WO1998035550A2 - Recepteurs d'ecdysteroide d'insecte - Google Patents

Recepteurs d'ecdysteroide d'insecte Download PDF

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Publication number
WO1998035550A2
WO1998035550A2 PCT/NZ1998/000018 NZ9800018W WO9835550A2 WO 1998035550 A2 WO1998035550 A2 WO 1998035550A2 NZ 9800018 W NZ9800018 W NZ 9800018W WO 9835550 A2 WO9835550 A2 WO 9835550A2
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Prior art keywords
vector
receptor
ligand
ecdysteroid
dna
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PCT/NZ1998/000018
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English (en)
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WO1998035550A3 (fr
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Sue Qiu Huang
Allen Charles Godfrey Heath
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New Zealand Pastoral Agriculture Research Institute Limited
The New Zealand Wool Board
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Priority to AU60077/98A priority Critical patent/AU6007798A/en
Publication of WO1998035550A2 publication Critical patent/WO1998035550A2/fr
Publication of WO1998035550A3 publication Critical patent/WO1998035550A3/fr

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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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • This invention relates to insect ecdysteroid receptors.
  • it relates to ecdysteroid receptors from the Australian blowfly Lucilia cuprina.
  • Flies including blowflies, are carriers of disease affecting humans and other animals. These animals include economically important stock such as sheep, cattle, poultry and horses.
  • flystrike Of the many known species of blowfly a few are responsible for serious conditions such as flystrike. This condition is caused by the larvae of blowflies feeding on the skin of an animal. Sheep are particularly susceptible to flystrike. Flystrike losses are currently estimated to cost about $40 million annually in New Zealand alone. These losses arise through lost production in meat and wool, pelt damage, deaths and associated production and treatment costs. Lucilia cuprina, or the Australian blowfly, is the main cause of flystrike in New Zealand.
  • An alternative or additional method is chemical treatment of sheep including methods such as dipping, showering, back-line treatments and the like.
  • chemicals are subject to a number of disadvantages. Firstly, blowflies develop tolerance to chemicals over time producing insecticide resistant populations. Secondly, chemical residues pose environmental hazards from discharged dips and scour products, as well as health concerns. In particular, wool can have high insecticide residues, exposing shearers and other wool handlers to risk.
  • SUBSTITUE SHEET (Rule 26)
  • One approach to biological control requires the identification and characterisation of insect genes or gene products which may serve as suitable targets for insect control agents. Insect steroid hormones and receptors are likely candidate targets for control agents. Of particular interest are the ecdysteroids.
  • the ecdysteroids are steroid hormones which form part of the endocrine systems of insects. Ecdysones such as 20-OH ecdysone (also known as ⁇ -ecdysone) have been shown to control the timing of development in many insects including species of fly.
  • ecdysone Pulses, or rises and falls in ecdysone concentration are observed at various stages of fly development. Concentrations of the ecdysone are usually constant during a feeding and growing phase but decline during transition phases such as from egg to larva, larva to pupa and pupa to adult (Ecdysone: from Chemistry to Mode of Action; Thieme Medical Pub, N. Y. (1989)).
  • EcRE ecdysteroid response elements
  • Similar genetic regulatory hierarchies may also detemiine the metamorphic changes in development of the imaginal tissues that are also targets of ecdysone, as well as the changes in tissue development induced by the pulses of ecdysone that occur at other developmental stages.
  • SUBSTITUE SHEET (Rule 26) Various structural data have also been derived from vertebrate steroid and other lipophilic receptor proteins. A "superfamily" of such receptors has been defined on the basis of their structural similarities. See Evans, “The Steroid and Thyroid Hormone Receptor Superfamily,” Science 240: 889-895 (1988); Green and Chambon, “Nuclear Receptors Enhance Our Understanding of Transcription Regulation” Trends in Genetics 4:309-314 (1988).
  • the structure of the superfamily of receptors reveals the existence of five receptor domains, for convenience identified herein as regions A/B, C, D, E and F. These five domains include a DNA binding domain (C) and a hormone binding domain (E).
  • the DNA binding domain is configured into two "zinc-binding fingers", with a zinc ion in each finger forming a tetrahedral coordination complex with cysteine residues.
  • these superfamily receptors complexed with their respective hormones, regulate the transcription of their primary target genes, as proposed for the ecdysone receptor in the above model.
  • Ecdysteroid receptors from Drosophila melanogaster were described by Handler et al. Mol Cell Endo 63: 103-9, 1989, Koelle et al.; Cell, 67:59-77, 1991 and in US Patent Number 5,514,578.
  • Corresponding receptors from Aedes aegypti (AaEcR) and Chironomus tentans (CtEcR) have been described by Cho et al.; and Imhof et al.; in Insect Biochem Molec. Biol. 25: 19-27, 1995 and 23: 115-124, 1993 respectively.
  • Ecdysteroid receptors for the blowfly Calliphora vicina were characterized by Lehmann et al., Eur J. Biochem 181:577-82, 1989.
  • SUBSTITUE SHEET (Rule 26) The applicant has now identified an ecdysteroid receptor for the Australian blowfly Lucilia cuprina. It is broadly to this receptor that the present invention is directed.
  • the present invention may broadly be said to consist in an isolated Lucilia cuprina ecdysteroid receptor (LcEcR) polypeptide which has the amino acid sequence set out in Figure 4, or a variant thereof having substantially equivalent receptor activity thereto.
  • LcEcR Lucilia cuprina ecdysteroid receptor
  • the present invention provides an isolated peptide which comprises or consists of the amino acid sequence for one or more of receptor domains A/B, C, D, E or F as set out in Figure 4.
  • the peptide comprises the amino acid sequences for domain C or E.
  • the receptor polypeptide of the invention is obtained by expression of a DNA sequence coding therefore in a host cell or organism.
  • the present invention provides an isolated nucleic acid molecule including a receptor polypeptide or peptide of the invention.
  • This nucleic acid molecule can be an RNA or cDNA molecule but is preferably a DNA molecule.
  • Also provided by the present invention are recombinant expression vectors which contain a DNA molecule of the invention, and hosts transformed with the vector of the invention capable of expressing a polypeptide or peptide of the invention.
  • the present invention provides an inducible expression system comprising:
  • the invention provides a method of producing a polypeptide or peptide of the invention comprising the steps of:
  • SUBSTITUE SHEET (Rule 26) (a) culturing a host cell which has been transformed or transfected with a vector as defined above to express the encoded polypeptide or peptide;
  • An additional aspect of the present invention provides a ligand that binds to a polypeptide or peptide of the invention.
  • the ligand is a phytoecdy steroid or an antibody or antibody binding fragment.
  • Such ligands also form a part of this invention.
  • the present invention provides methods of assaying samples for the presence of ligands; test kits suitable for use in such assays; test kits comprising the inducible expression system of the invention, methods of modulating the development of insects by administering selected products of the invention to an insect, and compositions and agents useful in such methods.
  • a method for screening for ecdysteroid ligands comprising:
  • Figure 1 is schematic diagram depicting the cloning strategy, stmcture and function of the ecdysteroid receptor from Lucilia cuprina.
  • Figure 2 is a pileup analysis of LcEcR, AaEcR, CtEcR and DmEcR.
  • the DNA binding domain in bold between 317-382.
  • the hormone binding domain is in bold between 492- 713.
  • Degenerated primers are underlined with arrow indicating forward primers and with arrow indicating reverse primers.
  • FIG. 3 depicts the results of the 557 BP Fragment amplification of LcEcR by RT-PCR.
  • the PCR products were electrophoresed in a 1% agarose gel and 1 x TAE buffer. DNA bands were visualised by staining with EtBr.
  • Lane 1 shows Lambda phage Hind III digestion markers.
  • Lanes 2-4 are the first-run PCR products with 503 and 302 primers as a set.
  • Lanes 5-7 are the second-run PCR with 503 and 304 primers as a set.
  • Lanes 2 and 5 are positive controls using DM RNA.
  • Lanes 4 and 7 are negative controls without adding any RNA.
  • Lanes 3 and 6 are PCR products from LcRNA. Lane 6 shows a DNA band with a size of 557bp.
  • Figure 4 represents both the deduced amino acid sequence of the open reading frame of the LcEcR polypeptide of the invention and the nucleotide sequence coding therefore.
  • the domains are identified as follows:
  • the DNA and hormone binding domains (domains C and E respectively) are in bold. Primers 511 and 512 and 3'end were boxed and labelled.
  • the nuclear localisation signals (NLS) are underlined.
  • the helix-turn-zipper motif is underlined with alternating single and double lines.
  • Figure 5 depicts the results of the 1.8kb fragment amplification of LcEcR by 3'RACE.
  • the PCR products were shown in 1% agarose gel.
  • Lane 1 shows Lambda Hind III digestion markers.
  • Lane 2 is on first-run PCR product with primer 511 and PCR anchor as a set. It shows a 1.8 kb band.
  • Figure 6 depicts the results of the 2.8 kb fragment of the open reading frame of LcEcR by RT-PCR.
  • the PCR products were shown in 1% agarose gel.
  • Lane 11 shows Lambda Hind in digestion markers.
  • Lane 2 is first- run PCR product with primers ORF1 and PCR anchor as a set.
  • Lane 3 is the second run PCR product with primers ORF1 and 3 'end as a set. It shows a 2.8 kb band.
  • Figure 7 depicts the baculoviral vector system, Trichophsia ni nuclear polyhedrosis virus, TnNPV, and the vector incorporating LcEcR cDNA, TnNPV-EcR.
  • Tn-5B1 cells infected with wild-type TnNPV and TnNPV-EcR were lysed and processed and electrophoresed on a 7.5% SDS-PAGE gel. The gel was then transferred onto nitrocellulose blotting membrane. The first antibody (anti HSV-gD with 1:5000 dilution) was used and then the second antibody (anti-mouse IgG) was applied to detect HSV-gD epitope.
  • Right Lane shows Tn-5B 1 cells infected with wild-type TnNPV.
  • Left lane shows Tn-5B 1 cells infected with TnNPV-EcR.
  • EcR deficient Drosophila L57-3-11 cells were transfected with control plasmid pUC19, with the ecdysone response reporter plasmid pEcR/Adh/ ⁇ gal alone or with the reporter plasmid plus an LcEcR expression plasmid, pAct/EcR. Transfected cells were left untreated, or treated with 20-hydroxyecdysone and cell extracts were assayed for ⁇ -gal activity.
  • the present invention provides an isolated Lucilia cuprina ecdysteroid receptor (LcEcR) polypeptide which has the amino acid sequence set out in Figure 4, or a variant thereof having substantially equivalent receptor activity thereto.
  • LcEcR Lucilia cuprina ecdysteroid receptor
  • the open reading frame of the LcEcR contains 757 amino acids with a molecular weight of approximately 84 kD.
  • the deduced amino acid sequence of LcEcR exhibits a high level (up to 72%) identity to DmEcR, AaEcR, and CtEcR.
  • variant refers to a polypeptide wherein the amino acid sequence exhibits substantially 75% or greater homology with the amino acid sequence set out Figure 4.
  • the variant may be arrived at by modification of the native amino acid sequence by such modifications as insertion, substitution or deletion of one or more amino acids.
  • the invention also provides an isolated peptide which comprises or consists of the amino acid sequence for one or more of receptor domains A/B, C, D, E or F shown diagrammatically in Figure 1 and with the sequences set out in Figure 4, or fragments thereof.
  • SUBSTITUE SHEET (Rule 26) Domain C is a 66 amino acid sequence coding for the DNA binding domain.
  • the DNA binding domain comprises two C2C2 zinc fingers which are responsible for the recognition and binding of the receptor to an ecdysteroid response element. These are specific DNA sequences that are recognised by the receptors.
  • Two amino acid motifs within the DNA binding domain confer receptor specificity for a particular ecdysteroid response element. These amino acid motifs are depicted as the proximal box (P-box) and the distal box (D-box) in Figure 4.
  • the peptide of the invention comprises or consists of the amino acid sequence for the P-box and/or the D-box of domain C.
  • Domain E shown in Figure 1 comprises the 221 amino acid hormone-binding domain which lies to the carboxy-terminal side of the DNA-binding domain. The hormone binding domain is responsible for the affinity binding of the ecdysteroid hormone to the receptor.
  • LcEcR A/B domain has 2 amino acid deletions at 177, 178 positions and 3 amino acid insertions at 135,136 and 152 positions
  • the ecdysteroid receptor possesses a unique amino-terminal domain through which contact is made with other transcription factors (region A B in Figure 1), although other domains also interact with regulatory proteins.
  • the ecdysteroid receptor being the nuclear transcriptional factor, possesses a nuclear localisation signal (NLS, region D in Figure 1), which are short amino acid sequences that modulate nuclear transfer.
  • NLS nuclear localisation signal
  • MKRREKK and QKEKDKI Two potential NLS's, MKRREKK and QKEKDKI, were found in this hinge domain.
  • the peptide comprises the amino acid sequence for domain C or E.
  • SUBSTITUE SHEET (Rule 26)
  • the reader will appreciate that modifications of the polypeptides and peptides of the invention are possible. The production of peptide fragments is also well within the capabilities of an art skilled worker.
  • polypeptide and peptides of the invention can be prepared in a variety of ways. For example, they can be produced by isolation from natural source, by synthesis using any suitable known techniques (such as by stepwise, solid phase, synthesis described by Merryfield (1963), J.Amer.Chem.soc. Vol 85:2149-2156 or as preferred, through employing recombinant DNA techniques.
  • variants of both the polypeptide and peptides can similarly be made by any of those techniques known in the art.
  • variants can be prepared by site-specific mutagenesis of the DNA encoding the native amino acid sequence as described by Adelman et al. DNA 2: 183 (1983).
  • the first step is to obtain DNA encoding the desired product.
  • DNA comprises a still further aspect of this invention.
  • the DNA of the invention may encode a native or modified polypeptide or peptide of the invention or an active fragment thereof.
  • the DNA comprises at least nucleotides 901-2022 of the sequence of Figure 4, or the nucleotide sequence extending from nucleotides 901-1098 (domain C) or 1360-2022 (domain E) of the sequence of Figure 4.
  • the DNA can be isolated from any appropriate natural source or can be produced as intron free cDNA using conventional techniques. DNA can also be produced in the form of synthetic oligonucleotides where the size of the active fragment to be produced permits.
  • the Triester method of Matteucci et al. J. Am.Chem.Soc. Vol 103:3185-3191 (1981) may be employed.
  • the DNA of the invention can also code for a fusion protein comprising the polypeptide or peptide of the invention and a carrier protein.
  • This carrier protein will generally be cleavable from the polypeptide, peptide or fragment under controlled conditions. Examples of commonly employed carrier proteins are ⁇ -galactosidase and glutathione-S-transferase.
  • variants of the polypeptide or peptide which differ SUBSTITUE SHEET (Rule 26) from the native amino acid sequence by insertion, substitution or deletion of one or more amino acids.
  • the nucleotide sequence of the native DNA is altered appropriately. This alteration can be made through elective synthesis of the DNA or by modification of the native DNA by, for example, site-specific or cassette mutagenesis.
  • site-specific primer directed mutagenesis is employed using techniques standard in the art.
  • the present invention consists in replicable transfer vectors suitable for use in preparing a polypeptide or peptide of the invention. These vectors may be constructed according to techniques well known in the art, or may be selected from cloning vectors available in the art.
  • the cloning vector may be selected according to the host or host cell to be used.
  • Useful vectors will generally have the following characteristics:
  • (c) desirably, carry genes for a readily selectable marker such as antibiotic resistance.
  • a readily selectable marker such as antibiotic resistance.
  • Two major types of vector possessing these characteristics are plasmids and bacterial viruses (bacteriophages or phages).
  • Presently preferred vectors include plasmids pMOS- Blue, pGem-T and pUC8.
  • the DNA molecules of the invention may be expressed by placing them in operable linkage with suitable control sequences in a replicable expression vector.
  • Control sequences may include origins of replication, a promoter, enhancer and transcriptional terminator sequences amongst others.
  • the selection of the control sequence to be included in the expression vector is dependent on the type of host or host cell intended to be used for expressing the DNA.
  • procaryotic, yeast, insect or mammalian cells are useful hosts.
  • plasmid vectors include E. coli Bacillus species and various species of Pseudomonas. Commonly used promoters such as ⁇ -lactamase (penicillinase) and lactose (lac) promoter systems are all well known in the art. Any available promoter system compatible with the host of choice can be used. Vectors used in yeast are also available and well known. A suitable example is the 2 micron origin of replication plasmid.
  • vectors for use in mammalian cells are also well known.
  • Such vectors include well known derivatives of SV-40, adenovirus, retro virus-derived DNA sequences, Herpes simplex viruses, and vectors derived from a combination of plasmid and phage DNA.
  • the expression vectors useful in the present invention contain at least one expression control sequence that is operatively linked to the DNA sequence or fragment to be expressed.
  • the control sequence is inserted in the vector in order to control and to regulate the expression of the cloned DNA sequence.
  • useful expression control sequences are the lac system, the tr . system, the tac system, the tic system, major operator and promoter regions of phage lambda, the glycolytic promoters of yeast acid phosphatase, e.g. Pho5, the promoters of the yeast alpha-mating factors, and promoters derived from polyoma, adenovirus, retrovirus, and simian virus, e.g. the early and late promoters of SV40, and other sequences known to control the expression of genes of prokaryotic and eucaryotic cells and their viruses or combinations thereof.
  • Preferred promoters for use herein include Psyn, Pxiv and Ppolh.
  • the dual promoter Psyn and Pxiv is especially preferred as it is a much stronger promoter than any of Psyn, Pxiv or Ppolh alone.
  • the vector In the construction of a vector it is also an advantage to be able to distinguish the vector incorporating the foreign DNA from unmodified vectors by a convenient and rapid assay.
  • Reporter systems useful in such assays include reporter genes, and other detectable labels which produce measurable colour changes, antibiotic resistance and the like.
  • the ⁇ -galactosidase reporter gene is used, which gene is detectable by clones exhibiting a blue phenotype on X-gal plates. This facilitates selection.
  • the ⁇ -galactosidase gene may be replaced by a polyhedrin-encoding gene; which gene is detectable by clones exhibiting a white phenotype when stained with X-gal.
  • This blue-white color selection can serve as a useful marker for detecting recombinant
  • the vectors may be isolated from the culture using routine procedures such as freeze-thaw extraction followed by purification.
  • vectors containing the DNA of the invention to be expressed and control signals are inserted or transformed into a host or host cell.
  • Some useful expression host cells include well-known prokaryotic and eucaryotic cells.
  • Some suitable prokaryotic hosts include, for example, E.coli, such as E. coli. S G-936, E. coli HB 101, E. coli W3110, E.coli X1776, E. coli. X2282, E. coli. DHT, and E. coli. MR01, Pseudomonas.
  • Bacillus such as Bacillus subtilis. and Streptomvces.
  • Suitable eucaryotic cells include yeast and other fungi, insect, animal cells, such as COS cells and CHO cells, human cells and plant cells in tissue culture.
  • the baculovirus gene expression vector system is ideal for the expression of LcEcR as it assures appropriate folding and post-translational modifications of LcEcR.
  • the baculoviral vector system Trichoplusia ni nuclear polyhedrosis virus, TnNPV, developed in Dr. Yi Pang's lab in State Key lab for Biocontrol in Zhongshan University in China has three unique advantages: 1)
  • the recombinant virus (TnVPV-EcR, as shown in Figure 7 is designed to express both the viral polyhedrin-encoding gene and a heterologous gene such as LcEcR. These recombinants form polyhedral occlusion bodies which serve as visible markers of recombination.
  • the recombinants can also be used to facilitate oral infection of insect larvae for mass-scale protein productions.
  • the dual promoter, Psyn and Pxiv, which drives LcEcR gene expression, is a much stronger promoter compared with Psyn, Pxiv or Ppolh alone.
  • the ⁇ -galactosidase gene expressed by the parental polyhedral inclusion body minus vector, TnNPV-SVI ' G as shown in Fig.7, can be replaced by polyhedrin-encoding gene by the recombinant virus.
  • TnNPV-EcR shows white phenotype when stained with X-gal. This blue-white color selection can serve as a useful marker for detecting recombinant viruses.
  • transformation is performed according to standard techniques appropriate to such cells.
  • the calcium treatment process (Cohen, S N Proceedings, National Academy of Science, SUBSTITUE SHEET (Rule 26) USA 69 2110 (1972)) may be employed.
  • the calcium phosphate precipitation method of Graeme and Van Der Eb, Virology 52:546 (1978) is preferred. Transformations into plants may be carried out using Agrobacterium tumefaciens (Shaw et al., Gene 23:315 (1983) or into yeast according to the method of Van Solingen et al. J.Bact. ISO: 946 (1977) and Hsiao et al. Proceedings, National Academy of Science, 76: 3829 (1979).
  • polypeptide or peptide encoded can be produced, often in the form of fusion protein, by culturing the host cells.
  • the polypeptide or peptide of the invention may be detected by rapid assays as indicated above.
  • the polypeptide or peptide is then recovered and purified as necessary. Recovery and purification can be achieved using any of those procedures known in the art, for example by absorption onto and elution from an anion exchange resin. This method of producing a polypeptide or peptide of the invention constitutes a further aspect of the present invention.
  • Host cells transformed with the vectors of the invention also form a further aspect of the present invention.
  • Steroid-responsive expression of other genes is also possible using an inducible expression system.
  • the steroid receptor gene will typically be co- transfected with a recombinant construct comprising a desired gene for expression operably linked to the steroid-responsive enhancer or promoter element.
  • a single inducible expression system will typically comprise a combination of (1) a controlling element responsive to a ligand of an ecdysteroid receptor of the invention; (2) a desired gene for expression, operably linked to the controlling element; and (3) an ecdysteroid receptor which can bind to the controlling element.
  • this system will be within a cell, but an in vitro system is also possible.
  • the ecdysteroid receptor will typically be provided by expression of a nucleic acid encoding it, though it need not be expressed at particularly high levels.
  • the system will be achieved through co- transfection of a cell with both the regulatable construct and another segment encoding the ecdysteroid receptor.
  • the controlling element will be an enhancer element, but it may work in reverse and be used to repress expression.
  • the ligand for the ecdysteroid receptor will be provided or withheld as appropriate for the desired expression properties.
  • this steroid-inducible expression system could be useful for the production of large amounts of protein.
  • the production of large quantities of foreign proteins makes some cells unhealthy, which may affect unfavourably the yield of the desired protein.
  • these cells are orchestrated so that they do not express the foreign protein, and therefore are not unhealthy, until an inducing agent (e.g. steroid) is added to the growth medium.
  • an inducing agent e.g. steroid
  • a particularly useful genetic construct comprises an alcohol dehydrogenase promoter operably linked to an easily assayable reporter gene, e.g. ⁇ -galactosidase.
  • an easily assayable reporter gene e.g. ⁇ -galactosidase.
  • a multiplicity of copies of the ecdysteroid receptor is used.
  • operable linkage of controlling elements responsive to ecdysteroid receptor e.g., EcR to the alcohol dehydrogenase (ADH) promoter, or others as described above
  • ADH alcohol dehydrogenase
  • This controlling element responsive to the construct provides a very sensitive system for the detection of responsive expression. This will be used in sensitive assays for the presence of a receptor-ligand interaction, allowing for detection of either ligand or receptor or both.
  • Test kits comprising the inducible expression system of the invention also form a part of the present invention.
  • the test kits may additionally comprise agents including for example, inducing agents, reagents suitable for use with such an expression system, buffers, diluents, standards and other agents known in the art which are commonly employed in such test kits.
  • a further aspect of the present invention provides a ligand that binds to a polypeptide or peptide of the invention.
  • Ligands may be of two functional types.
  • the first functional type of ligand is a molecule which binds to the ecdysteroid receptor and stimulates it in performing its normal function (an agonist ligand).
  • the second functional type of ligand is a molecule which binds to the ecdysteroid receptor and inhibits or prevents it performing its normal function (an antagonist ligand).
  • agonists and antagonists can be used to produce insecticidal compositions which interfere with or disrupt insect development.
  • the ligand may be an antibody or antibody binding fragment raised against the polypeptide or peptide.
  • Such antibodies may be polyclonal, but are preferably monoclonal.
  • Polyclonal antibodies may be produced according to the method used by Koelle el al.; Cell 67:59-77, 1991 incorporated herein by reference. Useful antibody production protocols are outlined in US Patent 5,514,578 incorporated herein by reference. Monoclonal antibodies may be produced by methods known in the art. These methods include the immunological method described by Kohler and Milstein in Nature 256:495- 497 (1975) as well as by the recombinant DNA method described by Huse et al. Science 246: 1275-1281 (1989). Any of the assay methods detailed in US Patent 5,514,578 are also incorporated for use herein by reference.
  • the ligand may comprise molecules that bind to the polypeptide or peptide of the invention which are derived from natural sources, including plants, animals and insects. Plant extracts which produce phytoecdysteroids are of particular interest.
  • the present invention provides a method of assaying samples for the presence of ligands.
  • Assaying processes using polypeptides or peptides as a ligand binding agent or probe are well within the capacity of the art skilled worker.
  • SUBSTITUE SHEET (Rule 26) The selection of the segment to be used as a probe will allow particular functionally associated segments to be isolated. For example, if a segment of the DNA binding domain is used as a probe, other nucleic acid segments encoding other DNA binding domains will be isolated.
  • Samples of material to be screened may be prepared in the form of substrate solutions, then exposed to the ligand binding agent or probe.
  • the presence of a ligand binding agent/ligand complex may be detected according to methods also known in the art. Examples of such methods include agglutination, radio immunoassay, fluorescence or enzyme immunoassay techniques.
  • a suitable screening test is an ELISA assay. In this method of the invention it is presently preferred that the hormone binding domain be used as the ligand binding agent.
  • the steroid responsive expression system can be employed in vivo to screen ligands which have specific ecdysteroid activity by operable linkage of a reporter system to an ecdysteroid responsive element (EcRE) which binds to the ecdysteroid receptor, and where binding is functionally linked to protein induction a sensitive assay for the presence of a ligand or receptor results.
  • EcRE ecdysteroid responsive element
  • a plasmid comprising an EcRE linked with a promoter such as the alcohol dehydrogenase promoter, (ADH) and a reporter gene (for example ⁇ -galactosidase ( ⁇ -gal)) can be transfected with another plasmid encoding the ecdysteroid receptor into the insect cells.
  • the EcRE, promoter, reporter gene and ecdysteroid receptor may be present in a single plasmid, or a multiplicity of plasmids.
  • the expression of ⁇ -gal activity would be induced.
  • This functional assay can provide a sensitive way to screen ligands that bind to the ecdysteroid receptor.
  • Example 6 An example of such a functional assay is provided in Example 6 below.
  • cells which do not support ecdysone-responsive transactivation SUBSTITUE SHEET can be used.
  • some cell lines of mammalian origin such as CV1 do not support ecdysone-responsive transactivation. Therefore, the LcEcR expression vector (pAcT/EcR) and DmUSP expression vector such as CMX-usp (Oro et al., 1990, Yao et al., 1992 both incorporated herein by reference) can be cotransfected into CV1 cells.
  • the reporter plasmid pEcRE/Adh/ ⁇ gal can also be cotransfected into CV1 cells.
  • LcEcR DmUSP forms a specific complex with the EcRE in CV1 cells, transactivating the ⁇ -galactosidase reporter gene expression, ⁇ -gal activity can be readily detected with a fluorescent reagent.
  • the hormone binding domain will be used as the ligand binding agent.
  • ligand binding methods are disclosed in US 5,514,578 which is specifically incorporated herein by reference.
  • test kits suitable for use in such assays are provided.
  • An example of such a test kit is an ELISA assay test kit including a ligand binding agent of the invention.
  • a still further aspect of the present invention provides methods of modulating the development of insects.
  • a first method it is desirable to bring about the over expression of the ecdysteroid protein in insect larvae, particularly blowfly larvae, by the induction of ecdysteroid hormone or its analogues to prematurely turn on the normal developmental moulting process leading to the death of the insect.
  • Another way in which the moulting hormone activity may be disrupted is by introducing the "anti-sense" RNA acid of the ecdysteroid receptor into an insect.
  • This anti-sense RNA binds to the sense receptor RNA produced by the insect larvae, thereby blocking the receptor protein translation and further signal transduction pathways. This would delay and eventually te ⁇ ninate normal moulting stages in larvae, again leading to death.
  • Delivery of the ecdysteroid receptor gene or anti-sense RNA to the insect may be achieved by way of vectors described above, either to the larvae or adult insect.
  • SUBSTITUE SHEET (Rule 26) Dissemination through adult flies may also be achieved by placing the vector in a bait.
  • a vector in such a case may be viral. The adult will then have to be capable of transovarial transmission (Smith 1967) so that larvae would be infected through the eggs that they developed from.
  • germline transformation may be effected by transforming the DNA encoding the ecdysteroid receptor or its antisense DNA according to techniques known in the art. See for example Loukeris et al. referenced above and Oakesshott J., Molecular Approaches to Fundamental and Applied Entomology, Chapter 12: Prospects and Possibilities for Gene Transfer Techniques in Insects, 451-488.
  • the ecdysteroid receptor DNA over expression of the ecdysteroid receptor in transgenic flies should prematurely turn on different developmental stages, the intereference with such developmental stages leading to the death of the flies.
  • the antisense DNA being transformed this should result in low levels of ecdysteroid expression in the transgenic flies preventing the flies advancing to the next developmental stage, also leading to the death of the flies.
  • This transposon-mediated germline transformation will not only facilitate studies on modulation of blowfly physiology and development by the ecdysteroid receptor, but also facilitate studies and developments, on genetic sexing, sterile-insect techniques and sterilization for sterile male release programs; conferring insecticide resistance to beneficial insects; and increasing insect susceptibility to control agents already in use.
  • sterile-insect techniques currently employed are often of limited use because of the existence of large quantities of sterile females mating with sterile males and essentially cancelling out the effectiveness of sterile males mating with wild type females.
  • An alternate strategy to reduce the number of viable females would be desirable.
  • One such strategy is to use a female-specific promoter to express an L. Cuprina DNA binding domain - estrogen receptor ligand binding domain hybrid protein in females only. In the presence of estrogen, the hybrid protein should activate the L.Cuprina receptor and other ecdysone responsive genes. If the estrogen is then added to the fly nutrient medium SUBSTITUE SHEET (Rule 26) diiring early larval growth, the premature induction of the moulting process should be both female specific and lethal.
  • FIG. 1 shows a schematic diagram of the amplification strategy.
  • the first 557 bp fragment covering part of DNA and hormone binding domains of Lucilia cuprina ecdysteroid receptor (LcEcR) was amplified by RT- PCR.
  • Degenerate primers 503, 302 and 304 were derived from the regions of DNA and hormone binding domains which are highly conserved among Drosophila melanogaster ecdysteroid receptor (DmEcR, Koelle et al, 1991), Aedes aegypti ecdysteroid receptor (AaEcR, Cho et al, 1995) and Chironomus tentans ecdysteroid receptor (CtEcR, Imhof et al, 1993) sequences.
  • DmEcR Drosophila melanogaster ecdysteroid receptor
  • AaEcR Aedes aegypti ecdysteroid receptor
  • CtEcR Chironomus tentans ecdysteroid receptor
  • the forward sense primers 511 and 512 were derived from the 557 bp sequence information of LcEcR.
  • the putative full length open reading frame of LcEcR was successfully amplified by RT-PCR.
  • the degenerate forward sense primer "ORFl " was based on DmEcR and CtEcR sequence homology at the very beginning of their open reading frame.
  • the specific reverse antisense primer "3 end” was derived from the 1. 8 kilobases (kb) sequence information of LcEcR.
  • Lucilia cuprina and Drosophila melanogaster were reared by standard methods in the Wallaceville Animal Research Centre (WARC) insectary.
  • Total RNA was isolated using TRIzol LS reagent (GIBCO-BRL) with improvement on the original Chomczynski and Sacchi's method (1987).
  • SUBSTITUE SHEET (Rule 26) Four late third instar larvae ( 0.2 g, 200 ⁇ l) of L. cuprina or 40 of late third instar larvae of D. melanogaster were washed in diethylpyrocarbonate treated H 2 0 (DEPC-H 2 0) to remove sand or food debris etc. Then DEPC-H 2 0 was added up to the volume of 0.75 mis. Then 2.25 mis of TRIzol LS reagent were added into a 15 ml polypropylene tube and the mixture was homogenised with polytran (Cat. No. IKA 0594000) in an Ultia-turrax T25 homogeniser at 2/3 of maximum speed. The subsequent procedures of phase separation, RNA precipitation, RNA washing and redissolving RNA were followed as in Chomczynski and Sacchi's method.
  • RT Reverse transcription
  • PCR nested polymerase chain reaction
  • the forward sense primer 503 was based on the region of the DNA-binding domain which is conserved among members of thyroid hormone/retinoic acid receptor family.
  • the reverse antisense primers 302 and 304 were designed based on the hormone-binding domain sequences which share 70-80% homology among the same domains of DmEcR, AaEcR and CtEcR.
  • cDNA was obtained through reverse transcription with 5 ⁇ g of total RNA prepared from whole bodies of blowfly L. cuprina at the late 3rd instar larvae stage. Five ⁇ g of total RNA with 2 pmols of 302 reverse primer in DEPC-H 2 0 were added up to 11.2 ⁇ l. This mixture was heat-denatured at 100 C for 4 minutes and then quickly chilled on ice and spun down briefly. Then the rest of the following reagents were added to the above RNA and primer mixture to make up the total volume of 19 ⁇ l : 4 ⁇ l of 5 x first strand buffer,
  • SUBSTITUE SHEET (Rule 26) 2 ⁇ l of 0.1 M DTT, 1 ⁇ l of 10 mM dNTP mix and 0.8 ⁇ l (32 units) Rnase-inhibitor (Boehringer Mannheim, Cat. No. 799 017). The contents of the tube were mixed gently and incubated at 42 °C for 2 minutes. 1 ⁇ l (200 units) of Superscript II reverse transcriptase (GIBCO-BRL, Cat. No. 18064-014) was added and mixed by pipetting gently and then incubated at 42 °C for 50 min. The reaction was heat-inactivated at 70 °C for 15 min. The cDNA product was used for PCR amplification directly or stored at - 20 °C if necessary.
  • the detailed protocol is as follows: In the primer mix, 2 ⁇ l of 10 mM dNTPs (final concentration: 200 ⁇ M) were mixed with 3 ⁇ l of 10 ⁇ M forward and reverse primers (final concentration: 300 nM) and sterile redistilled JJ20 up to 48 ⁇ l.
  • cDNA product (the template mix) were added to 48 ⁇ l of the primer mix and 50 ⁇ l of the enzyme mix in a 0.2 ml thin-walled tubes.
  • Thermal conditions in a Perkin-Elmer 480 thermal cycler were as follows: one cycle at 94°C for 2 min; 35 cycles at 94°C for 15 s, 55 °C for 30 s and 72°C for 1 min.
  • 1/lOth of the PCR product was electrophoresed in a 1% agarose gel in 50 mM Trisacetate-1 mM EDTA (pH 8.0). DNA bands were visualised by staining with ethidium bromide. Lambda phage DNA digested with Hindlll was used for size measurement and quantitation.
  • the successful amplification of the 557 bp fragment covering part of the DNA and hormone binding domains provided us with tools for subsequent 3 'RACE amplification.
  • the forward primers 511 and 512 were derived from this 557 bp sequence information while reverse primers were designed taken advantages of the natural poly (A)-tail of mRNAs.
  • 3' RACE kit was purchased from Boeringer Mannheim (Cat. No. 1734 792).
  • the first strand cDNA synthesis was initiated at the poly (A)-tail of mRNA using the oligo dT-anchor primer.
  • the nested PCR was performed by 511 and PCR anchor primer as a set for the first run followed by 512 and PCR anchor primer as a set for the second run.
  • the first strand cDNA synthesis was initiated by oligo dT anchor primer (Table 2) which effectively selected for poly (A)-tailed mRNA.
  • oligo dT anchor primer Table 2 which effectively selected for poly (A)-tailed mRNA.
  • Table 2 oligo dT anchor primer
  • PCR reactions were set up in a volume of 50 ⁇ l using the 3 master mixes protocol as above, The final primer concentration and dNTP concentrations were 250 nM and 100 ⁇ M respectively.
  • the optimisation of MgCl 2 concentration was conducted with the series of MgCl 2 at 1.0 mM 1.5 mM, 2.75 mM, 4.0 mM and 5.5 mM. It was found that the final concentration of MgCl 2 at 1.5 mM resulted in the most optimal outcome.
  • the PCR thermal conditions were as follows: one cycle at 94°C for 2 min; 10 cycles at 94°C for 15 s, 65°C for 30 s and 72°C for 2 min; 25 cycles at 94°C for 15 s, 65 °C for 30 s and 72°C for 2 min + additional 20 s elongation for each cycle; one cycle 94°C for 15 s, 65 °C for 30 s and 72 °C for 7 min.
  • SUBSTITUE SHEET (Rule 26) Amplification of the 1.8 kb fragment spanning the whole hormone binding domain and the poly (A) tail by 3' RACE is shown in Figure 5.
  • the nested PCR reactions were performed with forward primer 511 and reverse PCR anchor primer as a set for the first PCR run followed by forward primer 512 and PCR anchor primer as a set for the second PCR run.
  • This 1.8 kb DNA fragment was then isolated from a 0.8 % agarose gel and subsequently purified with a Prep-A-Gene DNA purification Matrix (Bio-Rad, Cat. No. 732-6011). The ligation between this purified 1.8 kb fragment with pMOSBlue T-vector (Amersham Life Science, Cat. No.
  • RPN 1719 was conducted according to the instructions provided with the pMOSBlue T-vector kit.
  • the recombinant plasmid was designated as pLC3 and the plasmid DNA was purified and sequenced.
  • Figure 4 shows the detailed DNA sequence and its deduced amino acid sequence.
  • the 1.8 kb 3'RACE amplification enabled us to design the reverse primer 3 'end (Table 2) at the very end of the poly (A) tail from LcEcR.
  • Table 2 GCG database analysis showed that DmEcR shares a few amino acid homologies at the very beginning of the open reading frame with AaEcR, but not with CtEcR. If the beginning of the open reading frame of LcEcR shares homology with DmEcR and AaEcR, then the putative full length open reading frame of LcEcR can theoretically be amplified.
  • the degenerate forward primer ORFl (Table 2) was designed based on his homology between DmEcR and AaEcR.
  • RT-PCR was used to amplify the putative full length open reading frame of LcEcR.
  • the first stand cDNA synthesis was initiated at the poly (A)-tail of mRNA using the oligo dT-anchor primer.
  • the nested PCR was performed by ORFl primer and PCR anchor primer as a set for the first run followed by ORFl and 3' end primer as a set for the second run.
  • the first strand cDNA synthesis followed the exact conditions as for the 1. 8 kb LcEcR cDNA synthesis above.
  • PCR reactions were also set up in a volume of 50 ⁇ l using the 3 master mixes protocol as well.
  • the final primer concentration and dNTP concentration were 250 nM and 100 ⁇ M respectively.
  • the optimisation of MgC ⁇ 2 concentration was conducted with the series of MgCl 2 at 1.0 mM 1.5 mM, 2.75 mM 4.0 mM and 5.5 mM. It was found that a concentration of MgCl 2 at 2.75 mM resulted in the most optimal outcome.
  • the PCR thermal conditions were as follows: one cycle at 94 °C for 2 min; 10 cycles at 94°C for 15 s, 60°C for 30 s and 72°C for 2 min; 25 cycles at 94°C for 15 s,
  • SUBSTITUE SHEET (Rule 26) 94°C for 15 s, 60°C for 30 s and 72°C for 7 min.
  • the amplification result for the putative full length open reading frame (2.8 kb) is shown in Figure 6.
  • This 2.8 kb DNA fragment was isolated from the 0.8 % agarose gel and subsequently purified by Prep-A-Gene DNA purification Matrix. The ligation between this purified 2.8 kb fragment and pMOSBlue T-vector were conducted according to the manufacturers instructions.
  • the recombinant plasmid was designated as pEcRO and the plasmid DNA was purified and sequenced.
  • Figure 4 shows the detailed DNA sequence and its deduced amino acid sequence.
  • the baculovirus gene expression vector system is ideal for the expression of LcEcR as it assures appropriate folding and post-translational modifications of LcEcR.
  • the baculoviral vector system Trichoplusia ni nuclear polyhedrosis virus, TnNPV, developed in Dr. Yi Pang's lab in State Key lab for Biocontrol in Zhongshan University in China (Wang et al, 1991b) has three unique advantages: 1)
  • the recombinant virus (TnVPV- EcR, as shown in Figure 7 is designed to express both the viral polyhedrin-encoding gene and a heterologous gene such as LcEcR. These recombinants form polyhedral occlusion bodies which serve as visible markers of recombination.
  • the recombinants can also be used to facilitate oral infection of insect larvae for mass-scale protein production.
  • the dual promoter, Psyn and Pxiv, which drives LcEcR gene expression, is a much stronger promoter compared with Psyn, Pxiv or Ppolh alone.
  • the ⁇ -galactosidase gene expressed by the parenteral polyhedral inclusion body minus vector, EnNPV-SVI " G as shown in Figure 7, can be replaced by the polyhedrin-encoding gene in the recombinant virus.
  • the recombinant virus TnNPV-EcR shows white phenotype when stained with X-gal. This blue-white color selection can serve as a useful marker for detecting recombinant viruses.
  • SUBSTITUE SHEET (Rule 26) The baculovirus transfer plasmid pSXIWI + X3/2 was chosen as the expression vector for the 2.8 kb LcEcR cDNA subcloning since pSXIWTX3/2 contains an efficient translational initiation signal (ATG) and it simplifies the LcEcR cDNA fusion to N- terminal codons in the correct reading frame (Wang et al, 1991a).
  • the resulting plasmid pSXIVVrX3/2-EcR was cotransfected into Sf-9 cells with the parental polyhedral inclusion body minus vector, TnNPV-SVTG, which expresses the ⁇ -galactosidase gene (Summers and Smith, 1989).
  • TnNPV-SVTG the parental polyhedral inclusion body minus vector
  • TnNPV-SVTG the parental polyhedral inclusion body minus vector
  • TnNPV-SVTG which expresses the ⁇ -galactosidase gene (Summers and Smith, 1989).
  • TnNPV-LcEcR- occ+ clones were obtained with the LcEcR gene under the dual control of synthetic and XIV promoters.
  • These three TnNPV-EcR clones all formed polyhedral inclusion bodies in Sf-9 and Tn-5B1 cells and showed white phenotype when stained with X-gal.
  • Tn-5B 1 cells infected with wild-type TnNPV and TnNPV-EcR were lysed, processed and electrophoresed on a 7.5% SDS- PAGE gel. The gel was then transferred onto a nitrocellulose blotting membrane. The first antibody (anti HSV-gD with 1:5000 dilution) was used and then the second antibody (anti-mouse IgG) was applied to detect HSV-gD epitope.
  • the results are shown in Figure 8 in which the right Lane shows tN-5B 1 cells infected with wild-type TnNPV. Left lane shows tn-5Bl cells infected with TnNPV-EcR. More specifically, the left lane shows a polypeptide with a molecular weight in excess of approximately 90 kD as detected by the western blot.
  • the ecdysteroids bind to an ecdysteroid receptor (EcR) which leads to the formation of a heterodimer with the ultraspiracle protein (USP).
  • EcR ecdysteroid receptor
  • USP ultraspiracle protein
  • This complex activates developmental SUBSTITUE SHEET (Rule 26) genes which contain an ecdysteroid response element (EcRE) (Koolman, 1990, Henrich, 1995).
  • a reporter plasmid containing the ⁇ -galactosidase ( ⁇ -gal) gene controlled by a promoter containing an ecdysteroid response element (EcRE, derived from Drosophila heat shock protein 27) can be co-transferred into this line. Therefore, in the presence of 20-hydroxyecdysone, LcEcR DmUSP forms a specific complex with the EcRE, trans-activiating the ⁇ -galactosidase reporter gene expression, ⁇ -gal activity can be readily detected with chromogenic or fluorescent reagents.
  • EcRE ecdysteroid response element
  • the plasmid pAct/EcR was constructed by inserting a 2.8 kb BamH 1 fragment of LcEcR cDNA from pSXIVVI + X3.2-EcR into the BamH 1 site of pMK26 containing the Drosophila actin 5C promoter (pMK26 was kindly provided by Dr. Hogness' group) (Koelle, et al, 1991). Two plasmids (pAct/EcR and pEcRE/Adh/ ⁇ gal) were then cotransfected into L-57-3-11 cells by the non-liposomal formulation FuGene 6 transfection reagent (Boeliringer-Mannheim, Cat.
  • L57-3-11 cells were cultured in HyQ CCM3 media (HyClone, Cat. SH30061.03) with 5% bovine fetal serum. Detailed protocol approximating that in the FuGene 6 manual from Boeliringer-Mannheim was employed. We found the optimal conditions for transfecting 80% confluency L57-3- 11 cells in a 50 ml tissue culture flask to be as follows: 14 ⁇ g plasmid DNA (11.48 ⁇ g of pAct EcR: 2.1 ⁇ g of pMK43.2F) with 12 ⁇ l FuGene 6 transfection reagent.
  • ecdysone induction and ⁇ -gal activity detection were carried out as follows: 24 hours after transfection, each dish of cells was split in half: one half was treated with 2 x lO ⁇ M of 20-Hydroxyexdysone (Sigma) and another received no hormone SUBSTITUE SHEET (Rule 26) treatment. Cells were then cultured for 24 hours after the addition of the hormone. Tissue culture cells were scraped and spun down at 2000 rpm, for 10 min.
  • Cell pellets were washed once in 1 ml of PBS buffer in an eppendorf tube and then resuspended in 100 ⁇ l of freeze/thaw buffer (0.25 M sucrose, lOmM Tris (pH 704), lOmM EDTA). Cells were then repeatedly frozen in liquid nitrogen and thawed in a 37° C water bath for a total of three freeze/thaw cycles. Cell debris was removed by a 10 min centrifugation in a microcentrifuge at 4°C.
  • the concentration of protein in the supernatant (cell extract) was dete ⁇ nined by the method of Bradford (1976), with bovine serum albumin as a standard, and typically 1.5-2.5 mg/ml Extracts were assayed immediately or frozen and assayed up to 2 weeks later with no loss in activity.
  • Assay buffer 0.1 Sodium phosphate (pH 8.0), 5 mM MgCl 2 .6H 2 0, 0.6 mM 4- methylumbelliferyl- ⁇ -D-galactoside (dissolve in DMSO first and then add into assay buffer)).
  • the Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily. Cells 67, 59-77.
  • Drosophila ultraspiracle modulates ecdysone receptor function via heterodimer formation. Cell 71:63-72.

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Abstract

On a isolé, séquencé, et exprimé un récepteur d'ecdystéroïde de Lucilia cuprina. L'invention concerne des dosages permettant de détecter des ligands se fixant au récepteur. Ces ligands et récepteurs sont utiles pour moduler le développement des insectes.
PCT/NZ1998/000018 1997-02-14 1998-02-16 Recepteurs d'ecdysteroide d'insecte WO1998035550A2 (fr)

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EP1373470A2 (fr) * 2001-02-20 2004-01-02 Rohm And Haas Company Nouveaux recepteurs de mutants de substitution et utilisation de ceux-ci dans un systeme d'expression genique inductible a base de recepteurs nucleaires
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US6767721B2 (en) * 1998-11-06 2004-07-27 Nancy Wisnewski Flea ecdysone nucleic acid molecules and uses thereof
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US7935510B2 (en) 2004-04-30 2011-05-03 Intrexon Corporation Mutant receptors and their use in a nuclear receptor-based inducible gene expression system
CN103555727A (zh) * 2013-10-14 2014-02-05 中国水产科学研究院淡水渔业研究中心 一种中华绒螯蟹性早熟个体活体分子检测方法
CN103555727B (zh) * 2013-10-14 2015-06-10 中国水产科学研究院淡水渔业研究中心 一种中华绒螯蟹性早熟个体活体分子检测方法

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