Insect muscle actin promoter

Abstract

Provided is a gene expression system, suitable for expression in an insect, comprising an insect muscle actin pro-moter operably linked to a marker gene, which overcomes or ameliorates one or more of: cost of rearing; amount of handling; errors in identification due to human error or loss of marker by the insect; and health concerns related to the effects of marker powders on workers in mass rearing facilities.

1 - 13 . (canceled) 
     
     
         14 . A polynucleotide expression system, comprising:
 a polynucleotide sequence encoding a fluorescent marker; and   an insect muscle actin promoter operably linked to the polynucleotide sequence, wherein the insect muscle actin promoter is not an indirect flight muscle actin promotor, and   wherein expression of the fluorescent marker of the polynucleotide expression system in an insect results in a phenotype that is visible to the naked eye when the insect is exposed to light.   
     
     
         15 . The polynucleotide expression system of  claim 14 , wherein the phenotype visible to the naked eye is detectable in at least two developmental stages selected from the group consisting of the egg, larval, pupal, and adult developmental stages of the insect. 
     
     
         16 . The polynucleotide expression system of  claim 15 , wherein the phenotype is visible to the naked eye at the larval, pupal, and adult stages of the insect. 
     
     
         17 . The polynucleotide expression system of  claim 14 , wherein the fluorescent marker is expressed across at least two body segments of the insect. 
     
     
         18 . The polynucleotide expression system of  claim 17 , wherein the body segments are selected from the group consisting of the head, thorax, and abdomen. 
     
     
         19 . The polynucleotide expression system of  claim 14 , wherein the polynucleotide expression system confers no substantial fitness disadvantage on the insect. 
     
     
         20 . The polynucleotide expression system of  claim 14 , wherein the fluorescent marker comprises green fluorescent protein (GFP), EGFP, ZsGreen, TurboGFP, AcGFPI, yellow fluorescent protein (YFP), mCitrine, DsRed, DsRed2, DsRed-Express, DsRed monomer, mCherry, mStrawberry, mRaspberry, mPlum, AmCyan, CyPet, tdTomato, AsRed2, and/or E2-Crimson. 
     
     
         21 . The polynucleotide expression system of  claim 14 , wherein the insect muscle actin promoter is derived from an insect of the order Diptera or Lepidoptera. 
     
     
         22 . The polynucleotide expression system of  claim 14 , wherein the insect muscle actin promoter is derived from an insect of the family Culicidae, Drosophilidae, Tephritidae, or Bombycidae. 
     
     
         23 . The polynucleotide expression system of  claim 14 , wherein the insect muscle actin promoter is derived from  Aedes aegypti  (also known as  Stegomyia aegyptae ),  Stegomyia albopicta  (also known as  Aedes albopictus ),  Anopheles stephensi, Anopheles albimanus, Anopheles gambiae,  Medfly ( Ceratitis capitata ), Mexfly ( Anastrepha ludens ), Oriental fruit fly ( Bactrocera dorsalis ), Olive fruit fly ( Bactrocera oleae ), Melon fly ( Bactrocera cucurbitae ), Natal fruit fly ( Ceratitis rosa ), Cherry fruit fly ( Rhagoletis cerasi ), Queensland fruit fly ( Bactrocera tyroni ), Peach fruit fly ( Bactrocera zonata ), Caribbean fruit fly ( Anastrepha suspensa ), West Indian fruit fly ( Anastrepha obliqua ), codling moth ( Cydia pomonella ), silk worm ( Bombyx mori ), pink bollworm ( Pectinophora gossypiella ), diamondback moth ( Plutella xylostella ), Gypsy moth ( Lymantria dispar ), Navel Orange Worm ( Amyelois transitella ), Peach Twig Borer ( Anarsia lineatella ), rice stem borer ( Tryporyza incertulas ), a moth of the family Noctuidae, a moth of the subfamily Heliothinae, or  Drosophila melanogaster.    
     
     
         24 . The polynucleotide expression system of  claim 14 , wherein the insect muscle actin promoter comprises the polynucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 6, or a variant or mutant thereof. 
     
     
         25 . The polynucleotide expression system of  claim 24 , wherein the variant or mutant has at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity with SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 6. 
     
     
         26 . The polynucleotide expression system of  claim 14 , wherein the insect in which the fluorescent marker is to be expressed is of the order Diptera or Lepidoptera. 
     
     
         27 . The polynucleotide expression system of  claim 14 , wherein the insect in which the fluorescent marker is to be expressed is of the family Culicidae, Tephritidae, or Bombycidae. 
     
     
         28 . The polynucleotide expression system of  claim 14 , wherein the insect in which the fluorescent marker is to be expressed is  Aedes aegypti  (also known as  Stegomyia aegyptae ),  Stegomyia albopicta  (also known as  Aedes albopictus ),  Anopheles stephensi, Anopheles albimanus, Anopheles gambiae,  Medfly ( Ceratitis capitata ), Mexfly ( Anastrepha ludens ), Oriental fruit fly ( Bactrocera dorsalis ), Olive fruit fly ( Bactrocera oleae ), Melon fly ( Bactrocera cucurbitae ), Natal fruit fly ( Ceratitis rosa ), Cherry fruit fly ( Rhagoletis cerasi ), Queensland fruit fly ( Bactrocera tyroni ), Peach fruit fly ( Bactrocera zonata ), Caribbean fruit fly ( Anastrepha suspensa ), West Indian fruit fly ( Anastrepha obliqua ), codling moth ( Cydia pomonella ), silk worm ( Bombyx mori ), pink bollworm ( Pectinophora gossypiella ), diamondback moth ( Plutella xylostella ), Gypsy moth ( Lymantria dispar ), Navel Orange Worm ( Amyelois transitella ), Peach Twig Borer ( Anarsia lineatella ), rice stem borer ( Tryporyza incertulas ), a moth of the family Noctuidae, or a moth of the subfamily Heliothinae. 
     
     
         29 . The polynucleotide expression system of  claim 14 , wherein the insect in which the fluorescent marker is to be expressed is a pest. 
     
     
         30 . The polynucleotide expression system of  claim 14 , wherein the insect in which the fluorescent marker is to be expressed is a beneficial insect. 
     
     
         31 . The polynucleotide expression system of  claim 14 , further comprising an enhancer associated with the insect muscle actin promoter. 
     
     
         32 . The polynucleotide expression system of  claim 14 , further comprising a 5′ untranslated region (UTR) and/or a 3′ UTR for the polynucleotide sequence. 
     
     
         33 . The polynucleotide expression system of  claim 32 , wherein the 3′ UTR is from an insect muscle actin gene. 
     
     
         34 . The polynucleotide expression system of  claim 33 , wherein the 3′ UTR is from the insect muscle actin gene of Mexfly ( Anastrepha ludens ). 
     
     
         35 . The polynucleotide expression system of  claim 14 , further comprising a transgene, wherein expression of the fluorescent marker in the insect indicates the presence of the transgene or a trait associated with the transgene in the insect. 
     
     
         36 . The polynucleotide expression system of  claim 14 , wherein expression of the fluorescent marker in the insect is inducible, repressible, and/or derepressible. 
     
     
         37 . The polynucleotide expression system of  claim 14 , which is selected from the group consisting of construct #4014, #4143, #4247, #4133, #4676, #4705, #4751, and #4718. 
     
     
         38 . An insect comprising, in its genome, the polynucleotide expression system of  claim 14 . 
     
     
         39 . A method, comprising:
 transforming an insect with the polynucleotide expression system of  claim 14 .   
     
     
         40 . A method of identifying a transgenic insect, comprising:
 exposing one or more insects to light of an excitation wavelength, and   identifying a phenotype with the naked eye present in the one or more insects, thereby identifying the insect(s) as containing a polynucleotide expression system of  claim 14  in its genome.   
     
     
         41 . The method of  claim 40 , further comprising separating the identified insect(s) comprising the polynucleotide expression system in its genome from a population of insects. 
     
     
         42 . The method of  claim 41 , wherein the identifying step and/or the separating step is automated. 
     
     
         43 . The method of  claim 40 , wherein the polynucleotide expression system further comprises a transgene, wherein expression of the fluorescent marker in the insect indicates the presence of the transgene or a trait associated with the transgene in the insect. 
     
     
         44 . The method of  claim 43 , wherein the transgene is a lethal gene or a gene, when expressed in the insect, results in sex separation, and the method further comprises monitoring the insect comprising the transgene in a Sterile Insect Technique (SIT) program. 
     
     
         45 . The method of  claim 44 , wherein the sex separation results from the group consisting of female elimination, insect lethality, and male sterility, due to the expression of the transgene.