MUTATED NUCLEOTIDE MOLECULE, AND TRANSFORMED PLANT CELLS AND PLANTS COMPRISING THE SAME
The present invention relates to a method for producing male sterile plant, a mutated nucleotide molecule comprising a nucleotide sequence of the transcription factor bHLH142 and an inserted T-DNA segment, and a novel transformed plant cell and a male-sterile mutant plant comprising the mutated nucleotide molecule, in which the transcription factor bHLH142 is not expressed. The present invention also relates to a novel reversible male sterile transgenic plant, wherein the transcription factor bHLH142 is overexpressed, and its preparation method. The bHLH gene is tissue specifically expresses in the anther and it plays a pivotal role in pollen development. Both the male sterile and reversible male sterile transgenic plants showed a completely male sterile phenotype, but the fertility of the reversible male sterile transgenic plant can be restored under low temperature.
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Rice (Oryza sativa) 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. Several approaches are currently adopted to increase rice yields, such as heterosis breeding, population improvement, wide hybridization, genetic engineering, and molecular breeding1. Among these, 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 has long been used in hybrid corn production, while both CMS and GMS are currently used for hybrid rice production3, 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. However, 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.
On the other hand, genetic MS (GMS), controlled by nuclear genes, offers an alternative hybrid seed production system. For the two-line hybrid system, it is beneficial to use photoperiod- or temperature-inducible MS (PGMS or TGMS) mutants to maintain seed stocks for hybrid seed production. Currently, in China, PA64S is the most widely used maternal line in two-line hybrid rice breeding, and it is crossed with paternal line 93-11 to generate superhybrid rice, LYP94. 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 RNA5. In the case of another rice genic MS mutant discovered recently, Carbon Starved Anther (CSA), the mutation on the R2R3 MYB transcription regulator defects pollen development6 and further study shows that csa is a new photoperiod-sensitive mutant, exhibiting MS under short-day conditions but male fertility under long-day conditions7. 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 restorers8. Recently, 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 setting8. 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. Alternatively, an inducible promoter by chemicals or other factors (e.g. photoperiod or temperature) can be directly used to regulate the expression of a GMS gene (e.g., bHLH142) and control pollen development in transgenic plants, eliminating the costly need to maintain MS lines.
Rice anthers are composed of four lobes attached to a central core by connective and vascular tissue. When anther morphogenesis is completed, microsporocytes form in the middle, surrounded by four anther wall layers: an epidermal outer layer, endothecium, middle layer, and tapetum9. 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 development10. The tapetum undergoes programmed cell death (PCD) during the late stage of pollen development11; this PCD causes tapetal degeneration and is characterized by cellular condensation, mitochondria and cytoskeleton degeneration, nuclear condensation, and internucleosomal cleavage of chromosomal DNA. Tapetal PCD must occur at a specific stage of anther development for normal tapetum function and pollen development, and premature or delayed tapetal PCD and cellular degeneration can cause male sterility3,12-14.
Genetic and functional genomic studies of MS in Arabidopsis have shown that many transcription factors (TFs) play an essential role in pollen development and the regulation of tapetal PCD, such as mutations in DYSFUNCTIONAL TAPETUM 1 (DYT1), Defective in Tapetal Development and Function 1 (TDF1, AtMYB35), ABORTED MICROSPORES (AMS, homolog of TDR1 in rice), and MALE STERILITY 1 (MS1); and mutations in these factors all result in MS phenotype. The genetic regulatory pathway of pollen development suggests that DYT1, TDF115 and AMS16 function at early tapetum development, while MS18817 and MS115,18,19 play important roles in late tapetum development and pollen wall formation. Whilst, in rice, several TFs, such as Undeveloped Tapetum1 (UDT1, homolog of DYT1), are known to be key regulators of early tapetum development20. In addition, mutations in TAPETUM DEGENERATION RETARDATION (TDR1)14, GAMYB21,22, ETERNAL TAPETUM 1 (EAT1)23 and DELAYED TAPETUM DEGENERATION (DTD)24 all cause MS associated with tapetal PCD. TDR1, ortholog of the Arabidopsis AMS gene, plays an essential role in tapetal PCD in rice; and tdr1 shows delayed tapetal degeneration and nuclear DNA fragmentation as well as abortion of microspores after release from the tetrad. Molecular evidences indicate that TDR1 directly binds the promoter of CP1 and C6 for their transcription14. C6 encodes a lipid transfer protein that plays a crucial role in the development of lipidic orbicules and pollen exine during anther development17. CP1 is involved in intercellular protein degradation in biological system and its mutant shows defected pollen development25. EAT1 acts downstream of TDR1 and directly regulates the expression of AP25 and AP37, which encode aspartic proteases involved in tapetal PCD23.
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 genome26,27 and more than 167 bHLH genes in Arabidopsis genome28,29. Generally, 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 genes30. The bHLH TFs play many different roles in plant cell and tissue development as well as plant metabolism3. The HLH domain promotes protein-protein interaction, allowing the formation of homodimeric or heterodimeric complexes31. They bind as dimers to specific DNA target sites and are important regulatory components in diverse biological processes29. 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).
From a screening of T-DNA tagged rice mutant pool of TNG6732, we isolated a novel MS-related gene encoding for another member of the bHLH TFs (bHLH142). In this invention, 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.
SUMMARY OF THE INVENTIONThe 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. Preferably, 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.
In one preferred embodiment of the mutated nucleotide molecule, the T-DNA segment has comprises a single copy of T-DNA.
In one preferred embodiment of the mutated nucleotide molecule, 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. Preferably, a DNA sequence having at least 80% similarity to SEQ ID No: 1; more preferably, a DNA sequence having at least 90% similarity to SEQ ID No: 1; even more preferably, a DNA sequence having at least 95% similarity to SEQ ID No: 1; and most preferably, a DNA sequence of SEQ ID No: 1. In addition, 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. Preferably, 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 present invention provides a transformed plant cell, which comprises the above-mentioned mutated nucleotide molecule. Preferably, 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.
In one preferred embodiment of the transformed plant cell comprising the mutated nucleotide molecule, 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. Preferably, a DNA sequence having at least 80% similarity to SEQ ID No: 1; more preferably, a DNA sequence having at least 90% similarity to SEQ ID No: 1; even more preferably, a DNA sequence having at least 95% similarity to SEQ ID No: 1; and most preferably, a DNA sequence of SEQ ID No: 1. In addition, 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. Preferably, 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 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. Preferably, 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.
In one preferred embodiment of the male sterile mutant plant, 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. Preferably, a DNA sequence having at least 80% similarity to SEQ ID No: 1; more preferably, a DNA sequence having at least 90% similarity to SEQ ID No: 1; even more preferably, a DNA sequence having at least 95% similarity to SEQ ID No: 1; and most preferably, a DNA sequence of SEQ ID No: 1. In addition, 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. Preferably, 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.
In one preferred embodiment of the male sterile mutant plant, the male sterile mutant plant of the present invention is a homozygous mutant.
In one preferred embodiment of the male sterile mutant plant, the plant is a monocot; preferably, the monocot is rice, maize, wheat, millet, sorghum or Brachypodium distachyon.
In one preferred embodiment of the male sterile mutant plant, 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.
In one preferred embodiment of the transformed plant cell comprising the sequence of the transcription factor bHLH142, 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. Preferably, a DNA sequence having at least 80% similarity to SEQ ID No: 1; more preferably, a DNA sequence having at least 90% similarity to SEQ ID No: 1; even more preferably, a DNA sequence having at least 95% similarity to SEQ ID No: 1; and most preferably, a DNA sequence of SEQ ID No: 1.
In one preferred embodiment of the transformed plant cell comprising the sequence of the transcription factor bHLH142, 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.
In one preferred embodiment of the reversible male sterile transgenic plant, 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. Preferably, a DNA sequence having at least 80% similarity to SEQ ID No: 1; more preferably, a DNA sequence having at least 90% similarity to SEQ ID No: 1; even more preferably, a DNA sequence having at least 95% similarity to SEQ ID No: 1; and most preferably, a DNA sequence of SEQ ID No: 1. In addition, 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. Preferably, 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.
In one preferred embodiment of the reversible male sterile transgenic plant, 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.
In one preferred embodiment of the reversible male sterile transgenic plant, the pollen fertility of the plant is recovered under low temperature. Particularly, the pollen fertility of the plant is recovered at 21-23° C.
In one preferred embodiment of the reversible male sterile transgenic plant, the plant is a monocot; preferably, the monocot is rice, maize, wheat, millet, sorghum or Brachypodium distachyon.
In one preferred embodiment of the reversible male sterile transgenic plant, 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:
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- (a) constructing a plasmid comprising the DNA sequence of bHLH142 and a strong promoter, and
- (b) introducing the plasmid into a target plant.
In one preferred embodiment of the preparation method, the DNA sequence of bHLH142 is SEQ ID No: 1 or a DNA sequence having at least 60% similarity to SEQ ID No: 1. Preferably, a DNA sequence having at least 80% similarity to SEQ ID No: 1; more preferably, a DNA sequence having at least 90% similarity to SEQ ID No: 1; even more preferably, a DNA sequence having at least 95% similarity to SEQ ID No: 1; and most preferably, a DNA sequence of SEQ ID No: 1.
In one preferred embodiment of the preparation method, 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.
In one preferred embodiment of the preparation method, the plasmid is introduced into calli of the target plant via Agrobacterium tumefaciens.
The following examples are presented to demonstrate the present invention. These examples are in no way to be construed as a limitation on the invention. The disclosure would enable those skilled in the art to practice the present invention without engaging in undue experimentation. All recited publications are incorporated herein by reference in their entirety.
Plant Materials and Growth ConditionsThe 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.
Anther AnatomySpikelets 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.
Total RNA Isolation and PCR.Total RNA was isolated from rice tissues using MaestroZol™ RNA PLUS (Invitrogen) as described by the supplier. 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. The stages of anthers were classified into the following categories according to spikelet length: 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). Total RNA was treated with DNase (Promega), and 1 μg RNA was used to synthesize the oligo(dT) primed first-strand cDNA using the M-MLV reverse transcriptase cDNA synthesis kit (Promega). One μL of the reverse transcription products was used as template in PCR reactions. Ubiquitin-like 5 and 18srRNA were used as normalizer control. Each sample has three biological repeats.
qRT-PCR Analysis.
Fifteen μL of RT-PCR reaction contained 4 μL, of ¼ diluted cDNA, 3 μM of primers, and 7.5 μL 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.
Spikelets of TNG67 and ms142 at various developmental stages were fixed in PFA [4% paraformaldehyde, 4% dimethylsulfoxide 0.25% glutaraldehyde, 0.1% Tween 20, 0.1% Triton X-100 in diethyl pyrocarbonate (DEPC)-treated H2O] at 4° C. overnight immediately after collection, and the tissue processor, KOS Rapid Microwave Lab station, was used for dehydration and wax infiltration. After embedding, sections of 10 μm thickness were prepared by a rotary microtome (MICROM, 315R) and mounted on APS adhesive microscope slides (FINE FROST). 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 (additionally including 25% RNAmate, BioChain) and hybridization were performed according to the previous protocols39. Hybridization was performed at 59° C. in hybridization solution: 50% formamide, 4×SSPE, 1×Denhardt's (Fluka), 250 μg/mL fish sperm DNA (Genemarker), 250 μg/mL yeast tRNA (Sigma), 10% dextran sulfate, 40 U/mL RNasin (Promega) and 40 ng of DIG-labeled RNA probe/per slide. 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). Antisense 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):
We have some 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) was obtained from 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.
TUNEL AssayPCD is characterized by cellular condensation, mitochondria and cytoskeleton degeneration, nuclear condensation, and internucleosomal cleavage of chromosomal DNA33. To investigate the nature of the tapetal breakdown in ms142, the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was performed using DeadEnd Fluorometric TUNEL system (Promega). This assay detects in situ DNA cleavage, a hallmark feature of apoptosis-like PCD, by enzymatically incorporating fluorescein-12-dUTP into the 3′-OH ends of fragmented DNA. Stage of anther development was based primarily on spikelet size and developmental stages.
Subcellular Localization of bHLH142
For subcellular localization of bHLH142, the coding sequences of the gene were subcloned into p2FGW7 (Invitrogen) to generate 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 previously34. After incubation at room temperature for 16 h in light, protoplasts were observed with a Zeiss LSM 780 laser scanning confocal microscope.
Phylogenetic Analysis of the bHLH142 Subfamily
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 5.0535. 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.
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 (EAT1Δ, amino acids 1-254) and a truncated TDR1 (TDRΔ, 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, EAT1Δ, and TDRΔ 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-α-Gal induction to confirm positive interaction. This system provides a transcriptional assay for detecting and confirming protein interactions in vivo in yeast.
Bimolecular Fluorescence Complementation (BiFC) AssayBiFC 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. Next, the tested genes were introduced into pSAT5-DEST_CYN1 and pSAT4(A)-DEST_NYN1. Ballistic bombardment-mediated transient transformation in rice protoplasts was carried out following a previously published protocol36. Florescence images were photographed on a LSM 780 Plus ELYRA S.1 confocal microscope with Plan-Apochromat 40×/1.4 oil objective lens (Zeiss, Germany).
Co-Immunoprecipitation AssayRecombinant 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 Ni2+ resin. For immunoprecipitation, extracts were pre-cleared by 30 min incubation with 20 μl 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 μL 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).
RNAi-Mediated Gene Silencing of bHLH142
To generate an 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 system37. Transgenic plants were regenerated from transformed calli by selection on hygromycin-containing medium.
Examples Identification of a New Male Sterility Rice MutantFrom the T2 population of Taiwan Rice Insertional Mutants (TRIM) (http://trim.sinica.edu.tw) lines we identified a T-DNA-tagged rice mutant (denoted ms142) with a completely MS phenotype. In the field, this mutant produced no viable seeds but maintained a normal vegetative growth (
Sequence Analysis of the T-DNA-Tagged Gene in ms142 Mutant
To determine T-DNA insertion copy number, Southern blot analysis of T2 mutant lines using hptII as a probe was conducted, and only a single band was detected in the mutant lines (
Agronomic Traits of ms142 Mutant and Genetic Study
The agronomic traits of the mutant were examined in the selfed progenies of heterozygous mutant grown in the outdoor GMO net house. Heterozygous plants behaved similarly to WT in terms of vegetative and reproductive growth and produced fertile seeds. However, homozygous ms142 mutant plants exhibited similar plant height, panicle number, and panicle length to the WT, but produced no viable seeds.
To understand whether the sterility in ms142 is due to male sterility or female sterility, homozygous mutant was backcrossed with WT pollen; and all F1 plants displayed WT-like phenotype in growth and fertility (data not shown). These results imply that the female organs of ms142 develop normally. When the ms142 BCF1 was selfed, the BCF2 progenies segregated into fertile and sterile plants in a ratio of 3:1 (Table 3), suggesting the MS trait is controlled by a recessive gene. Consistent with mutant phenotype, backcross segregants showed MS only in the homozygous plants, indicating that the MS phenotype co-segregated with the genotype. Moreover, when the selfed seeds derived from heterozygous plants of T2, T3, T4, and BCF2 generations were planted in different years and different cropping seasons, the scoring of phenotype indicated that MS in ms142 is stable and not affected by cropping season or year. Again, the fertile and sterile plants segregated approximately in a 3:1 ratio, supported by Chi-square analysis (data for T4 and BCF2 shown in Table 4). Taken together, these genetic analyses validate that the MS in ms142 is controlled by a single recessive locus.
Defects in Anther Wall and Pollen Development in the ms142 Mutant
To determine the defects in the anthers of ms142, we examined the anatomy of anther in WT and homozygous mutant. At the microspore mother cell (MMC) stage, the WT anther walls contained epidermal cell layer, endothecial cell layer, middle layer and tapetal cell layer (
At the MMC stage, there were no visible differences in the anthers between WT and ms142. The ms142 anther consisted of normal epidermis, endothecium, middle layer and tapetum (
Mutated bHLH142 Causes Defects in Tapetal PCD
Histological analysis indicated that ms142 has abnormal anther morphology and aborted degradation of tapetal cells (
bHLH142 is a Nuclear Protein
The gene structure of bHLH142, shown in
The nucleotide sequence of bHLH142 is shown below:
The amino acid sequence of bHLH142 is shown below:
Since the bHLH proteins are characterized as TFs, we assumed that bHLH142 is localized in the nucleus. To verify its subcellular localization, we constructed a fusion gene of the green fluorescent protein gene (GFP) and bHLH142 under the control of the 35S promoter and the nos terminator for transient expression in rice leaf mesophyll protoplasts (
To understand the evolutionary relationship of bHLH142 among various organisms, we used full-length bHLH142 protein sequence to NOM BLAST database and retrieved 21 homologs containing bHLH domain from 10 diverse terrestrial plants. The phylogenetic tree shows that UDT1 (bHLH164) and TDR1 (bHLH5) are in the same cluster, while bHLH142 and EAT1 (bHLH141) evolved and diversified into two separate clades. Phylogenetic analysis also suggests that bHLH142 is descended from a common ancestor of monocots. 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 (
Expression Pattern of bHLH142
Both RT-PCR and qRT-PCR analyses with WT showed that the 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). In particular, high levels of transcripts were found in developing panicles (
In addition, the expression patterns of various known pollen regulatory genes in the anther of WT versus ms142, as examined by qRT-PCR, confirmed the knockout of bHLH142 transcript in the ms142 null mutant (
bHLH142 and TDR1 Coordinately Regulate EAT1 Promoter Activity
Based on the alternations in expression of known pollen regulatory genes in the different mutants (
Protein Interactions Among bHLH142, TDR1 (bHLH5) and EAT1 (bHLH141)
We performed yeast two-hybrid analysis to determine whether bHLH142, as bait, interacts with the prey, TDR1 or EAT1. As previously reported that full-length EAT1 and TDR1 proteins possess self-activation activity in nature23,24, our Y2H study also confirmed this phenomenon (
RNAi Transgenic Rice Lines Validate the Role of bHLH142 in Pollen Development
To further validate the biological function of bHLH142, we generated an RNA interference (RNAi) construct to suppress the expression of bHLH142 in rice. The gene specific region from the 5′UTR of bHLH142 was, amplified, fused with β-glucuronidase (GUS) intron and introduced into WT calli via Agrobacterium tumefaciens. 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 (
Overexpression bHLH142 Caused Male Sterility
For functional genomic study, we constructed overexpressing bHLH142 driven by constitutively express Ubiquitin promoter (
Interesting to observe that overexpressing bHLH142 TO transgenic plants all showed grain sterility (
By using RT-PCR, we detected some regulatory genes associated with pollen development in rice. As expected, overexpression line constitutively express abundant of bHLH142 transcripts during various stages of anther development. Interestingly, Udt1 was simultaneously upregulated in the overexpression line. EAT1 mRNA also prematurely upregulated before meiosis stage but decrease its expression at latter stage of anther development. However, MS2 was significantly downregulated in the overexpressing line (
Heterologuos Overexpression bHLH142 Confers Male Sterility in Maize
Since bHLH142 shares high identity with maize38, 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 (
Therefore, we use the similar construct of overexpression bHLH142 in
Overexpression bHLH142 Induces Reversible Male Sterility in Low Temperature
bHLH142-overexpressed plants also showed a completely male sterile phenotype during summer season (
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
So far, three of the bHLH TFs have been shown to be involved in pollen development in rice and mutations of these TF genes all lead to complete MS, including UDT1 (bHLH164)20, TDR1 (bHLH514, and EAT1/DTD1 (bHLH141)23,24. They all play an important role in pollen development by regulating tapetal PCD. In this invention, we identified a novel rice MS mutant, ms142 (
Our analysis of expression profile of known regulatory genes involved in pollen development demonstrates the down-regulation of several genes, such as TDR1, EAT1, AP37, CP1, C6, MS2, etc. in ms142 during pollen development (
This invention uncovers bHLH142 as another critical factor in the bHLH TF family for pollen development, besides UDT1 (bLHL164), TDR1 (bLHL5) and EAT1 (bHLH141). Our mutagenesis analysis suggests that the gene hierarchy of bHLH142 is in the downstream of UDT1 (bHLH164) but upstream of TDR1 (bHLH5) and EAT1 (bHLH141) (
Also, we noticed a lower suppression in expression of TDR1 in ms142, compared to other downstream genes in the regulatory network, which may be attributed to the fact that TDR1 is also known to be regulated by another TF GAMYB22. In agreement, we also found that the expression of GAMYB is not altered in ms142 (
bHLH142 Functions Coordinately with TDR1 to Regulate EAT1 Promoter
Since 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 (
Our molecular studies provide solid in vivo (Y2H, BiFC) and in vitro (co-IP) evidences that both bHLH142 and TDR1 can form protein interaction (
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Claims
1. A mutated nucleotide molecule, comprising a nucleotide sequence of the transcription factor bHLH142 and an inserted T-DNA segment.
2. The mutated nucleotide molecule according to claim 1, wherein 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.
3. The mutated nucleotide molecule according to claim 1, wherein the T-DNA segment is inserted in the third intron of the nucleotide sequence of the transcription factor bHLH142.
4. The mutated nucleotide molecule according to claim 2, wherein the T-DNA segment is inserted at +1257 bp of the nucleotide sequence of the transcription factor bHLH142.
5. A transformed plant cell, which comprises the mutated nucleotide molecule according to claim 1.
6. The transformed plant cell according to claim 5, wherein 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.
7. The transformed plant cell according to claim 5, wherein the T-DNA segment is inserted in the third intron of the nucleotide sequence of the transcription factor bHLH142.
8. The transformed plant cell according to claim 6, wherein the T-DNA segment is inserted at +1257 bp of the nucleotide sequence of the transcription factor bHLH142.
9. A male sterile mutant plant, which comprises the mutated nucleotide molecule according to claim 1, and the transcription factor bHLH142 is not expressed.
10. The male sterile mutant plant according to claim 9, wherein 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.
11. The male sterile mutant plant according to claim 9, wherein the T-DNA segment is inserted in the third intron of the nucleotide sequence of the transcription factor bHLH142.
12. The male sterile mutant plant according to claim 10, wherein the T-DNA segment is inserted at +1257 bp of the nucleotide sequence of the transcription factor bHLH142.
13. The male sterile mutant plant according to claim 9, which is a homozygous mutant.
14. The male sterile mutant plant according to claim 9, wherein the plant is a monocot plant.
15. The male sterile mutant plant according to claim 14, wherein the monocot plant is rice, maize, wheat, millet, sorghum or Brachypodium distachyon.
16. The male sterile mutant plant according to claim 9, wherein the plant is a dicot plant.
17. The male sterile mutant plant according to claim 16, wherein the dicot plant is Arabidopsis or Brassica species.
18. A transformed plant cell, which comprises a plasmid comprising the sequence of the transcription factor bHLH142 and a strong promoter.
19. The transformed plant cell according to claim 18, wherein 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.
20. The transformed plant cell according to claim 18, wherein the strong promoter is Ubiquitin promoter, CaMV 35S promoter, Actin promoter, an anther tapetum-specific promoter or a pollen-specific promoter.
21. A reversible male sterile mutant plant, wherein the transcription factor bHLH142 is overexpressed.
22. The reversible male sterile transgenic plant according to claim 21, wherein 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.
23. The reversible male sterile transgenic plant according to claim 21, wherein the expression of the transcription factor bHLH142 is controlled by a strong promoter.
24. The reversible male sterile transgenic plant according to claim 21, wherein the fertility of the plant is recovered under low temperature.
25. The reversible male sterile transgenic plant according to claim 21, wherein the plant is a monocot plant.
26. The reversible male sterile transgenic plant according to claim 25, wherein the monocot plant is rice, maize, wheat, millet, sorghum or Brachypodium distachyon.
27. The reversible male sterile transgenic plant according to claim 21, wherein the plant is a dicot plant.
28. The reversible male sterile transgenic plant according to claim 27, wherein the dicot plant is Arabidopsis or Brassica species.
29. A method for preparing the reversible male sterile transgenic plant according to claim 21, comprising:
- (a) constructing a plasmid comprising the sequence of the transcription factor bHLH142 and a strong promoter; and
- (b) introducing the plasmid into a target plant.
30. The method according to claim 29, wherein the DNA sequence of bHLH142 is SEQ ID No: 1 or a DNA sequence having at least 60% similarity to SEQ ID No: 1.
31. The method according to claim 29, wherein the strong promoter is Ubiquitin promoter, CaMV 35S promoter, Actin promoter, an anther tapetum-specific promoter or a pollen-specific promoter.
32. The method according to claim 29, wherein the plasmid is introduced into calli of the target plant via Agrobacterium tumefaciens.
Type: Application
Filed: Jan 14, 2015
Publication Date: Nov 5, 2015
Applicant:
Inventors: SWEE-SUAK KO (Taipei), MIN-JENG LI (Taipei)
Application Number: 14/596,419