US20150315607A1 - Mutated nucleotide molecule, and transformed plant cells and plants comprising the same - Google Patents

Mutated nucleotide molecule, and transformed plant cells and plants comprising the same Download PDF

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US20150315607A1
US20150315607A1 US14/596,419 US201514596419A US2015315607A1 US 20150315607 A1 US20150315607 A1 US 20150315607A1 US 201514596419 A US201514596419 A US 201514596419A US 2015315607 A1 US2015315607 A1 US 2015315607A1
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bhlh142
seq
plant
male sterile
transcription factor
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Swee-Suak Ko
Min-Jeng Li
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Academia Sinica
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    • 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
    • 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

Definitions

  • Rice is one of the most important staple crops in the world, feeding almost half of the world's population, and it serves as a model for monocots, which include many important agronomic crops (e.g. wheat, maize, sorghum, millet). Food and Agriculture Organization (FAO) predicts that rice yield will have to be increased 50-70% by 2050 to meet the demands.
  • FEO Food and Agriculture Organization
  • Several approaches are currently adopted to increase rice yields, such as heterosis breeding, population improvement, wide hybridization, genetic engineering, and molecular breeding 1 .
  • hybrid rice is being considered the most promising one (15-20% increases in yield) 2 .
  • Crops produced from F1 hybrid seeds offer significant benefits in terms of yield improvement, agronomic performance and consistency of end-use quality. This is due to the ‘hybrid vigor’ generated by combining carefully selected parent lines. Hybrid crops are responsible for a dramatic increase in global crop yields in the past decades, and male sterility (MS) has played a significant role in this advancement. Male sterile traits can be divided into cytoplasmic male sterility (CMS), which is determined by cytoplasmic factors such as mitochondria, and genetic male sterility (GMS), which is determined by nuclear genes.
  • CMS cytoplasmic male sterility
  • GMS genetic male sterility
  • CMS has long been used in hybrid corn production, while both CMS and GMS are currently used for hybrid rice production 3 , due to the convenience of controlling sterility expression by manipulating the gene-cytoplasm combinations in any selected genotype. Most importantly, it evades the need for emasculation in cross-pollinated species, thus encouraging cross breeding and producing pure hybrid seeds under natural conditions.
  • commercial seed production must be simple and inexpensive, and the requirement for a maintainer line to produce the seed stocks of CMS line increases the production cost for this 3-line hybrid system.
  • GMS genetic MS
  • PGMS photoperiod- or temperature-inducible MS
  • LYP9 4 paternal line 93-11 to generate superhybrid rice
  • PA64S derived from a spontaneous PGMS japonica mutant NK58S (long day->13.5 h; Shi, 1985), is also a TGMS indica rice, whose MS is promoted by temperatures greater than 23.5° C., but recovers its fertility at temperatures between 21 ⁇ 23° C.
  • Recent mapping analyses demonstrate that the P/TGMS in these MS lines is regulated by a novel small RNA 5 .
  • CSA Carbon Starved Anther
  • the mutation on the R2R3 MYB transcription regulator defects pollen development 6 and further study shows that csa is a new photoperiod-sensitive mutant, exhibiting MS under short-day conditions but male fertility under long-day conditions 7 .
  • the molecular basis of its MS sensitivity to day length remains to be addressed.
  • Transgenic male sterility has been generated using a number of transgenes, but its application in commercial production of hybrid seeds is limited due to the lack of an efficient and economical means to maintain the MS lines, or the lack of suitable restorers 8 .
  • a reversible MS system has been demonstrated in transgenic Arabidopsis plants by manipulating a R2R3 MYB domain protein (AtMYB103) 8 . Blocking the function of AtMYB103 using an insertion mutant or an AtMYB103EAR chimeric repressor construct under the control of the AtMYB103 promoter resulted in complete MS without seed setting 8 .
  • a restorer containing the AtMYB103 gene driven by of a stronger anther-specific promoter was introduced into pollen donor plants and crossed into the MS transgenic plants for the repressor. The male fertility of F1 plants is restored.
  • the chimeric repressor and the restorer constitute a reversible MS system for hybrid seed production. The successful application of this system for large scale hybrid seed production depends on whether the MS female parent lines can be multiplied efficiently and economically.
  • an inducible promoter by chemicals or other factors e.g. photoperiod or temperature
  • Rice anthers are composed of four lobes attached to a central core by connective and vascular tissue.
  • microsporocytes form in the middle, surrounded by four anther wall layers: an epidermal outer layer, endothecium, middle layer, and tapetum 9 .
  • the tapetum is located in the innermost cell layer of the anther walls and plays an important role in supplying nutrients such as lipids, polysaccharides, proteins, and other nutrients for pollen development 10 .
  • PCD programmed cell death
  • TFs transcription factors
  • DYSFUNCTIONAL TAPETUM 1 DYSFUNCTIONAL TAPETUM 1
  • TDF1 Defective in Tapetal Development and Function 1
  • MS1 MALE STERILITY 1
  • TDR1 Undeveloped Tapetum1
  • EAT1 ETERNAL TAPETUM 1
  • DTD DELAYED TAPETUM DEGENERATION
  • TDR1 directly binds the promoter of CP1 and C6 for their transcription 14 .
  • C6 encodes a lipid transfer protein that plays a crucial role in the development of lipidic orbicules and pollen exine during anther development 17 .
  • CP1 is involved in intercellular protein degradation in biological system and its mutant shows defected pollen development 25 .
  • EAT1 acts downstream of TDR1 and directly regulates the expression of AP25 and AP37, which encode aspartic proteases involved in tapetal PCD 23 .
  • the basic helix-loop-helix (bHLH) proteins are a superfamily of TFs and one of the largest TF families in plants. There are at least 177 bHLH genes in the rice genome 26,27 and more than 167 bHLH genes in Arabidopsis genome 28,29 .
  • eukaryotic TFs consist of at least two discrete domains, a DNA binding domain and an activation or repression domain that operate together to modulate the rate of transcriptional initiation from the promoter of target genes 30 .
  • the bHLH TFs play many different roles in plant cell and tissue development as well as plant metabolism 3 .
  • the HLH domain promotes protein-protein interaction, allowing the formation of homodimeric or heterodimeric complexes 31 .
  • bHLH TFs bind as dimers to specific DNA target sites and are important regulatory components in diverse biological processes 29 . So far, three of the bHLH TFs have been shown to be involved in rice pollen development—UDT1 (bHLH164), TDR1 (bHLH5), and EAT1/DTD1 (bHLH141).
  • bHLH142 a novel MS-related gene encoding for another member of the bHLH TFs.
  • the molecular mechanism of MS in this mutant is elucidated, and it suggests that bHLH142 is specifically expressed in the anther and bHLH142 coordinates with TDR1 in regulating EAT1 promoter activity in transcription of protease genes required for PCD during pollen development. That is to say, bHLH142 plays an essential role in rice pollen development by controlling tapetal PCD.
  • Both null mutant and overexpression transgenic plants showed a completely male sterile phenotype. Most interestingly, the overexpression plants have restored the fertility under low temperature. Homologs of SEQ ID NO: 2 with high similarity are found in other major cereal crops, and its use may increase the productivity of cereal crops by manipulating the bHLH gene for development of male sterility and production of hybrid crops.
  • the object of the present invention is developing a mutated nucleotide molecule, and a transformed plant cell and a male sterile mutant plant comprising the mutated nucleotide molecule; in which the male sterile mutant plant can be used as a female parent to produce F1 hybrid seeds, thereby improving yield and quality of crops.
  • the present invention provides a mutated nucleotide molecule, comprising a nucleotide sequence of the transcription factor bHLH142 and an inserted T-DNA segment.
  • the T-DNA segment is inserted in the third intron of the nucleotide sequence of the transcription factor bHLH142; more preferably, the T-DNA segment is inserted at +1257 bp.
  • the T-DNA segment has comprises a single copy of T-DNA.
  • the nucleotide sequence of the transcription factor bHLH142 has a DNA sequence of SEQ ID No: 1 or a DNA sequence having at least 60% similarity to SEQ ID No: 1.
  • the transcription factor bHLH142 has a polypeptide sequence of SEQ ID No: 2 or a polypeptide sequence having at least 60% similarity to SEQ ID No: 2.
  • the present invention provides a transformed plant cell, which comprises the above-mentioned mutated nucleotide molecule.
  • the T-DNA segment is inserted in the third intron of the nucleotide sequence of the transcription factor bHLH142; more preferably, the T-DNA segment is inserted at +1257 bp.
  • the nucleotide sequence of the transcription factor bHLH142 has a DNA sequence of SEQ ID No: 1 or a DNA sequence having at least 60% similarity to SEQ ID No: 1.
  • the transcription factor bHLH142 has a polypeptide sequence of SEQ ID No: 2 or a polypeptide sequence having at least 60% similarity to SEQ ID No: 2.
  • the present invention also provides a male sterile mutant plant comprising the above-mentioned mutated nucleotide molecule, and the transcription factor bHLH142 is not expressed; particularly, not expressed in anthers.
  • the T-DNA segment is inserted in the third intron of the nucleotide sequence of the transcription factor bHLH142; more preferably, the T-DNA segment is inserted at +1257 bp.
  • the nucleotide sequence of the transcription factor bHLH142 has a DNA sequence of SEQ ID No: 1 or a DNA sequence having at least 60% similarity to SEQ ID No: 1.
  • the transcription factor bHLH142 has a polypeptide sequence of SEQ ID No: 2 or a polypeptide sequence having at least 60% similarity to SEQ ID No: 2.
  • a polypeptide sequence having at least 80% similarity to SEQ ID No: 2 more preferably, a polypeptide sequence having at least 90% similarity to SEQ ID No: 2; even more preferably, a polypeptide sequence having at least 95% similarity to SEQ ID No: 2; and most preferably, a polypeptide sequence of SEQ ID No: 2.
  • the male sterile mutant plant of the present invention is a homozygous mutant.
  • the plant is a monocot; preferably, the monocot is rice, maize, wheat, millet, sorghum or Brachypodium distachyon.
  • the plant is a dicot; preferably, the dicot is Arabidopsis or Brassica species.
  • the present invention also provides a transformed plant cell, which comprises a plasmid comprising the sequence of the transcription factor bHLH142 and a strong promoter.
  • the transcription factor bHLH142 has a DNA sequence of SEQ ID No: 1 or a DNA sequence having at least 60% similarity to SEQ ID No: 1.
  • the strong promoter is Ubiquitin promoter, CaMV 35S promoter, Actin promoter, an anther tapetum-specific promoter or a pollen-specific promoter; preferably, the anther tapetum-specific promoter is Osg6B or TA29, and the pollen-specific promoter is LAT52 or LAT59.
  • the present invention also provides a reversible male sterile transgenic plant, wherein the transcription factor bHLH142 is overexpressed; particularly, overexpressed in anthers.
  • the nucleotide sequence of the transcription factor bHLH142 has a DNA sequence of SEQ ID No: 1 or a DNA sequence having at least 60% similarity to SEQ ID No: 1.
  • the transcription factor bHLH142 has a polypeptide sequence of SEQ ID No: 2 or a polypeptide sequence having at least 60% similarity to SEQ ID No: 2.
  • the expression of the transcription factor bHLH142 is controlled by a strong promoter; preferably, by an Ubiquitin promoter, CaMV 35S promoter, Actin promoter, an anther tapetum-specific promoter or a pollen-specific promoter; preferably, the anther tapetum-specific promoter is Osg6B or TA29, and the pollen-specific promoter is LAT52 or LAT59.
  • the pollen fertility of the plant is recovered under low temperature.
  • the pollen fertility of the plant is recovered at 21-23° C.
  • the plant is a monocot; preferably, the monocot is rice, maize, wheat, millet, sorghum or Brachypodium distachyon.
  • the plant is a dicot; preferably, the dicot is Arabidopsis or Brassica species.
  • the present invention also provides a method for preparing the above-mentioned reversible male sterile transgenic plant, comprising:
  • the DNA sequence of bHLH142 is SEQ ID No: 1 or a DNA sequence having at least 60% similarity to SEQ ID No: 1.
  • the strong promoter is Ubiquitin promoter, CaMV 35S promoter, Actin promoter, an anther tapetum-specific promoter or a pollen-specific promoter; preferably, the anther tapetum-specific promoter is Osg6B or TA29, and the pollen-specific promoter is LAT52 or LAT59.
  • the plasmid is introduced into calli of the target plant via Agrobacterium tumefaciens.
  • FIG. 1 Phenotypic Analyses of ms142 Mutant.
  • A Comparison of the wild-type plant (left) and ms142 mutant (right) after bolting;
  • B comparison of the wild-type panicle (left) and ms142 mutant (right) at heading stage;
  • C comparison of the wild-type panicle (left) and ms142 mutant (right) at seed maturation stage;
  • D phenotype of the wild-type spikelet (left) and ms142 mutant (right) one day before anthesis;
  • E phenotype of the wild-type spikelet (left) and ms142 mutant (right) after anthesis;
  • F comparison of the wild-type grain (left) and ms142 mutant (right) at harvest stage;
  • G phenotype of the wild-type grain (left) and ms142 mutant (right) after removing rice husk;
  • H phenotype of the wild-type anther (left) and ms142 mutant (right) one day before an
  • FIG. 2 Evidences of T-DNA insertion in ms142 mutant.
  • A Southern blotting to hptII probe confirmed single T-DNA insertion (marked with arrow) in ms142 mutant.
  • B T-DNA tagged construction map of pTag4 vector.
  • C Gene structure and T-DNA insertion site in the mutant at 3rd intron (+1257 bp from ATG).
  • D Genotyping heterozygous T4 mutant progeny. Genotype: WT, wild-type like; He, heterozygous; and Ho, homozygous. Grain fertility: F, fertile; and S, sterile.
  • FIG. 3 Transverse anatomical comparison of the anther development in the wild type (TNG67) and ms142 mutant.
  • FIG. 4 TUNEL assay showed defect tapetal program cell death in ms142 mutant.
  • DNA fragmentation signals yellow fluorescence
  • PI propidium iodide
  • FIG. 5 Scheme of bHLH142 gene, multiple alignment, and subcellular localization of bHLH142 fused with GFP.
  • A Scheme of the bHLH142 gene and T-DNA insertion position. Gray boxes represent exons and interventing lines represent introns. The ATG start codon and TGA stop codon are indicated.
  • bHLH basic helix-loop-helix domain (amino acids 182 to 228); NLS, two nuclear localization signals (amino acids 159 to 165 and 235 to 240, respectively).
  • B Alignment of bHLH domains. The bHLH142 protein was aligned with the bHLH domains of the analogous proteins from other species.
  • C Schemes of fusion constructs. P35S, cauliflower mosaic virus 35S promoter; Trios, nopaline synthase gene terminator. The NLS domain of VirD2 fused with mRFP was used as the nuclear marker.
  • FIG. 6 Phylogenetic analysis of bHLH142 related proteins.
  • Aral Arabidopsis lyrata ; Aegt, Aegilops tauschii ; At, Arabidopsis thaliana ; Brad, Brachypodium distachyon ; Os, Oryza sativa ; Sei, Setaria italica ; Sb, Sorghum bicolor ; SeMa, Selaginella moellendorffii ; Triu, Triticum urartu ; and Zm, Zea may.
  • FIG. 7 Spatial and temporal gene expression of bHLH142 in various tissues of TNG67 (WT) by (A) RT-PCR and (B) qRT-PCR, and (C) in TNG67 spikelet at various developmental stages; and (D) ISH of bHLH142 antisense (left panel) and sense (right panel) probes in spikelet of TNG67 at meiosis stage.
  • Scale bar 1 mm in (C), 50 ⁇ m in (D).
  • FIG. 10 Gene hierarchy of bHLH142, as determined by the expression profiles of the key regulatory genes involved in pollen development in ms142 and eat1 mutants.
  • A Phenotype of spikelet in TNG67 (WT) and ms142.
  • B Phenotype of spikelet in Hitomebore and H0530 (eat1 mutant).
  • C Real-time RT-PCR of bHLH142 in ms142 and eat1 mutants and their respective wild-types.
  • MMC meiocyte mother cell; Mei, meiosis; YM, young microspore; and VP, vacuolated pollen.
  • FIG. 11 Coordinated Regulation of EAT1 Promoter by bHLH142 and TDR1.
  • A Schematic diagrams of the reporter, effector, and internal control plasmids used in the transient transactivation assay in rice leaf protoplasts.
  • the reporter plasmid contains the CaMV35S minimal promoter and the EAT1 promoter sequence (2 Kb) fused to the firefly luciferase gene (Luc).
  • bHLH142, TDR1, and EAT1 genes were driven under the control of the CaMV35S promoter. Nos and t35s denote the terminators of nopaline synthase and CaMV35S, respectively.
  • the pBI221 vector contains a CaMV35S promoter driving the expression of GUS as the internal control.
  • B Transactivation of the Luc reporter gene by bHLH142 and TDR in rice protoplasts. Different effectors were co-transfected with the reporter and internal control plasmid (pBI221). The data represent means of three independent transient transformations. Error bars indicate SD. Transient transformation without the effector plastnid (mini35p) was used as a negative control.
  • FIG. 13 The Interactions between bHLH142 Protein and TDR1, and between TDR1 and EAT1.
  • A Yeast two-hybrid (Y2H) assays. Constructs expressing the full length bHLH142 were cloned into the prey vector pGADT7 (AD), and truncated forms of TDR and EAT1 were prepared in the bait vector pGBKT7 (BD).
  • B BiFC in rice protoplasts expressing the indicated constructs. Bars represent 10 ⁇ m.
  • C Co-IP assay of HA fused TDR and bHLH142 recombinant proteins expressed in E. coli using anti-HA antibody.
  • D Co-IP assay of HA fused TDR and bHLH142 recombinant proteins expressed in E. coli using bHLH142 antibody.
  • FIG. 14 RNAi Knockdown (KD) of bHLH142 Inhibited Pollen Development.
  • KD RNAi Knockdown
  • A Construct of RNAi vector.
  • B RT-PCR showed down regulation of bHLH142 in the anthers of four RNAi knock down lines.
  • FIG. 15 Overexpression rice bHLH142 driven by Ubiquitin promoter in transgenic rice.
  • A Construction of bHLH142 (LOC_Os01g18870) driven by Ubiquitin promoter in pCAMBIA1301 vector,
  • B Genomic PCR confirmed T-DNA insertion of target gene (upper panel) and selection marker hygromycin (lower panel) in the transgenic rice.
  • FIG. 16 Phenotype of TNG67 (WT) and Ubi::bHLH142 transgenic lines.
  • A Plant type of WT (left) and transgenic line (right), (13) panicles of WT (bottom) and transgenic line (top panel),
  • C panicles of WT (left) and different transgenic lines,
  • D spikelet of WT (left) and different transgenic lines at one day before anthesis,
  • E mature seed of WT (left) and different transgenic lines, and
  • F removed husk rice seed of WT (left) and different transgenic lines.
  • Scale bars 20 cm in (A), 3 cm in (B), 7 cm in (C) and 1 cm in (F).
  • FIG. 17 Overexpression bHLH142 prematurely up-regulated Udt1 and EAT1 before meiosis stage but significantly down-regulated MS2 that associated in pollen exine development.
  • SC sporogenous cell
  • MMC meiocyte mother cell
  • Mei meiosis
  • YM young microspore
  • PM pollen mitosis
  • MP mature pollen.
  • FIG. 18 Gene expression pattern of bHLH142 homolog in various organs of maize by using RT-PCR. Floret size by measuring the length of floret. 10G, floret length at 10 mm with green color, 1 DBA, one day before anthesis.
  • FIG. 19 Heterologus overexpressing Ubi::bHLH142 caused male sterility in transgenic maize
  • A The transgenic line has smaller angle of tassel branch (right panel) than the WT (left panel).
  • B Closed up tassel during anthesis stage, WT has large and anther opened during anthesis stage (left panel), while anther of transgenic maize were significantly smaller in size and anther no dehiscence (right panel).
  • C Morphology of spikelet of WT (left panel) and transgenic line (right panel)
  • FIG. 20 The male sterility of Overexpression Ubi::bHLH142 transgenic line is sensitive to environment.
  • the bHLH142 overexpressed lines produced no pollen grains during summer season (Left panel) but had fertile pollens during winter seasons. Pollen grains were stained by I 2 /KI solution.
  • FIG. 21 A proposed model for the molecular function of bHLH142 in rice anther development, relative to other key regulators.
  • bHLH142 is in the downstream of UDT1 but upstream of TDR1, and bHLH142 interacts with TDR1 protein and coordinately regulate the promoter of EAT1.
  • EAT1 regulates AP37 and CP1 and promotes tapetal PCD.
  • Evidences from previous works were indicated by black arrows, while data demonstrated in the present study were indicated by red arrows.
  • the seed of ms142 mutant was obtained from TRIM library. Seedlings of ms142 mutant and its WT (TNG67) were raised in half strength Kimura solution for 3 weeks and then transplanted into soil in AS-BCST GMO screen house located in Tainan, Taiwan.
  • Spikelets and anthers of the WT and ms142 mutant were sampled at various stages of development and fixed overnight in phosphate buffer, pH 7.0, that contained 4% paraformaldehyde and 2.5% glutaraldehyde. They were then rinsed with the same buffer and post fixed for 30 min in phosphate buffer, pH 7.0, containing 1% osmium tetroxide. After dehydration, the specimens were embedded in Spurr's Resin (EMS). The processor, KOS Rapid Microwave Labstation, was chosen for post fixation, dehydration, resin infiltration, and embedding. For TEM, ultrathin sections (90 to 100 nm thick) collected on coated copper grids were stained with 6% uranyl acetated and 0.4% lead citrate and examine using transmission electron microscope.
  • EMS Spurr's Resin
  • Various rice organs at different developmental stages were harvested for RNA isolation: root, shoot, flag leaf, internode, panicles of 0.5 cm, 1 cm, 5 cm, 9 cm, and 20 cm length, spikelet at 1 day before anthesis (1 DBA), lemma, palea, anthers, ovary, seed at 5 days after pollination (S1), 15 days after pollination (S3), 25 days after pollination (S5), and callus.
  • microspore mother cell MMC with spikelet length of approximately 2 mm, meiosis (4 mm), young microspore (YM, 6 mm), vacuolated pollen (VP, 8 mm), mitosis pollen (MP, 8 mm with light green lemma), and mature pollen at one day before anthesis (1 DBA).
  • MMC microspore mother cell
  • VP vacuolated pollen
  • MP mitosis pollen
  • MP 8 mm with light green lemma
  • RT-PCR Fifteen ⁇ L of RT-PCR reaction contained 4 ⁇ L, of 1 ⁇ 4 diluted cDNA, 3 ⁇ M of primers, and 7.5 ⁇ L of 2 ⁇ KAPA SYBR FAST master mix (KAPA Biosystems, USA). Quantitative Real-Time PCR (qRT-PCR) was performed using a CFX96 Real-Time PCR detection system (Bio-Rad, USA). Quantification analysis was carried out using CFX Manager Software (Bio-Rad, USA). Primers used for qPCR are listed in Table 1.
  • Tissue sections were deparaffinized with xylene, rehydrated through an ethanol series, and pre-treated with proteinase K (2 mg/mL) in 1-phosphate buffered saline (PBS) at 37° C. for 30 min.
  • Pre-hybridization additionalally including 25% RNAmate, BioChain
  • Hybridization was performed at 59° C.
  • RNA probes were synthesized by in vitro transcription of the RT-PCR fragment in pGEM-T easy vector using the DIG RNA labeling kit (SP6/T7, Roche).
  • RNA probes were synthesized by SP6 RNA polymerase, while sense RNA probes were synthesized by T7 RNA polymerase and used as control. Sequence of fragment to synthesize RNA probe (SEQ ID No. 119):
  • T-DNA/Tos17 knock out mutant lines in hands such as: in udt1 (TRIM), bHLH142 (ms142, TRIM), and eat1 (bHLH141)
  • Tos17 mutant line H0530 background of Hitomebore
  • Rice Tos17 Insertion Mutant Database http://tos.nias.affrc.go.jp/. Flanking sequences were confirmed by genotyping PCR amplification with specific primers (Table 1). We will verify their gene hierarchy using these mutants. Spikelet samples at various developmental stages were collected, isolated RNA, and performed qRT-PCR analysis.
  • PCD is characterized by cellular condensation, mitochondria and cytoskeleton degeneration, nuclear condensation, and internucleosomal cleavage of chromosomal DNA 33 .
  • TUNEL terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling
  • bHLH142-GFP fusion genes driven by the CaMV 35S promoter.
  • Rice protoplasts were isolated and transformed using the polyethylene glycol (PEG) method following procedures described previously 34 . After incubation at room temperature for 16 h in light, protoplasts were observed with a Zeiss LSM 780 laser scanning confocal microscope.
  • the bHLH142 protein sequence was used to search for the closest homologues from their plant species using BLASTP programs. Multiple sequence alignment of full-length protein sequences was performed using ClustalW online (http://www.ch.embnet.org/software/ClustalW.html), and the alignment was used to perform neighbor-joining analysis using Mega 5.05 35 .
  • the numbers at the nodes represent percentage bootstrap values based on 1000 replications.
  • the length of the branches is proportional to the expected numbers of amino acid substitutions per site.
  • Gene identification numbers used to generate the phylogenetic trees and the alignment are listed in Table 2.
  • Peptide sequence (N′ ⁇ C′) ID BradXP_003567568 myhpqcellmphesldmdavvgqshlaasgvsaipaelnfhllhhsfvdtaaspqpptvdyffpgtdpppaa SEQ ID vqfeqlaatnhhamsmlrdyygqqypaetylrggprtttagssslvfgvahddesaaynmvgpfvesspttr No.
  • the MATCHMAKER GAL4 Two-Hybrid System (Clontech, USA) was used for Y2H assays. Since both full-length EAT1 and TDR1 proteins were reported having self-activation (Ji et al., 2013), we made a truncated EAT1 (EAT1 ⁇ , amino acids 1-254) and a truncated TDR1 (TDR ⁇ , amino acids 1-344) to reduce self activation.
  • the full length cDNA of bHLH142 was cloned into pGAD-T7 (Clontech, USA), and full length bHLH142, EAT1, TDR, EAT1 ⁇ , and TDR ⁇ were cloned into pGBK-T7 (Clontech, USA), respectively.
  • the pairs of constructs to be tested were co-transformed into AH109 yeast cells and selected on plates containing Leu (for pGADT7 plasmid) and Trp (for pGBKT7 plasmid) dropout medium for 3-4 days at 30° C.
  • Transformants were tested for specific protein interactions by growing on SD/-Leu/-Trp/-His plates with 30 mM 3-amino-1,2,4 triazole (3AT), and tested after X- ⁇ -Gal induction to confirm positive interaction.
  • This system provides a transcriptional assay for detecting and confirming protein interactions in vivo in yeast.
  • BiFC assay allows visualization of protein-protein interactions in living cells and the direct detection of the protein complexes in subcellular compartments, providing insights into their functions.
  • Full-length cDNAs of bHLH142, UDT1, TDR1, and EAT1 were independently introduced into pJET1.2 (Thermo Scientific).
  • the sequence for the N-terminal amino acid residues 1-174 of YFP was then in-frame fused to the sequence of the C-terminal region of the tested proteins, while the sequence of the C-terminal amino acid residues 175-239 of YIP was in-frame fused to the sequence of the N-terminal end of the proteins.
  • the tested genes were introduced into pSAT5-DEST_CYN1 and pSAT4(A)-DEST_NYN1.
  • Recombinant proteins of bHLH142 and TDR1 fused with hemagglutinin (HA) tag were expressed in bacteria harboring pET-53-DEST (HIS-tag), and cell extracts after lysis were cleared by centrifugation at 12,000 rpm for 15 min, suspended in binding buffer (20 mM Tris-HCl, pH 7.9, 500 mM NaCl), and sonicated on ice for 30 s using an ultrasonic homogenizer (Misonix XL Sonicator Ultrasonic Cell Processor). The supernatants were purified using Ni 2+ resin.
  • extracts were pre-cleared by 30 min incubation with 20 ⁇ l of Pure Proteome Protein G Magnetic Beads (Millipore Co., Billerica, Mass.) at 4° C. with rotation.
  • the antibodies (anti-bHLH142 or anti-HA) were then added to the pre-cleared extracts. After incubation for 4 h at 4° C., 40 ⁇ L of PureProteome Protein G Magnetic Beads was added, and the extracts were further incubated for 10 min at room temperature with rotation. After extensive washing, bound proteins were analyzed by western blotting.
  • Rabbit antiserum against rice bHLH142 was produced using a synthetic peptide (CSPTPRSGGGRKRSR, SEQ ID No. 116) as antigen (GenScript Co).
  • RNA intereference (RNAi) construct for suppressing the expression of bHLH142
  • a 149 bp fragment from 5′UTR region of bHLH142 was amplified by PCR with specific primers (Table 1) and cloned into pENTR (Invitrogen) to yield an entry vector pPZP200 hph-Ubi-bHLHI42 RNAi-NOS (12,483 bp).
  • the RNAi construct was transformed into WT (TNG67) rice calli via Agrobacterium tumefaciens -mediated transformation system 37 . Transgenic plants were regenerated from transformed calli by selection on hygromycin-containing medium.
  • Taiwan Rice Insertional Mutants http://trim.sinica.edu.tw) lines we identified a T-DNA-tagged rice mutant (denoted ms142) with a completely MS phenotype.
  • this mutant produced no viable seeds but maintained a normal vegetative growth ( FIG. 1A ), with panicles and spikelet developing similarly to those of the wild-type (WT) ( FIGS. 1B to 1E ).
  • the ms142 mutant exhibits normal opening of spikelets and elongation of anther filaments, and its anthers are exerted completely in the husk ( FIG. 1E ). However, the anthers of ms142 were significantly smaller in size and appeared yellowish white ( FIG.
  • the protein encoded by the gene is annotated as a basic helix-loop-helix dimerization region bHLH domain containing protein (RiceXPro Version 3.0).
  • the bHLH142 gene comprises of four exons and three introns.
  • genotyping by PCR using specific primers acrossing the T-DNA insertion site verified its FST ( FIG. 2 ).
  • heterozygous mutant plants behaved similarly to WT in terms of vegetative and reproductive growth and produced fertile seeds.
  • homozygous ms142 mutant plants exhibited similar plant height, panicle number, and panicle length to the WT, but produced no viable seeds.
  • the WT anther walls contained epidermal cell layer, endothecial cell layer, middle layer and tapetal cell layer ( FIG. 3A ).
  • MMC microspore mother cell
  • the microspore mother cells underwent meiosis to form tetrads of haploid microspores; the tapetal cells differentiated to form large vacuole; and the middle layer cells began to degenerate ( FIG. 3B ).
  • the meiocytes formed tetrads ( FIG. 3C ).
  • the ms142 anther consisted of normal epidermis, endothecium, middle layer and tapetum ( FIG. 3F ).
  • ms142 microspore mother cells did not enter meiosis and formed abnormal organelles ( FIG. 3G , indicated by arrows).
  • Abnormal endoplasmic reticulum structure and apoptosis was also observed by transmission electron microscopy.
  • the ms142 tapetal cells continuously became vacuolated and elongated, with some cells divided into two tapetum layers ( FIG. 3G ).
  • the mid-layers of the mutant tapetum maintained their initial shapes, but failed to divide into four cells at tetrad stage ( FIG. 3H ).
  • the ms142 mutant's microspores finally degenerated during the vacuolated pollen stage.
  • the tapetal and middle layer cells contained a large vacuole, and the middle layer cells did not degenerate ( FIG. 3I ). Consequently, there were no mature pollen grains formed in the locules at the mature stage.
  • the mutant anther wall still retained four to five layers of cells, i.e. epidermis, endothecium, middle layer, and one or two layers of tapetum cells. By contrast, the endothecial cell layer did not become thickened in the mutant even at the latter stage of anther development ( FIG. 3J ).
  • ms142 has abnormal anther morphology and aborted degradation of tapetal cells ( FIG. 3 ).
  • Tapetal PCD is characterized by cellular condensation, mitochondria and cytoskeleton degeneration, nuclear condensation, and internucleosomal cleavage of chromosomal DNA 33 . Therefore, we performed the TUNEL assay to detect DNA fragmentation in the anthers of WT and ms142.
  • a TUNEL positive signal began to appear in the tapetal cells of WT during meiosis and a strong TUNEL signal was detected during the young microspore stages ( FIG. 4 ). In contrast, no DNA fragmentation was observed in the tapetal layer in ms142 throughout anther development ( FIG. 4 ).
  • bHLH142 is a Nuclear Protein
  • bHLH142 The gene structure of bHLH142, shown in FIG. 5A , indicates that bHLH domain contains bipartite nuclear localization signal (NLS), and the gene is predicted to encode a protein of 379 amino acids with a theoretical molecular mass of 40.7 kDa and pI of 6.2.
  • NLS nuclear localization signal
  • bHLH142 The nucleotide sequence of bHLH142 is shown below:
  • amino acid sequence of bHLH142 is shown below:
  • bHLH142 is localized in the nucleus.
  • GFP green fluorescent protein gene
  • bHLH142 under the control of the 35S promoter and the nos terminator for transient expression in rice leaf mesophyll protoplasts.
  • RFP red fluorescent protein
  • Rice bHLH142 shares a high similarity with the related proteins from Brachypodium distachyon , millet ( Setaria italica ), Triticum urartu , maize ( Zea may ), Sorghum ( Sorghum bicolor ) and Aegilops tauschii ( FIG. 6 ).
  • the maize homolog, GRMZM2G021276, is highly expressed in immature tassel and meiotic tassel and anther.
  • bHLH142 mRNA is accumulated in young rice panicle and anther only, but not in other tissues (e.g. root, shoot, leaf, lemma, palea, ovary, and seed).
  • high levels of transcripts were found in developing panicles ( FIGS. 7A and 7B ).
  • bHLH142 transcripts were highly expressed in meiocyte mother cells (MMC) and extremely highly expressed in the anther at meiosis stage ( FIG. 7C ).
  • ISH in-situ hybridization
  • EAT1 was expressed slightly later (at young microspore stage) than bHLH142 (at meiosis stage) ( FIG. 10 ).
  • ms142 anther exhibited a similar amount of EAT1 mRNA to WT anthers at MMC, but tended to decline after young microspore stage ( FIG. 10 ).
  • bHLH142 plays a role in the downstream of UDT1, but upstream of EAT1.
  • EAT1 promoter assays in that both bHLH142 and TDR1 are required for the transcription of EAT1 ( FIG. 11 ).
  • Bimolecular fluorescent complementation (BiFC) assay showed that yellow fluorescent protein (YFP) signals are detected only in the nucleus of the rice cells co-expressing both NYN1-bHLH142 and CYN1-TDR1 and in the cells co-expressing both NYN1-TDR1 and CYN1-EAT1, but not in the cells co-expressing both NYN1-bHLH142 and CYN1-EAT1 ( FIG. 13B ).
  • YFP yellow fluorescent protein
  • RNA interference RNA interference construct to suppress the expression of bHLH142 in rice.
  • the gene specific region from the 5′UTR of bHLH142 was, amplified, fused with ⁇ -glucuronidase (GUS) intron and introduced into WT calli via Agrobacterium tumefaciens .
  • GUS ⁇ -glucuronidase
  • All 16 TO RNAi transgenic lines obtained had a MS phenotype similar to the T-DNA mutant ms142.
  • These RNAi lines showed reduced expression of bHLH142, as examined by RT-PCR, and produced poorly developed anthers without pollen grains ( FIG. 14 ). This result further supports the notion that bHLH142 plays a key role in rice anther and pollen development.
  • the RNAi fragment SEQ ID No. 120:
  • bHLH142 shares high identity with maize 38 , and gene specific primer sets were designed from homolog of maize ZmLOC100283549 (denoted Zm-142). RT-PCR indicated that Zm-142 was not expressed in vegetative organs of maize such as leaf, root, shoot, and stamen. Interestingly, Zm-142 specifically expressed in floret of 1 mm to 7 mm length but not detectable at later stage and in the mature pollen ( FIG. 18 ). The expression pattern of Zm-142 was similar to bHLH142 38 .
  • FIG. 15A we use the similar construct of overexpression bHLH142 in FIG. 15A was transformed into maize (in cultivar Crystal White background) using agrobacterium -mediated pollen transformation method.
  • One transgenic maize showed obvious male sterility phenotype with smaller angle of tassel branch ( FIG. 19A , right panel) than the WT ( FIG. 19A , left panel). Closed up tassel during anthesis stage, WT has larger anther than transgenic line and it has normal opening of spikelets and elongation of anther filaments during anthesis stage ( FIG. 19B , left panel). Whilst, anther of transgenic maize were significantly smaller in size and anther was completely no elongation of anther filaments ( FIG.
  • FIG. 19B right panel
  • Morphology of spikelet of WT at one day before anthesis was shown in FIG. 19C (left panel) with long and fat anther, but anther of transgenic line was short and shrinkage ( FIG. 19C , right panel).
  • pollens of transgenic line could not be stained and transparent due to no starch accumulation. That implied transgenic line was male sterile ( FIG. 19D ).
  • bHLH142-overexpressed plants also showed a completely male sterile phenotype during summer season ( FIG. 20 , left panel).
  • anthers of overexpression transgenic line produce many pollen grains inside the locules and their pollen grains can be stained by I 2 /KI solution, indicating that the plants have restored the fertility ( FIG. 20 , right panel). Therefore, this novel functionality nature of our target bHLH142 has a big advantage over other genetic MS (GMS) genes for hybrid crop production.
  • GMS genetic MS
  • This reversible pollen fertility trait makes it more desirable in producing hybrid crop seeds just in one cross without the need to maintain the seed stocks of the MS lines as with cytoplasmic MS (CMS).
  • CMS cytoplasmic MS
  • biotech companies are known to prefer adopting overexpression over suppression approach in generating transgenic lines because overexpression lines are more stable than RNAi or antisense knock-down lines.
  • Rice bHLH142 have homologous in maize, sorghum and wheat, and they share more than 70% similarity in amino acid sequence to the rice counterpart (Table 3). This will benefit to genetic engineering male sterile for F1 hybrid seed production and generating hybrid vigor (heterosis) in terms of growth and grain yield in cereal crops.
  • bHLH142 is a New Major Regulator of Rice Anther Development
  • bHLH142 as another critical factor in the bHLH TF family for pollen development, besides UDT1 (bLHL164), TDR1 (bLHL5) and EAT1 (bHLH141).
  • UDT1 bLHL164
  • TDR1 bLHL5
  • EAT1 bHLH141
  • FIG. 10 and FIG. 9 show that the gene hierarchy of bHLH142 is in the downstream of UDT1 (bHLH164) but upstream of TDR1 (bHLH5) and EAT1 (bHLH141) ( FIG. 10 and FIG. 9 ).
  • all these 4 bHLH TFs are tissue specifically expressed in the anther and participate in the important process of sequential pollen development events, particularly in tapetal PCD.
  • bHLH142 Functions Coordinately with TDR1 to Regulate EAT1 Promoter
  • TDR1 and EAT1 mRNA are both down-regulated in ms142, we hypothesize that TDR1 interacts with bHLH142 and positively regulate EAT1 promoter for transcriptional activities of AP25 and AP37, encoding aspartate proteases for tapetal PCD.
  • Our promoter transient assay provides solid evidence that bHLH142 and TDR1 work coordinately in regulating EAT1 promoter ( FIG. 11 ).
  • EAT1 protein significantly reduced EAT1-Luc promoter strength from a 30 fold down to 18 fold increase ( FIG. 11 ), which may be attributed to the competition between bHLH142 and EAT to interact with TDR1.
  • EAT1 favors TDR1-EAT1 interaction and might consequently reduce the interaction between bHLH142 and TDR1, therefore reducing EAT1 transcriptional activation ( FIG. 11 ). It is likely that bHLH142 interacts with TDR1 and TDR1 in turn interacts with EAT1 and bHLH142 does not directly interact with EAT1 ( FIG. 13 ). Whether some other TFs may be required to regulate the transcription of bHLH142 is worth further investigation to unravel the entire regulatory gene hierarchy.
  • N-terminal truncated forms of TDR ⁇ aa(1-344) and EAT1 ⁇ aa(1-254) were used in our experiment to reduce self activation. These two N-terminal truncated protein forms did not exhibit self activation in yeast cells ( FIG. 12A ). Therefore, we are confident that bHLH142 interacts with TDR1 by using these truncated proteins to eliminate the bias ( FIG. 13 ).
  • Our data indicate that bHLH142 interacts with TDR1 in the C-terminal ( FIG. 13 ), and support the conclusion of the previous study in that DTD/EAT1 (bHLH141) interacts with TDR1 in the C-terminal region 24 .
  • both bHLH142 and EAT1 can interact with TDR1 in the C′ terminal of TDR1.
  • This finding also supports the result of our EAT1 promoter assay, where additional EAT1 protein reduces EAT1 promoter activity, presumably due to the competition between bHLH142 and EAT1 proteins in the C′ terminal of TDR1.
  • the current regulatory network for rice pollen development is presented in FIG. 21 .
  • Previous works with various rice MS mutants suggest that UDT1 and GAMYB may positively regulate the transcription of TDR1 22 and TDR1 in turn controls the transcription of C6 and CP1 14 .
  • TDR1 interacts with EAT1 for its direct regulation of the expression of two aspartate proteinase genes for initiation of tapetal PCD 23 .
  • bHLH142 acts downstream of UDT1 but upstream of TDR1 and EAT1, and then bHLH142 interact with TDR1 in activating EAT1 transcription (indicated by red arrows in FIG. 21 ).
  • EAT1 also positively regulate the transcription of AP37 and CP1 directly, two proteins involved in tapetal PCD at late pollen development stage.

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3490365A4 (en) * 2016-07-29 2020-04-29 Elsoms Developments Ltd WHEAT
CN111593135A (zh) * 2020-05-07 2020-08-28 海南波莲水稻基因科技有限公司 一种鉴别转基因材料及其自交、杂交、回交后代中内、外源基因的检测引物和方法
CN115948416A (zh) * 2022-10-20 2023-04-11 安徽农业大学 一种玉米花期调控的转录因子ZmCIB1基因及其应用
CN116875580A (zh) * 2023-09-08 2023-10-13 北京首佳利华科技有限公司 利用人工突变创制玉米msp1雄性不育系

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106566834A (zh) * 2016-11-04 2017-04-19 西北大学 瘿椒树dyt1基因
CN107384928B (zh) * 2017-09-01 2020-09-18 北京市农林科学院 小麦花药绒毡层特异表达启动子及其应用
WO2019136174A2 (en) * 2018-01-03 2019-07-11 Uwm Research Foundation, Inc. Sterile mutant and two line breeding system
CN112501178B (zh) * 2020-10-16 2022-10-14 上海师范大学 一种水稻温敏不育突变体tms18及其应用
CN112522283B (zh) * 2020-12-22 2023-01-03 浙江大学 一种花粉发育相关基因及其应用
CN112961231B (zh) * 2021-03-12 2023-07-14 北京科技大学 雄性不育基因ZmbHLH122及其在创制玉米雄性不育系中的应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1041988C (zh) * 1993-10-21 1999-02-10 中国科学院昆明植物研究所 稻类可逆转两用雄性核不育系的选育方法
FR2757524B1 (fr) * 1996-12-19 1999-01-29 Rhone Poulenc Rorer Sa Polypeptides de la famille bhlh, sequences d'acides nucleiques correspondantes
CN1565154A (zh) * 2003-06-24 2005-01-19 华中农业大学 一种保存和繁殖水稻光、温敏核不育系的方法
EP1765058A4 (en) * 2004-06-15 2008-05-28 Univ Latrobe NUCLEIC ACID MOLECULES AND THEIR USE FOR POLLENSTERILITY IN PLANTS

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Abe et al (2003, The Plant Cell 15:63-78) *
Hsing et al (2007, Plant Molecular Biology 63:351-364) *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3490365A4 (en) * 2016-07-29 2020-04-29 Elsoms Developments Ltd WHEAT
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CN115948416A (zh) * 2022-10-20 2023-04-11 安徽农业大学 一种玉米花期调控的转录因子ZmCIB1基因及其应用
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