WO2017121411A1 - 一种与植物雄性育性相关的蛋白及其编码基因与应用 - Google Patents

一种与植物雄性育性相关的蛋白及其编码基因与应用 Download PDF

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WO2017121411A1
WO2017121411A1 PCT/CN2017/073142 CN2017073142W WO2017121411A1 WO 2017121411 A1 WO2017121411 A1 WO 2017121411A1 CN 2017073142 W CN2017073142 W CN 2017073142W WO 2017121411 A1 WO2017121411 A1 WO 2017121411A1
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plant
protein
gene
sequence
male
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陈化榜
赵丽
张华�
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中国科学院遗传与发育生物学研究所
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility
    • 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
    • A01H6/4684—Zea mays [maize]
    • 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 invention relates to the field of plant genetic engineering, in particular to a protein related to plant male fertility and a coding gene thereof and application thereof.
  • Maize is a crop with very obvious heterosis. Hybrids are commonly used in production, so seeding is required every year. In the process of seed production, since the corn is a cross-pollinated crop, in order to ensure the purity of the seed, the female parent used in the seed production needs to be emasculated. If the mother is male sterile, there is no need to go to the male, which saves labor costs and avoids the reduction of seed purity due to incomplete emasculation.
  • Male sterility in maize including male-sterile sterility and nuclear male sterility. In the process of growth and development of male male tissues until the production of active pollen, mutation of any of the nuclear male fertility genes may lead to male sterility. Male sterility caused by mutations in these nuclear male fertility genes can be divided into dominant sterility and recessive nuclear sterility, and most of them are recessive sterility.
  • the currently cloned maize nuclear male fertility genes include MS8 (Wang et al., 2013, Plant Reprod. 26: 329-338), MS9 (Patent No.: US20150191743), MS22/MSCA1 (Chaubal et al., 2003, Planta 216).
  • the protein provided by the present invention entitled ZmIAP1, derived from Zea mays L., is as follows (a) or (b):
  • Sequence Listing 1 is the amino acid sequence of ZmIAP1, including 582 amino acids.
  • 263 are hydrophobic amino acids
  • 266 are hydrophilic amino acids
  • 70 are basic amino acids
  • 52 are acidic amino acids.
  • the molecular weight was 63.43 KD and the isoelectric point was 8.73.
  • a label shown in the following table may be attached to the amino terminus or the carboxy terminus of a protein consisting of the amino acid residue sequence of SEQ ID NO: 1 in the Sequence Listing.
  • the protein in the above (b) can be artificially synthesized, or the encoded gene can be synthesized first, and then obtained by biological expression.
  • the gene encoding the protein in (b) above may be deleted by one or several amino acid residues in the DNA sequence shown in SEQ ID NO: 2 in the sequence listing, and/or one or several base pairs may be missed. mutation.
  • Nucleic acid molecules encoding the proteins are also within the scope of the invention.
  • the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA, hnRNA or tRNA.
  • the nucleic acid molecule is specifically a gene encoding the protein (designated as ZmIAP1), and the gene may specifically be a DNA molecule according to any one of the following 1) to 6):
  • the gene is a DNA molecule of any of the following 1) to 6):
  • sequence 2 is the sequence of the ZmIAP1 gene in the maize genome
  • sequence 3 is the cDNA sequence of the ZmIAP1 gene
  • position 249-1997 is the CDS.
  • Recombinant vectors, expression cassettes, transgenic cell lines, recombinant microorganisms or transgenic plants containing the above nucleic acid molecules are also within the scope of the present invention.
  • the recombinant vector may be a recombinant expression vector or a recombinant cloning vector.
  • the recombinant expression vector can be constructed using existing plant expression vectors.
  • the plant expression vector includes a dual Agrobacterium vector and a vector which can be used for plant microprojectile bombardment, and the like, such as pGreen0029, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other derivative plant expression vectors.
  • the plant expression vector may further comprise a 3' untranslated region of a foreign gene, i.e., comprising a polyadenylation signal and any other DNA fragment involved in mRNA processing or gene expression.
  • Polyadenylic acid The signal directs the addition of polyadenylation to the 3' end of the mRNA precursor.
  • any enhanced, constitutive, tissue-specific or inducible promoter may be added before the transcription initiation nucleotide, such as cauliflower mosaic virus (CaMV) 35S promoter.
  • a ubiquitin gene Ubiquitin promoter (pUbi), a stress-inducible promoter rd29A, etc. which can be used alone or in combination with other plant promoters; in addition, when a recombinant expression vector is constructed using the gene of the present invention, it can also be used.
  • Enhancers including translational enhancers or transcriptional enhancers, may be ATG start codons or contiguous region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence.
  • the sources of the translational control signals and initiation codons are broad and may be natural or synthetic.
  • the translation initiation region can be from a transcription initiation region or a structural gene.
  • the recombinant expression vector used can be processed, such as a gene encoding a color-changing enzyme or luminescent compound that can be expressed in plants, and a resistant antibiotic marker. Or anti-chemical reagents, etc. Transformed plants can also be screened directly under adverse conditions without any selectable marker genes.
  • the recombinant expression vector is a recombinant plasmid obtained by inserting the ZmIAP1 gene into a multiple cloning site (such as Kpn I and Hind III) of the pCAMBIA3300 vector. More specifically, a recombinant plasmid obtained by replacing the small fragment between the cleavage sites Kpn I and Hind III of the pCAMBIA3300 vector with the DNA fragment shown at positions 1406 to 6601 of SEQ ID NO: 4 in the sequence table (designated p3300- ZmIAP1).
  • the expression cassette consists of a promoter capable of initiating expression of the gene, the gene, and a transcription termination sequence.
  • the transgenic cell line is a non-propagating material that is transferred into the gene.
  • the transgenic plant may specifically be a seed, callus, whole plant or cell into which the gene is transferred.
  • the application in (a) may be transgenic or non-transgenic.
  • the application of "non-transgenic” may be: cultivating the sterile line using the ZmIAP1 gene.
  • the male sterility trait of the sterile line is caused by a loss or decrease in the ability of the plant to express a functional ZmIAP1 gene which is homozygous aa.
  • the application of "transgenic” may be: combining the maintainer line and the sterile line into a dual-use system, and introducing the normally functional ZmIAP1 gene based on the sterile line, and the sterile line can be isolated in the offspring. And dual-use.
  • the application may be the utilization of the ZmIAP1 gene (normally functioning) or the utilization of the ZmIAP1 gene (reduced or lost function).
  • the regulating plant male fertility may be to increase the male fertility of the plant or reduce the male fertility of the plant.
  • the invention also provides methods of growing transgenic plants.
  • the method for cultivating a transgenic plant provided by the present invention may be as follows (A) or (B):
  • a method of cultivating a male fertile transgenic plant comprising the steps of:
  • the coding gene can be introduced into the recipient plant by the above recombinant expression vector p3300-ZmIAP1;
  • the expression of the coding gene in the recipient plant is inhibited by: encoding a genome encoding the ZmIAP1 protein in the recipient plant using a CRISPR/Cas9 nuclease; The DNA sequence undergoes specific cleavage, causing the recipient plant to lose or reduce its ability to express a functional ZmIAP1 protein.
  • the target fragment when the CRISPR/Cas9 nuclease specifically cleaves the genomic DNA sequence encoding the ZmIAP1 protein in the recipient plant is 5' in the genomic DNA sequence encoding the ZmIAP1 protein in the recipient plant -N X -NGG-3' or 5'-CCN-N X -3' sequence alignment rule segment; N represents any of A, G, C and T, 14 ⁇ X ⁇ 30, and X is an integer (If X is 20), N X represents X consecutive deoxyribonucleotides. More specifically, the target fragment is "5'-GAACGCGCGGGCCCGGGTGCTGG-3' (ie, positions 701-723 of SEQ ID NO: 2) in the genomic DNA sequence encoding the ZmIAP1 protein in the recipient plant.
  • the male sterility is "the flowering period is not attached to the anther" and/or "the anther is not identified as a viable pollen by 1% I 2 -KI staining".
  • the plant may be either a monocot or a dicot.
  • the monocotyledonous plant is a gramineous plant, such as corn.
  • the maize mutant ms*-6044 can be used as the recipient plant to obtain a corresponding male fertile transgenic maize; of course, as in one embodiment of the present invention, The vector of ZmIAP1 gene was transformed into maize variety HiII, and then the transgenic positive plants were crossed twice with the maize mutant ms*-6044. The plants with the genome background of ms*-6044 and positive transgene were selected in the corresponding interval. Male fertile genetically modified corn.
  • the recipient plant used in cultivating a male sterile transgenic plant is specifically the maize variety HiII.
  • Transgenic plants obtained by the methods are also within the scope of the invention.
  • the transgenic plant may specifically be a seed, callus, whole plant or cell into which the gene is transferred.
  • Figure 1 is a phenotypic comparison of the maize mutant ms*-6044 with the wild type. Among them, A, C, E, and G are wild type, and B, D, F, and H are ms*-6044 mutants. A and B, plant morphology; C and D, tassel anthers; E and F, anther morphology; G and H, pollen 1% I 2 -KI staining.
  • Figure 2 is a map of the ZmIAP1 gene map.
  • Figure 3 is a schematic representation of the ZmIAP1 gene structure and its insertion site in the maize mutant ms*-6044.
  • Figure 4 is a diagram of an agarose gel electrophoresis of a complementary vector PCR product.
  • Figure 5 is an electropherogram of the PCR product after double digestion with KpnI and HindIII.
  • Figure 6 is a genetic complementation verification diagram of the ZmIAP1 gene.
  • A schematic diagram of the preparation of transgenic progeny population
  • B male fertile phenotype of genotype aaBb plant
  • C transgenic detection (3, 4, 5 is a genotype aaBb plant, identified as transgenic positive
  • 1 is The control, that is, the p3300-ZmIAP1 plasmid used for transforming the maize receptor HiII was used as a template for amplification of the positive control)
  • D the genotype was pollen 1% I 2 -KI staining of the aaBb plants.
  • Figure 7 is a ZmIAP1 gene knockout verification map.
  • A gene knockout target sequence
  • B PCR digestion of transgenic plants, 1-4 are transgenic plants, CK is wild type, PCR product size is 805 bp, and is 574 bp and 231 bp after SmaI digestion. Two fragments
  • C wild type and transgenic plants PCR product sequencing and coding amino acid sequence comparison
  • D transgenic positive plants full bloom tassel
  • E transgenic positive plants anther 1% I 2 -KI staining
  • F transgenic negative plants Splendid tassel
  • G pollen-negative plant pollen 1% I 2 -KI staining.
  • Maize Mutant ms*-6044 Saved by Maize Genetics Cooperation Stock Center, Co-op ID: 104G; Description: 104G ms*-6044.
  • pBUN411 vector in the literature "Hui-Li Xing, Li Dong, Zhi-Ping Wang, Hai-Yan Zhang, Chun-Yan Han, Bing Liu, Xue-Chen Wang, Qi-Jun Chen. BMC plant biology. 14:327- It is disclosed in 338 (2014), and the public can obtain it from the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences, and can only be used to repeat the experiments of the present invention.
  • This plasmid can be used for both transcription of the guide RNA and expression of the Cas9 protein.
  • the maize mutant ms*-6044 had no abnormalities in the overall morphology of the plants (A and B in Figure 1), but the anthers of the tassels were not plugged (C and D in Figure 1), and the anthers were dissected. Dry and thin (E and F in Figure 1), the inside of the anther was stained with 1% I 2 -KI solution and no full-fledged pollen grains (G and H in Figure 1) were observed, which showed complete male sterility. This phenomenon is consistent in the observation of phenotypes in Beijing and Hainan for 4 consecutive years, and the infertility characteristics are stable, and it is possible to distinguish male sterility by the naked eye under field conditions.
  • the maize mutant ms*-6044 was used as the female parent, and the maize inbred line B73 or Zheng 58 was used as the male parent to match F 1 , and the F 1 field phenotype was male fertile. Then the maize mutant ms*-6044 was used as the female parent, and the F 1 was used as the male parent to construct the backcross population (BC 1 F 1 ).
  • the BC 1 F 1 population can be divided into male fertile individuals and male sterile individuals in the field. Types, and the ratios of the two types were 1:1 (Table 1) by chi-square test. It is speculated that the phenotype of maize mutant ms*-6044 is controlled by a recessive single gene, and the BC 1 F 1 population is used as a genetic mapping population.
  • the genomic DNA of maize mutant ms*-6044 and maize inbred lines B73 and Zheng 58 were used as templates, and maize whole genome primers were used to screen primers with polymorphism between mutants and inbred lines.
  • 10 male sterile plants and 10 male sterile plants were selected from the BC 1 F 1 population to verify whether the polymorphic primers were linked to male fertility traits.
  • the linkage primers were screened for the genotype of 1060 individuals in the BC 1 F 1 population, and the male fertility phenotype was selected to select the phenotype and genotype inconsistent with each other, and selected according to different primers.
  • the number of individual plants was exchanged, and the localization interval was determined according to the decreasing trend, thereby locating the ZmIAP1 gene between the primer markers K204 (80.92 Mb) and K265 (81.93 Mb) located on chromosome 1.
  • K204 80.92 Mb
  • K265 81.93 Mb
  • chromosome 1 chromosome 1
  • the 140 kb range of the localization interval contains 4 genes, namely GRMZM2G434514, GRMZM2G133968, GRMZM2G434500, GRMZM2G133943.
  • the four genes were amplified and their sequence differences were compared. It was found that only GRMZM2G434500 gene exists between male fertile plants and male sterile plants. The difference in sequence.
  • the male sterile individual had a 1331 bp insertion at the 691th base of the third exon of the GRMZM2G434500 gene relative to the male fertile individual (Fig. 3). Therefore, it is speculated that the candidate gene GRMZM2G434500 is the ZmIAP1 gene to be cloned.
  • the genomic DNA of maize inbred line B73 was used as a template, and the primers were used for PCR amplification of F1/R1.
  • the sequence of the obtained PCR product was sequence 2 in the sequence listing, and sequence 2 was the sequence of ZmIAP1 gene in maize genome.
  • the total RNA of maize inbred line B73 was extracted and reverse transcribed into cDNA.
  • the primers were used for PCR amplification of F2/R2.
  • the sequence of the obtained PCR product was sequence 3 in the sequence listing, and sequence 3 was the cDNA sequence of ZmIAP1 gene.
  • the 249-1997 is the CDS. Both Sequence 2 and Sequence 3 encode the ZmIAP1 protein shown in SEQ ID NO: 1 in the Sequence Listing.
  • P1-F is located 3323 bp upstream of the ATG of the ZmIAP1 gene
  • P1-R is located 1404 bp downstream of the stop codon of ZmIAP1 gene.
  • the genomic DNA of maize inbred line B73 was used as a template, and the primers were used for PCR amplification of P1-F/P1-R.
  • the amplified PCR products were detected as single bands by 1.0% agarose gel electrophoresis (Fig. 4). .
  • the target strip gel was further recovered and sequenced, and the sequence was shown as shown in SEQ ID NO: 4 in the Sequence Listing.
  • the PCR product (SEQ ID NO: 4) was digested with Kpn I and Hind III, and the digested product was detected by 1.0% gel electrophoresis. As shown in Fig. 5, two bands were obtained, which were 5.2 kb and 1.3 kb in length, respectively. Left and right, the target band of 5.2 kb (upstream 1919 bp + 1919 bp of ZmIAP1 gene + 1358 bp downstream, ie, position 1405-6602 of sequence 4 in the sequence listing) was recovered and ligated to the large fragment of pCAMBIA3300 which was digested by the same enzyme. Identification and sequencing identified, constructed into a complementary vector p3300-ZmIAP1.
  • the genetic transformation of the recombinant expression vector p3300-ZmIAP1 to the maize hybrid Hi II was completed by Tianjin Jinuowo Biotechnology Co., Ltd.
  • the specific transformation method was conventional Agrobacterium-mediated genetic transformation of maize immature embryos.
  • the transfer vector p3300-ZmIAP1 T 0 of transgenic plants formulated into groups.
  • the normal ZmIAP1 gene on the maize genome is represented by genotype AA
  • the mutated zmiap1 gene is represented by genotype aa.
  • the maize receptor HiII is a male fertile material, so the genotype of the ZmIAP1 gene in HiII is AA, and the genotype in maize mutant ms*-6044 is aa.
  • the genotype of the transgenic negative or non-transgenic plants is represented by bb
  • the transgenic positive plants ie, the complementary vector fragment containing the ZmIAP1 gene has been integrated into the maize genome
  • B genotype of the transgenic hybrid plant
  • the genotype of the transgenic homozygous plant is BB.
  • the preparation of the transgenic progeny population (A in Figure 6) firstly uses the T 0 generation transgenic positive plant (AABb) as the male parent, and the maize mutant ms*-6044 (aabb) as the female parent, and the F 1 is combined.
  • the F 1 population contains two genotypes, AaBb and Aabb.
  • the genomic DNA of each individual strain of F 1 was extracted to determine whether it was transgenic positive (B).
  • the transgenic positive plants, ie, the AaBb genotype were selected as the male parent, and the maize mutant ms*-6044 (aabb) was used as the female parent to re-hybridize.
  • the progeny produced four genotypes: AaBb, Aabb, aabBb, and aabb.
  • the genotype of the aaBb genotype in the maize genome was aa in the location of chromosome 1 (the method was determined by linking polymorphic molecular markers on both sides of the localization interval, ie K10 and localization interval at the right end of the localization interval).
  • K57 polymorphic molecular markers on both sides of the localization interval
  • the band band type is the same as that of HiII. It is considered that this segment is from HiII, and the band band type obtained is the same as that of ms*-6044, and this segment is considered to be from ms*- 6044.
  • the molecular markers on both sides are also the band type of ms*-6044 material, and it is determined that this genomic DNA is from the mutant ms*-6044, if The genomic DNA double strands are from ms*-6044, then the genotype of the target gene in this interval is aa), so the corresponding phenotype should be male sterility, but the maize genome is due to transgenic positive (Bb)
  • the ZmIAP1 gene is integrated, and the phenotype of the aaBb genotype should be male fertile.
  • the method for determining whether the transgene is positive is as follows: genomic DNA is extracted from a single plant in the transgenic progeny population, and the target band of 1396 bp is amplified by using P2-F and P2-R as primers. Transgenic positive, no such band is negative for transgene. At the same time, the p3300-ZmIAP1 plasmid before transformation was used as a template for amplification, and it was used as a positive control.
  • P2-F 5'-CTCCACCATGTTATCACATCAATCC-3' (on the CaMV35S promoter of the promoter gene on the pCAMBIA3300 vector)
  • P2-R 5'-CGTCTTTGTCTTTCGCGTAGC-3' (located on the insert between the p1-3300-ZmIAP1 cleavage site Kpn I and Hind III, ie, the reverse complement of position 2185-2205 of SEQ ID NO: 4).
  • Lanes 3, 4, and 5 of C in Fig. 6 are plants identified as genotype-positive genotype aaBb, and lane 1 is a result of using the p3300-ZmIAP1 plasmid before transformation as a template, and is a positive control.
  • the plant phenotype is male fertile, specifically identified by the following two methods: 1. Observe whether the anther of the tassel at the flowering stage is externally attached, and the male sterility is not externally hanged, and the male is fertile. 2. The inside of the anther was stained with 1% I 2 -KI solution. No black round full and mature pollen grains were observed for male sterility. It was observed that the black round full and mature pollen grains were male fertile.
  • the four transformation events detected by the present invention have a total of 63 aaBb genotypes, and the phenotypes are male fertile (B and D in Fig. 6).
  • the figure shows that the anthers are full and externally attached, and the anthers are 1% I 2 in the anther.
  • - KI staining all pollen showed a black round fullness, identified as viable pollen), while the 33 aabb genotypes isolated were phenotypically male sterile (flowering period anthers dry and not hanging, anthers There was no viable pollen identified by 1% I 2 -KI staining).
  • the transformed ZmIAP1 gene can complement the male fertility phenotype of the homozygous mutation of ms*-6044.
  • the inventors of the present invention also designed experiments to edit the ZmIAP1 gene in plants with normal male fertility in maize.
  • the genome sequence of the ZmIAP1 gene in the maize recipient plant was subjected to site-by-point editing using the CRISPR-Cas9 (Clustered regular interspaced short palindromic repeats associated 9) gene editing system.
  • the CRISPR-Cas9 technology cleaves DNA at a specific locus on the genome, and the repair of the DNA strand by the organism cannot guarantee 100% correct characteristics every time. The rejoined DNA strand will differ in sequence from before being cut. Thus, the sequence of the gene changes, and the encoded protein also changes.
  • the specific sequence containing the SmaI restriction site in the second exon of the ZmIAP1 gene was selected as the sgRNA (single guide RNA) target sequence (Fig. 7 A).
  • sgRNA single guide RNA
  • pBUN411 vector herbicide resistance
  • ZmIAP1 gene knockout vector ligated into the pBUN411 vector (herbicide resistance), constructed into a ZmIAP1 gene knockout vector, and transformed the maize receptor HiII by Agrobacterium-mediated method.
  • This part of the vector construction and corn genetic transformation work was commissioned by Tianjin Genovo Biotechnology Co., Ltd.
  • the specific operations are as follows:
  • the target site sequence contains the Sma I restriction recognition sequence (the sequence underlined in italics), it can be cleaved by the Sma I restriction enzyme.
  • the target sequence region is recognized and cleaved by CRISPR-Cas9 nuclease. After self-repair, if a mutation occurs, the Sma I restriction recognition sequence is destroyed and will not be cleaved by the restriction endonuclease SmaI; if no mutation occurs, it will be Restriction enzyme Sma I cleavage.
  • the pBUN411 vector was digested with restriction endonuclease BsaI, and a carrier skeleton of about 12.4 kb was recovered and designated as BUN411.
  • ZmIAP1F and ZmIAP1R were annealed to form a double-stranded DNA having a sticky end, which was ligated with the gum recovery product BUN411 in the step (2) to obtain a recombinant plasmid pBUN411-ZmIAP1.
  • the structure of the recombinant plasmid pBUN411-ZmIAP1 was described as follows: after the fragment (about 1.1 kb) between the recognition sequences of the two restriction endonucleases BsaI of the pBUN411 plasmid was replaced with the DNA fragment "5'-GAACGCGCGGG CCCGGG TGC-3'" The resulting recombinant plasmid.
  • the recombinant plasmid pBUN411-ZmIAP1 was introduced into the maize variety HiII by Agrobacterium transformation.
  • HiII immature embryos and callus were used as transforming receptors, and after transformation, tissue culture was carried out to obtain whole regenerated plants.
  • the genomic DNA of the transgenic plant was extracted, and T3-F/T3-R was amplified by a specific primer capable of amplifying the sequence containing the target site, and the PCR product was digested with SmaI. .
  • T3-F 5'-CGCCCCTGGTGTCGCAGTACA-3' (positions 484-504 of sequence 2);
  • T3-R 5'-CGCCGACAGGATCACCTCGTTC-3' (reverse complement of positions 1267-1288 of SEQ ID NO: 2).
  • T3-F is located 231 bp upstream of the Sma I restriction site
  • T3-R is located 574 bp downstream of the Sma I restriction site.
  • the amplified PCR products were digested with Sma I and detected by 1.0% agarose gel electrophoresis. If the PCR product can be digested by Sma I, it means that the site does not mutate, which is called transgenic negative; if the PCR product can not be digested by Sma I, the sequence has changed, and the ZmIAP1 gene has been successfully edited by the site. It is called positive for transgene (B in Figure 7). The PCR products that could not be cleaved by Sma I were sequenced. One of them was used as an example. Compared with the wild type, the transgenic plants were deleted by 7 bp on the second exon of the ZmIAP1 gene, resulting in amino acid frameshift and translation. Early termination (C in Figure 7).
  • transgenic positive plants were examined for fertility by the method of Example 2.
  • a transgenic negative control was set, a control of pBUN411 empty vector was introduced into maize variety HiII, and a non-transgenic maize variety HiII wild type control was also introduced.
  • the number of tested plants per experimental material is not less than 80 strains.
  • the cloned ZmIAP1 gene is a gene for controlling male fertility in maize by map cloning, gene complementation experiments and knockout experiments, and the mutation can lead to complete male sterility. And there is no obvious negative impact on other traits, which can be utilized in the corn seed production process.
  • the present invention utilizes the strategy of map cloning to control the gene of this mutant trait by using the maize male sterile mutant ms*-6044 with the maize inbred line B73 and/or the inbred line Zheng 58 with the BC 1 F 1 population. Positioned on the maize chromosome 80 between 80.96cM and 81.1cM, the published B73 genome sequencing results as a reference physical distance of about 140kb, including a total of four genes. The sequence with the gene number GRMZM2G434500 differed between the mutant and the maize inbred line B73/Zheng 58. The mutant had a 1.3 kb insertion, and B73 and Zheng 58 did not have this insertion.
  • the gene-encoded protein could be translated correctly.
  • This gene was named ZmIAP1.
  • the ZmIAP1 gene was complementarily expressed in the above-mentioned maize male sterile mutant by transgenic technology, and its male fertility can be restored. Knockout of the ZmIAP1 gene of the male fertile material HiII of the maize using the CRISPR-Cas9 gene editing technique can cause the male fertility of Hi II to be lost and unable to produce active pollen.
  • the ZmIAP1 gene of the present invention is capable of controlling male fertility in maize, that is, homozygous mutation or deletion of the gene enables male male sterility, and normal expression of the ZmIAP1 gene in the mutant or deleted material of the gene can restore male fertility.
  • the invention provides a new genetic resource for the male fertility research of corn, which can play an important role in the application of the nuclear male fertility gene to create a sterile line and a maintainer line in the field of corn seed production.

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Abstract

一种与植物雄性育性相关的蛋白及其编码基因与应用。该蛋白是如下(a)或(b)：(a)由序列表中序列1所示的氨基酸序列组成的蛋白质；(b)将序列1的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加，且与植物雄性育性相关的由序列1衍生的蛋白质。

Description

一种与植物雄性育性相关的蛋白及其编码基因与应用 技术领域
本发明涉及植物基因工程领域，特别涉及一种与植物雄性育性相关的蛋白及其编码基因与应用。
背景技术
玉米是一种杂种优势十分明显的作物，生产上普遍使用杂交种，因此每年都需要制种。在制种过程中，由于玉米是异花授粉作物，为了保证种子纯度，制种所用的母本需要去雄。如果母本自身雄性不育，则无需去雄，既节省人力成本又避免了由于去雄不彻底所带来的制种纯度降低。玉米的雄性不育，包括质核互作的雄性不育和细胞核基因雄性不育。在玉米雄性组织的生长发育直至产生有活性花粉的过程中，任何一个细胞核雄性育性基因发生突变，都有可能导致雄性不育。这些细胞核雄性育性基因突变导致的雄性不育可分为显性核不育和隐性核不育，且以隐性核不育居多。
目前克隆的玉米细胞核雄性育性基因包括MS8(Wang et al.,2013,Plant Reprod.26:329-338)、MS9(专利号：US20150191743)、MS22/MSCA1(Chaubal et al.,2003,Planta 216:778-788，专利号：US20090038028、EP2631243A2)、MS26(专利号：US7098388)、MS32(Moon et al.,2013,Plant Journal76:592-602)、MS45(Albertsen et al.,1993,Proc Annu Corn Sorghum Ind Res Conf 48:224-233)、AM1(Pawlowski et al.,2009,Proc Natl Acad Sci USA 106:3603-3608)、MAC1(Wang et al.,2012,Development 139:2594-2603)、OCL4(Vanessa et al.,2009,Plant Journal 59:883-894)。其中有些基因由于其突变导致雄性不育严格彻底且对玉米其他性状没有明显的负面影响，可以在玉米制种过程中得以利用。例如MS22、MS26、MS45已获得美国或欧洲专利保护。在我国，以MS1、MS7、MS30为基础的不育系保持和繁殖方法专利也已公开(专利号：CN104823840A、CN105039316A、CN105018475A)。
发明公开
本发明的目的是提供一种与植物雄性育性相关的蛋白及其编码基因与应用。
本发明所提供的蛋白质，名称为ZmIAP1，来源于玉米属的玉米(Zea mays L.)，是如下(a)或(b)：
(a)由序列表中序列1所示的氨基酸序列组成的蛋白质；
(b)将序列1的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加，且与植物雄性育性相关的由序列1衍生的蛋白质。
序列表序列1为ZmIAP1的氨基酸序列，包括582个氨基酸，在该蛋白质序列中，疏水氨基酸占263个，亲水氨基酸占266个，碱性氨基酸占70个，酸性氨基酸占52个，该蛋白质的分子量为63.43KD，等电点为8.73。
为了便于上述(a)中所示蛋白质的纯化，可在由序列表中序列1的氨基酸残基序列组成的蛋白质的氨基末端或羧基末端连接上如下表所示的标签。
表：标签的序列
标签 残基 序列
Poly-Arg 5-6(通常为5个) RRRRR
Poly-His 2-10(通常为6个) HHHHHH
FLAG 8 DYKDDDDK
Strep-tag II 8 WSHPQFEK
c-myc 10 EQKLISEEDL
上述(b)中的蛋白质可人工合成，也可先合成其编码基因，再进行生物表达得到。上述(b)中的蛋白质的编码基因可通过将序列表中序列2所示的DNA序列中缺失一个或几个氨基酸残基的密码子，和/或进行一个或几个碱基对的错义突变。
编码所述蛋白质的核酸分子也属于本发明的保护范围。
所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA、hnRNA或tRNA等。
在本发明的一个实施例中，所述核酸分子具体为编码所述蛋白质的基因(命名为ZmIAP1)，所述基因具体可为如下1)-6)中任一的DNA分子：
所述基因为如下1)-6)中任一的DNA分子：
1)序列表中序列2所示的DNA分子；
2)序列表中序列3所示的DNA分子；
3)序列表中序列3的第249-1997位所示的DNA分子；
4)序列表中序列4所示的DNA分子；
5)在严格条件下与1)-4)中任一限定的DNA分子杂交且编码与植物雄性育性相关的由序列1衍生的蛋白质的DNA分子；
6)与1)-5)中任一限定的DNA序列具有90％以上同一性，且编码与植物雄性育性相关的由序列1衍生的蛋白质的DNA分子。
其中，序列2为ZmIAP1基因在玉米基因组中的序列；序列3为ZmIAP1基因的cDNA序列，第249-1997位为CDS。
含有上述核酸分子的重组载体、表达盒、转基因细胞系、重组微生物或转基因植物也属于本发明的保护范围。
所述重组载体可为重组表达载体，也可为重组克隆载体。
所述重组表达载体可用现有的植物表达载体构建。所述植物表达载体包括双元农杆菌载体和可用于植物微弹轰击的载体等，如pGreen0029、pCAMBIA3301、pCAMBIA1300、pBI121、pBin19、pCAMBIA2301、pCAMBIA1301-UbiN或其它衍生植物表达载体。所述植物表达载体还可包含外源基因的3’端非翻译区域，即包含聚腺苷酸信号和任何其它参与mRNA加工或基因表达的DNA片段。所述聚腺苷酸 信号可引导聚腺苷酸加入到mRNA前体的3’端。使用所述基因构建重组表达载体时，在其转录起始核苷酸前可加上任何一种增强型、组成型、组织特异型或诱导型启动子，例如花椰菜花叶病毒(CaMV)35S启动子、泛素基因Ubiquitin启动子(pUbi)、胁迫诱导型启动子rd29A等，它们可单独使用或与其它的植物启动子结合使用；此外，使用本发明的基因构建重组表达载体时，还可使用增强子，包括翻译增强子或转录增强子，这些增强子区域可以是ATG起始密码子或邻接区域起始密码子等，但必需与编码序列的阅读框相同，以保证整个序列的正确翻译。所述翻译控制信号和起始密码子的来源是广泛的，可以是天然的，也可以是合成的。翻译起始区域可以来自转录起始区域或结构基因。为了便于对转基因植物细胞或植物进行鉴定及筛选，可对所用重组表达载体进行加工，如加入可在植物中表达的编码可产生颜色变化的酶或发光化合物的基因、具有抗性的抗生素标记物或是抗化学试剂标记基因等。也可不加任何选择性标记基因，直接以逆境筛选转化植株。
在本发明中，所述重组表达载体为将ZmIAP1基因插入到pCAMBIA3300载体的多克隆位点(如Kpn Ⅰ和Hind Ⅲ)处后得到的重组质粒。更加具体的，为将pCAMBIA3300载体的酶切位点Kpn Ⅰ和Hind Ⅲ之间的小片段替换为序列表中序列4的第1406-6601位所示DNA片段后得到的重组质粒(命名为p3300-ZmIAP1)。
所述表达盒由能够启动所述基因表达的启动子，所述基因，以及转录终止序列组成。
所述转基因细胞系为转入所述基因的非繁殖材料。
所述转基因植物具体可为转入所述基因的种子、愈伤组织、完整植株或细胞。
所述蛋白质或所述核酸分子或所述重组载体、表达盒、转基因细胞系或重组微生物在如下任一中的应用也属于本发明的保护范围：
(a)植物育种和/或制种；
(b)调控植物雄性育性。
所述(a)中的应用可以是转基因的，也可以是非转基因的。其中“非转基因”的应用，可为：利用所述ZmIAP1基因培育不育系。不育系的雄性不育性状是由于植物体内表达有功能的所述ZmIAP1蛋白的能力丧失或降低引起的，所述ZmIAP1基因为纯合态aa。“转基因”的应用可为：将保持系和不育系合二为一成为两用系，需要以不育系为基础导入所述正常有功能的ZmIAP1基因，在后代中可分离出不育系和两用系。所述应用可以是对所述ZmIAP1基因(正常有功能)的利用，也可以是所述ZmIAP1基因(降低或丧失功能)的利用。
所述(b)中，所述调控植物雄性育性可为提高植物雄性育性或降低植物雄性育性。
本发明还提供了培育转基因植物的方法。
本发明所提供培育转基因植物的方法，可为如下(A)或(B)：
(A)培育雄性可育转基因植物的方法，包括如下步骤：
(a1)向雄性不育的受体植物中导入ZmIAP1蛋白的编码基因，得到表达所述编码基因的转基因植物；所述受体植物的雄性不育性状是由于所述受体植物表达有功能的所述ZmIAP1蛋白的能力丧失或降低引起的；
(a2)从步骤(a1)所得转基因植物中得到雄性可育的转基因植物；
(B)培育雄性不育转基因植物的方法，包括如下步骤：
(b1)抑制雄性可育的受体植物中ZmIAP1蛋白的表达，得到转基因植物；
(b2)从步骤(b1)所得转基因植物中得到雄性不育的转基因植物。
在所述方法(A)的步骤(a1)中，所述编码基因可通过以上重组表达载体p3300-ZmIAP1导入所述受体植物；
在所述方法(B)步骤(b1)中，是通过如下实现抑制所述受体植物中所述编码基因的表达的：采用CRISPR/Cas9核酸酶对所述受体植物中编码ZmIAP1蛋白的基因组DNA序列进行特异性剪切，使所述受体植物丧失或降低表达有功能的ZmIAP1蛋白的能力。
其中，所述CRISPR/Cas9核酸酶对所述受体植物中编码ZmIAP1蛋白的基因组DNA序列进行特异性剪切时的靶标片段为所述受体植物中编码ZmIAP1蛋白的基因组DNA序列中符合5’-NX-NGG-3’或5’-CCN-NX-3’序列排列规则的片段；N表示A、G、C和T中的任一种，14≤X≤30，且X为整数(如X为20)，NX表示X个连续的脱氧核糖核苷酸。更加具体的，所述靶标片段为所述受体植物中编码ZmIAP1蛋白的基因组DNA序列中的“5’-GAACGCGCGGGCCCGGGTGCTGG-3’(即序列2的第701-723位)”。
在本发明中，所述雄性不育为“盛花期花药不外挂”和/或“花药内没有经1％I2-KI染色鉴定为有活力的花粉”。
在本发明中，所述植物既可为单子叶植物，也可为双子叶植物。其中，所述单子叶植物如禾本科植物，具体如玉米。
在培育雄性可育转基因植物时，可将玉米突变体ms*-6044作为所述受体植物，获得相应的雄性可育转基因玉米；当然也可以像本发明的一个实施例一样，将含有所述ZmIAP1基因的载体转化到玉米品种HiII中，然后再将转基因阳性植株与玉米突变体ms*-6044杂交两次，选择定位区间内基因组背景为ms*-6044且转基因为阳性的植株，即为相应的雄性可育转基因玉米。
在本发明的另一个实施例中，培育雄性不育转基因植物时采用的所述受体植物具体为玉米品种HiⅡ。
由所述方法培育获得的转基因植物也属于本发明的保护范围。所述转基因植物具体可为转入所述基因的种子、愈伤组织、完整植株或细胞。
附图说明
图1为玉米突变体ms*-6044与野生型的表型对比图。其中，A、C、E、G为野生型，B、D、F、H为ms*-6044突变体。A和B，植株形态；C和D，雄穗花药外挂情况；E和F，花药形态；G和H，花粉1％I2-KI染色。
图2为ZmIAP1基因图位克隆图。
图3为ZmIAP1基因结构及其在玉米突变体ms*-6044中插入位点示意图。
图4为构建互补载体PCR产物的琼脂糖凝胶电泳图。
图5为PCR产物经KpnⅠ和HindⅢ双酶切后的电泳图。
图6为ZmIAP1基因遗传互补验证图。其中，A：转基因后代群体配制示意图；B：基因型为aaBb植株的雄性可育表型；C：转基因检测(3、4、5是基因型为aaBb的植株，经鉴定为转基因阳性，1是对照，即转化玉米受体HiII时用的p3300-ZmIAP1质粒作为模板扩增所得的阳性对照)；D：基因型为aaBb植株的花粉1％I2-KI染色。
图7为ZmIAP1基因敲除验证图。其中，A：基因敲除靶点序列；B：转基因植株的PCR酶切验证，1-4为转基因植株，CK为野生型，PCR产物大小为805bp，被SmaⅠ酶切后产生大小为574bp和231bp的两个片段；C：野生型与转基因植株PCR产物测序和编码氨基酸序列比较；D：转基因阳性植株盛花期雄穗；E：转基因阳性植株花药1％I2-KI染色；F：转基因阴性植株盛花期雄穗；G：转基因阴性植株花粉1％I2-KI染色。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
玉米突变体ms*-6044：由Maize Genetics Cooperation Stock Center保存，Co-op ID:104G；Description:104G ms*-6044。该玉米突变体在该保藏中心的具体网页链接如下：http://www.maizegdb.org/data_center/stock？id＝130311。
pBUN411载体：在文献“Hui-Li Xing,Li Dong,Zhi-Ping Wang,Hai-Yan Zhang,Chun-Yan Han,Bing Liu,Xue-Chen Wang,Qi-Jun Chen.BMC plant biology.14:327-338(2014)”中公开过，公众可从中国科学院遗传与发育生物学研究所获得，仅可用于重复本发明实验。该质粒可同时用于转录向导RNA和表达Cas9蛋白。
实施例1、玉米雄性育性基因ZmIAP1的图位克隆
一、玉米突变体ms*-6044的表型
与正常植株相比，玉米突变体ms*-6044在植株整体形态方面没有异常(图1中A和B)，但是其雄穗的花药不外挂(图1中C和D)，经解剖其花药干瘪瘦小(图1中E和F)，花药内部用1％I2-KI溶液染色未观察到饱满成熟的花粉粒(图1中G和H)，表现为彻底的雄性不育现象。该现象在北京和海南连续4年观察表型一致，不育特性稳定，且在大田条件下肉眼即可区分是否为雄性不 育。
二、遗传定位群体的构建
以玉米突变体ms*-6044为母本，以玉米自交系B73或郑58为父本组配F1，F1田间表型均为雄性可育。而后以玉米突变体ms*-6044为母本，以F1为父本构建回交群体(BC1F1)，BC1F1群体在田间可分为雄性可育个体和雄性不育个体两种类型，且两种类型的比例经卡方检验符合1:1(表1)。由此推测玉米突变体ms*-6044的表型由隐性单基因控制，并将BC1F1群体做为遗传定位群体。
表1BC1F1群体不同表型个体的比例统计
Figure PCTCN2017073142-appb-000001
Χ0.05 2(1)＝3.84
三、ZmIAP1基因的定位
首先，以玉米突变体ms*-6044和玉米自交系B73、郑58的基因组DNA为模板，用玉米全基因组引物筛选在突变体和自交系之间有多态性的引物。然后，从BC1F1群体中选取雄性可育单株和雄性不育单株各10株，验证多态性引物是否与雄性育性性状连锁。筛选出连锁引物，用于对BC1F1群体的1060个单株测定基因型，从而结合雄性育性表型筛选出表型与基因型不符的为交换单株，并根据不同引物筛选出的交换单株个数的不同，依照减少趋势确定定位区间，由此将ZmIAP1基因定位在位于一号染色体的引物标记K204(80.92Mb)和K265(81.93Mb)之间。在K204和K265之间继续开发多态性分子标记，并用于检测BC1F1群体的所有单株，最终将ZmIAP1基因定位在K168(80.96Mb)和K163(81.1Mb)之间，参考已公布的玉米自交系B73基因组测序结果，物理距离约为140kb(图2)。其中，用于基因定位的分子标记引物序列如表2所示。
表2用于基因定位的分子标记引物序列
Figure PCTCN2017073142-appb-000002
Figure PCTCN2017073142-appb-000003
四、ZmIAP1基因的克隆
参考玉米基因组测序信息，定位区间的140kb范围内包含4个基因，分别是GRMZM2G434514、GRMZM2G133968、GRMZM2G434500、GRMZM2G133943。以雄性可育单株和雄性不育单株的基因组DNA为模板，扩增这4个基因并比较其序列差异，发现只有GRMZM2G434500基因在雄性可育单株和雄性不育单株之间存在DNA序列上的差异。相对于雄性可育单株，雄性不育单株在GRMZM2G434500基因的第三外显子的第691位碱基处，存在一个1331bp的插入(图3)。因此推测候选基因GRMZM2G434500即为要克隆的ZmIAP1基因。
以玉米自交系B73的基因组DNA为模板，采用引物对F1/R1进行PCR扩增，所得PCR产物的序列为序列表中序列2，序列2即为ZmIAP1基因在玉米基因组中的序列。提取玉米自交系B73的总RNA，反转录为cDNA，采用引物对F2/R2进行PCR扩增，所得PCR产物的序列为序列表中序列3，序列3即为ZmIAP1基因的cDNA序列，其中第249-1997位为CDS。序列2和序列3均编码序列表中序列1所示的ZmIAP1蛋白。
Figure PCTCN2017073142-appb-000004
实施例2、玉米雄性育性基因ZmIAP1的功能验证(互补实验)
一、互补载体的构建
设计针对ZmIAP1基因的基因组序列的特异引物。具体序列为：
Figure PCTCN2017073142-appb-000005
其中，P1-F位于ZmIAP1基因起始密码子ATG上游3323bp，P1-R位于ZmIAP1基因终止密码子下游1404bp。
以玉米自交系B73的基因组DNA为模板，采用引物对P1-F/P1-R进行PCR扩增，扩增获得的PCR产物经1.0％琼脂糖凝胶电泳检测为单一条带(图4)。进一步将目标条带胶回收测序，显示其序列如序列表中序列4所示。
将此PCR产物(序列4)用Kpn Ⅰ和Hind Ⅲ双酶切，酶切产物经过1.0％的凝胶电泳检测，如图5所示，得到两条条带，长度分别为5.2kb和1.3kb左右，将5.2kb(上游1919bp+ZmIAP1基因1922bp+下游1358bp，即序列表中序列4的第1405-6602位)的目的条带回收，与经同样双酶切的pCAMBIA3300的骨架大片段相连，经酶切和测序鉴定，构建成为互补载体p3300-ZmIAP1。
重组表达载体p3300-ZmIAP1的结构描述：将pCAMBIA3300载体的酶切位点Kpn Ⅰ和Hind Ⅲ之间的小片段替换为序列表中序列4的第1406-6601位所示DNA片段后得到的重组质粒。
二、玉米遗传转化实验
由天津吉诺沃生物科技有限公司完成重组表达载体p3300-ZmIAP1向玉米杂交种Hi Ⅱ的遗传转化，具体的转化方法是常规的农杆菌介导的玉米幼胚的遗传转化。
三、转基因后代群体配制
为鉴定ZmIAP1基因的功能，将转p3300-ZmIAP1载体的T0代转基因植株配制成群体。
在此，将玉米基因组上正常的ZmIAP1基因以基因型AA表示，突变的zmiap1基因以基因型aa表示。玉米受体HiⅡ是雄性育性正常的材料，因此ZmIAP1基因在HiⅡ中的基因型是AA，而在玉米突变体ms*-6044中的基因型是aa。同时，将转基因阴性植株或非转基因植株的基因型以bb表示，将转基因阳性植株(即含有ZmIAP1基因的互补载体片段已整合到玉米基因组上)以B表示，那么转基因杂合植株的基因型是Bb，转基因纯合植株的基因型是BB。
转基因后代群体的配制(图6中A)，首先以T0代转基因阳性植株(AABb)为父本，玉米突变体ms*-6044(aabb)为母本，组配F1。F1群体包含两种基因型，即AaBb和Aabb。提取F1各个单株的基因组DNA鉴定其是否为转基因阳性(B)。选择转基因阳性植株即AaBb基因型单株为父本，以玉米突变体ms*-6044(aabb)为母本，再次杂交，其后代共产生四种基因型：AaBb、Aabb、aaBb、aabb。其中aaBb基因型的单株，在玉米基因组上一号染色体定位区间内的基因型为aa(确定方法是通过在定位区间两侧的连锁的多态性分子标记，即定位区间右端K10和定位区间左端K57，来扩增单株的基因组DNA，得到的条带带型与HiII一样，就认为这一段来自HiII，得到的条带带型与ms*-6044一样，就认为这一段来自ms*-6044。我们将ZmIAP1基因两侧的分子标记都用来做这个检测，两侧的分子标记都同时是ms*-6044材料的带型，就确定这一段基因组DNA来自突变体ms*-6044，如果基因组DNA双链都是来自ms*-6044，那么在这一区间内的目的基因的基因型就是aa)，由此对应的表型应为雄性不育，然而由于转基因阳性(Bb)使得玉米基因组上整合了ZmIAP1基因，aaBb基因型的单株的表型实则应为雄性可育。
其中，确定是否为转基因阳性的方法具体如下：将转基因后代群体中的单株提取基因组DNA，以此为模板，以P2-F和P2-R为引物，扩增获得大小1396bp的目的条带为转基因阳性，无此条带为转基因阴性。同时以转化前的p3300-ZmIAP1质粒为模板进行扩增，做为阳性对照。
P2-F：5’-CTCCACCATGTTATCACATCAATCC-3’(位于pCAMBIA3300载体上启动标记基因的CaMV35S启动子上)
P2-R：5’-CGTCTTTGTCTTTCGCGTAGC-3’(位于p3300-ZmIAP1的酶切位点Kpn Ⅰ和Hind Ⅲ之间的插入片段上，即序列4的第2185-2205位的反向互补序列)。
图6中C的泳道3、4、5为鉴定为转基因阳性的基因型为aaBb的植株，泳道1是以转化前的p3300-ZmIAP1质粒为模板的结果，是阳性对照。
其中，植株表型是否为雄性可育，具体通过如下两种方法鉴定：1、观察盛花期雄穗的花药是否外挂，不外挂为雄性不育，外挂为雄性可育。2、花药内部用1％I2-KI溶液染色，未观察到黑色圆形饱满成熟的花粉粒为雄性不育，观察到黑色圆形饱满成熟的花粉粒为雄性可育。
最终，本发明检测的4个转化事件共63个aaBb基因型单株其表型均为雄性可育(图6中B和D，图中显示盛花期花药饱满且外挂，花药内经1％I2-KI染色全部花粉均呈现黑色圆形饱满状，鉴定为有活力的花粉)，同时分离出的33个aabb基因型单株的表型均为雄性不育(盛花期花药干瘪且不外挂，花药内没有经1％I2-KI染色鉴定为有活力的花粉)。由此得出，转化的ZmIAP1基因可以互补ms*-6044纯合突变的雄性育性表型。
实施例3、玉米雄性育性基因ZmIAP1的功能验证(敲除实验)
除了上述互补实验，本发明的发明人还设计实验将玉米雄性育性正常的植株中的ZmIAP1基因进行编辑。具体是采用CRISPR-Cas9(Clustered regularly interspaced short palindromic repeats associated 9)基因编辑系统，对玉米受体植株中ZmIAP1基因的基因组序列进行定点编辑。CRISPR-Cas9技术可针对基因组上特定位点DNA进行切割，利用生物体对DNA链的修复无法每次都保证100％正确的特点，重新连接的DNA链在序列上与未被切割前会产生差异，从而使得基因序列发生变化，其编码的蛋白质也随之变化。
具体到本实验中，根据CRISPR-Cas9系统的特点，选取ZmIAP1基因的第二外显子上含有SmaⅠ酶切位点的特定序列做为sgRNA(single guide RNA)靶点序列(图7中A)，并将其连入到pBUN411载体(除草剂抗性)，构建成为ZmIAP1基因敲除载体，并采用农杆菌介导的方法转化玉米受体HiⅡ。该部分的载体构建和玉米遗传转化工作是委托天津吉诺沃生物科技有限公司完成的。具体操作如下：
(1)设计靶位点
5’-GAACGCGCGGGCCCGGGTGCTGG-3’(即序列2的第701-723位)。
由于该靶位点序列中含有Sma Ⅰ酶切识别序列(斜体下划线的序列)，可以被Sma Ⅰ限制性内切酶切割。靶序列区域被CRISPR-Cas9核酸酶识别切割，自身修复后，如果发生了突变，则Sma Ⅰ酶切识别序列被破坏，将不能被限制性内切酶SmaⅠ切割；如果没有发生突变，将可以被限制性内切酶Sma Ⅰ切割。
(2)用限制性内切酶BsaI酶切pBUN411载体，回收约12.4kb的载体骨架，命名为BUN411。
(3)根据步骤(1)设计的靶位点序列，合成如下带有粘性末端(下划线部分)的引物：
Figure PCTCN2017073142-appb-000006
(4)将ZmIAP1F和ZmIAP1R进行退火，形成有粘性末端的双链DNA，将其和步骤(2)中的胶回收产物BUN411连接，得到重组质粒pBUN411-ZmIAP1。重组质粒pBUN411-ZmIAP1的结构描述为：将pBUN411质粒的两个限制性内切酶BsaI的识别序列之间的片段(约1.1kb)替换为DNA片段“5’-GAACGCGCGGGCCCGGGTGC-3’”后所得的重组质粒。
(5)将构建好的重组质粒pBUN411-ZmIAP1通过农杆菌转化的方法导入玉米品种HiII。以HiII未成熟胚及愈伤组织为转化受体，转化后经过组织培养获得完整再生植株。
经过转化pBUN411-ZmIAP1获得完整的转基因植株后，提取转基因植株基因组DNA，用能够扩增含有靶位点序列的特异引物对T3-F/T3-R进行PCR扩增，PCR产物用SmaⅠ单酶切。
T3-F：5'-CGCCCCTGGTGTCGCAGTACA-3'(序列2的第484-504位)；
T3-R：5'-CGCCGACAGGATCACCTCGTTC-3'(序列2的第1267-1288位的反向互补序列)。
其中，T3-F位于Sma Ⅰ酶切位点上游231bp，T3-R位于Sma Ⅰ酶切位点下游574bp。
以转基因植株的基因组DNA为模板，扩增获得的PCR产物用Sma Ⅰ酶切后经1.0％琼脂糖凝胶电泳检测。如果PCR产物可以被Sma Ⅰ酶切，则说明该位点没有发生变异，称之为转基因阴性；如果PCR产物不能被Sma Ⅰ酶切，则说明该序列已经发生变化，ZmIAP1基因被定点编辑成功，称之为转基因阳性(图7中B)。将无法被Sma Ⅰ切开的PCR产物测序，以其中一株为例，发现其与野生型相比，转基因植株在ZmIAP1基因的第二个外显子上缺失7bp，导致氨基酸移码和翻译的提前终止(图7中C)。
参照实施例2中的方法观察检测转基因阳性植株的雄性是否可育。实验同时设置了转基因阴性的对照，向玉米品种HiII导入了pBUN411空载体的对照，以及未转基因的玉米品种HiII野生型对照。每种实验材料的供试植株数不少于 80株。
结果显示：在表型上，转基因阳性植株花药干瘪不外露，无可被1％I2-KI溶液染色的花粉，表现为彻底的雄性不育；而转基因阴性植株花药饱满且外挂，散出的花粉可以被1％I2-KI溶液染色，表现为雄性可育(图7中D、E、F和G)。而空载对照和野生型对照的花药表型及1％I2-KI溶液染色结果均与转基因阴性植株结果相似，无显著差异。由此得出，ZmIAP1基因被敲除会导致玉米雄性不育的表型。另外，转基因阳性植株除了雄性不育外，其他表型和性状与未转基因的对照玉米植株相比基本上没有差异。
综合以上各实施例的研究结果，可见：通过图位克隆、基因的互补实验和敲除实验，本发明克隆的ZmIAP1基因是控制玉米雄性育性的基因，其突变可导致彻底的雄性不育，且对其他性状没有明显的负面影响，可以在玉米制种过程中得以利用。
工业应用
本发明利用图位克隆的策略，用玉米雄性不育突变体ms*-6044与玉米自交系B73和/或自交系郑58组配BC1F1群体，将控制这一突变性状的基因定位到玉米一号染色体80.96cM到81.1cM之间，以公布的B73基因组测序结果为参考物理距离约为140kb，共包括四个基因。其中基因号为GRMZM2G434500的序列在突变体与玉米自交系B73/郑58之间存在差异，突变体有一个1.3kb的插入，B73和郑58没有这一插入，基因编码蛋白可以正确翻译，将此基因命名为ZmIAP1。用转基因技术将ZmIAP1基因在上述玉米雄性不育突变体中互补表达，可以恢复其雄性育性。用CRISPR-Cas9基因编辑技术将玉米雄性育性正常的材料HiⅡ的ZmIAP1基因敲除，可以使Hi Ⅱ的雄性育性丧失，不能产生有活性的花粉。
本发明的ZmIAP1基因在玉米中能够控制雄性育性，即该基因的纯合突变或缺失能够使玉米雄性不育，在该基因突变或缺失的材料中正常表达ZmIAP1基因能够恢复雄性育性。
本发明为玉米雄性育性研究提供了新的基因资源，其可在玉米制种领域利用细胞核雄性育性基因创制不育系和保持系的应用中发挥重要作用。

Claims (15)

  1. 蛋白质，是如下(a)或(b)：
    (a)由序列表中序列1所示的氨基酸序列组成的蛋白质；
    (b)将序列1的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加，且与植物雄性育性相关的由序列1衍生的蛋白质。
  2. 编码权利要求1所述蛋白质的核酸分子。
  3. 根据权利要求2所述的核酸分子，其特征在于：所述核酸分子为编码权利要求1所述蛋白质的基因，所述基因为如下1)-6)中任一的DNA分子：
    1)序列表中序列2所示的DNA分子；
    2)序列表中序列3所示的DNA分子；
    3)序列表中序列3的第249-1997位所示的DNA分子；
    4)序列表中序列4所示的DNA分子；
    5)在严格条件下与1)-4)中任一限定的DNA分子杂交且编码与植物雄性育性相关的由序列1衍生的蛋白质的DNA分子；
    6)与1)-5)中任一限定的DNA序列具有90％以上同一性，且编码与植物雄性育性相关的由序列1衍生的蛋白质的DNA分子。
  4. 含有权利要求2或3所述核酸分子的重组载体、表达盒、转基因细胞系、重组微生物或转基因植物。
  5. 根据权利要求4所述的重组载体，其特征在于：所述重组载体为重组表达载体或重组克隆载体。
  6. 权利要求1所述蛋白质或权利要求2或3所述核酸分子或权利要求4或5所述的重组载体、表达盒、转基因细胞系或重组微生物在如下任一中的应用：
    (a)植物育种和/或制种；
    (b)调控植物雄性育性。
  7. 根据权利要求6所述的应用，其特征在于：所述植物为单子叶植物或双子叶植物。
  8. 根据权利要求7所述的应用，其特征在于：所述单子叶植物为禾本科植物。
  9. 根据权利要求8所述的应用，其特征在于：所述禾本科植物为玉米。
  10. 培育转基因植物的方法，为如下(A)或(B)：
    (A)培育雄性可育转基因植物的方法，包括如下步骤：
    (a1)向雄性不育的受体植物中导入权利要求1所述蛋白质的编码基因，得到表达所述编码基因的转基因植物；所述受体植物的雄性不育性状是由于所述受体植物表达有功能的权利要求1所述蛋白质的能力丧失或降低引起的；
    (a2)从步骤(a1)所得转基因植物中得到雄性可育的转基因植物；
    (B)培育雄性不育转基因植物的方法，包括如下步骤：
    (b1)抑制雄性可育的受体植物中权利要求1所述蛋白质的表达，得到转基因植物；
    (b2)从步骤(b1)所得转基因植物中得到雄性不育的转基因植物。
  11. 根据权利要求10所述的方法，其特征在于：步骤(a1)中，所述编码基因是通过权利要求5中的所述重组表达载体导入所述受体植物的；
    步骤(b1)中，是通过如下实现抑制所述受体植物中所述编码基因的表达的：采用CRISPR/Cas9核酸酶对所述受体植物中编码权利要求1所述蛋白质的基因组DNA序列进行特异性剪切，使所述受体植物丧失或降低表达有功能的权利要求1所述蛋白质的能力。
  12. 根据权利要求10或11所述的方法，其特征在于：所述植物为单子叶植物或双子叶植物。
  13. 根据权利要求12所述的方法，其特征在于：所述单子叶植物为禾本科植物。
  14. 根据权利要求13所述的方法，其特征在于：所述禾本科植物为玉米。
  15. 由权利要求10-14中任一所述方法培育获得的转基因植物。
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