(也称为TaGS)的基因编辑信息和转基因小麦植株相应碱胁迫的表型。(A)CRISPR/Cas9构建体中T-DNA结构的示意图。(B)CRISPR/Cas9诱导的靶基因诱变。靶序列和PAM序列分别用蓝色和红色显示。突变位点以虚线显示。(C)野生型小麦TaWT和TaAT1敲除(TaAT1ko)植株在碱胁迫下的表型分析。小麦种子播种在无或有125mM混合碱的土壤中，在播种后第21天拍照，刻度条表示5cm。(D)(C)中小麦品系的相对存活率统计学分析。(E)在播种后第21天在无或有125mM混合碱胁迫处理下TaAT1遗传植株的代表性幼苗。(F)(E)中小麦品系的相对株高统计学分析。数据为每个品系三个代表性植株的平均值±SEM。(G)野生型小麦TaWT和TaAT1敲除(TaAT1ko)植株叶片的DAB染色。刻度条表示1cm。将10天的幼苗用或不用250mM混合碱处理60小时，用于分析。(H)在TaAT1相关遗传植株的根尖用ROS检测探针(H2DCFDA)检测的H2O2量。将10天的幼苗用或不用250mM混合碱处理48小时，用于分析。刻度条表示100μm。(I)(H)中测得的H2O2浓度的统计学分析。数据为平均值±SEM(n＝6个植株)。
图22.OsGS3无功能等位基因有助于水稻的耐碱性。(A)用或不用75mM混合碱处理的KYNIL(GS3)与KYNIL(gs3-)幼苗在播种后第21天的表型。刻度条表示5cm。右图显示与无碱处理相比较在碱处理下水稻的相对株高统计学分析。(B)在土壤中生长的水稻KYNIL(GS3)与KYNIL(gs3-)表型。刻度条表示5cm。右图显示相抵存活率统计学分析。数据为每个处理四次重复的平均值±SEM，每次重复检测40个植株。(C)在中国吉林省温室中的自然碱性土壤(pH9.45)和接近中性土壤(pH7.74)中生长的水稻NIL的相对 存活率和籽粒产量。穗数表示每个植株的稻穗数量，数据为平均值±SEM(n＝3小块试验田)。(D)2021年在中国吉林省碱地(pH9.17)的KYNIL(GS3)与KYNIL(gs3-)的幼苗表型。(E)2021年在中国吉林省碱地(pH9.17)的KYNIL(GS3)与KYNIL(gs3-)的代表性稻穗。刻度条表示2cm。(F)2021年在中国黑龙江省田地(pH5.58)种植的KYNIL(GS3)与KYNIL(gs3-)的籽粒数目/稻穗。数据为平均值±SEM(n＝28)。(G和H)2022年在中国吉林省碱地(pH9.10)和黑龙江省田地(pH5.58)种植的KYNIL(GS3)与KYNIL(gs3-)的籽粒产量。数据为平均值±SEM(n＝5小块试验田)。(I和J)2022年在中国北京无碱田地(pH7.20)和吉林省碱地(pH9.10)种植的水稻KYNIL(GS3)与KYNIL(gs3-)的粒长(I)和粒宽(J)。数据为平均值±SEM(n＝100)。(K)具有OsGS3无功能等位基因的改良优良水稻品种Zhongkefa5(ZKF5)的籽粒产量。该水稻在2021年在中国吉林省的高钠土壤(pH8.5-8.7)和低钠土壤(pH7.4-7.6)种植。(L)在中国吉林省温室中的自然碱土(pH9.45)和接近中性土壤(pH7.74)种植的OsWT和OsGS3ko的相对存活率统计学分析。数据为平均值±SEM(n＝3小块试验田)。
图23.AT1/GS3敲除和自然无功能等位基因提盐碱地作物产量。(A)2021年在中国吉林省碱土(pH9.17)种植的水稻KYNIL(GS3)与KYNIL(gs3-)的表型和籽粒产量。第一个图显示在生殖期(在田间种植后3个月)水稻植株的表型。穗数表示每个水稻植株的稻穗数量。数据为平均值±SEM(n＝3小块试验田)。(B)2021年在中国宁夏自治区碱土(pH9.10)种植的高粱SbWT和SbAT1ko的表型、存活率、籽粒产量和总生物量。数据为平均值±SEM(n＝3小块试验田)。(C)2021年在中国宁夏自治区碱土(pH9.10)种植的高粱NIL-SbAT1和NIL-Sbat1幼苗的表型和鲜重。(D)2021年在中国宁夏自治区碱土(pH9.10)种植的谷子品系的表型、存活率和籽粒产量。数据为平均值±SEM(n＝3小块试验田)。(E)2021年在中国宁夏自治区碱土(pH9.10)种植的玉米ZmWT和ZmGSko植株的存活率统计学分析。数据为平均值±SEM(n＝3小块试验田)。在(A-E)中，统计学显著性通过双尾不配对t检验确定。*P<0.05和**P<0.01，***P<0.001和****P<0.0001。
图24.推测的植物中Gγ亚基AT1-介导的碱应激反应的模型。在碱胁迫下，PIP2s作用为H2O2输出蛋白。Gγ亚基AT1可能与Gβ配对负调控PIP2s的磷酸化，由此降低PIP2s的H2O2输出能力，导致H2O2过度积聚，并导致植物对碱胁迫的敏感性。截短形式的AT1，即at1，进一步抑制H2O2输出活性，并导致植物对碱胁迫的高敏感性。然而， AT1的自然无功能形式或敲除AT1同源基因释放对PIP2s的抑制作用并有效地提高作物的碱胁迫耐受性。
图25.高粱(Sb)、水稻(Os)、谷子(Si)、栽培大豆(Gm)、野生大豆(Gs)的AT1同源基因的氨基酸序列比对。红色方框框出的部分是预测的保守的GGL结构域。
序列表说明
表A.本发明中序列的简要说明

发明详述
本领域技术人员应该理解，本发明不限于本文中描述的特定方法学、实施方案和试剂，因为这些是示例性说明。还应理解本文中使用的术语仅为了描述具体实施方案，而并不意图限制本发明的范围，本发明的范围仅由所附权利要求书限定。
除非另外定义，本文中使用的所有技术和科学术语与本发明所属领域中普通技术人员通常的理解具有相同的含义。
此外，除非上下文另有要求，单数形式的术语应包括复数形式，复数形式的术语应包括单数形式。更具体地，如在本说明书和所附权利要求中所使用的，除非上下文另外明确指出，否则单数形式“一种”和“这种”包括复数指示物。
定义
提供以下定义和方法用以更好地界定本申请以及在本申请实践中指导本领域普通技术人员。除非另作说明，术语按照相关领域普通技术人员的常规用法理解。本文所引用的 所有专利文献、学术论文、行业标准及其他公开出版物等，其中的全部内容整体并入本文作为参考。
如本文所用，“植物”在广义上包括对整株植物、植物器官、植物组织、种子和植物细胞以及它们的子代的标引。植物细胞包括但不限于来自种子、悬浮培养物、胚芽、分生区域、愈伤组织、叶、根、苗、配子体、孢子体、花粉和小孢子的细胞。“子代”包含植物的任何后续世代。“水稻”或“玉米”是任何水稻或玉米植物并包括可以与水稻或玉米育种的所有植物品种，包括整株植物、植物细胞、植物器官、植物原生质体、植物可以从中再生的植物细胞组织培养物、植物愈伤组织、植物或植物部分中完整的植物细胞，所述植物部分例如胚、花粉、胚珠、种子、叶、花、枝、果实、茎杆、根、根尖、花药等。
如本文所用，术语“盐碱地”、“含钠土壤”或“碱性土壤”是指盐类集积的土壤种类，指土壤里面所含的盐分影响到植物(例如，作物)的正常生长。碱土和碱化土壤的形成，大部分与土壤中碳酸盐的累计有关，因而碱化度普遍较高，严重的盐碱土壤地区植物几乎不能生存。根据土壤含盐量和pH值，盐碱地分为轻盐碱地、中度盐碱地和重盐碱地。其中，轻盐碱地是指种植作物时的出苗率在70％-80％，含盐量在千分之三以下；重盐碱地是指含盐量超过千分之六，出苗率低于50％；介于轻盐碱地与重盐碱地之间的是中度盐碱地；用pH值表示为：轻度盐碱地pH值为：7.1-8.5，中度盐碱地pH值为：8.5-9.5，重度盐碱地pH值为：9.5以上。在实验室中通常用混合钠盐(例如，碳酸钠和碳酸氢钠)来模拟“含钠土壤”。
在本申请中，词语“包括”、“包含”或其变体应理解为除所描述的元素、数或步骤外，还包含其它元素、数或步骤。
除非另有所指，核酸以5’至3’方向从左向右书写；氨基酸序列以氨基至羧基方向从左向右书写。氨基酸在本文可以用其通常所知的三字母符号或IUPAC-IUB生物化学命名委员会推荐的单字母符号来表示。同样地，可以用通常接受的单字母码表示核苷酸。数字范围包括限定该范围的数字。
如本文所用，“核酸”包括涉及单链或双链形式的脱氧核糖核苷酸或核糖核苷酸多聚物，并且除非另有限制，包括具有天然核苷酸基本性质的已知类似物(例如，肽核酸)，所述类似物以与天然存在的核苷酸类似的方式与单链核酸杂交。
如本文所用，术语“编码”或“所编码的”用于特定核酸的上下文时，指该核酸包含指导 该核苷酸序列翻译成特定蛋白的必需信息。使用密码子表示编码蛋白的信息。如本文所用，涉及特定多核苷酸或其所编码的蛋白的“全长序列”指具有天然(非合成)内源序列的整个核酸序列或整个氨基酸序列。全长多核苷酸编码该特定蛋白的全长、催化活性形式。
本文可互换地使用术语“多肽”、“多肽”和“蛋白”，以指氨基酸残基的多聚物。该术语用于氨基酸多聚物，其中一个或多个氨基酸残基是相应天然存在的氨基酸的人工化学类似物。该术语还用于天然存在的氨基酸多聚物。
本文可互换地使用术语“残基”或“氨基酸残基”或“氨基酸”，以指被并入蛋白、多肽或肽(统称“蛋白”)的氨基酸。氨基酸可以是天然存在的氨基酸，并且除非另有限制，可以包括天然氨基酸的已知类似物，所述类似物可以与天然存在的氨基酸相似的方式起作用。
在一些实施方案中，可以对本申请的核苷酸序列进行改变，以进行保守氨基酸替换。保守氨基酸替换的原则和实例在下文中进一步描述。在某些实施方案中，可以依照公开的单子叶密码子偏好性对本申请的核苷酸序列进行不改变氨基酸序列的替换，例如可以用单子叶植物偏好的密码子替换编码同一氨基酸序列的密码子，而不改变该核苷酸序列所编码的氨基酸序列。在一些实施方案中，以编码同一氨基酸序列的不同密码子替换本申请中的部分核苷酸序列，从而在改变核苷酸序列的同时不改变其编码的氨基酸序列。保守变体包括由于遗传密码子简并性而编码实施方案的蛋白中的一种的氨基酸序列的那些序列。在一些实施方案中，根据单子叶植物偏好密码子替换本申请中的部分核苷酸序列。本领域技术人员会认识到氨基酸添加和/或取代通常基于氨基酸侧链取代基的相对相似性，例如，所述取代基的疏水性、电荷、大小等等。具有各种前述所考虑性质的示例性氨基酸取代基团为本领域技术人员所公知，并且包括精氨酸与赖氨酸；谷氨酸和天门冬氨酸；丝氨酸和苏氨酸；谷氨酰胺和天冬酰胺；以及缬氨酸、亮氨酸和异亮氨酸。关于不影响目的蛋白生物学活性的适当氨基酸取代的指南可以在Dayhoff等人(1978)Atlas of Protein Sequence and Structure(Natl.Biomed.Res.Found.,Washington,D.C)(通过引用并入本文)的模型中找到。可以进行诸如将一个氨基酸换作具有相似性质的另一个氨基酸的保守性取代。“保守氨基酸取代”是将氨基酸取代为不同氨基酸的那些取代，其中预测该取代对参考多肽的特性干扰最少。换言之，保守氨基酸取代基本上保留了参考多肽的结构和功能。下表B提供了本文考虑的示例性保守氨基酸取代的列表.
表B.示例性保守氨基酸取代
关于蛋白质，“缺失”是指导致一个或多个氨基酸残基的不存在的氨基酸序列的变化。缺失可以去除至少1、2、3、4、5、10、20、50、100、200或更多个氨基酸残基。缺失可包括内部缺失和/或末端缺失(例如，参考多肽的N-末端截短、C-末端截短或两者)。参考多肽序列的“变体”、“突变体”或“衍生物”可以包括相对于参考多肽序列的缺失。
关于蛋白质，“片段”是与参考序列在序列上相同但长度比参考序列短的氨基酸序列的一部分。片段可以包含多至参考序列的整个长度，减去至少一个氨基酸残基。例如，片段可分别包含参考多肽的5至1000个连续氨基酸残基。在一些实施方案中，片段可包含参考多肽的至少5、10、15、20、25、30、40、50、60、70、80、90、100、150、250或500个连续氨基酸残基。片段可以优先选自分子的某些区域。术语“至少一个片段”包括全长多肽。相对于全长蛋白质，片段可以包括N-末端截短、C-末端截短或这两种截短。参考多肽序列的“变体”、“突变体”或“衍生物”可以包括参考多肽序列的片段。
关于蛋白质，术语“插入”和“添加”是指导致一个或多个氨基酸残基的添加的氨基酸序列的变化。插入或添加可指1、2、3、4、5、10、20、30、40、50、60、70、80、90、100、150、200或更多个氨基酸残基。参考多肽序列的“变体”、“突变体”或“衍生物”可以包括相对于参考多肽序列的插入或添加。蛋白质的变体可具有N-末端插入、C-末端插入、内部插入或N-末端插入、C-末端插入和内部插入的任意组合。
关于蛋白质，术语“同一性百分比”和“％同一性”是指使用标准化算法比对的至少两个氨基酸序列之间的残基匹配的百分比。氨基酸序列比对的方法是众所周知的。一些比对方法考虑了保守的氨基酸取代。下文更详细解释的此类保守取代通常保留取代位点处的电荷和疏水性，从而保留多肽的结构(并因此保留功能)。氨基酸序列的同一性百分比可以如本领域所理解的那样确定(参见，例如，美国专利号7,396,664，其通过引用整体并入本文)。美国国家生物技术信息中心(NCBI)基本局部比对搜索工具(BLAST)提供了一套常用且可免费获得的序列比较算法，其可从多个来源获得，包括NCBI，Bethesda，Md.，在其网站上获得。BLAST软件套件包括各种序列分析程序，包括“blastp”，其用于将已知氨基酸序列与来自各种数据库的其他氨基酸序列进行比对。
关于蛋白质，同一性百分比可以在整个确定的多肽序列(例如，如由特定的SEQ ID号确定的)的长度上测量，或可以在较短的长度上测量，例如，在取自更大的、确定的多肽序列的片段的长度(例如至少15、至少20、至少30、至少40、至少50、至少70或至少150个连续残基的片段)上测量。此类长度仅是示例性的，并且应理解，本文在表格、附图或序列表中所示序列支持的任何片段长度可用于描述在其上可测量同一性百分比的长度。
关于核酸和蛋白质，术语“相似性(similarity)”是指所检测的序列与参考序列之间相同的碱基或氨基酸占整个序列的比例(相对宏观的描述)。在氨基酸序列比对中，相似性还包括，除了完全相同的残基外，在对应位置的两个残基是否具有相似的特性，如侧链基团的大小、电荷性、亲疏水性等。换言之，对于蛋白质而言，“同一性”要求比对的位置处的氨基酸完全相同，而“相似性”不要求所比对的位置处的氨基酸完全相同，如果所比对位置处的氨基酸属于保守取代的残基，则认为该位置处的氨基酸是相似的。
“蛋白标签(protein-tag)”是指利用DNA体外重组技术，与目的蛋白一起融合表达的一种多肽或者蛋白，以便于目的蛋白的表达、检测、示踪和/或纯化。蛋白标签包括但 不限于Flag标签、His标签、MBP标签、HA标签、myc标签、GST标签和/或SUMO标签等。
如本文所用，“核酸序列同一性”是指两个多核苷酸序列之间的序列相似性。当两个比较序列中的位置均被相同碱基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同一的。两个序列之间的同一性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100的函数。
核酸序列同一性的鉴定包括杂交技术。例如，将已知核苷酸序列的全部或部分用作与其它相应核苷酸序列选择性杂交的探针，所述其它相应核苷酸序列存在于来自所选生物体的已克隆基因组DNA片段或cDNA片段群(即基因组文库或cDNA文库)。所述杂交探针可以是基因组DNA片段、cDNA片段、RNA片段或其它寡核苷酸，并且可以用诸如32P的可检测基团或其它可检测标志物来标记。因而，例如，可以通过标记基于实施方案序列的合成寡核苷酸制备杂交探针。制备杂交探针和构建cDNA及基因组文库的方法通常为本领域已知。可以在严谨条件下进行所述序列的杂交。如本文所用，术语“严谨条件”或“严谨杂交条件”表示如下条件，即在该条件下，相对于与其它序列杂交，探针将以可检测的更大程度(例如，背景的至少2倍、5倍或10倍)与其靶序列杂交。严谨条件是序列依赖性的并且在不同环境中有所不同。通过控制杂交严谨性和/或控制清洗条件，可以鉴定与所述探针100％互补的靶序列(同源探针法)。备选地，可以调节严谨条件，以允许一些序列错配，以便检测较低的相似度(异源探针法)。通常，探针长度少于约1000或500个核苷酸。通常，严谨条件是如下的条件，即在该条件中，盐浓度为pH 7.0至8.3下，少于约1.5M Na离子，通常约0.01M至1.0M Na离子浓度(或其它盐)，并且温度条件为：当用于短探针时(例如10到50个核苷酸)，至少约30℃；当用于长探针时(例如大于50个核苷酸)，至少约60℃。还可以通过添加诸如甲酰胺的去稳定剂来实现严谨条件。示例性的低严谨条件包括37℃下使用30％至35％的甲酰胺缓冲液、1M NaCl、1％SDS(十二烷基硫酸钠)杂交，50℃至55℃下在1×至2×SSC(20×SSC＝3.0M NaCl/0.3M柠檬酸三钠)中清洗。示例性的中度严谨条件包括37℃下在40％至45％甲酰胺、1.0M NaCl、1％SDS中杂交，55℃至60℃下在0.5×至1×SSC中清洗。示例性的高严谨条件包括37℃下在50％甲酰胺、1M NaCl、1％SDS中杂交，60℃至65℃下在0.1×SSC中最后清洗至少约20分钟。任选地，清洗缓冲液可以包含约0.1％至约1％SDS。杂交持续时间通常少于约24小时，通常为约4小时至约12小时。特异性通常依赖杂交后的清 洗，关键因素在于最后清洗溶液的离子强度和温度。DNA-DNA杂合体的Tm(热力学熔点)可以近似自Meinkoth and Wahl(1984)Anal.Biochem.138:267-284的公式：Tm＝81.5℃+16.6(logM)+0.41(％GC)-0.61(％甲酰胺)-500/L；其中M是一价阳离子的克分子浓度，％GC是DNA中鸟苷和胞嘧啶核苷酸的百分数，“甲酰胺％”是杂交溶液的甲酰胺百分数，而L是杂合体的碱基对长度。Tm是(确定的离子强度和pH下)50％的互补靶序列与完全匹配的探针杂交时的温度。通常将清洗至少进行至达到平衡，并且达到低的杂交背景水平，诸如进行2小时、1小时或30分钟。每1％的错配对应使Tm降低约1℃；因而，可以调节Tm、杂交和/或清洗条件，从而与所需一致性的序列杂交。例如，如果需要≥90％一致性的序列，可以将Tm降低10℃。通常，将严谨条件选择为比确定离子强度和pH下的特异序列及其互补序列的Tm低约5℃。然而，在非常严谨的条件下，可以在比所述Tm低4℃下进行杂交和/或清洗；在中度严谨条件下，可以在比所述Tm低6℃下进行杂交和/或清洗；在低严谨条件下，可以在比所述Tm低11℃下进行杂交和/或清洗。
术语“移码突变”是指DNA片段中某一位点插入或丢失一个或几个(非3或3的倍数)碱基对时，造成插入或丢失位点以后的一系列编码顺序发生错位的一种突变。它可引起该位点以后的遗传信息都出现异常。发生了移码突变的基因在表达时可使组成多肽链的氨基酸序列发生改变，从而严重影响蛋白质或酶的结构与功能。
除非另外指明，本说明书和权利要求书中使用的表示成分的量、反应条件等的所有数字应被理解为在所有情况下用术语“约”来修饰。如本文所使用的术语“约”，当指代可测量的值例如质量、重量、时间、体积、浓度或百分比的量时，意味着涵盖在一些实施例中与规定量相比±20％的变化、在一些实施例中与规定量相比±10％的变化、在一些实施例中与规定量相比±5％的变化、在一些实施例中与规定量相比±1％的变化、在一些实施例中与规定量相比±0.5％的变化、以及在一些实施例中与规定量相比±0.1％的变化，因为此类变化适合于执行所披露的方法和/或使用所披露的组合物、核酸、多肽等。因此，除非相反地指出，在本说明书和所附权利要求书中所列出的数值参数是可以取决于试图通过本申请披露的主题获得的期望特性而变化的近似值。
实施例
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理 解，下列实施例仅用于说明本发明，而不用来限制本发明的范围。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本申请的范围。若无特别指明，实施例按照常规实验条件，如Sambrook等人的分子克隆实验手册(Sambrook J&Russell D W,Molecular cloning:a laboratory manual,2001)，或按照制造厂商说明书建议的条件。若未特别指明，实施例中所用的化学试剂均为常规市售试剂，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1.高粱耐盐碱基因AT1的发现及遗传分析
1.1高粱自然群体SAP(Sorghum Association Panel)材料的耐盐碱性鉴定
自美国农业部植物研究服务署USDA-ARS(United States Department of Agriculture-Agricultural Research Service)种质资源系统搜集到352份籽粒高粱和38份甜高粱自然群体材料。将刚收获的种子晾晒、自然风干后，将其放在55℃烘箱中烘5-7天，以打破种子的休眠。
为了模拟可能在田地盐碱土壤中存在的各种盐碱条件，我们首先测试了不同浓度(0、25、50、75、100、125和150mM)的两种碱盐混合物(NaHCO3和Na2CO3)并检测其对高粱幼苗存活率的影响。使用两种碱盐的混合物可以在整个处理期间产生相对稳定的pH范围，利于实验的进行。在处理中采用不同的处理时间。土壤基质中无(对照)或有不同浓度(0、25、50、75、100、125和150mM)两种碱盐混合物，记录播种后第21天经处理的种子的植株存活率(图1A和图2)。数据为每个处理三个重复的平均值，每个重复检测9个植株。相对存活率分析(在碱胁迫处理下存活数量/没有碱胁迫处理的存活数量)显示在21天处理后，75mM混合碱溶液(即，摩尔比5：1的NaHCO3：Na2CO3，pH＝9.2-9.4)表现出最宽范围的变化并且是评价高粱耐碱性的最可靠的处理浓度(图1A-C)。
后续处理选择75mM混合碱溶液(即，摩尔比5：1的NaHCO3：Na2CO3，pH＝9.2-9.4)。首先，将种子播种到按1：1比例混匀的蛭石和营养土的土壤基质中，每穴播种12粒种子，设置3次重复。接着，用75mM混合碱溶液(摩尔比5：1的NaHCO3：Na2CO3，pH＝9.2-9.4)浇灌至土壤饱和作为处理，用清水浇灌的作为对照(CK)，置于平地上使其均匀吸收，每组处理重复三次。将充分吸收后的穴盘放入模式植物玻璃温室中，后期用清水补水。幼苗生长环境如下：光照/黑暗时间为16h/8h，昼夜温度范围为28/26℃，相对湿度为60％到70％。培养至第23天时，统计成苗个数，计算相对存活率，实验结果发现不同品种之间萌芽期的耐盐碱性差异极显著(图1B-F)。
同时，通过随机选取的方式对其中16份籽粒高粱品种在萌芽期时处于盐碱胁迫状态下的种子发芽率、萌芽指数和其耐碱指数进行动态监测，结果发现：在不同浓度条件下各高粱材料的耐碱性表现出明显差异，随着碱处理浓度的增加，所有供试高粱材料的成苗率均显著下降，但是下降的程度有所不同。通过统计分析发现，对照条件下这16份高粱材料成苗率约95％、标准差0.04、变异系数0.04，在50mM混合碱处理时，以上三项统计数据没有明显差异。而在75mM混合碱处理时，随机选取的16份高粱材料的成苗率均值、标准差和变异系数分别为0.66、0.20和0.30。在高于75mM混合碱处理时，所种的成苗率骤降，甚至趋近于0。可见，在75mM混合碱处理(图2中75mM mixed Alkaline所示)时材料之间的表型变异程度最大(图2A)。因此，75mM混合碱处理适宜作为自然群体萌芽期耐碱程度的鉴定。成苗率的动态监测是耐盐碱鉴定过程中最简单快捷有效的方式，省时省力。因此，在后续实验中，选择相对成苗率这一指标作为萌芽期耐碱性评价的代表性指标来评价耐盐碱的程度。后续发现碱处理下的高粱材料在前两周时苗子的长势较好，但是在碱处理的后期有烧苗现象，第23天是该现象的“分水岭”，因此，选择第23天作为GWAS鉴定的时间节点(图2B)。
对352份籽粒高粱自然群体材料在对照(CK)和碱处理条件(75mM混合碱处理)下进行相对成苗率的统计分析发现，该群体材料的相对存活率分布属于正态分布(图1D)，碱处理(图1E的T/CK所示)之后的存活率受到很大影响(图1E)。经QQ-plot分析，发现所得到的观测值与期望值的吻合度高(图1F)，表明该结果较为理想，表型数据是可靠的，可以进行进一步的分析。该群体材料的基因型数据来源于开放性获取的GBS测序原始数据(Morris et al.,2013)。使用GATK软件的SelectVariants和VariantFiltration方法(QD<2.0，FS>200.0，ReadPosRankSum<20.0)进行筛选鉴定各品种的SNPs(DePristo et al.,2011)。处于杂合状态的SNP位点记为缺失，删除缺失率超过20％且最小等位基因频率(MAF)低于5％的SNP。最终对检测到的82,430个SNP标记。
1.2 GWAS分析发现高粱耐碱性状相关基因SbAT1
对检测到的82,430个SNP标记对该群体进行GWAS后续分析，并使用cMLM模型计算结果(Wen et al.,2018)。通过Bonferroni校正结果以及计算显著性相关P值(Ranstam,2016)。通过对352份籽粒高粱耐碱性状的GWAS分析，在籽粒高粱自然群体的1号染色体55Mb左右的位置检测到两个主效位点与耐碱性状的相对成苗率极显著相关(-log10 P>5.0)(图3A和表1)。其中，在该群体中显著的SNP：S1_5577933和S1_55779336直接 定位到了Sobic.001G341700基因的内部，将其命名SbAT1(Alkali Tolerance 1，或简称为AT1)(图3B)，初步判断SbAT1是一个控制高粱盐碱胁迫下相对成苗率的主效基因。
高粱AT1的基因组基因序列如SEQ ID NO:1所示，AT1基因包含5个外显子，其中第一外显子位于SEQ ID NO:1的第1-111位；第二外显子位于SEQ ID NO:1的第2447-2499位；第三外显子位于SEQ ID NO:1的第2846-2890位；第四外显子位于SEQ ID NO:1的第2972-3025位；第五外显子位于SEQ ID NO:1的第4084-4417位。AT1的cDNA基因的序列如SEQ ID NO:2所示，其编码序列(CDS)如SEQ ID NO:3所示。AT1编码一个高度保守的蛋白，由198个氨基酸组成(SEQ ID NO:4)，对蛋白产物进行序列比对，发现该基因与谷子(Setaria italica)中的同源AT1蛋白氨基酸总长度接近，且N端保守序列相似度很高，因此认为高粱AT1是谷子SiAT1的同源基因。谷子SiAT1基因的基因组核苷酸序列为SEQ ID NO:5所示，谷子SiAT1蛋白的氨基酸序列如SEQ ID NO:8所示，SiAT1蛋白的编码cDNA序列如SEQ ID NO:6所示，其编码序列(CDS)如SEQ ID NO:7所示。
表1. 352份籽粒高粱自然群体材料检测到的与耐碱性状相关的显著SNP位点
1.3 AT1基因的自然变异影响高粱的耐碱程度
为了验证该候选基因的准确性和AT1调控高粱耐碱性，接下来调取了AT1从5’-UTR到3’-UTR的所有变异，包括SNP和Indel的变异，序列比对分析表明，在第5个外显子有三个变异，其中在SEQ ID NO:1的第3271位核苷酸处出现了一个5bp的插入，造成蛋白翻译提前终止，表明该5bp的插入是可能引起AT1蛋白功能性发生变异的位点，含有该变异位点的高粱为at1突变体(图3C)。根据图3D表格的这8处变异将随机选取的38份高粱材料分为两种单倍型：单倍型I(Hap1，AT1基因野生型所对应的单倍型)高粱在碱处理条件下的存活率显著高于单倍型II(Hap2，at1突变体中AT1基因所对应的单倍型)的高粱(P值＝2.43×10-10)(图3E)，且其与耐碱的表型相关性P值高达3.31×10-10，因此，单倍型I和单倍型II分别为耐碱(AT1)和碱敏感(at1)的等位基因(图3D)。随后，我们在籽粒高粱自然群体中随机选择了8个耐碱和8个碱敏感的品系，测定碱胁迫5天和8天后AT1的表达水平，发现在耐碱品系(ARL)和碱敏感(ASL)品系之间检测到的AT1基因的表达没有任何的显著差异。这表明，与在AT1的5’-UTR没有发现强相关信号一致，基于单倍型的变异确实与基于RNA水平的AT1表达水平不相关(图3F)。这些 数据表明两种AT1单倍型中碱处理的耐碱和碱敏感表型与AT1及其变体at1的转录水平无关，而更可能是由于导致编码区内蛋白编号的突变所致。
最终，检测到SbAT1基因一种等位的自然突变序列形式，Sbat1基因(也简称为at1基因)，它是野生型AT1基因第5个外显子插入五个碱基(GTGGC)(即在SEQ ID NO:1的第3271-3272位之间插入了GTGGC五个核苷酸)，导致AT1基因3’端发生移码突变，蛋白翻译提前终止，形成137个氨基酸(图4中“at1-a”所示)。at1基因的cDNA的核苷酸序列如SEQ ID NO:9所示，AT1基因对应的突变后的at1蛋白的氨基酸序列如SEQ ID NO:10所示。
1.4 AT1基因在耐盐碱性中的作用
为了评估AT1基因对高粱耐碱性的等位基因作用，我们构建并选择一对近等基因系材料(NIL)：NIL-AT1和NIL-at1，它们仅在1号染色体上有58kb的区间的基因型是不同的，其余背景区域标记基因均一致。NIL-AT1材料的AT1基因的氨基酸序列为SEQ ID NO:4所示；NIL-at1材料的at1基因的氨基酸序列为SEQ ID NO:10所示。
NIL来自两个高粱种质SN010和M-81E的杂交。SN010是单倍型Hap1(包含野生型AT1)，M-81E是单倍型Hap2(包含at1)，并且根据我们的存活率和株高数据(图5A和图6A-C)，相较于M-81E(NIL-at1)，SN010(NIL-AT1)表现出更高的耐碱性。我们用75mM混合碱处理这两个NIL，在碱处理下，相较于M-81E(NIL-at1)，SN010(NIL-AT1)表现出56.1％更高的相对存活率和更好的生长，但在中性pH土壤中种植时，两个NIL没有表现出显著差异(图5D-E)。
由图5A可知，这对近等基因系材料在没有盐碱胁迫的正常生长条件下(对照，图5中CK所示)没有明显的表型差异，而在75mM混合碱胁迫处理(pH值9.32)第23天后(图5中75mM Alkali所示)，NIL-at1植株基本全部死亡，而NIL-AT1只有少量叶片干枯，植株仍然处于存活状态。接着，通过对它们的相对存活率、株高、单株鲜重和相对叶绿素含量(SPAD)这几项表型和生理指标分析(图5B，C，J)，结果表明：NIL-AT1的耐碱性远远超过NIL-at1，证明AT1确实是参与碱胁迫的应答。
近等基因系材料单盐NaCl处理
本领域技术人员容易理解，用75mM混合碱处理处理不仅施加碱胁迫，而且对植物施加Na+胁迫(即，盐胁迫)。为了区分高粱AT1-相关的胁迫表型是单独由高pH引起还是单独由高钠离子(Na+)浓度引起，我们使用75、100、150和200mM的NaCl溶液替换混 合碱(NaHCO3和Na2CO3)在中性pH土壤中进行了相似处理。结果发现：随着盐胁迫程度的加剧，这两种NIL都对高NaCl敏感，NIL系材料的相对存活率和单株鲜重均明显降低。但在不同程度盐胁迫处理条件下，两者之间没有显著差异(P＞0.05)(图5D-F)。这一结果表明AT1/at1-相关的胁迫表型更可能是碱特异性反应，而不是盐应激反应。
为了证实这种可能性，我们在Wheatland背景(SbWT，包含完整的野生型(WT)SbAT1基因，来源于美国种质资源库USDA惠赠，Population genomic and genome-wide association studies of agroclimatic traits in sorghum.(2013).Proceedings of the National Academy of Science of the United States of America 110:453-458.)下生成了另外的转基因高粱植株：过表达SbAT1基因或通过基因编辑技术敲除SbAT1基因。过表达或敲除通过qRT-PCR或序列分析证实(图5G)。出乎意料地，我们发现SbAT1过表达(SbAT1-OE)植株的耐碱性降低，而具有SbAT1基因敲除(SbAT1ko)的植株表现出极大提高的耐碱性(图5H-J，图6D-F)。在75mM混合碱胁迫下，SbAT1过表达(SbAT1-OE)植株的存活率比Wheatland植株(即，SbWT植株)低13.95％，而SbAT1基因敲除(SbAT1ko)的植株的存活率比Wheatland植株(即，SbWT植株)高17.93％(图5F)。这些转基因植物的表型促使我们重新考虑自然变异情形下SbAT1 C端基因突变的功能性(图3C-D)，认为截短的突变蛋白(at1)可能在耐碱性方面起负作用。为了证实我们的推测，我们首先在植物细胞中瞬时表达了两种GFP融合蛋白：SbAT1-GFP和Sbat1-GFP，以检测具有更高蛋白水平积聚的蛋白状态(图5K)，从而表明突变体at1基因的翻译能力。
田间试验
为了进一步验证所得结论，在自然环境中盐碱胁迫条件下开展了田间试验，分别在低碱胁迫(pH 8.13，全盐含量7.68‰，碱化度10.71％)和较高碱胁迫(pH 9.07，全盐含量3.18‰，碱化度20.92％)的大田中种植上述两种高粱NIL系材料，发现在低碱胁迫和较高碱胁迫下，NIL-AT1材料较NIL-at1材料长势好，发现所得出的田间试验结果(图7)和温室内结果一致。
为了贴合生产实际，于2020年4月到10月在中国宁夏回族自治区石嘴山市平罗县撂荒的盐碱地惠威村(北纬38°57′29″，东经106°32′39″，海拔1090m)和东风村(北纬38°56′20″，东经106°35′26″，海拔1100m)开展田间区域试验。该地区紧挨黄河，水源充足，适宜农业发展，但区域内由于长期大水漫灌、地下水位高、蒸发量大和不合理施肥等自然和人为因素，导致灌区内土壤次生盐渍化严重，农业耕地也出现了不同程度的次生盐 碱化。采用单因素完全随机区组的试验设计，每地块选取三个重复共六个小区，小区行长7m，行宽5m，行距0.6m，株距0.2m，走道0.8m，小区面积35m2。双粒穴播播种，播种深度约3cm。播种前精选籽粒饱满、大小均一的种子，施基肥(N：P2O5：K2O＝15：15：15)，整地耙平。整个生育期内按时进行中耕、除草及施肥等管理项目。供试土壤的理化性质如图8A和B所示。由于盐碱地分布不均匀，有“碱斑”存在，我们发现不同地块的高粱长势具有较大差异，但是整体的趋势一致，均表现为NIL-AT1的生长状态优于NIL-at1，且六个小区的成苗率统计数据均表明NIL-AT1远远超过NIL-at1(P＜0.05)(图8C和D)。
其中，本实施例中所用的方法如下所述：
(I)高粱AT1基因过表达载体的构建
植物表达载体pCAMBIA2300-Ubi-Myc由本实验室保存(参见Liu Y,Sun J,Wu Y.Arabidopsis ATAF1 enhances the tolerance to salt stress and ABA in transgenic rice.J Plant Res.2016 Sep；129(5):955-962.doi:10.1007/s10265-016-0833-0.Epub 2016 May 23.PMID:27216423，其中第2页右栏Materials and methods中Constructs and transformation的第1-3行所述的载体，即在pCAMBIA2300商业载体上添加了Ubi启动子和OCS终止子，同时还带有Myc标签序列的重组载体，公众可从申请人处获得以重复本发明)。首先，采用限制性内切酶Spe I和BamH I(均购自New England Biolabs公司)置于PCR仪上37℃酶切2h，使该载体线性化得到线性化的载体。接着，再用携带接头的引物来扩增加上终止密码子的高粱AT1基因的CDS编码区(核苷酸序列为SEQ ID NO:3所示)得到带接头的目的片段，利用无缝克隆试剂盒pEASY-Uni Seamless Cloning and Assembly Kit(CU101-01)把得到的带接头的目的片段(基因片段)和得到的线性化载体进行连接。无缝克隆试剂盒连接反应的体系(10μL)：5μL的2×Assembly Mix，将线性化载体和带接头的目的片段按照摩尔比1:2混合后，加ddH2O补齐至10μL。轻轻混合后，50℃反应15min。反应结束后，将离心管置于冰上冷却数秒，之后可将重组产物转化大肠杆菌XL1-Blue，提取质粒，酶切确定阳性克隆重组质粒pCAMBIA2300-Ubi-Myc-AT1，送睿博公司进一步测序验证。
(II)AT1基因过表达植株的获得
重组农杆菌的获得
取1μg制备的重组载体质粒pCAMBIA2300-Ubi-Myc-AT1转化农杆菌EHA105的 感受态细胞，经PCR鉴定得到的阳性菌命名为重组农杆菌EHA105/pCAMBIA2300-Ubi-Myc-AT1，-80℃保存。
重组农杆菌EHA105/pCAMBIA2300-Ubi-Myc-AT1含有高粱AT1基因的CDS核苷酸序列(SEQ ID NO:3)
高粱AT1基因过表达植株的获得
将重组农杆菌EHA105/pCAMBIA2300-Ubi-Myc-AT1通过农杆菌介导的方法转入到高粱的受体材料Wheatland中。
高粱遗传转化方法如下：
(1)高粱受体材料的准备：
在温室中种植Wheatland高粱植株，待生长至开花期15天左右，将穗子剪断，并在无菌超净台台中，尽可能地将灌浆的籽粒中的幼胚挤出，均匀地放置到愈伤培养基上进行愈伤组织的培养。
(2)侵染菌液的制备：
将保存于-80℃的重组农杆菌在添加Kan的YEP固体培养基中划线培养。长出的单菌落接种于10mL YEP液体培养基(含有Kan)中进行一次活化(28℃，220rpm震荡过夜)。取一次活化的菌液按1:1000的比例接种于80mL YEP液体培养基(含Kan)，28℃，220rpm震荡培养至OD600nm为0.8-1.0。
(3)侵染过程：将达到所需OD值的农杆菌菌液转移至50mL离心管中，5000rpm离心10min以富集菌体。然后用重悬缓冲液将其菌体充分重悬，并将长出的愈伤组织加入到重悬的侵染液中，进行侵染转化，侵染后的愈伤转移到分化再生培养基上，再生出苗后移到生根培养基(含有Kan)上，进行生根培养，若出现培养基褐化，则需要多次转移生根培养基。
(4)将株高约5-7cm且根部生长良好的T0代高粱转化幼苗移苗到土里，在温室中培养，并保持一定湿度，保证再生苗的成活率，待培养至成熟后，收获其成熟后的种子即为T0代转基因种子。
(III)AT1基因突变体植株的构建
重组CRISPR-Cas9载体的构建
利用基因编辑技术对高粱受体材料品种Wheatland中的AT1基因进行了敲除。该单子叶植物基因编辑CRISPR-Cas9的原始载体包括gRNA表达盒pYLsgRNA-OsU6a/LacZ 载体和CRISPR-Cas9载体pYLCRISPR/Cas9Pubi-B是由华南农业大学刘耀光教授惠赠(Ma X,Zhang Q,Zhu Q,et al..A Robust CRISPR/Cas9 System for Convenient,High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants.Mol Plant.2015 Aug；8(8):1274-84.doi:10.1016/j.molp.2015.04.007.Epub 2015 Apr 24.PMID:25917172.)。
AT1基因突变体植株的获得
首先将含有pYLsgRNA-OsU6a/LacZ载体和CRISPR-Cas9载体pYLCRISPR/Cas9Pubi-B的菌种活化和提取质粒。接着是制备靶点接头和sgRNA载体酶切后进行连接sgRNA表达盒。sgRNA靶点的核苷酸序列为5’-ACGCCTGAAAAGTTGACAGCTG-3’，靶向于AT1基因的SEQ ID NO:1的第2317-2338位。具体操作方法如下：
1)菌种活化、质粒提取制备：将pYLCRISPR/Cas9Pubi-B的菌种(TOP10F)和pYLsgRNA-OsU6a/LacZ的菌种(DH10B)分别在含有卡那霉素(25μg/mL)和氨苄青霉素(50μg/mL)平板培养基划线培养过夜，挑取单菌落进行扩大培养用于提取质粒。
2)靶点接头制备：将设计合成的接头引物AT1-Target-F：5’-GCCGCAAGTCGCCGCCTGCCTCGC-3’和AT1-Target-R：5’-AAACGCGAGGCAGGCGGCGACTTG-3’加ddH2O溶解成100μM母液，各取左右引物混合并稀释到终浓度1μM。在PCR仪上进行加热(约90℃30s)，于室温冷却完成退火从而形成双链的靶点接头。
3)sgRNA载体酶切：取pYLsgRNA-OsU6a/LacZ质粒1μg，在25μL酶切反应体系(中用10U Bsa I酶切20min得到酶切后的质粒，冷冻保存。
4)sgRNA表达盒连接反应：酶切后的质粒与对应双链靶点接头连接反应(10μL体系)：1μL 10×T4 DNA连接酶缓冲液、10ng酶切后的质粒、0.05μM双链靶点接头、18 U T4 DNA连接酶，用超纯水补足到10μL；室温连接20min。
5)第一轮扩增：15μL反应体系：取0.5μL连接产物为模板，使用正向引物U-F：5’-CTCCGTTTTACCTGTGGAATCG-3’和接头反向引物gRNA-R：5’-CGGAGGAAAATTCCATCCAC-3’，各0.2μM，1μL KOD Fx Neo酶；PCR程序为：30个循环：94℃ 10s，60℃ 15s，68℃ 20s。
6)第二轮扩增：取第一轮PCR产物稀释10倍后，再取1μL为第二轮PCR的模板。同样使用适量KOD Fx Neo酶，用正向引物B1’：5’- TTCAGAggtctcTctcgCACTGGAATCGGCAGCAAAGG-3’和反向引物BL：5’-AGCGTGggtctcGaccgGGTCCATCCACTCCAAGCTC-3’。扩增30个循环：95℃ 10s，58℃ 15s，68℃ 20s。通过跑胶检测目的条带大小(831bp)，并进行乙醇沉淀纯化，得到的PCR纯化产物即为带有OsU6a启动子的sgRNA：OsU6a-sgRNA。
7)取约2μg的pYLCRISPR/Cas9Pubi-B质粒在酶切体系中(30U Bsa I)酶切2h后，用DNA胶电泳回收酶切的质粒片段。取100ng Bsa I酶切后的pYLCRISPR/Cas9Pubi-B质粒，同时加入已用Bsa I酶切好的步骤6)第二轮PCR纯化产物OsU6a-sgRNA(20ng)，在10μL连接反应中加35U的T4连接酶，通过变温循环：10℃ 5min，20℃ 5min；10-15个循环)连接2-3h。
8)取1μL连接产物电激转化E.coli DH10B感受态细胞，电激后加入1mL SOC，37℃培养2h。涂板培养基为LB(25μg/mL Kan，0.5mM IPTG和适量X-gal)。挑取单克隆菌落通过菌落PCR方法，用载体引物SP1：5’-CCCGACATAGATGCAATAACTTC-3’和SP2：5’-GCGCGGTGTCATCTATGTTACT-3’对特定靶点进行PCR扩增和测序，扩增产物条带大小约1kb左右，经测序该扩增产物含有OsU6a-sgRNA。获得的阳性克隆即为重组成功的质粒pYLCRISPR/Cas9Pubi-B-AT1-sgRNA。
9)取1μg制备的重组载体质粒pYLCRISPR/Cas9Pubi-B-AT1-sgRNA转化农杆菌EHA105的感受态细胞，28℃条件下于YEP培养基(含卡那霉素50mg/L)上培养两天，挑选阳性克隆，同样利用上述引物SP1和SP2进行PCR鉴定。将经PCR鉴定得到的阳性农杆菌命名为重组农杆菌EHA105/pYLCRISPR/Cas9Pubi-B-AT1-sgRNA，-80℃保存。
将获得的阳性农杆菌侵染高粱(Wheatland)和谷子(Ci846)的愈伤组织。高粱的遗传转化步骤同步骤4.2.2，谷子受体材料Ci846由中国农业科学院作物科学研究所隋毅教授惠赠(相关文献：Cheng Z,Sun Y,Yang S,Zhi H,Yin T,Ma X,Zhang H,Diao X,Guo Y,Li X,Wu C,Sui Y.Establishing in planta haploid inducer line by edited SiMTL in foxtail millet(Setaria italica).Plant Biotechnol J.2021 Jun；19(6):1089-1091.)。谷子遗传转化参照下述专利文献中的谷子遗传转化方法进行：获得谷子用于遗传转化的胚性愈伤组织和遗传转化的方法，公开号CN108588002A。
具体步骤如下：
(1)谷子成熟胚的消毒：将谷子种子成熟胚用10％的次氯酸钠溶液中消毒10-15min，然后用无菌水反复冲洗3-5遍,最后用吸水纸把水吸干备用。
(2)谷子初生愈伤组织的诱导：在诱导培养基上进行种子成熟胚愈伤组织的诱导，于28℃下暗培养约30天。
(3)谷子胚性愈伤组织细胞悬浮系的制备和大量扩增：将诱导获得的初生愈伤组织转移到悬浮培养基中悬浮培养，培养条件为28-30℃，转速为150r/min的摇床上振荡培养。培养过程中去除褐化组织，选择胚性状态较好的细胞，扩大培养，获得大量的胚性细胞愈伤组织。
(4)农杆菌介导的谷子胚性细胞的遗传转化：提前活化好重组农杆菌EHA105/pYLCRISPR/Cas9Pubi-B-AT1-sgRNA，将准备好的愈伤组织与农杆菌在预培养基上进行共培养，并将共培养后的愈伤组织转移至含有相应抗性的筛选培养基上进行继代培养，继代培养三次后将抗性愈伤转移至分化再生培养基，直至长出幼苗。
(5)将幼苗转移至生根培养基进行生根培养，长出较为健壮的根系后移栽到土中，温室培养，生长后期鉴定阳性苗即为转基因T0代植株幼苗，并收获T0代种子。
待获得转基因T0植株后进行打靶效果检测，即以靶点为中心，在两侧各约200-300bp处合成引物PCR扩增。在靶点上游约150-250bp处设计内部引物为测序引物，对PCR产物直接测序。如果测序峰图无重叠峰，可以被判定为野生型或纯合突变，如果靶点附近出现双峰的植株，即为阳性AT1基因突变体植株。
分别以高粱转基因T0代植株和Wheatland(野生型高粱)的基因组DNA为模板，PCR扩增包括剪切位点的基因组DNA片段，对扩增产物进行测序检测AT1基因的敲除情况。之前预测的高粱中AT1蛋白N端的22到88位氨基酸(SEQ ID NO:4的第22-88位氨基酸)是高度保守的结构域。CRISPR-Cas9转基因植株用引物F:5’-GTTGACAGCTGAACACATGGCT-3’和R：5’-ATACATCGTTAGGAATGGATCCG-3’进行PCR扩增，选择测序结果表明AT1基因被编辑，导致AT1基因功能丧失的CRISPR-Cas9转基因植株。将AT1基因被编辑的转基因植株命名为AT1KO(图9C)。AT1基因缺失AT1KO植株的引物F和R扩增产物与野生型高粱Wheatland的引物F和R扩增产物比对结果显示，AT1基因缺失AT1KO植株在AT1基因相应区域(SEQ ID NO:3的第129位核苷酸发生突变)丢失一个碱基T，并造成在AT1蛋白相应区域(SEQ ID NO:4的第43位氨基酸发生突变，造成了后续翻译的提前终止)产生截短蛋白，破坏了保守结构域(图9C)，从而将AT1基因敲除。AT1KO植株表达的编码序列如SEQ ID NO:11所示，蛋白质的氨基酸序列如SEQ ID NO:12所示。
之前也预测了谷子中SiAT1蛋白(SEQ ID NO:8)的N端的25-91位氨基酸(SEQ ID NO:8的第25-91位氨基酸)编码的也是一个保守的结构域，其对应的编码序列为SEQ ID NO:7的第73-273位核苷酸序列所示。使用上述同样基因编辑的方法得到谷子SiAT1的突变体植株命名为SiAT1KO(图16C)，谷子SiAT1KO是在SEQ ID NO:7(野生型SiAT1基因)的第14-15位核苷酸之间插入了一个碱基T，造成了基因移码突变，产生42个氨基酸的肽段，蛋白翻译提前终止，因此，基因编辑的材料是AT1蛋白完全缺失了保守结构域的材料(图10)。SiAT1KO植株表达的AT1的编码序列如SEQ ID NO:13所示，蛋白质的氨基酸序列如SEQ ID NO:14所示。
其中，谷子SiAT1基因的sgRNA靶点的核苷酸序列为5’-TATAATGGCTGCTGCGCCGG-3’，靶向于SiAT1基因即SEQ ID NO:5的第393-412位；靶点接头制备时所使用的接头引物为SiAT1-Target-F：5’-GGCGTATAATGGCTGCTGCGCCGG-3’和SiAT1-Target-R：5’-AAACCCGGCGCAGCAGCCATTATA-3’。
高粱T0代筛选到阳性转基因值株后，将该植株进行自交获得T1代植株，待收获后的T1代时再次用引物F和R进行靶点鉴定，将PCR产物测序后将靶点确实被编辑且测序为单峰的保留，作为纯合植株。把该植株再用终载引物SP1：5’-CCCGACATAGATGCAATAACTTC-3’和SP2：5’-GCGCGGTGTCATCTATGTTACT-3’进行PCR扩增，若无法获得扩增产物即为把Cas9筛选掉的纯合植株，可将本材料用于后续实验。筛选后的T1代纯合植株经过自交得到T2代纯合植株。
通过基因编辑技术，在转基因高粱和谷子的T2代均鉴定和筛选到CRISPR-Cas9载体分离掉的、在靶点位置造成提前终止突变类型的纯合株系SbAT1KO和SiAT1KO。
实施例2.遗传转化AT1基因产生的高粱和谷子抗性植株的应用
转基因高粱和谷子阳性植株鉴定和萌芽期耐盐碱表型观察
在AT1-MYC过表达转基因高粱T0代共获得了54株幼苗，通过PCR鉴定获得12株T0代转基因阳性株，再通过qPCR鉴定到其中有10个株系(分别命名为AT1-MYC-3～AT1-MYC-12)的AT1基因表达量远远高于野生型Wheatland，倍数在92到303之间(图9B)，以SbEIF作为内参基因。接下来，为了选择合适的浓度来鉴定上述构建成功的转基因高粱和谷子的耐碱程度，设置三个碱浓度梯度(50mM混合碱溶液、75mM混合碱溶液和100mM混合碱溶液)，同时设置对照(水)处理。用来做实验的种子均是相同条件下 生长、同一时间收获和相同条件下储存的种子。将种子播种到按1：1比例混匀的蛭石和营养土的土壤基质中，每穴播种12粒种子，再覆2cm左右的土层，与盘面铺平，设置重复2次。接着，用处理液(水或者碱溶液)浇灌至土壤饱和作为处理，用清水浇灌的作为对照，置于平地上使其充分均匀吸收到土壤中，再将其置于模式植物玻璃温室中(幼苗生长环境如下：昼夜温度控制为28℃/22℃，光照/黑暗时间为16h/8h，相对湿度为60％至70％)，后期用清水补齐，每组处理重复三次。待种子的胚芽顶出土壤表层时，就认为该种子已经发芽。其中，存活率＝成苗种子粒数(14天)/供试种子数×100％；相对存活率＝碱处理存活率/相应对照存活率×100％。数据统计方法如下：首先，将Excel 2016进行数据整理与图表制作，利用DPS 7.5软件进行数据统计，采用最小显著差异法(LSD)和单因素方差分析(ANOVA)对结果进行显著性方差分析。若P＜0.05，标为小写字母，差异显著；若P＜0.01，标为大写字母，差异极显著。
通过观察其碱处理后的长势情况和分析相对成苗率，我们认为在75mM混合碱处理时的表型变异程度最高，适合选择该浓度做后期的表型鉴定(图11)。图11中Sorghum代表高粱；Millet代表谷子。
其中，50mM混合碱溶液(摩尔比5：1的NaHCO3：Na2CO3)的配置方法是称取12.32g NaHCO3和3.11g Na2CO3溶于2升水，溶液pH 10.03，土壤充分吸收溶液后土壤pH值为9.19；75mM混合碱溶液(摩尔比5：1的NaHCO3：Na2CO3)的配置方法是称取18.48g NaHCO3和4.66g Na2CO3溶于2升水，溶液pH 10.04，土壤充分吸收溶液后土壤pH值为9.32；100mM混合碱溶液(摩尔比5：1的NaHCO3：Na2CO3)的配置方法是称取24.64g NaHCO3和6.22g Na2CO3溶于2升水，溶液pH 10.03，土壤充分吸收溶液后土壤pH值为10.10。
表型观察结果表明：在对照(CK)正常生长条件下，野生型高粱Wheatland、高粱AT1基因过表达株系AT1-OE和高粱AT1基因缺失突变体株系AT1KO之间存活率无显著差异(图5H，图6E)，野生型谷子Ci846(图12B中SiWT)、谷子SiAT1基因过表达SiAT1-OE和缺失突变体株系SiAT1KO之间存活率也无显著差异(图12B)；75mM混合碱处理条件(土壤pH值为9.32)下的高粱AT1-OE株系表现为对碱极度敏感，而基因功能缺失的高粱突变体AT1KO和谷子突变体SiAT1KO均较野生型高粱Wheatland和谷子Ci846表现为耐盐碱程度增加(图6E-F和图12B-C)。以上的结果均表明：高粱的SbAT1和谷子的SiAT1确实是一个负调控高粱和谷子耐盐碱程度的重要基因，其N端GGL保守结构 域(SEQ ID NO:15)突变后，有助于高粱和谷子耐盐碱程度的提高。
此外，实施例1和2中结果已经表明，相对于有198个氨基酸的野生型AT1蛋白(SEQ ID NO:1)而言，C端截短仅保留有136个氨基酸的NIL-at1突变体中的at1(SEQ ID NO:10)是AT1的自然变异等位基因对应的蛋白。对近等基因系材料分析发现，相较于野生型AT1，虽然缺失了C端但保留有完整的N端GGL结构域的at1的耐碱性却大大降低。因此，实施例1和实施例2上述所有结果表明：禾本科作物高粱和谷子中的AT1蛋白是一个负调控耐碱程度的关键因子，基因编辑AT1基因，敲除AT1后可以提高作物的耐盐碱性。
为了进一步证实上述观察，我们生成下述转基因植株：通过基因编辑在与高粱相同的位置产生终止密码子，以模拟谷子中潜在的C端截短SiAT1102蛋白。我们还生成了过表达具有截短的C端的SiAT1124(SiAT1124-OE)植株和敲除SiAT1的植株(SiAT1ko)(图12A，图13A-B)。
在本实施例中使用谷子进行测试是基于下述考虑：(1)与高粱相比，谷子的植株转化更容易和更快速，(2)谷子与高粱在分类学上密切相关，具有高基因组相似性和相似的环境生理表现，和(3)单拷贝的谷子SiAT1与高粱SbAT1在蛋白水平上具有75.24％的同一性。
与转基因高粱的结果类似，敲除SiAT1基因产生了对碱性条件具有更高耐受性的植株，如与其他基因型相比SiAT1ko植株观察到更高的存活率所示(图12B-C)。相反，C端截短的转基因谷子(SiAT1102)和过表达C端截短SiAT1(SiAT1124-OE)的谷子植株在碱胁迫处理下表现出降低的耐碱性，其中SiAT1124-OE植株最弱的响应碱度的生长(图12B-C，图13C)。这一结果表明：C端截短蛋白可以在植株中表达，并且增加量的C端截短蛋白对耐碱性具有负作用，而敲除AT1对植株的耐碱性具有正影响。联系在高粱中观察到的AT1过表达表型，我们能够得出AT1在高粱和谷子的耐碱性中表现出负调控作用，at1突变增强了这种负调控作用。
实施例3.AT1同源基因在水稻和玉米中的相似作用
我们进一步研究了AT1同源基因在其他主要单子叶作物水稻和玉米中耐碱性方面的作用。AT1在水稻中的直向同系物已鉴定为OsGS3，是调控籽粒大小的主要QTL(H.Mao,S.Sun,J.Yao,C.Wang,S.Yu,C.Xu et al.,Linking differential domain functions of the GS3 protein to natural variation of grain size in rice.Proc.Natl.Acad.Sci.USA 107,19579-19584(2010).doi:10.1073/pnas.1014419107；C.Fan,Y.Xing,H.Mao,T.Lu,B.Han,C.Xu  et al.,GS3,a major QTL for grain length and weight and minor QTL for grain width and thickness in rice,encodes a putative transmembrane protein.Theor.Appl.Genet.112,1164-1171(2006).doi:10.1007/s00122-006-0218-1)。
水稻GS完整(GS3-1)蛋白序列如SEQ ID NO:16所示，水稻GS3C端截短蛋白(GS3-4)序列如SEQ ID NO:17所示，玉米GS3蛋白序列如SEQ ID NO:18所示。
我们发现，在碱性土壤(75mM混合碱，pH9.0-9.2)中，过表达完整的OsGS3(OsGS3-1OE)和C端截短版本OsGS3(OsGS3-4OE)表现出较低的耐碱性，而敲除OsGS3(OsGS3ko)或RNAi OsGS3(OsGS3Ri)的水稻在相对存活率、以相对株高和相对叶绿素含量测定的植株生长方面比ZH11野生型水稻(OsWT)表现出更强的耐碱性(图12D-F和图13D-F)。在转基因植株中，OsGS3-1OE和OsGS3-4OE的相对存活率分别比OsWT低12.5％和26.4％，而OsGS3ko和OsGS3Ri的相对存活率分别比OsWT高8.3％和7.4％(图12F)。这些结果表明，抑制水稻OsGS3功能可以增强水稻的耐碱性，并提示Gγ亚基具有保守功能。此外，通过操纵或选择OsGS3的无功能等位基因，我们可以增强水稻的耐碱性。
AT1的玉米同源基因先前被鉴定为ZmGS3(Q.Li,X.Yang,G.Bai,M.L.Warburton,G.Mahuku,M.Gore et al.,Cloning and characterization of a putative GS3 ortholog involved in maize kernel development.Theor.Appl.Genet.120,753-763(2010).doi:10.1007/s00122-009-1196-x)。因此，我们将该基因命名为AT1/GS3，并以前缀表示物种；根据上下文，AT1和GS3也可以单独使用或互换使用。通过对玉米品系KN5585(ZmWT)进行基因编辑，获得了玉米ZmGS3敲除(ZmGS3ko)品系。玉米ZmGS3ko缺失34bp，在ZmGS3的第一个外显子发生碱基突变。这些突变导致移码突变和预测蛋白的过早翻译终止(图13G)。经过碱性处理后，与野生型玉米相比，敲除型玉米植株在培养第14天的生长性能表明它们具有更强的耐碱性(图12G-H，图13H)。碱胁迫处理50天后，二者的表型差异更加显著；几乎所有的野生型玉米幼苗都死亡了，而ZmGS3ko存活了下来，继续生长(图12I，图13I)。这一结果支持ZmGS3ko能够提高玉米的耐碱性，与我们在高粱、谷子和水稻中观察到的情况类似。
本实施例的方法如下：
GS3基因调控耐碱性的功能研究
3.1过表达GS3基因的转基因植株的构建
根据水稻基因组注释信息(GS3基因组序列如SEQ ID NO:19所示)设计引物，以水 稻品种广陆矮的GS3全长cDNA(Osigcea013f09t3)为模板，分别扩增GS3的2种等位基因型GS3-1以及GS3-4，大小分别为696bp(如SEQ ID NO:20所示)和450bp(如SEQ ID NO:21所示)。
为了扩增GS3基因编码区的cDNA，本发明设计如下引物：
GS3OEF的正向引物(SEQ ID NO:36)：5'-ggtaccACCATGGCAATGGCGGCGGCGCCC-3'(下划线序列为KpnⅠ识别位点)；
GS3-1OER的反向引物(SEQ ID NO:37)：5'-agatctCAAGCAGGGGGGGCAGCAACG-3'(下划线序列为BglⅡ识别位点)；
GS3-4OER的反向引物(SEQ ID NO:38)：5'-agatctACGCCGCCCCACATGAGGA-3'(下划线序列为BglⅡ识别位点)；
分别用引物组合GS3OEF、GS3-1OER和GS3OEF、GS3-4OER进行PCR扩增，获得GS3-1与GS3-4的目标片段。PCR反应总体积为50μl，包含cDNA模板2μl，2×GC I缓冲液25μl，10mM dNTP 5μl，10mM引物GS3OEF和GS3OER各1μl，ExTaq酶1μl，加去离子水至50μl(所用到的2×GC I缓冲液、dNTP、rTaq酶等均购自宝生物工程大连有限公司)。PCR反应条件如下：①94℃ 4min，②94℃ 30s，③58℃ 30s，④72℃ 1min，⑤从②－④循环33次，⑥72℃ 7min，⑦25℃保存。PCR产物在1％(质量/体积)的TBE琼脂糖凝胶上电泳检测，回收长度为696bp的GS3-1(681bp的目标DNA区段加上引物上附加的两个限制性酶切位点15bp)与462bp的GS3-4(447bp的目标DNA区段加上引物上附加的两个限制性酶切位点15bp)的DNA片段。将它们分别构建到常用的超量表达载体pCAMBIA1301U上[该载体是本实验室改造而来:其基本骨架是澳大利亚CAMBIA实验室的(http://www.cambia.org/daisy/cambia/materials/overview.html)的pCAMBIA1301，通过加入Ubi启动子实现对转化基因的表达调控]，转化水稻中花11(Zhonghua 11，ZH11)获得超量表达植株GS3-1OE和GS3-4OE。
载体结构如图14A所示。
3.2 RNAi干扰的植株的构建
构建GS3的表达抑制RNAi载体，分两步进行。首先将含有GS3全长cDNA(osigcea013f09t3)的质粒用BamHI和KpnI进行双酶切消化，连到经改造过的dsRNAi1301， 得到含有第一链(正向)GS3的载体；同时将BamHI和KpnI消化产物连到中间载体GZ-1(本实验室丁新华博士提供)上，然后用SacI和SpeI双酶切消化，再将产物连到已经含有第一链的dsRNAi1301(也用SacI和SpeI双酶切消化)上，使得两个相同序列的片段方向相反，由35S启动子驱动，转化Zhonghua11，获得抑制表达转基因植株GS3-1RNAi。载体结构示意图如图14B所示。
利用RNA干扰敲降植物中GS3基因或蛋白表达的方法，优选为将植物GS3基因的编码序列分别按照正向和反向插入至pDS1301，得到的重组载体转化植物，得到GS3基因或蛋白表达量降低的植株。向所述pDS1301载体中插入GS3基因的方法优选采用酶切方式完成。正向插入时，采用KpnⅠ和BamHⅠ双酶切和连接。反向插入时，采用SacⅠ和SpeⅠ双酶切和连接。所述GS3基因的编码序列正向插入时，核苷酸序列为SEQ ID NO:34所示，所述GS3基因的编码序列反向插入时，核苷酸序列为SEQ ID NO:35所示。其中，pDS1301载体是本实验室改造而来(参见现有技术：Yuan B，Shen X，Li X，Xu C，Wang S(2007)Mitogen-activated protein kinase OsMPK6negatively regulates rice disease resistance to bacterial pathogens.Planta226:953–960)；其基本骨架是澳大利亚CAMBIA实验室(http://www.cambia.org/daisy/cambia/materials/overview.html)的pCAMBIA1301，通过加入35S启动子实现对转化基因的表达调控。
3.3超量表达以及抑制表达转基因植株的表达量检测
(1)抽提GS3-1OE、GS3-4OE和GS3-1RNAi以及野生型植株幼穗分化期1cm长的幼穗的RNA，RNA抽提用的试剂购采用Invitrogen公司生产的Trizol抽提试剂盒(具体操作步骤按照试剂盒提供的说明书操作)。
(2)反转录合成cDNA第一链
步骤如下：
①取抽提的总RNA 3μg，加入DNaseI 1μl，10×DNaseI缓冲液1μl，加DEPC(焦碳酸二乙酯，RNA酶的强抑制剂，工作浓度为0.01％)处理过的水到10μl，混匀后室温放置15min以去除残留的基因组DNA；
②加入0.2M EDTA 1μl，并于65℃水浴中孵育10min以去除DNaseI的活性；
③加入oligo(dT)15引物1μl，并于65℃水浴中孵育10min以破坏RNA的二级结构，然后冰上放置2min；
④加入5×第一链缓冲液4μl，0.1M DTT(二硫苏糖醇)2μl，10mM dNTP混合物1μl，反转录酶1μl，混匀后置于42℃水浴锅内温浴1.5h；
⑤反应结束后将反转录产物置于85℃干浴10min以灭活反转录酶；
⑥向反转录产物中加入80μl水，混匀后-20℃保存反应最终产物。反应中用到的试剂全部购自Invitrogen公司。
(3)对得到的反转录产物用实时PCR的检测，GS3基因检测引物为GS3QRT-F和GS3QRT-R，并且用Ubiquitin基因(LOC_Os03g13170)作为内参(引物组合为UbiQRT-F和UbiQRT-R)，序列如下所示：
GS3QRT-F:5'-CCGCGAGATCGGATTCC-3'(SEQ ID NO:39)；
GS3QRT-R:5'-CGTGGATCCCTTCGATTGA-3'(SEQ ID NO:40)；
UbiQRT-F:5'-AACCAGCTGAGGCCCAAGA-3'(SEQ ID NO:41)；
UbiQRT-R:5'-ACGATTGATTTAACCAGTCCATGA-3'(SEQ ID NO:42)。
在10μL体系中进行，其中含有1μL反转录产物、0.3μL的正向、反向引物和5μL的FastStart Universal SYBR Green Master，补水至10μL。反应程序为：①95℃ 10min，②95℃ 10s，③60℃ 30s④从②-③循环40次。根据CT值计算相对表达量。
实时PCR检测结果表明，水稻植株GS3-1OE以及GS3-4OE相对于野生型ZH11而言，靶基因表达量明显上升。GS3RNAi植株相对于野生型ZH11而言，靶基因表达量明显下降，如图14C所示。
3.4 GS3基因耐碱性调控功能实验
将上述制备的过表达GS3基因的转基因植株和GS3表达量降低的植株分别进行碱处理，并比较主要农艺性状与非转基因受体的差异。具体如下：利用相同条件上生长，同一时间收获以及相同条件储存下的种子，进行了碱处理的实验。
将种子播种到等体积比的蛭石和营养土形成的土壤基质中，每穴播种12粒种子，再覆2cm左右的土层，与盘面铺平，设置重复2次。接着，用处理液(水或者75mM混合碱溶液)浇灌至土壤饱和作为处理，用清水浇灌的作为对照，置于平地上使其充分均匀吸收到土壤中，再将其置于模式植物玻璃温室中(幼苗生长环境如下：昼夜温度控制为28℃/22℃，光照/黑暗时间为16h/8h，相对湿度为60％至70％)，后期用 清水补齐，每组处理重复三次。待种子的胚芽顶出土壤表层时，就认为该种子已经发芽。按照公式I和公式II统计植株的存活率和相对存活率。
存活率＝成苗种子粒数(14天)/供试种子数×100％    (公式I)
相对存活率＝碱处理存活率/相应对照存活率×100％   (公式II)。
数据统计方法如下：首先，将Excel 2016进行数据整理与图表制作，利用DPS 7.5软件进行数据统计，采用最小显著差异法(LSD)和单因素方差分析(ANOVA)对结果进行显著性方差分析。若P＜0.05，标为小写字母，差异显著；若P＜0.01，标为大写字母，差异极显著。
表型观察结果表明：正常水处理的情况下，野生型ZH11、GS3基因过表达株系GS3-1OE、GS3-4OE以及GS3抑制表达植株GS3RNAi之间存活率无显著差异，而在75mM混合碱溶液处理下，GS3-1OE、GS3-4OE转基因植株相对于ZH11，存活率分别下降12.5％和26.4％，相反GS3RNAi存活率上升7.4％(图12F)，以上的结果均表明：水稻的GS3是一个负调控耐碱程度的重要基因。
结果显示，碱胁迫处理后，过表达转基因水稻的耐碱能力降低了，而抑制GS3显著提高耐碱能力，表明GS3是一个以前未曾发现的碱负调控基因。因此抑制蛋白表达有可能可以提高植物的耐碱性。
3.5利用CRISPR/Cas9基因编辑手段抑制水稻中的GS3蛋白的表达
针对水稻GS3基因设计基于CRISPR/Cas9的靶点，合成sgRNA序列，将含有编码所述sgRNA序列的DNA片段连接到携带CRISPR/Cas的载体上(载体信息参见：Ma X，Zhang Q，Zhu Q，et al.A Robust CRISPR/Cas9 System for Convenient，High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants.Mol Plant，2015，8(8):1274-84)中。
设计了2个靶点(如SEQ ID NO:24和25所示)，均位于GS3的第1个外显子上(图15A)。所述基因编辑载体pYL-Cas9-gRNA-GS3的具体构建方法如下：
(1)OsU6a-T1-gRNA-polyT和OsU6b-T2-gRNA-polyT片段的构建
第一轮PCR以pYL-U6a-gRNA质粒为模板，用引物B1’和T1R扩增OsU6a启动子与GS3基因20bp T1靶序列；同样以及pYL-U3-gRNA为模板，用引物T1F和B2 扩增GS3基因T1靶序列与gRNA-polyT；第二轮PCR是以第一轮PCR产物为模板，用引物B1’和B2扩增得到OsU6a-T1-gRNA-polyT片段；同样的方法获得OsU6a-T2-gRNA-polyT的片段。第一轮PCR以pYL-U6a-gRNA质粒为模板，用引物B2’和T2R扩增OsU6b启动子与GS3基因20bp T2靶序列；同样以pYL-U6b-gRNA为模板，用引物T2F和BL扩增GS3基因T2靶序列与gRNA-polyT；第二轮PCR是以第一轮PCR产物为模板，用引物B2’和BL扩增得到OsU6a-T2-gRNA-polyT片段(错误！未找到引用源。15B)。
其中步骤1)中使用的引物序列如下：
B1’:TTCAGAggtctcTctcgCACTGGAATCGGCAGCAAAGG-3(下划线为BsaⅠ酶切位点，SEQ ID NO:43)
B2:AGCGTGggtctcGtcagGGTCCATCCACTCCAAGCTC-3(下划线为BsaⅠ酶切位点，SEQ ID NO:44)
B2’:TTCAGAggtctcTctgaCACTGGAATCGGCAGCAAAGG-3(下划线为BsaⅠ酶切位点，SEQ ID NO:45)
BL:AGCGTGggtctcGaccgGGTCCATCCACTCCAAGCTC-3(下划线为BsaⅠ酶切位点，SEQ ID NO:46)
T1F:AACGGATTCAGCCGGTCTCGGTTTTAGAGCTAGAAATAGCA(下划线为靶点T1，SEQ ID NO:47)
T1R:CGAGACCGGCTGAATCCGTTTGCCACGGATCATCTGCACA(下划线为靶点T1，SEQ ID NO:48)
T2F:GGGACTTGAACGGATTCAGCGTTTTAGAGCTAGAAATAGCA(下划线为靶点T2，SEQ ID NO:49)
T2R:GCTGAATCCGTTCAAGTCCCCGGCAGCCAAGCCAGCACCCG(下划线为靶点T2，SEQ ID NO:50)。
PCR反应总体积为50μl，包含cDNA模板2μl，2×GC I缓冲液25μl，10mM dNTP 5μl，10mM引物GS3OEF和GS3OER各1μl，ExTaq酶1μl，加去离子水至50μl(所用到的2×GC I缓冲液、dNTP、ExTaq酶等均购自宝生物工程大连有限公司)；PCR反 应条件如下：①94℃ 4min，②94℃ 30s，③58℃ 30s，④72℃ 1min，⑤从②－④循环33次，⑥72℃ 7min，⑦25℃保存。
(2)pYL-Cas9-gRNA-OsGS3的构建
将OsU6a-T1-gRNA-polyT，OsU6b-T2-gRNA-polyT的PCR片段以及pYLCRISPR/Cas9-MT载体经过BsaI通过边切边连的方法，将OsU6-T1-gRNA-polyT，OsU6-T2-gRNA-polyT连接到pYLCRISPR/Cas9-MT载体中(图15C)，获得pYL-Cas9-gRNA-OsGS3载体，转化中花11(Zhonghua 11)获得转基因植株OsGS3ko。
3.6 OsGS3ko突变情况检测
在靶点上游111bp，下游72bp处设计引物，对实施例2得到的转基因植株OsGS3ko的DNA进行PCR扩增，对扩增片段进行测序，判断靶位点突变情况。用GS3CRJCF/GS3CRJCR引物进行PCR扩增，鉴定GS3突变效果的引物序列如下，片段大小为273bp。
GS3CRJCF：TACATAGCTGCTGCACCGTC(SEQ ID NO:51)；
GS3CRJCR：GAAGCAAGATCGAAGGAGTATG(SEQ ID NO:52)。
PCR反应总体积为20μl，包含DNA模板2μl，2×GC I缓冲液20μl，2mM dNTP 2μl，10mM引物GS3OEF和GS3OER各0.2μl，rTaq酶0.2μl，加去离子水至20μl(所用到的2×GC I缓冲液、dNTP、rTaq酶等均购自宝生物工程大连有限公司)；PCR反应条件如下：①94℃ 4min，②94℃ 30s，③58℃ 30s，④72℃ 30s，⑤从②－④循环33次，⑥72℃ 7min，⑦25℃保存。测序结果显示，本发明得到一个纯和突变体OsGS3ko。OsGS3ko发生了2bp的插入(见图16)，序列如SEQ ID NO:30所示。
利用类似的方法抑制了玉米自交系KN5585(品种权申请号20191002444)中的GS3蛋白(SEQ ID NO:18)。其中玉米GS3基因组序列如SEQ ID NO:22所示，cDNA序列如SEQ ID NO:23所示。靶点也是设计在第一外显子处，序列如SEQ ID NO:26所示，合成的sgRNA序列如SEQ ID NO:29所示。编辑载体构建和玉米遗传转化完成后，在靶点上游118bp，下游113bp处设计引物，PCR扩增和测序，判断靶位点突变情况。鉴定ZmGS3突变效果的引物序列如下，片段大小为255bp。
ZmGS36F：ACTATAACAATCGACGACGTG(SEQ ID NO:53)；
ZmGS36R：AGCAGTGCAGCGTAATCGAT(SEQ ID NO:54)。
PCR反应总体积为20μl，包含DNA模板2μl，2×GC I缓冲液20μl，2mM dNTP 2μl，10mM引物ZmGS36F和ZmGS36R各0.2μl，rTaq酶0.2μl，加去离子水至20μl(所用到的2×GC I缓冲液、dNTP、rTaq酶等均购自宝生物工程大连有限公司)；PCR反应条件如下：①94℃ 4min，②94℃ 30s，③58℃ 30s，④72℃ 1min，⑤从②－④循环33次，⑥72℃ 7min，⑦25℃保存。根据测序结果可知，本实验获得了一个纯和敲除突变体，记为ZmGS3ko，靶点处存在34bp的缺失(图17)。ZmGS3ko的突变基因序列如SEQ ID NO:31所示，突变基因序列推导的氨基酸序列如SEQ ID NO:32所示。
3.7将OsGS3ko的水稻植株和ZmGS3ko玉米植株分别进行耐碱性鉴定
利用75mM混合碱溶液对水稻野生型ZH11以及OsGS3ko、玉米野生型KN5585以及ZmGS3ko进行了处理。表型观察结果表明：正常水处理的情况下，水稻OsGS3ko相比较ZH11没有显著存活率的差异，玉米ZmGS3缺失突变体株系ZmGS3ko相比较野生型KN5585也没有明显差异，而在75mM碱溶液处理下，水稻OsGS3ko转基因植株相对于ZH11存活率显著上升(图12E-F)。同样玉米ZmGS3ko转基因植株的成活率相对于KN5585也显著上升(图12H-I)，结果表明：无论水稻还是玉米的GS3基因均是负调控耐盐碱程度的重要基因，这个功能在禾本科植物玉米与水稻中保守。
这些突变基因或突变蛋白可以通过常规杂交授粉的方式转育到其他水稻或玉米或其他可杂交的材料中，从而培育新的耐碱品系。
实施例4.AT1同源基因在小麦中的相似作用
普通小麦(Triticum aestivum)为六倍体植物，具有基因组A、B和D。AT1在小麦中的同源基因命名为TaGS或TaAT1(二者可互换使用)，有三个拷贝：TaGS-4A基因、TaGS-7A基因和TaGS-7D基因为小麦中的三个同源基因，基因组序列分别如SEQ ID NO:59、SEQ ID NO:60和SEQ ID NO:61所示。TaGS蛋白质包括TaGS-4A1蛋白、TaGS-4A2蛋白、TaGS-7A蛋白或TaGS-7D蛋白中的任意一种或多种。其中，TaGS-4A1蛋白和TaGS-4A2蛋白为小麦TaGS-4A基因的2个转录本，氨基酸序列分别为SEQ ID NO:55和SEQ ID NO:56。TaGS-7A基因表达如SEQ ID NO:57所示的TaGS-7A蛋白。TaGS-7D基因表达如SEQ ID NO:58所示的TaGS-7D蛋白。通过下述实验证明：通过调整上述多条核酸分子中的任意一种或多种，均能够改变相应的TaGS蛋白质的表达量， 从而实现对于小麦植物耐盐碱性的调控，能够应用于植物育种中。
4.1小麦TaGS靶位点的选择并构建敲除载体
1)小麦TaGS靶位点的选择
小麦中TaGS基因有三个同源基因TaGS-4A、TaGS-7A和TaGS-7D，对应基因ID号分别为：TraesCS4A02G474000、TraesCS7A02G017700和TraesCS7D02G015000。寻找合适的靶位点，利用一个保守靶序列靶向所述三个同源基因，构建敲除载体选择的靶位点位于第1外显子中(图18)。
利用CRISPR技术敲除的靶标双链中的一条链具有如下结构：5-Nx-NGG-3，PAM(NGG)中的N表示A、T、C和G中的任一种，Nx中的N表示A、T、C和G中的任一种，x＝20。本实施例中，选用的TaGS基因的靶序列如下，带下划线的碱基为PAM。
TaGS基因靶序列：AAGTCCCCGCTCGACCCCTGCGG(SEQ ID NO:70)。
该敲除载体转化小麦后，在sgRNA的介导下，Cas9蛋白在靶序列区域切割，形成DNA双链断裂，触发机体内的自我损伤修复机制，细胞自发修复该缺口的过程中会引入突变(本处“突变”指的是广义突变，包括插入、缺失、狭义突变等形式，这些突变中绝大多数为基因功能失活突变)。
2)重组载体的构建
(1)用限制性内切酶BsaI酶切pBUE411(addgen#62200)质粒，回收约12.5kb的载体骨架，命名为BUE411。
(2)根据设计的TaGS基因靶序列(SEQ ID NO:70)，合成如下带有粘性末端(下划线部分)的引物：
TaGS-1F：GGCGAAGTCCCCGCTCGACCCCTG(SEQ ID NO:71)。
TaGS-1R：AAACCAGGGGTCGAGCGGGGACTT(SEQ ID NO:72)。
(3)将TaGS-1F和TaGS-1R进行退火，形成有粘性末端的双链DNA命名为TaGS-1，将其和步骤1中的胶回收产物BUE411连接，得到重组质粒pBUE411-TaGS-1。重组质粒pBUE411-TaGS-1的结构描述为：将pBUE411质粒的两个限制性内切酶BsaI的识 别序列之间的小片段替换为SEQ ID NO:70的第1-20位所示DNA片段后所得的重组质粒。
本申请实施例中所使用的实验方法如无特殊说明，均为常规方法。
本申请实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
4.2转化小麦
利用携带实施例1构建的重组质粒pBUE411-TaGS-1的农杆菌EHA105遗传转化小麦幼胚愈伤组织，转化后经过组织培养获得完整再生植株(即T0代)。
经过传代在T2代，得到TaGS基因三个同源基因TaGS-4A、TaGS-7A和TaGS-7D功能缺失的转基因植株E5。Sanger测序发现，E5植株的TaGS基因TaGS-4A为纯合突变，TaGS-7A和TaGS-7D都为双等位突变(如图19所示)。E5植株的TaGS-4A突变是第6-18位的CCCGCTCGACCCC(SEQ ID NO:73)碱基缺失13bp；E5植株的TaGS-7A第一种突变是第18位的C碱基缺失1bp，第二种突变是第17-18位的CC碱基缺失2bp；E5植株的TaGS-7D第一种突变是第20位的G碱基缺失1bp，第二种突变是第19-20位的TG碱基缺失2bp。
4.3盐碱胁迫下小麦TaGS基因三突突变体的耐盐碱表型
首先，将实施例4.2获得的T2代TaGS基因三突突变体种子E5、野生型Fielder播种到按1：1比例混匀的蛭石和营养土的土壤基质中，每穴播种12粒种子，设置2次重复。接着，用75mM混合碱溶液(pH 9.2，摩尔比5：1的NaHCO3：Na2CO3)浇灌至土壤饱和作为处理，用清水浇灌的作为对照，置于平地上使其均匀吸收，每组处理重复三次。将充分吸收后的穴盘放入模式植物玻璃温室中，后期用清水补水。幼苗生长环境如下：光照/黑暗时间为16h/8h，昼夜温度范围为28/26℃，相对湿度为60％到70％。
结果显示：经盐碱胁迫(75mM混合碱溶液)处理，E5突变体株系耐盐碱性显著高于野生型，地上部分生物量明显高于野生型，地下部分根生长抑制程度明显弱于野生型(如图20所示)。
与上述操作相同，分别用100mM或125mM混合碱溶液(pH 9.2，摩尔比5：1的NaHCO3：Na2CO3)浇灌至土壤饱和作为处理，用清水浇灌的作为对照，E5突变体株系 耐盐碱性显著高于野生型，失绿萎蔫程度弱于野生型。
从以上的结果可以看出，本发明上述的实施例实现了如下技术效果：通过对小麦中的用于转录翻译TaGS蛋白质的核酸分子进行基因编辑，减少小麦中TaGS蛋白质的表达量，实现了小麦耐盐碱性的提高，获得了耐盐碱性提高的小麦新植株。
4.4小麦TaGS基因三突突变体的其他检测
我们用所有三个拷贝的TaAT1(也称为TaGS)基因生成了TaAT1 null突变体(即TaGS基因三突突变体)。表型分析显示，在125mM(104.2mM NaHCO3和20.8mM Na2CO3,pH 9.7-9.8)混合碱处理下，TaAT1ko-1和TaAT1ko-2的存活率分别比TaWT高122％和164％(图21C、D、E和F)。由此可见，敲除所有拷贝的TaAT1可大大增强小麦的耐盐碱性。
为了进一步研究TaAT1是否像高粱和水稻一样采用保守机制来抵御盐碱胁迫，采用DAB(3,3-二氨基联苯胺)和H2DCFDA染色法测定了TaAT1ko品系的ROS积累。从图21G和H中可以看出，盐碱处理下，TaAT1ko品系的细胞内H2O2水平明显低于TaWT，这与其他物种的AT1ko表型一致。结果表明，与AT1基因在高粱、玉米和水稻中的作用一样，TaAT1基因是负调控小麦耐盐碱程度的重要基因，TaAT1基因的基因修饰也能有效提高小麦在盐碱土壤中的生长性能，且机制保守。
实施例5.在高钠土中提高作物产量
为了评估AT1/GS3基因在作物生产中的有用性，我们在含天然碱的高钠土中对高粱、水稻、玉米和谷子进行了田间测试，这些高粱、水稻、玉米和小米具有不同的天然等位基因和基因修饰的AT1/GS3基因。这些田地位于中国盐碱地区的两个地区：吉林省大安地区(中国北部)和宁夏平楼地区(中国西北部)。这两个地区是中国的主要作物产地，但是由于存在大面积的盐碱地，作物产量受到限制。
KYNIL(GS3)是携带OsGS3-2(相对于OsGS3-1在C端有一个阅读框内3-bp插入)的优良水稻品种Kongyu131，OsGS3-2的功能与OsGS3-1相当。KYNIL(gs3-)是在Kongyu131背景下具有渗入的OsGS3-3，OsGS3-3是一个完全丧失功能的等位基因。田间试验前，对KYNIL(GS3)与KYNIL(gs3-)进行耐碱性试验。试验在幼苗期在温室中进行，混合碱浓度为75mM。正如预期的那样，KYNIL(gs3-)表现出比Kongyu131更高的耐碱性(图22A和B)。
然后，我们进行了一项实验，使用来自同一地区的高钠土壤和营养土壤混合，比较了pH值为9.45和7.74(对照)的两种不同土壤中的NIL。在pH为9.45的钠性土壤下， KYNIL(gs3-)除单株穗数(图22C)外，在相对成活率、每穗粒数、粒重和产量(图22C)等方面均显著优于KYNIL(GS3)。
我们还在中国吉林省大安进行了田间试验，大安的土壤pH值为9.17。KYNIL(gs3-)在幼苗期(图22D)和收获期(图23A，左图)的表现都比KYNIL(GS3)好得多，表明KYNIL(gs3-)增强的耐碱性。在收获期，KYNIL(gs3-)水稻产生了更大的稻穗(图22E)，每穗的籽粒数更高(图23A，第三幅图)，粒重增加(图23A，第四幅图)，导致每丛籽粒产量增加29.3％(图23A，第五幅图)KYNIL(gs3-)水稻的籽粒产量比对照高27.8％(图23A，右图)。在pH值为5.58的田地上进行同样的种植，KYNIL(gs3-)与对照之间每株产量的差异仅为10.3％(图22F)。第2年，在同一地点研究了NIL在钠性土壤和酸性土壤中的籽粒产量。碱土增产22.4％，对照田增产6.64％(图22G和H)，与之前观察到的结果相似。此外，在钠性土壤和中性土壤中，敲除OsGS3对粒长有贡献。我们还发现，OsGS3基因敲除在钠性土壤中对粒宽有贡献，但在中性土壤中没有贡献(图S22I和J)。这些结果表明，GS3的无功能等位基因可以在高钠性土壤中实现更高的作物产量。
由于品质优良、产量高，自2018年以来，改良优良水稻Zhongkefa5(ZKF5)已在中国北方种植超过10万公顷。ZKF5有一个GS3的无功能等位基因(OsGS3-3)，与KYNIL(gs3-)相似。然后在2021年夏季，在相对高钠土壤(pH 8.5-8.7)和低钠土壤(pH 7.4-7.6)中测试了ZKF5田间性能。在生长季节结束时，当地农民协会从30多公顷土地上获得了田间生产数据。我们发现，与中性田(低钠)相比，含钠田(高钠)的产量仅减少了7.8％(图22K)。这些大面积的田间生产数据还表明，在含钠土地的水稻生产中使用GS3-3等位基因(即，无功能等位基因)可以培育作物产量提高的水稻。
此外，我们还将Zhonghua 11(ZH11)水稻及其OsGS3敲除系OsGS3ko种植在温室中相同的两个pH值土壤中，对照为pH7.74，碱性土壤为pH9.45。虽然由于ZH11在中国吉林省种植时的光周期敏感性，我们未能收获其种子，但在OsGS3ko品系中观察到的较高相对存活率(图22L)，表明其对碱性条件的耐受性更强。
高粱、玉米和谷子在中国甘肃省平洛地区进行了测试。平洛地区位于中国西北部，是由高钠土壤的干旱土地组成的。在该地区，由于地下水位的变化，作物生长季节pH值自然升高(B.P.Singh,A.L.Cowie,K.Y.and Chan,Soil health and climate change.New York:Springer-Verlag Berlin Heidelberg 29,(2011).doi:10.1007/978-3-642-20256-8)。在同一地块种植的Wheatland野生型和SbAT1ko高粱，春季pH值为8.97，8月开花期pH值为 9.27。SbAT1ko品系的存活率超过60％，而Wheatland野生型的存活率仅为33％(图23B，第1和第2幅图)。叶焦(leaf burn)，一种通常发生在受高盐或高钠胁迫影响的单子叶作物中的症状，在大多数Wheatland野生型植物中观察到，但在SbAT1ko品系的植物中没有观察到(图23B，第一幅图)。在收获期，SbAT1ko品系的分蘖数和穗数(图23B，第四和第五幅图)较对照低，但产量却比对照高20.1％(图23B，第三幅图)。由于高粱全株通常用于青贮，我们测量了全株生物量的鲜重，发现SbAT1ko品系的植株鲜重比对照高30.5％(图23B，右图)。SbAT1ko品系的籽粒产量和全株生物量均高于野生型，表明在含钠土地(盐碱地)上SbAT1ko品系高粱的田间性能更好。在同一地区，我们还在夏季种植了NIL-SbAT1和NIL-Sbat1品系。在8月底，我们发现NIL-SbAT1优于NIL-Sbat1(图23C)，差异与温室实验记录的结果相似(图6B)。
谷子SiAT1ko及其野生型对照Ci846也在同一地区与高粱一起种植。SiAT1ko株系幼苗期成活率接近100％，而野生型Ci846的成活率仅为约75％(图23D，左图)。此外，在收获期，敲除系SiAT1ko的穗粒大小也比对照大(图23D，中间图)，SiAT1ko的产量比对照高大约19.5％(图23D，右图)。
在同一块土地上，我们还种植了玉米ZmGS3敲除系及其野生型对照系KN5585(ZmWT)。在幼苗期，种植一个月后，ZmGS3ko株系的相对存活率为约42.5％，而野生型对照的相对存活率仅为约18.5％(图23E)。经过3个月的生长，KN5585株系的大多数个体死亡，而敲除系的个体中有7.4％存活。虽然由于玉米对碱性条件的固有敏感性，这些玉米植物都没有成熟到可以生产籽粒，但ZmGS3敲除系显示出增强的耐碱性。
总之，我们能够得出的结论是，无论是从自然变异中获得还是通过基因编辑产生的AT1同源基因无功能性突变，都可以在钠性土壤(盐碱地)中种植时提高作物在生物量或产量方面的田间表现。
实施例6.AT1作用机制研究
基于我们的研究，我们设计了推测的植物中Gγ亚基AT1-介导的碱应激反应的模型(图24)。在碱胁迫下，PIP2s(PIP2水通道蛋白)作用为H2O2输出蛋白。Gγ亚基AT1可能与Gβ配对负调控PIP2s的磷酸化，由此降低PIP2s的H2O2输出能力，导致H2O2过度积聚，并导致植物对碱胁迫的敏感性。截短形式的AT1，即at1，进一步抑制H2O2输出活性，并导致植物对碱胁迫的高敏感性。然而，AT1的自然无功能形式或敲除AT1同源基因释放对PIP2s的抑制作用并有效地提高作物的碱胁迫耐受性。
实施例7.AT1大豆同源基因的相似功能
通过BLAST，在野生大豆(Glycine soja)和栽培大豆(Glycine max)中(均为四倍体)各检索到三个与高粱AT1蛋白序列同源性相对较高的基因(图25和表2，相似性为约40％至约50％)，它们均含有Gγ样结构域。但三种系统进化分析图均显示它们与高粱水稻玉米谷子和小麦的亲缘关系较远。
表2.全长氨基酸序列与高粱AT1比对(使用VectorBuilder的序列比对工具进行)
表3.GGL结构域与高粱AT1 GGL结构域比对(使用VectorBuilder的序列比对工具进行)
在实施例1-5中，我们已经证实了AT1/GS3基因在五种单子叶谷物(高粱、谷子、水稻、玉米和小麦)的盐碱应激反应中起保守且重要的作用。虽然对AT1/GS3基因进行基因修饰是否也能够调控双子叶植物(如大豆)的耐盐碱性还需要进一步研究，但考虑到大豆中AT1同源基因与高粱AT1基因具有一定的同一性，尤其是GGL结构域的同一性 较高(约40％至50％)，我们的初步试验结果(数据未显示)支持大豆中AT1同源基因将与高粱、谷子、水稻、玉米和小麦中同源基因起相似的调控耐盐碱性的保守作用，例如，减少大豆中所有拷贝的AT1同源基因的表达，尤其是敲除GGL结构域，或采用所有拷贝的无功能性等位基因，均能够提高大豆的耐盐碱性；反之，如果增加大豆中AT1同源基因的表达(例如，过表达)，或表达AT1同源基因的C端截短突变体，也可能获得盐碱敏感性大豆。
大豆中AT1同源基因序列见表4。
表4.大豆中AT1同源基因序列



表5.GGL结构域的氨基酸序列
本领域技术人员将进一步认识到，在不脱离其精神或中心特征的情况下，本发明可以以其他具体形式来实施。由于本发明的前述描述仅公开了其示例性实施方案，应该理解的是，其他变化被认为是在本发明的范围内。因此，本发明不限于在此详细描述的特定实施方案。相反，应当参考所附权利要求来指示本发明的范围和内容。

Claims (30)

  1. 一种核酸分子，其编码包含与SEQ ID NO:15具有至少40％同一性、优选至少约75％同一性、更优选至少约80％同一性或更优选至少约90％或约95％同一性、或至具有少约60％相似性、优选至少约70％、约80％或约85％相似性、更优选至少约90％或约95％相似性的氨基酸序列的蛋白，其用于调控植物的耐盐碱性，或用于培育具有耐盐碱性或盐碱敏感性的植物。
  2. 根据权利要求1所述的核酸分子，其中所述核酸分子编码包含SEQ ID NO:15和86-96中任一氨基酸序列的蛋白。
  3. 根据权利要求1所述的核酸分子，其编码选自下述的氨基酸序列：
    (i)与SEQ ID NO:4具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
    (ii)与SEQ ID NO:8具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
    (iii)与SEQ ID NO:16-17中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
    (iv)与SEQ ID NO:18具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
    (v)与SEQ ID NO:55-58中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；或
    (vi)与SEQ ID NO:75、77、79、81、83或85中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列。
  4. 一种突变蛋白质，其由编码下述氨基酸序列的核酸分子发生移码突变后的变体核酸分子编码：
    (i)与SEQ ID NO:4具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
    (ii)与SEQ ID NO:8具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
    (iii)与SEQ ID NO:16-17中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
    (iv)与SEQ ID NO:18具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
    (v)与SEQ ID NO:55-58中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；或
    (vi)与SEQ ID NO:75、77、79、81、83或85中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
    其中与核酸分子发生移码突变前所编码的蛋白质相比，所述突变蛋白质的活性降低或没有活性。
  5. 一种核酸分子，其编码权利要求4所述的突变蛋白质。
  6. 一种表达盒，其包含权利要求1-3或5中任一项所述的核酸分子。
  7. 一种重组载体，其包含权利要求1-3或5中任一项所述的核酸分子，或包含权利要求5所述的表达盒。
  8. 一种细胞，其包含权利要求1-3或5中任一项所述的核酸分子、或权利要求5所述的表达盒，或权利要求6所述的重组载体。
  9. 根据权利要求8所述的细胞，其中所述细胞选自原核细胞或真核细胞，所述原核细胞例如细菌细胞或真菌细胞，例如，大肠杆菌细胞、酵母菌细胞或农杆菌细胞；所述真核细胞例如植物细胞。
  10. 一种培育耐盐碱性植物的方法，所述方法包括：
    减少植物中编码包含与SEQ ID NO:15具有至少40％同一性、优选至少约75％同 一性、更优选至少约80％同一性或更优选至少约90％或约95％同一性、或至具有少约60％相似性、优选至少约70％、约80％或约85％相似性、更优选至少约90％或约95％相似性的氨基酸序列的蛋白的所有等位基因的表达水平或使所述所有等位基因不表达，
    其中所述耐盐碱性植物在盐碱性条件下的生长优于野生型植物，
    其中所述盐碱性条件包括pH>7.5、Na+浓度>75mM的生长条件或pH>8.0、Na+浓度>50mM的生长条件。
  11. 根据权利要求10所述的方法，其中通过基因编辑方法、靶向诱变法、化学诱导、射线诱导、自然突变、RNAi或添加抑制目的基因表达的物质来减少植物中所述所有等位基因的表达水平或使所述所有等位基因不表达。
  12. 根据权利要求10所述的方法，其中植物中所述所有等位基因被敲除或突变，例如，通过同源重组敲除所述基因的GGL结构域或GGL样结构域，或通过CRISPR技术编辑所述基因的GGL结构域或GGL样结构域。
  13. 根据权利要求10所述的方法，其中所述所有等位基因的N端第一个外显子部分被敲除或突变。
  14. 根据权利要求10所述的方法，其中所述所有等位基因的GGL结构域或GGL样结构域、或N端第一个外显子部分被敲除或突变。
  15. 根据权利要求10所述的方法，其中相较于野生型对照植物，所述所有等位基因的表达水平至少减少51％，优选减少60％、70％或80％，更优选85％、90％或95％，或甚至不表达。
  16. 根据权利要求10所述的方法，其中所述植物是单子叶植物或双子叶植物，例如，禾本科植物，优选高粱属植物、稻属植物、谷子、玉米、小麦或大豆。
  17. 根据权利要求10所述的方法，所述方法还包括鉴定包含所述所有等位基因的 敲除或突变、或包含无功能等位基因的亲本植物，进行自交或与另一包含所述所有等位基因的敲除或突变或包含无功能等位基因的亲本植物杂交得到的一代或多代后代植物，
    其中所述所有等位基因的敲除或突变使得所述所有等位基因编码的蛋白活性降低或没有活性。
  18. 根据权利要求10所述的方法，其中编码选自下述的氨基酸序列的基因的所有等位基因的GGL结构域或GGL样结构域、或N端第一个外显子部分被敲除或突变：
    (i)与SEQ ID NO:4具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
    (ii)与SEQ ID NO:8具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
    (iii)与SEQ ID NO:16-17中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
    (iv)与SEQ ID NO:18具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
    (v)与SEQ ID NO:55-58中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；或
    (vi)与SEQ ID NO:75、77、79、81、83或85中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列。
  19. 一种植物或植物材料，其中编码包含与SEQ ID NO:15具有至少40％同一性、优选至少约75％同一性、更优选至少约80％同一性或更优选至少约90％或约95％同一性、或至具有少约60％相似性、优选至少约70％、约80％或约85％相似性、更优选至少约90％或约95％相似性的氨基酸序列的蛋白的所有等位基因被敲除或突变，优选地，其中所述基因的所有等位基因的GGL结构域或GGL样结构域、或N端第一个外显子部分被敲除或突变；
    其中所述所有等位基因的敲除或突变使得所述所有等位基因编码的蛋白活性降低或没有活性。
  20. 根据权利要求19所述的植物或植物材料，其中所述植物为单子叶植物或双子叶植物，例如，禾本科植物，优选高粱属植物、稻属植物、谷子、玉米、小麦或大豆。
  21. 根据权利要求19所述的植物或植物材料，其中编码选自下述的氨基酸序列的基因的所有等位基因的GGL结构域或GGL样结构域、或N端第一个外显子部分被敲除或突变：
    (i)与SEQ ID NO:4具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
    (ii)与SEQ ID NO:8具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
    (iii)与SEQ ID NO:16-17中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
    (iv)与SEQ ID NO:18具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
    (v)与SEQ ID NO:55-58中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；或
    (vi)与SEQ ID NO:75、77、79、81、83或85中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列。
  22. 根据权利要求19所述的植物或植物材料，其中所述植物材料为植物部分、植物器官、植物组织、种子、植物原生质体或植物细胞，例如，胚、花粉、胚珠、种子、叶、花、枝、果实、茎杆、根、根尖、花药、植物细胞培养物或植物愈伤组织。
  23. 一种制备杂交种植物种子的方法，所述方法包括：
    (i)将第一亲本植株与第二亲本植株杂交，所述在第一亲本植株和第二亲本植株中，编码包含与SEQ ID NO:15具有至少40％同一性、优选至少约75％同一性、更优选至少约80％同一性或更优选至少约90％或约95％同一性、或至具有少约60％相似性、优选至少约70％、约80％或约85％相似性、更优选至少约90％或约95％相似性的氨 基酸序列的蛋白的所有等位基因被敲除或突变；以及
    (ii)收获杂交植株或其后代的种子。
  24. 一种制备常规种植物种子的方法，所述方法包括：
    扩繁亲本种子以收获其后代种子，其中在所述亲本种子中，编码包含与SEQ ID NO:15具有至少40％同一性、优选至少约75％同一性、更优选至少约80％同一性或更优选至少约90％或约95％同一性、或至具有少约60％相似性、优选至少约70％、约80％或约85％相似性、更优选至少约90％或约95％相似性的氨基酸序列的蛋白的所有等位基因已被敲除或突变，使得所述蛋白不被表达或与野生型植物相比表达水平减少。
  25. 根据权利要求23或24所述的方法，其中在所述亲本种子中，编码包含下述氨基酸序列的蛋白的所有等位基因的已被敲除或突变：
    (i)与SEQ ID NO:4具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
    (ii)与SEQ ID NO:8具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
    (iii)与SEQ ID NO:16-17中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
    (iv)与SEQ ID NO:18具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；
    (v)与SEQ ID NO:55-58中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列；或
    (vi)与SEQ ID NO:75、77、79、81、83或85中任一项具有至少80％、85％、90％、95％、99％或100％同一性并且具有相似或相同功能的氨基酸序列。
  26. 根据权利要求23所述的方法，其中所述第一亲本植株和/或第二亲本植株是近交系植株。
  27. 一种植物或其植物材料，所述植物由权利要求23-26中任一项所述的方法制备的 种子长成。
  28. 一种培育盐碱敏感性的植物的方法，所述方法包括：
    增加植物中编码包含与SEQ ID NO:15具有至少40％同一性、优选至少约75％同一性、更优选至少约80％同一性或更优选至少约90％或约95％同一性、或至具有少约60％相似性、优选至少约70％、约80％或约85％相似性、更优选至少约90％或约95％相似性的氨基酸序列的蛋白的基因的表达水平，或在植物表达或过表达所述基因突变体编码的C端截短蛋白。
  29. 根据权利要求28所述的方法，其中通过向植物中导入外源编码包含与SEQ ID NO:15具有至少40％同一性、优选至少约75％同一性、更优选至少约80％同一性或更优选至少约90％或约95％同一性、或至具有少约60％相似性、优选至少约70％、约80％或约85％相似性、更优选至少约90％或约95％相似性的氨基酸序列的核酸分子而增加目的基因的表达水平。
  30. 根据权利要求29所述的方法，其中向所述植物的细胞或组织中导入携带所述核酸分子的遗传物质，所述遗传物质在所述植物内，以游离或整合至所述植物的染色体的形式存在，再将导入所述遗传物质后的细胞或组织培养成完整植株，获得所述盐碱敏感植物。
PCT/CN2023/081663 2022-08-12 2023-03-15 植物耐盐碱基因及其用途 WO2024031987A1 (zh)

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CN202211198322.2 2022-09-29
CN202211198322.2A CN116162142B (zh) 2022-09-29 2022-09-29 一种植物gs3基因或蛋白在调控植物耐盐碱中的应用

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DATABASE Nucleotide 31 August 2009 (2009-08-31), ANONYMOUS : "Oryza sativa Indica Group GS3 gene for seed length and weight protein long form for short seed, complete cds, cultivar: Basmati 1", XP093138135, retrieved from NCBI Database accession no. AB488626.1 *
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DATABASE Protein 19 April 2021 (2021-04-19), ANONYMOUS : "guanine nucleotide-binding protein subunit gamma 3 [Glycine max] ", XP093138185, retrieved from NCBI Database accession no. XP_003534947.1 *
DATABASE Protein 19 April 2021 (2021-04-19), ANONYMOUS : "guanine nucleotide-binding protein subunit gamma 3 isoform X1 [Glycine max]", XP093138198, retrieved from NCBI Database accession no. XP_006593954.1 *
DATABASE Protein 19 April 2021 (2021-04-19), ANONYMOUS : "guanine nucleotide-binding protein subunit gamma 3 isoform X2 [Glycine max]", XP093138205, retrieved from NCBI Database accession no. XP_006593955.1 *
DATABASE Protein 2 December 2021 (2021-12-02), ANONYMOUS : "guanine nucleotide-binding protein subunit gamma 3 isoform X1 [Aegilops tauschii subsp. strangulata]", XP093138179, retrieved from NCBI Database accession no. XP_020199303.1 *
DATABASE Protein 20 August 2020 (2020-08-20), ANONYMOUS : "hypothetical protein CFC21_055613 [Triticum aestivum]", XP093138140, retrieved from NCBI Database accession no. KAF7046588.1 *
DATABASE Protein 25 October 2021 (2021-10-25), ANONYMOUS : "guanine nucleotide-binding protein subunit gamma 3-like [Triticum aestivum]", XP093138173, retrieved from NCBI Database accession no. XP_044426581.1 *
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