WO2000034476A2 - Molecules d"acide nucleique de crevette - Google Patents

Molecules d"acide nucleique de crevette Download PDF

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WO2000034476A2
WO2000034476A2 PCT/US1999/029571 US9929571W WO0034476A2 WO 2000034476 A2 WO2000034476 A2 WO 2000034476A2 US 9929571 W US9929571 W US 9929571W WO 0034476 A2 WO0034476 A2 WO 0034476A2
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tudglv3
tudglv
tudglvl
nucleic acid
dna
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PCT/US1999/029571
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WO2000034476A3 (fr
WO2000034476A9 (fr
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Acacia Alcivar-Warren
Zhenkang Xu
Arun K. Dhar
Yongjun Fan
Dawn Meehan
Denise K. Garcia
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Trustees Of Tufts College
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Publication of WO2000034476A9 publication Critical patent/WO2000034476A9/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
    • C07K14/43509—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from crustaceans

Definitions

  • the Giant Black Tiger Prawn (Penaeus monodon Fabrizio), one of the most economically important captured and cultured shrimp species, is widely distributed in the eastern hemisphere (Lucieu-Brun, H., World Aquaculture 28, 21-33 (1997)).
  • the sustainability of P. monodon commercial fisheries as well as aquaculture industry has been threatened by overfishing, viral epizootics, environmental contamination and habitat destruction (Lucieu-Brun, H., World Aquaculture 28, 21-33 (1997)).
  • a key strategy is to prevent the loss of genetic diversity of wild shrimp and to increase the genetic variability of cultured stocks (Nlcivar-Warren, N, pp.
  • Microsatellites and ESTs have been used in various species of animals to identify quantitative trait loci (QTL).
  • Microsatellites are sequence elements of tandemly repeating nucleotides (usually 2-6 basepair repeating motifs) usually less than 100 basepairs in any one region of repeated motifs, some of which are known to flank coding genes. The high rate of mutation at microsatellite loci leads to extensive allelic variation and high levels of heterozygosity.
  • microsatellites exhibit extremely high levels of allelic variation. This attribute makes microsatellites especially attractive in a variety of research contexts, including: (1) populations that are inbred (such as those that could occur in aquaculture); (2) recently derived or geographically proximate populations in which genetic differentiation may be limited; (3) pedigree analysis and paternity assessment, such as might be employed in aquaculture or studies of variation in reproductive success among individuals.
  • Microsatellites can be assayed rapidly compared with many other types of DNA markers. High numbers of samples can be assayed readily, and because of the highly allelic nature, microsatellites can confer more information per unit assay than other marker systems.
  • An additional advantage of microsatellites is that only minute amounts of tissue are required for analysis, because microsatellites are assayed using PCR. Microsatellites are ideal markers in shrimp because they are highly variable and widely distributed in the genome, inherited in a Mendelian fashion and expressed codominantly (O'Reilly, P. et al, Journal of Fish Biology 47 (Supplement A), 29-55 (1995)). Recently, microsatellites have been isolated from P. vannamei, P. monodon and P.
  • the invention encompasses isolated nucleic acid molecules or polynucleotides having nucleotide sequences shown herein, the complements thereof, and mutants and variants thereof which are characterized by having various degrees of nucleotide sequence identity to the nucleotide sequences shown herein, such that some mutants and variants can hybridize to a given nucleic acid molecule whose sequence is shown herein, under the appropriate hybridization conditions.
  • Also embodiments of the invention are contiguous portions of the above-described nucleic acid molecules, having lengths that can be at least about 10, 12, 15, 18, 20, 25 or 30 nucleotides, for example.
  • Nucleic acid constructs such as vectors, some of which can be useful for the production of a polypeptide which can be encoded in an open reading frame of a nucleic acid molecule, are also embodiments of the invention. Further embodiments are host cells that can harbor such nucleic acid constructs, for instance, for the purposes of replication or for expression of a gene or a portion of a gene to produce a polypeptide.
  • a further embodiment is an assay to detect the presence of a first nucleic acid molecule in a sample, which may be, for example, an extract of biological materials from an animal, or a mixture of purified or partially purified RNA or DNA, or some combination thereof. The assay comprises contacting the sample with a second nucleic acid molecule, wherein the second nucleic acid molecule can comprise a nucleotide sequence described herein or a variant or segment thereof as described herein, or the complement of any of these.
  • Also described herein is a method for mapping the genome of a species of shrimp, as in the genus Penaeus.
  • the method can be carried out by, for example, determining the nucleotide sequence of an isolated shrimp DNA comprising a microsatellite and non-repeated, non-polymorphic regions flanking the microsatellite; amplifying segments of DNA, from each of templates of genomic ⁇ A -
  • DNA or cloned DNA from parental animals and form a number of their offspring with primer pairs suffiiciently complementary to the non-repeated, non-polymorphic regions flanking the microsatellite; determining the length of the amplified DNAs from each animal to establish the genotype of each animal; and determining from the distribution of genotypes in the offspring animals the frequency of segregation of parental alleles, and thus, linkage groups and map distance between loci.
  • further crosses can be carried out and the distribution of genotypes among the progeny can be analyzed to determine the map location of the microsatellites.
  • a further embodiment of the invention is a method to determine whether an individual shrimp has a genotype associated with resistance to a particular phenotypic trait, especially one that is controlled by quantitative trait loci (QTLs).
  • the method can be characterized as comprising the following steps: isolating genomic DNA from a test shrimp; amplifying a segment of DNA from the genomic DNA of the test shrimp by using primers having a sequence complementary to that of regions that flank a microsatellite marker associated with the phenotypic trait, thereby obtaining amplified DNA products from the test shrimp; determining the lengths of the amplified DNA products from the test shrimp; and comparing the length of the amplified DNA products from the test shrimp to the length of the products obtained in a similar manner from a population of shrimp known to have a certain type of the phenotypic trait, wherein if the lengths of the amplified DNA products from the test shrimp are the same as the lengths of the products obtained from a population of shrimp known to have that type of phenotypic trait
  • Also described is a method for mapping the genome of a species of Penaeus comprising determining the nucleotide sequence of an isolated shrimp DNA comprising a microsatellite and non-repeated, non-polymorphic regions flanking the microsatellite; amplifying segments of DNA, from each of templates of genomic DNA or cloned DNA from parental animals and a number of their progeny, with primer pairs complementary to the non-repeated, non-polymorphic regions flanking the microsatellite; determining the length of the amplified DNAs from each animal to establish the genotype of each animal; determining from the distribution of genotypes in the offspring animals the frequency of segregation of parental alleles, and thus, linkage groups and map distance between alleles; and performing further crosses and analyzing the distribution of genotypes to determine map location of the microsatellites.
  • Polypeptides as encoded by the nucleotide sequences of the nucleic acid molecules described herein are also embodiments of the invention.
  • Figures 1A-1F are representations of the nucleotide sequences of DNA segments cloned from Litopenaeus vannemei, containing microsatellites. Shown are the DNA sequences of DNA segments named TUDGLvl-3.49R, TUDGLvl- 3.254R, TUDGLvl-3.387F, TUDGLv3-5.237F, TUDGLv3-5.271R, TUDGLv.3- 5.342R, TUDGLv3-5.391R, TUDGLv7-9.179R, TUDGLv7-9.226R, TUDGLvl- 3.6R, TUDGLvl-3.224R, TUDGLvl-3.319R, TUDGLv3-5.235R, TUDGLv3- 5.259R, TUDGLv5-7.33R, TUDGLv7-9.17R, TUDGLvl-3.339R, TUDGLv3- 5.147F, TUDGLv3-5.356R, TUDGLvl-3.66
  • nucleic acid As used herein, "nucleic acid,” “nucleic acid molecule,” “oligonucleotide” and “polynucleotide” include DNA and RNA and chemical derivatives thereof, including phosphorothioate derivatives and RNA and DNA molecules having a radioactive isotope or a chemical adduct such as a fluorophor, chromophor or biotin (which can be referred to as a "label”).
  • the RNA counterpart of a DNA is a polymer of ribonucleotide units, wherein the nucleotide sequence can be depicted as having the base U (uracil) at sites within a molecule where DNA has the base T (thymidine).
  • Isolated polynucleotides or nucleic acid molecules can be purified from a natural source or can be made recombinantly.
  • Polynucleotides referred to herein as "isolated” are polynucleotides purified to a state beyond that in which they exist in cells. They include polynucleotides obtained by methods described herein, similar methods or other suitable methods, and also include essentially pure polynucleotides produced by chemical synthesis or by combinations of biological and chemical methods, and recombinant polynucleotides that have been isolated.
  • isolated indicates that the molecule in question exists in a physical milieu distinct from that in which it occurs in nature.
  • an isolated polynuleotide may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, and may even be purified essentially to homogeneity, for example as determined by agarose or polyacrylamide gel electrophoresis or by A 260 /A 2g0 measurements, but may also have further cofactors or molecular stabilizers (for instance, buffers or salts) added.
  • recombinant DNA contained in a vector is included in the definition of "isolated” as used herein.
  • isolated nucleic acid molecules include recombinant DNA molecules in heterologous host cells, as well as partially or substantially purified DNA molecules in solution. "Isolated" nucleic acid molecules also encompass in vivo and in vitro RNA transcripts of the DNA molecules of the present invention.
  • the invention further provides variants of the isolated nucleic acid molecules of the invention.
  • variants can be naturally occurring, such as allelic variants (same locus), homologs (different locus), and orthologs (different organism), or may be constructed by recombinant DNA methods or by chemical synthesis.
  • non-naturally occurring variants can be made using well-known mutagenesis techniques, including those applied to polynucleotides, cells, or organisms.
  • variants can contain nucleotide substitutions, deletions, inversions and/or insertions in either or both the coding and non-coding region of the nucleic acid molecule. Further, the variations can produce both conservative and non-conservative amino acid substitutions.
  • variants have a substantial identity with a nucleic acid molecule described by sequence herein and the complements thereof.
  • nucleic acid molecules and fragments which have at least about 60%, preferably at least about 70, 80 or 85%. more preferably at least about 90%o, even more preferably at least about 95%. and most preferably at least about 98% identity with nucleic acid molecules described herein.
  • percent identity of two nucleotide or amino acid sequences can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence).
  • the length of a sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 60%, and even more preferably at least 70%o, 80% or 90% of the length of the reference sequence.
  • the actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm. A preferred, non-limiting example of such a mathematical algorithm is described in Karlin et al, Proc. Natl. Acad. Sci.
  • a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the CGC sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12 , and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include
  • the percent identity between two amino acid sequences can be accomplished using the GAP program in the CGC software package (available at http://www.cgc.com) using either a Blossom 63 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4.
  • the percent identity between two nucleic acid sequences can be accomplished using the GAP program in the CGC software package (available at http://www.cgc.com), using a gap weight of 50 and a length weight of 3.
  • the present invention also provides isolated nucleic acids that contain a fragment or portion that hybridizes under highly stringent conditions to a polynucleotide having a nucleotide sequence reported herein and the complements of these polynucleotides.
  • the nucleic acid has a portion of a nucleotide sequence shown herein or a portion of its complement.
  • the nucleic acid fragments of the invention are at least about 15, preferably at least about 18, 20, 23 or 25 nucleotides, and can be 30, 40, 50, 100, 200 or more nucleotides in length, depending on the length of the fragments as shown herein.
  • nucleotide sequences described herein can also be contigged to produce longer sequences (see, for example, http://bozeman.mbt.washington.edu phrap.docs/phrap.html).
  • the nucleic acid fragments of the invention are used as probes or primers in assays such as those described herein.
  • Probes are oligonucleotides that hybridize in a base-specific manner to a complementary strand of nucleic acid. Such probes include polypeptide nucleic acids, as described in Nielsen et al, Science, 254, 1497-1500 (1991).
  • a probe comprises a region of nucleotide sequence that hybridizes under highly stringent conditions to at least about 15, typically about 20-25, and more typically about 40, 50 or 75 consecutive nucleotides of a nucleic acid whose sequence is presented herein and the complements thereof.
  • the probe further comprises a label, e.g., radioisotope, fluorescent compound, enzyme, or enzyme co-factor. Allele: One of several alternative forms of a gene occupying a give locus on a chromosome.
  • Heterozygosity The fraction of individuals that have different alleles at a particular locus (as opposed to two copies of the same allele). Heterozygosity is usually expressed in percent. Heterozygosity values range from 0 to 100%).
  • Hybridization The process in which a strand of nucleic acid non-covalently joins with a complementary strand through base pairing.
  • Informativeness The informativeness of a DNA polymorphism is a measure of the utility of the polymo ⁇ hism. In general, higher informativeness means greater utility. Informativeness is usually defined in terms of either heterozygosity or Polymorphism Information Content (PIC).
  • PIC Polymorphism Information Content
  • a linkage group includes all loci that can be connected (directly or indirectly) by linkage relationships; equivalent to a chromosome.
  • a map distance is measured as cM (centiMorgans) and is determined by the percent recombination observed between loci (sometimes subject to adjustments).
  • PIC Polymorphism information content
  • Polymo ⁇ hism The simultaneous occurrence in the population of genomes showing allelic variations (as seen either in alleles producing different phenotypes or, for example, in changes in DNA affecting the restriction pattern or the length of a microsatellite resulting from a different number of repeats of a basic sequence unit.
  • the abundance of microsatellites in eukaryotic genomes makes them easily isolated by cloning methodologies. Briefly, genomic DNA of the shrimp can be digested in separate reactions by restriction endonucleases that recognize 4-5 bp sequences.
  • the DNA from these reactions is size fractionated by gel electrophoresis and restriction fragments of 300-800 bp are excised from the gel, purified and then cloned into an appropriate vector such as the plasmid pUC18/19 or the bacteriophage Ml 3.
  • an appropriate vector such as the plasmid pUC18/19 or the bacteriophage Ml 3.
  • the size-selected library is screened using radiolabelled oligonucleotides corresponding to common microsatellite repeat motifs, such as (GT) n or (GA) ⁇ . Double-stranded DNA from positively hybridizing clones is sequenced.
  • forward and reverse sequencing primers can determine the complete nucleotide sequence of the cloned DNA in two sequencing reactions. Once the sequence of a microsatellite locus is known, primers are designed for the non-repetitive, non-polymo ⁇ hic flanks, and PCR amplification of the locus can proceed.
  • a polymo ⁇ hic DNA marker based on length variations in blocks of microsatellite repeats involves a series of steps. First, the sequence of a segment of DNA containing the microsatellite repeats must be determined. This is accomplished most commonly by selecting a genomic DNA clone through hybridization to synthetic DNA comprising the microsatellite repeats and then subsequently sequencing that clone. This same step can also be accomplished simply by selecting a suitable sequence from the literature or from one of the DNA sequence databases such as GenBank, if such sequences are available.
  • a pair of appropriate primers can be synthesized which are at least partially complementary to non-repeated, non- polymo ⁇ hic sequences which flank the block of microsatellite repeats. These sequences are not satellite sequences, and can be unique in the genome, if chosen to be sufficiently long.
  • PCR polymerase chain reaction
  • PCR uses an exponential process of replication. PCR permits a target sequence of DNA to be multiplied as quickly as a millionfold within hours.
  • a target sequence of double-stranded DNA is denatured into single-stranded form by a process of heating.
  • Two small pieces of synthetic DNA each complementing a sequence at one end of the target sequence, serve as primers and bind with their complementary sequences on the single strand.
  • Polymerases start at each primer, copying the sequence of that strand and ultimately producing exact replicas of the target sequence.
  • the product of each cycle then serves as a template for succeeding cycles, resulting in an exponential process of replication.
  • the pool of pieces of DNA with the target sequence has been greatly multiplied. This amplified genetic material is then available for further analysis and use.
  • the DNA is preferably labeled during the amplification process by inco ⁇ orating radioactive nucleotides, for example.
  • the DNA so amplified can be used in screening a library of DNA by hybridization (cosmid, phage, YAC or BAC, for example) for the presence of microsatellites.
  • In situ hybridization can also be used to map the newly generated microsatellite marker loci to the shrimp chromosomes. See R. Fries, Animal Genetics 24:111-116 (1993) for a discussion of microsatellite mapping and other methods of genome mapping.
  • the amplified DNA is resolved by polyacrylamide or agarose gel electrophoresis in order to determine the sizes of these fragments (usually by comparison with size markers) and hence the genotypes of the genomic DNA donor.
  • Some or all of the polynucleotide primers can be 32 P or 35 P labeled in any conventional manner, such as end labeling, interior labeling, or post reaction labeling.
  • Alternative methods of labeling are also within the contemplation of the invention such as biotin labeling or enzyme labeling (Matthews and Kricka 1988, Anal Biochem. 169:1-25).
  • the practical outer limits of the length of the amplified DNA fragment are generally limited only by the resolving power of the particular separation system employed.
  • Thin, denaturing polyacrylamide gels that can be used to separate amplified DNA fragments are capable of resolving fragments differing by as little as 2 basepairs up to a total fragment length of about 300 bp.
  • Use of longer gels and longer electrophoresis times can extend the resolving power up to perhaps 600 bp or even more. However the longer the fragment the lower the proportion of its length will be made up of the microsatellite sequences, and hence the more difficult the resolution.
  • Species homologs of the disclosed polynucleotides and proteins are also provided by the present invention.
  • a "species homologue" is a protein or polynucleotide with a different species of origin from that of a given protein or polynucleotide, but with significant sequence similarity to the given protein or polynucleotide, as determined by those of skill in the art.
  • Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.
  • the invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally-occurring alternative forms. Variants include mutants differing by the addition, deletion or substitution of one or more residues, modified nucleic acids in which one or more residues are modified (e.g., DNA or RNA analogs), and mutants comprising one or more modified residues.
  • a polynucleotide variant can also encode a polypeptide (which may have activity) which is identical, homologous, or related to that encoded by the "non-variant" polynucleotide.
  • the invention also includes polynucleotides with sequences complementary to those of polynucleotides disclosed herein.
  • the present invention also includes polynucleotides capable of hybridizing under reduced stringency conditions, more preferably stringent conditions, and more preferably highly stringent conditions, to polynucleotides described herein.
  • stringency conditions are shown in the tables below: highly stringent conditions are at least as stringent as, for example, conditions A-F (Table 1); stringent conditions are at least as stringent as, for example, conditions G-L (Table 2); and reduced stringency conditions are at least as stringent as, for example, conditions M-R (Table 3).
  • the hybrid length is that anticipated for the hybridized region(s) of the hybridizing polynucleotides.
  • the hybrid length is assumed to be that of the hybridizing polynucleotide.
  • the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity.
  • SSPE 0.15 M NaCl, 10 mM NaH 2 PO 4 , and 1.25 mM EDTA, pH 7.4
  • SSC 0.15 M NaCl and 15 mM sodium citrate
  • T m melting temperature
  • each such hybridizing polynucleotide has a length that is at least
  • sequence identity is determined by comparing the sequences of the hybridizing polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • an isolated polynucleotide may be operably linked to an expression control sequence such as those disclosed in Kaufman et al, Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce a polypeptide recombinantly.
  • an expression control sequence such as those disclosed in Kaufman et al, Nucleic Acids Res. 19, 4485-4490 (1991)
  • Many suitable expression control sequences are known in the art.
  • General methods of expressing recombinant polypeptides are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990).
  • operably linked means that a polynucleotide and an expression control sequence are situated within a vector or cell in such a way that the polypeptide is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.
  • Expressed sequence tags are cDNA fragments that can be used to detect, identify, locate, map and isolate complete genes (that is coding regions as well as flanking regions that may, with interacting proteins and or RNA, provide a means of regulating expression of genes.
  • Genes isolatable by using ESTs can be used to produce enzymes or other proteins having utilities corresponding to those of characterized homologs or orthologs, and can be used in the production of host cells comprising recombinant polypeptide genes using methods familiar to persons of skill in the art.
  • a nucleic acid molecule, or sufficient portions thereof, whether isolated and/or recombinant or synthetic, including fragments produced by PCR, can be used to detect and or recover genes related by sequence similarity from the species that the nucleic acid was originally derived from, or from other related species (e.g., as probes for hybridization or primers for PCR, or in other suitable techniques), from genomic DNA, from an ordered cosmid library or from other suitable sources (e.g., libraries constructed in bacteriophages and plasmids), according to suitable methods.
  • a nucleic acid described herein, or a sufficient portion thereof can be used to detect, identify, locate, map and isolate partial or complete genes from other species.
  • the identification of additional genes sharing sequence similarity can be accomplished by an extension of the methods used to clone ESTs. Pairs of degenerate oligonucleotides that were successfully used in a PCR amplification to identify ESTs can be used in PCRs using the reaction conditions described herein or other suitable conditions. Primer pairs can be produced based upon DNA sequence information from the ESTs. It is expected that they can be used to amplify a PCR product using template nucleic acid from other closely related species, as the genes are expected to be most closely related in DNA sequence to each other within a genus.
  • the sequence information generated for one gene can also be used to design more accurately biased degenerate primers, which can be used alone or in combination with other primers for amplifying ortholog genes of other species.
  • the following exemplary PCR reaction conditions can be used: 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 2.5 mM MgCl 2 , 200 ⁇ M each dinucleotide triphosphate (dNTP), 10-30 ng of genomic or library DNA of the shrimp species, 100 pmole of each of the primers, and 2.5 units of Taq polymerase (Boehringer Mannheim).
  • Cycling conditions can be, for example, 30 cycles of denaturing at 95°C for 30 seconds, annealing at 50°C for 30 seconds, and elongation at 72°C for 2 minutes. If it is found that lower stringency is needed, lower annealing and washing temperatures can be used, such as 40-45°C. If extraneous PCR fragments are produced under these conditions, higher stringency conditions can be used (for example, by raising the annealing and washing temperatures to about 55°C or higher as needed) to eliminate any artifactual PCR products.
  • a fragment of a gene can be sequenced, and the sequence of the product can be compared to other DNA sequences, for example, by using the BLAST Network Service at the National Center for Biotechnology Information. The boundaries of an open reading frame can then be identified using semi-specific PCR or other suitable methods such as library screening. Once the 5' initiator methionine codon and the 3' stop codon have been identified, a PCR product encoding a full-length gene can be generated using genomic DNA as a template, with primers complementary to the extreme 5' and 3' ends of the gene or to their flanking sequences. The full-length genes can then be cloned into expression vectors for the production of functional proteins.
  • Genes of a species of shrimp or portions thereof can be used as probes to identify DNA fragments encoding the corresponding gene from other species of shrimp or from less related species by specific hybridization (e.g., by Southern blot). It is predicted that the genes encoding orthologs from other shrimp species have a high degree of similarity to the originally isolated "probe" gene.
  • a systematic, stepwise series of washes of the Southern blot filter can be done in order of increasing stringency conditions, from low to high, as described below.
  • a filter can be prepared bound with fragmented DNA from the species of interest as well as with DNA from P. vannemei (as a positive control) and DNA from a suitable species that is not closely related (e.g., yeast or rat, as a negative control).
  • the filter can be probed with the radioactively labeled full-length or partial P. vannemei gene under medium stringency conditions, such as 37°C for 16 hr in 50% formamide, 5x SSC, IX Denhardt's solution, 0.1% sodium dodecyl sulfate (SDS), and 100 ⁇ g/ml sheared salmon sperm DNA.
  • the probed blot can then be washed with wash buffers of increasing stringency, with monitoring for decreasing background while maintaining a positive signal (presence of a band at the expected molecular weight).
  • An example of the progression of wash buffers is: 2x SSC/0.1% SDS at 37°C (low stringency wash), then lx SSC/0.1% SDS at 37°C (or 42°C), then finally 0.2x SSC/0.1 % SDS at 42°C (moderate stringency wash).
  • Each wash can be followed by monitoring the signal to noise ratio, e.g., with a Geiger counter.
  • washing can be terminated and the blot exposed to X-ray film.
  • P. vannemei genes or gene fragments when used as probes, can hybridize to the corresponding genes from other organisms within the genus Penaeus, using 0.2x SSC/0.1% SDS wash buffer at a temperature of60°C - 65°C.
  • primer refers to a single-stranded oligonucleotide which acts as a point of initiation of template-directed DNA synthesis using well-known methods (e.g., PCR, LCR) including, but not limited to those described herein.
  • the appropriate length of the primer depends on the particular use, but typically ranges from about 15 to 30 nucleotides.
  • primer site refers to the area of the target DNA to which a primer hybridizes.
  • primer pair refers to a set of primers including a 5' (upstream) primer that hybridizes with the 5' end of the nucleic acid sequence to be amplified and a 3' (downstream) primer that hybridizes with the complement of the sequence to be amplified.
  • nucleic acid molecules of the invention such as those described herein can be identified and isolated using standard molecular biology techniques and the sequence information provided herein.
  • nucleic acid molecules can be amplified and isolated by the polymerase chain reaction using synthetic oligonucleotide primers designed based on one or more of the sequences provided by nucleotide sequence herein and the complements thereof See generally PCR Technology Principles and Applications for DNA Amplification (ed H A Er ch, Freeman Press, NY, NY, 1992), PCR Protocols A Guide to Methods and Applications (Eds Innis, et al . Academic Press, San Diego, CA, 1990), Mattila et al , Nucleic Acids Res , 19 4967 (1991), Eckert et al , PCR Methods and
  • nucleic acid molecules can be amplified using cDNA, mRNA or genomic DNA as a template, cloned into an approp ⁇ ate vector and characte ⁇ zed by DNA sequence analysis
  • suitable amplification methods include the hgase chain reaction
  • the amplified DNA can be radiolabelled and used as a probe for screemng, for example, a cDNA library
  • Corresponding clones can be isolated, DNA can obtained following in vivo excision, and the cloned insert can be sequenced m either or both o ⁇ entations by art recognized methods to identify the correct reading frame encoding a protein of the appropnate molecular weight
  • the direct analysis of the nucleotide sequence of nucleic acid molecules of the present invention can be accomplished using well-known methods that are commercially available See, for example, Sambrook et al , Molecular Cloning, A Laboratory Manual (2nd Ed , CSHL, New York 1989), Zyskind et al , Recombinant DNA Laboratory Manual, (Acad Press, 1988))
  • Antisense nucleic acids of the invention can be designed using the nucleotide sequences given herein, and constructed using chemical synthesis and enzymatic gation reactions using procedures known m the art
  • an antisense nucleic acid e g , an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, and 2,6-diaminopurine.
  • antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest.
  • nucleic acid molecules of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorganic & Medicinal Chemistry, 4:5).
  • peptide nucleic acids or "PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • PNAs The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA, 95:14670.
  • PNAs can be further modified, e.g., to enhance their stability, specificity or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • the polynucleotides provided by the present invention can be used by the research community for various pu ⁇ oses.
  • the polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively of at a particular stage of tissue differentiation or stage of development or in disease states); as molecular weight markers on Southern gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in individual animals to identify potential genetic disorders; as probes to hybridize and thus discover related DNA sequences; as a source of information to derive PCR primers for genetic fmge ⁇ rinting; as a probe to "subtract-out" known sequences in the process of discovering other novel polynucleotides; and for selecting and making oligomers for attachment to a "gene chip” or other support, including for examination of expression patterns.
  • the polynucleotide encodes a polypeptide or protein which binds or potentially binds to another polypeptide or protein (such as, for example, in a receptor-ligand interaction)
  • the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al, Cell 75:791:803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.
  • a vector of the invention has a polynucleotide of the invention inserted in a sense or antisense orientation.
  • a vector refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector is another type of vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • expression vectors are capable of directing the expression of genes to which they are operably linked.
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors).
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) that serve equivalent functions.
  • Expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein .
  • the recombinant expression vectors of the invention can be designed for expression of a polypeptide of the invention in prokaryotic or eukaryotic cells, e.g., bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, supra.
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three pu ⁇ oses: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson (1988) Gene, 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase
  • GST maltose E binding protein
  • protein A protein A
  • E. coli expression vectors examples include pTrc (Amann et al, (1988) Gene, (59:301-315) and pET 1 Id (Studier et al, Gene Expression Technology: Methods in Enzymology, 185, Academic Press, San
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid t ⁇ -lac fusion promoter.
  • Target gene expression from the pET l id vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl).
  • This viral polymerase is supplied by host strains BL21(DE3) or
  • HMS174(DE3) from a resident ⁇ prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
  • the expression vector is a yeast expression vector.
  • yeast S. cerevisiae examples include pYepSecl (Baldari et al. (1987) EMBO J., (5:229-234), pMFa (Kurjan and Herskowitz. (1982)
  • a nucleic acid of the invention can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al (1983) Mol. Cell Biol, 5:2156-2165) and the pVL series (Lucklow and
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed (1987) Nature, 329:840) and pMT2PC
  • the expression vector's control functions are often provided by viral regulatory elements.
  • promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook et al. , supra.
  • host cell and "recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • a nucleic acid of the invention can be expressed in bacterial cells (e.g., E. coli), insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • bacterial cells e.g., E. coli
  • insect cells e.g., yeast or mammalian cells
  • mammalian cells such as Chinese hamster ovary cells (CHO) or COS cells.
  • Other suitable host cells are known to those skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (supra), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that nucleic acid of the invention or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have inco ⁇ orated the selectable marker gene will survive, while the other cells die).
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a polypeptide of the invention encoded in a an open reading frame of a polynucleotide of the invention.
  • the invention further provides methods for producing a polypeptide using the host cells of the invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a polypeptide of the invention has been introduced) in a suitable medium such that the polypeptide is produced.
  • the method further comprises isolating the polypeptide from the medium or the host cell.
  • the host cells of the invention can also be used to produce non-human transgenic animals.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which a nucleic acid of the invention have been introduced.
  • Such host cells can then be used to create non-human transgenic animals in which exogenous nucleotide sequences have been introduced into their genome or homologous recombinant animals in which endogenous nucleotide sequences have been altered.
  • Such animals are useful for studying the function and/or activity of the nucleotide sequence and polypeptide encoded by the sequence and for identifying and/or evaluating modulators of their activity.
  • transgenic animal is a non-human animal, in which one or more of the cells of the animal includes a transgene.
  • Other examples of transgenic animals include sheep, dogs, cows, goats, chickens, amphibians, fish, mollusks, etc.
  • a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • homologous recombinant animal is a non-human animal in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
  • a number of types of cells may act as suitable host cells for expression of a polypeptide encoded by an open reading frame in a polynucleotide of the invention.
  • Mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
  • Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe,
  • Kluyveromyces strains Candida, or any yeast strain capable of expressing heterologous proteins.
  • Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis,
  • Salmonella typhimurium or any bacterial strain capable of expressing heterologous polypeptides. If the polypeptide is made in yeast or bacteria, it may be necessary to modify the polypeptide produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain a functional polypeptide, if the polypeptide is of sufficient length and conformation to have activity. Such covalent attachments may be accomplished using known chemical or enzymatic methods.
  • a polypeptide may also be produced by operably linking an isolated EST to suitable control sequences in one or more insect expression vectors, and employing an insect expression system.
  • Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac® kit), and such methods are well known in the art.
  • an insect cell capable of expressing a polynucleotide of the present invention is "transformed.”
  • a polypeptide may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein.
  • the resulting expressed polypeptide or protein may then be purified from such culture (e.g., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography.
  • the purification of the polypeptide or protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A- agarose, heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffmity chromatography.
  • a polypeptide or protein may also be expressed in a form which will facilitate purification.
  • it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits for expression and purification of such fusion proteins or fusion polypeptides are commercially available from New England BioLabs (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively.
  • a polypeptide can also be tagged with an epitope and can subsequently be purified by using specific antibody directed to such epitope.
  • One such epitope (“Flag") is commercially available from Kodak (New Haven, CT).
  • RP-HPLC reverse-phase high performance liquid chromatography
  • hydrophobic RP-HPLC media e.g., silica gel having pendant methyl or other aliphatic groups
  • Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant polypeptide.
  • the protein or polypeptide thus purified is substantially free of other proteins or polypeptides and is defined in accordance with the present invention as "isolated.”
  • Polypeptides or proteins of the present invention can be used as molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns using art-recognized methods.
  • the polypeptides of the present invention can be used to raise antibodies or to elicit an immune response.
  • the polypeptides can also be used as a reagent, e.g., a labeled reagent, in assays to quantitatively determine levels of the protein or a molecule to which it binds (e.g., a receptor or a ligand) in biological fluids.
  • the polypeptides can also be used as markers for tissues in which the co ⁇ esponding protein is preferentially expressed, either constitutively, during tissue differentiation, or in a diseased state.
  • the polypeptides can be used to isolate a co ⁇ esponding binding partner, e.g., receptor or ligand, such as, for example, in an interaction trap assay, and to screen for peptide or small molecule antagonists or agonists of a binding interaction.
  • the invention provides antibodies to the polypeptides and polypeptide fragments of the invention, e.g., having an amino acid encoded by a nucleic acid comprising all or a portion of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-49, for example, or other nucleic acids described herein.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen.
  • a molecule that specifically binds to a polypeptide of the invention is a molecule that binds to that polypeptide or a fragment thereof, but does not substantially bind to other molecules in a sample, e.g., a biological sample, which naturally contains the polypeptide.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab') 2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies that bind to a polypeptide of the invention.
  • Polyclonal antibodies can be prepared by immunizing a suitable subject with a desired immunogen, e.g., polypeptide of the invention or fragment thereof.
  • a desired immunogen e.g., polypeptide of the invention or fragment thereof.
  • the antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules directed against the polypeptide can be isolated from the animal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature, 25(5:495-497 '.
  • the technology for producing hybridomas is well known (see generally Current Protocols in Immunology (1994) Coligan et al. (eds.) John Wiley & Sons, Inc., New York, NY).
  • an immortal cell line typically a myeloma
  • lymphocytes typically splenocytes
  • the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds a polypeptide of the invention.
  • An exemplary method for detecting the presence or absence of proteins or nucleic acids of the invention in a biological sample involves obtaining a biological sample and contacting the biological sample with a compound or an agent capable of detecting the protein, or nucleic acid (e.g., mRNN genomic D ⁇ A) that encodes the protein, such that the presence of the protein or nucleic acid is detected in the biological sample.
  • a preferred agent for detecting mR ⁇ A or genomic D ⁇ A is a labeled nucleic acid probe capable of hybridizing to mR ⁇ A or genomic D ⁇ A sequences described herein.
  • the nucleic acid probe can be, for example, a full-length nucleic acid, or a portion thereof, such as an oligonucleotide of at least 10, 12, 15, 18.
  • nucleic acid probe can be all or a portion of a polynucleotide whose sequence is described herein or the complement of such polynucleotide.
  • suitable probes for use in the assays of the invention are described herein.
  • a method for assaying for the presence of a first nucleic acid molecule in a sample can comprise contacting the sample with a second nucleic acid molecule having a nucleotide sequence selected from the group consisting of a) SEQ ID NO:l; b) the complement of SEQ ID NO:l; c) a portion of SEQ ID NO:l which is at least 10 nucleotides in length; and d) a portion of the complement of SEQ ID NO:l which is at least 10 nucleotides in length, under conditions appropriate for specific hybridization.
  • biological sample is intended to include tissues, cells and biological fluids isolated from a subject animal, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect mRNA, protein, or genomic DNA of the invention in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations.
  • in vitro techniques for detection of protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • In vitro techniques for detection of genomic DNA include Southern hybridizations.
  • in vivo techniques for detection of protein include introducing into a subject a labeled anti-protein antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the biological sample contains protein molecules from the test subject.
  • the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.
  • the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting protein, mRNA, or genomic DNA of the invention, such that the presence of protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of protein, mRNA or genomic DNA in the control sample with the presence of protein, mRNA or genomic DNA in the test sample.
  • Methods can be used to detect genetic alterations in genes or nucleic acid molecules of the present invention.
  • the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding a particular protein, or the mis-expression of the gene.
  • such genetic alterations can be detected by ascertaining the existence of at least one of (1) a deletion of one or more nucleotides; (2) an addition of one or more nucleotides; (3) a substitution of one or more nucleotides, (4) a chromosomal rea ⁇ angement; (5) an alteration in the level of a messenger RNA transcript; (6) abe ⁇ ant modification, such as of the methylation pattern of the genomic DNA; (7) the presence of a non-wild type splicing pattern of a messenger RNA transcript; (8) a non-wild type level; (9) allelic loss; and (10) inappropriate post-translational modification.
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from an animal, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to the gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and compa ⁇ ng the length to a control sample.
  • nucleic acid e.g., genomic, mRNA or both
  • PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
  • Alternative amplification methods include: self sustained sequence replication (Guatelli, J.C. et al (1990) Proc. Natl. Acad. Sci. USA, 57:1874-1878), transcriptional amplification system (Kwoh, D.Y. et al, (1989) Proc. Natl Acad. Sci. USA, 86:1173-1177), Q-Beta Replicase (Lizardi, P.M.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence the gene and detect mutations by comparing the sequence of the gene from the sample with the co ⁇ esponding wild-type (control) gene sequence.
  • Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert ((1997) PNAS, 74:560) or Sanger ((1977) PNAS, 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized.
  • Other methods for detecting mutations include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science, 230:1242).
  • RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S 1 nuclease to enzymatically digest the mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation.
  • control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in cDNAs obtained from samples of cells.
  • DNA mismatch repair enzymes
  • the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis, 75:1657-1662).
  • a probe based on an nucleotide sequence of the invention is hybridized to a cDNA or other DNA product from a test cell(s).
  • the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Patent No. 5,459,039.
  • alterations in electrophoretic mobility can be used to identify mutations in genes.
  • SSCP single strand conformation polymo ⁇ hism
  • Single-stranded DNA fragments of sample and control nucleic acids will be denatured and allowed to renature.
  • the secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments can be labeled or detected with labeled probes.
  • the sensitivity of the assay can be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al (1991) Trends Genet., 7:5).
  • kits for detecting the presence of proteins or nucleic acid molecules of the invention in a biological sample can comprise a labeled compound or agent capable of detecting protein or mRNA in a biological sample; means for determining the amount of in the sample; and means for comparing the amount of in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect protein or nucleic acid. Microsatellites can be used as a measure of the level of heterozygosity, and hence, genetic diversity level in a population to be used in a breeding program, for example.
  • Microsatellites can also be used in a method for testing an animal for a particular genetic trait, including those traits which can be classified as quantitative trait loci (QTL).
  • QTL quantitative trait loci
  • WSSV White Spot Syndrome Virus
  • TSV Taura Syndrome Virus
  • growth performance Resistance to TSV has been associated with low growth rate.
  • Methods for mapping the species of shrimp discussed herein are also embodiments of the invention. Because of similarities in the structure and a ⁇ angement of genes, and similarities in DNA sequence among species of shrimp, methods of mapping the shrimp genome are not confined to those that employ a set of microsatellite alleles to produce mapping data within only one species. Microsatellite markers of one species of shrimp can be used in methods to produce mapping data of another species of shrimp, for example, Penaeus. For example, PCR primers designed based on the sequences flanking microsatellite markers discovered in P. monodon can be used to also amplify microsatellites at co ⁇ esponding loci of P. stylirostris, L. vannemei and P. chinensis.
  • initial steps include those to identify microsatellite loci.
  • An appropriate oligonucleotide containing a repeated sequence can be used as a probe to hybridize to a library or to clones, to identify those clones containing a microsatellite.
  • DNA sequencing methods can then be used to determine the nucleotide sequence across the repeat region and into flanking regions of non-microsatellite sequence.
  • An oligonucleotide sequence unique to the clone should be chosen for the production of a primer, for each region flanking the microsatellite.
  • primer oligonucleotides sufficient to amplify DNA by PCR or by LCR are known in the art. It is best to choose primer sequences that are as close in to the repeated region as possible; it is easier to detect differences in the length of PCR or other amplified products, the shorter the product.
  • the length of the amplified DNA products can be determined after separation and staining of the DNA on polyacrylamide or agarose gels, for example. DNA products can be detected by a variety of means known in the art, such as by ethidium bromide staining or by autoradiography of the products formed with radioactively labeled primers or substrate dNTPs.
  • Markers can be used in a variety of methods to create a map of a genome of a species of shrimp. See, for examples of methods of genome mapping, including crosses and inte ⁇ retations of data generated from those crosses, L.M. Silver, Mouse Genetics: Concepts and Applications, Oxford University Press, New York, 1995, especially chapters 9 and 10. See also Rafalski, J.A. and T.V. Tingey, TIG 9:275-279 (March, 1993) and Poompuang, S. and E.M. Hallerman, Reviews in Fisheries Science 5(3):253-211 (1997). EXAMPLES
  • Example 1 Microsatellites of P. monodon
  • Genomic DNA was extracted from tail muscle of P. monodon (Garcia, D.K., et al, Molecular Marine Biology and Biotechnology 5, 71-83 (1996)) and size fractionated libraries were constructed as described earlier (Garcia D.K., et al, Proceedings of the XXVth International Conference on Animal Genetics, 21-25 July, Tours, France, Abstract C061, p. 113 (1996)) with some modifications. Briefly, Sau3A I digested genomic DNA of 200-400 bp and 400-600 bp were eluted from the agarose (0.8%>) gel using Spin-X column (Costar, MA) and ligated to BamEl digested pBluescript II SK+ (Stratagene, CA).
  • target (17-200 ng) or vector (100-400 ng) DNA was dephosphorylated using calf intestine alkaline phosphatase (Gibco BRL, MD), whereas for the 200-400 bp library only the target DNA (50-300 ng) was dephosphorylated.
  • Recombinant clones were sequenced at the Tufts University Sequencing Facility using Ml 3 reverse primers. Clones with ambiguous sequences were manually sequenced using the ffnol sequencing kit (Promega, Madison, Wl).
  • a total of 83 clones were directly sequenced and 49 contained 99 microsatellite a ⁇ ays with 3 or more repeats. Assuming that microsatellites are evenly distributed in P. monodon genome, a frequency of 1/0.2 kb was obtained by dividing the number of microsatellites by the total length of sequenced DNA
  • TUZXPm236 ACAGCATC IT (IC) 3 i G l c IGG i GG Perfect AF077554 "1 UZXPm241 TTAT AAACI r ⁇ A) come (CDj I AGTCTC I GG 2 Peifect AF077555 TUZXPm259 TATTAAGGAC (CT) 4 cccc ⁇ c:r ⁇ c Perfect AF077556 TUZXPm269 CA'IGAGCATG (TA) 3 ⁇ A(TA)( I G)C l'l ( I A)( I G) 4 AA ⁇ CAAA ⁇ T Compound imperfect AF077557 lUZXPm271 TCG ⁇ TTTG (GT) ⁇ (TG) 3 (AG) 3 AAGAAC ⁇ GGG 3 Perfect AF077558 TUZXP111274 CTTTTCCTAC (TCC) 3 .
  • Tl Tufts U ⁇ iveisity, followed by the initials of the researcher that cloned or characterized the microsatellite (e.g. ZX), the species name and clone number.
  • 'Flanking sequences refer to nucleotides immediately before and after the fiist and last microsatellite.
  • (TC) 3 is a shorthand version of "TCTCTC” (i.e., the repeat motif in TUZXPm2.26 in Table 4).
  • the overall abundance of di-, tri-, tetra- and hexanucleotide repeats in 99 microsatellites were 67% (1/0.3 kb), 20% (1/1.0 kb), 9% (1/2.1 kb) and 3% (1/6.2 kb), respectively.
  • one octanucleotide repeat (ATTTATTC) 5 was detected within a large repeat sequence.
  • the dinucleotide microsatellites with 3 or more repeats included 36 (CT) n , 31 (GT) n , 17 (AT), and 3 (CG) n .
  • CT 36
  • GT 31
  • GT 17
  • CG 3
  • the order was different for microsatellites with 5 or more [10 (GT) n , 7 (AT) n and 5
  • the frequency of (GT) n and (CT) n in microsatellites of 3 or more repeats was 1/0.8 kb and 1/0.7 kb, in microsatellites of 5 or more was 1/2.5 kb and 1/4.9 kb and in microsatellites of 10 or more repeats was 1/5 kb and 1/25 kb, respectively.
  • Tassanakajon et al. (1998) reported that the frequency of (GT) n and (CT) n repeats (n>6) in P. monodon was 1/93 kb and 1/164 kb.
  • the reverse primer was labeled with ⁇ - 32 P and PCR was performed in a thermocycler (PTC- 100, MJ Research, MA) with the reaction mixture (25 ⁇ l) containing 100 ng DNA, 7.5 ng of reverse primer, 50 ng of forward primer, 2 mM MgCl 2 , 0.2 mM dNTPs and 2.5 units of Taq polymerase (Promega, Madison, WT) (Wolfus, G.M., et al, Aquaculture 152, 35-47 (1997)).
  • the thermal profiles for PCR were: 94°C for 3 min followed by 22 cycles of 94°C for 1 min, experimental annealing temperature for 1 min and 72°C for 2 min.
  • P. monodon microsatellite primers also amplified P. vannamei alleles (Table 5).
  • the PIC values for these microsatellites ranged from 0.18 to 0.64. Additional microsatellites with a higher number of samples need to be tested before determining the usefulness of P. monodon microsatellites for genetic analyzes in P. vannamei.
  • Miuosatellile Annealing size alleles size alleles clone Primer sequences 5'— >3' St-Q ID NO temp (°C) in bpj (# of samples) I' in bp (# of samples) IC
  • Example 2 Determining genotype at Ml microsatellite locus to characterize shrimp for susceptibility/resistance to Taura Syndrome Virus
  • the optimum amplification reaction conditions for Ml were 50 ng DNA, 7.5 ng Primer 1 (MIR), 50 ng Primer 2 (B202FB), 1 X Thermo buffer (Promega, Madison, Wl), 2 mM MgCl 2 , 0.2 mM dNTPs, and 2.5 units Taq polymerase in 25 ⁇ l total volume.
  • Primer MIR was 5'-end labeled using ⁇ 32 P-dATP, T4 polynucleotide kinase and the exchange reaction with exchange buffer following the manufacturer's instructions (Gibco, BRL).
  • Microsatellite amplification was performed using a Thermal cycler (MJ Research).
  • the cycler program began with 94°C for 3 min, followed by 22 cycles of 94°C for 1 min, 52°C for 1 min and 72°C for 2 min.
  • the loading buffer for the polymerase chain reaction (PCR) was 10 mM NaOH, 95% formamide, 0.05% bromophenol blue and 0.05% xylene cyanol. Samples were heated to 90°C for 10 min before loading onto the polyacrylamide gel. Amplified samples were run on a 7.6% acrylamide/bis acrylamide (19:1), 7.6
  • Tabie Macrosatellite MlGenotypes in shrimp of TSV susceptible (Kona stocks of USMSFP) or TSV resistant (Batch 10 of USMSFP breeding program)
  • Example 3 Isolation of ESTs from Taura Syndrome Virus (TSV)-challenged shrimp (Litopenaeus vannamei) by mRNA differential display
  • mRNADD mRNA differential display
  • the objectives of this study were to use the mRNA differential display method (Liang, P. and Pardee, A.B., Science, 257:967-971 (1992)) to isolate differentially expressed genes in shrimp challenged with TSV, with the hope of obtaining expressed sequence tags (ESTs) that could be used to map the quantitative trait loci (QTL) responsible for TSV resistance.
  • ESTs expressed sequence tags
  • RT-PCR reverse transcriptase- polymerase chain reaction
  • Sense and antisense primers for TSV were used to amplify the cDNA synthesized with oligo-d(T) 16 primer using the SuperscriptTM Preamplification System (Gibco BRL).
  • PCR was performed in a 25 ⁇ l reaction volume containing 2.0 ⁇ l cDNA, lx PCR buffer (Promega), 2.0 mM MgCl 2 , 50 ng each of TSV sense and antisense primers, 1.25 mM dNTP and 2.5 U to Taq DNA polymerase (Promega).
  • the temperature profile for PCR was 95°C for 7 min, followed by 95°C 45 sec, 55°C 1 min, and 72°C 2 min for 30 cycles with a final extension of 72°C for 7 min using a M.J. Research thermocycler PTC- 100TM.
  • the amplified products were electrophoresed in a 1% agarose gel using TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA) and photographed.
  • the expected TSV band of ⁇ 1.0 kb was amplified by PCR in most of the samples. TSV was successfully detected by RT-PCR as early as 6 h after TSV feeding. Some samples showed variable results in TSV amplification. For example, one sample from both crosses #2 and #10 collected at 12 h showed faint TSV bands and the 36 h samples of cross #10 did not amplify the TSV cDNA. Since the RNA obtained from these samples was of good quality, it may be possible that these animals did not ingest TSV infected food for the virus to be detected by RT-PCR.
  • RNA differential display assay Total RNA obtained from one animal of each of crosses #2 and #10 before (0 h) and after (6 h, 12 h, 24 h and 36 h) TSV challenge was used for mRNA differential display assay. These crosses were used because they showed the most extreme differences in the percent survival (4.2% and 87.5%, respectively).
  • the mRNA differential display assay was performed following RNAimageTM protocol (GenHunter Corporation, Nashville, TN).
  • Amplified cDNAs were separated in a 6%o polyacrylamide gel in TBE (89 mM Tris, 89 mM boric acid and 20 mM EDTA, pH 8.3) and differentially expressed cDNAs were eluted from the gel, reamplified and cloned into pCR-TRAP cloning vector (GenHunter Corporation).
  • RNA fingerprint was done using samples of crosses #10 and #2 before and after TSV challenge using the H-T n G with AP-5 and AP-6 primers.
  • a total of 41 bands (cDNAs) were eluted from the gels. Twenty-seven out of the 41 bands were from cross #10 and designated as H (High survival), and 14 were from cross #2 and named as L (Low survival) (Table 8).
  • the recombinant plasmids were sequenced using an ABI automated sequencer at Tufts University DNA Sequencing Facility (Boston, MA) or manually using the/mo/ TM sequencing kit (Promega).
  • TU Tufts llniveisily, followed by the initials of the researcher that cloned or characterized the cDNA (e.g.AD), the species name and clone number. Clones with II were isolated from Cross # 10 (87% siuvivul) and clones wilh were isolated fiom CrossW2 (4 2% survival). Only sequence omologies of known genes and selected sequences -60% in more than 85% length of the s aied sequences were included.
  • the sizes of the amplified cDNAs ranged from 43 to 294- bp.
  • the 49 sequences were submitted to GerLBank, 39 of them as ESTs (Adams, M.D. et al, Nature, 377 (supplement) 3-174 (1995)), representing -7,000 bp of expressed shrimp genes. Similarities to known sequences in GenBank are presented in Table 8. Only sequence homologies of -60% in more than 85%) of the shared sequences were included in the table, unless it is indicated otherwise.
  • RNAs were electrophoresed in a 1% formaldehyde agarose gel and transferred to nitrocellulose membranes (MSI, Westborough, MA) (Alcivar, A.A. et al, Developmental Biology, 35:263-271 (1989)).
  • the cDNA insert was recovered by digesting with Hindlll restriction enzyme (GenHunter Corporation, Nashville, TN), after electrophoresis in a 1% agarose gel and elution from the gel using Spin- X columns (5 Prime ->3Prime Inc). The insert was used as probe by labeling with ⁇ -[ 32 P]dCTP (Amersham Inc.) using the random primers DNA labeling system (Gibco BRL). Hybridizations were performed following a published protocol (Garcia. D.K. et al, Molecular Marine Biology and Biotechnology, 5:71-83 (1996)).
  • the membranes were washed first at medium stringency (2% SSC, 0.1 % SDS) for 15 min at 50°C and then at high stringency (0.1 % SSC and 0.1 % SDS) twice for 15 min at 50°C before exposing them to Kodak Biomax films at -80°C.
  • Brine shrimp (Artemia spp) and mouse testis RNA were used as controls.
  • Hybridization signals were detected for H30.45, L21.1 and LI.12 clones (not shown). This indicates that the cloned genes have a potential functional role in shrimp development and survival.
  • a single mRNA transcript was observed with clone H30.45 and appears to be up- (6 h and 24 h) and down-regulated (12 h and 36 h) in the low surviving cross #2.
  • Two mRNA transcripts were detected in all RNA samples tested (0, 6, 12, 24, and 36 h from crosses #1 and #5) using the insert for LI.12 clone. A single band was detected in PL10 (low signal) and juvenile (strong signal) shrimp using L21.1 (actin-like) suggesting it is developmentally and differentially expressed.
  • tri- and tetra- nucleotide microsatellites were isolated from a genomic library of L. vannemei by probe hybidization. Clones were obtained by ligating target DNA to vector DNA at different ratios. A total of 1479 positive clones were obtained and used for hybridization with each of the following probes (TAT) 10 , (CTC) 10 , (CTTT) 8 , and (TGTA) 8 . One hundred seventy-eight out of the 1479 clones tested positive to either one or more of the tri-nucleotide and tetra-nucleotide probes and were sequenced.
  • One hundred sixteen clones contained microsatellite arrays and two pairs of clones in this group are identical. Forty-seven out of 116 clones have tri- nucleotide and/or tetra-nucleotide arrays with three or more repeats. Within these 47 clones, a total of 94 trinucleotide and 23 tetranucleotide arrays have been identified. Fifteen out of the 116 clones contained penta-nucleotide repeats either alone or together with tri-nucleotide or tetra-nucleotide repeats.
  • the 94 tri-nucleotide microsatellites were classified as perfect (60%o), compound imperfect (28%), imperfect (7%) and compound perfect (5%).
  • the 23 tetra-nucleotide microsatellites were classified as perfect (43%), compound perfect (26%), compound imperfect (22%), and imperfect (9%). All clones also contained di-nucleotides in addition to the larger repeats.
  • microsatellites of I. vannemei are characterized by nucleotide sequences in Figs. 1A-1E. These microsatellite sequences are further characterized as to their repeated sequences in Table 9. Table ' Summary of vannamei irucrosatellilos characterized, including then 5' .i l V flrinkiiij; sequences and Ihe c ⁇ le ⁇ ;o ⁇ y of ⁇ ik msalellite accoirling lo Webei (1990)
  • TUDGLv 1-3224 RB ⁇ C ⁇ I ⁇ CA ⁇ AG ( ⁇ CAG) 4 ⁇ (G ⁇ ) Z1 ⁇ (G ⁇ ) 10 CT ⁇ AC ⁇ IGC ⁇ 1 Perfec 1, 1 liupei feet

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Abstract

La présente invention concerne des molécules d"acide nucléique pouvant être définies comme des microsatellites de Penaeus monodon. L"invention concerne également des molécules d"acide nucléique pouvant être définies comme des microsatellites ou des séquences EST de Litopenaeus vannemei. Ces molécules d"acide nucléique, leurs compléments, ainsi que les molécules partageant une séquence de nucléotides similaire, y compris celles partageant une séquence de nucléotides contigüe, et d"autres molécules présentées par l"invention, peuvent être utilisées pour identifier, cartographier et définir des segments du génome de diverses espèces de crevettes.
PCT/US1999/029571 1998-12-10 1999-12-10 Molecules d"acide nucleique de crevette WO2000034476A2 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002031157A2 (fr) * 2000-10-10 2002-04-18 Biotec Pharmacon Asa Phosphatase alcaline de crevette
WO2007057915A1 (fr) * 2005-11-21 2007-05-24 Bose Institute Marqueur adn de type microsatellite utilise dans l'identification de populations de penaeus monodon resistantes aux maladies
CN101880721A (zh) * 2010-07-20 2010-11-10 中国水产科学研究院黄海水产研究所 中国对虾es15微卫星标记的检测方法
EP3153030A1 (fr) 2007-11-29 2017-04-12 Monsanto Technology LLC Produits de viande ayant des niveaux accrus d'acides gras bénéfiques
CN111225977A (zh) * 2017-09-19 2020-06-02 本-古里安大学B.G.内盖夫技术和应用公司 小龙虾的性连锁基因组标记及其用途
CN112048486A (zh) * 2020-07-23 2020-12-08 中国水产科学研究院南海水产研究所 一种斑节对虾PmGFPT2基因及其应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108841941B (zh) * 2018-05-22 2021-11-02 广西壮族自治区水产引育种中心 利用线粒体nadh5基因精准鉴别金边鲤的方法

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BALL, A. O. (1) ET AL: "Characterization of (GT)n microsatellites from native white shrimp ( Penaeus setiferus)." MOLECULAR ECOLOGY, (SEPT., 1998) VOL. 7, NO. 9, PP. 1251-1253., XP000892438 *
GARCIA, D. K. ET AL: "Identification and organization of microsatellites in Penaeus vannamei shrimp." ANIMAL GENETICS, (1996) VOL. 27, NO. SUPPL. 2, PP. 71. MEETING INFO.: 25TH INTERNATIONAL CONFERENCE ON ANIMAL GENETICS TOURS, FRANCE JULY 21-25, 1996, XP000892381 cited in the application *
GARCIA, DENISE K. ET AL: "Molecular analysis of a RAPD marker (B20) reveals two microsatellites and differential mRNA expression in Penaeus vannamei." MOLECULAR MARINE BIOLOGY AND BIOTECHNOLOGY, (1996) VOL. 5, NO. 1, PP. 71-83., XP000673936 cited in the application *
TASSANAKAJON, ANCHALEE (1) ET AL: "Isolation and characterization of microsatellite markers in the black tiger prawn Penaeus monodon." MOLECULAR MARINE BIOLOGY AND BIOTECHNOLOGY, (MARCH, 1998) VOL. 7, NO. 1, PP. 55-61., XP000892382 *
VONAU, V. ET AL: "Three polymorphic microsatellites in the shrimp Penaeus stylirostris." ANIMAL GENETICS, (JUNE, 1999) VOL. 30, NO. 3, PP. 234-235., XP000892442 *
WOLFUS, GREG M. ET AL: "Application of the microsatellite technique for analyzing genetic diversity in shrimp breeding programs." AQUACULTURE, (1997) VOL. 152, NO. 1-4, PP. 35-47., XP000892713 *
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002031157A2 (fr) * 2000-10-10 2002-04-18 Biotec Pharmacon Asa Phosphatase alcaline de crevette
WO2002031157A3 (fr) * 2000-10-10 2002-08-01 Norwegian Inst Of Fisheries & Phosphatase alcaline de crevette
US7323325B2 (en) 2000-10-10 2008-01-29 Biotec Pharmacon Asa Shrimp alkaline phosphatase
WO2007057915A1 (fr) * 2005-11-21 2007-05-24 Bose Institute Marqueur adn de type microsatellite utilise dans l'identification de populations de penaeus monodon resistantes aux maladies
EP3153030A1 (fr) 2007-11-29 2017-04-12 Monsanto Technology LLC Produits de viande ayant des niveaux accrus d'acides gras bénéfiques
CN101880721A (zh) * 2010-07-20 2010-11-10 中国水产科学研究院黄海水产研究所 中国对虾es15微卫星标记的检测方法
CN111225977A (zh) * 2017-09-19 2020-06-02 本-古里安大学B.G.内盖夫技术和应用公司 小龙虾的性连锁基因组标记及其用途
CN111225977B (zh) * 2017-09-19 2024-03-26 本-古里安大学B.G.内盖夫技术和应用公司 小龙虾的性连锁基因组标记及其用途
CN112048486A (zh) * 2020-07-23 2020-12-08 中国水产科学研究院南海水产研究所 一种斑节对虾PmGFPT2基因及其应用

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