Insect Chymotrypsin and Inhibitors Thereof

Abstract

The present invention relates generally to a novel chymotrypsin that exhibits resistance to a plant serine proteinase inhibitor. More particularly, the present invention provides a chymotrypsin which is up-regulated in the gut of Helicoverpa armigera and Helicoverpa punctigera insect larvae when fed the serine proteinase inhibitors of Nicotiana alata . The novel chymotrypsin represents, therefore, a target for the identification of antagonists including inhibitors which are proposed to be useful in the control of Helicoverpa spp. populations that have become resistant to serine proteinase inhibitors produced in plants. The antagonists of the chymotrypsin may be topically applied to the plants or, when in proteinaceous form, may be produced by genetic means in plant cells. The antagonists may act at the level of gene expression or protein activity.

1 . An isolated nucleic acid molecule comprising a sequence encoding a chymotrypsin from  Helicoverpa  spp. or a variant, derivative, homolog or analog of said chymotrypsin, wherein said chymotrypsin exhibits resistance to a proteinase inhibitor (PI) from  Nicotiana alata , said chymotrypsin comprising the amino acid sequence set forth in SEQ ID NO:2 or an amino acid sequence having at least 75% similarity to SEQ ID NO:2 after optimal alignment. 
     
     
         2 . The isolated nucleic acid molecule of  claim 1 , wherein the nucleotide sequence encodes an amino acid sequence set forth in SEQ ID NO:2. 
     
     
         3 . The isolated nucleic acid molecule of  claim 1 , wherein the sequence is as set forth in SEQ ID NO:4 or SEQ ID NO:6 or a nucleotide sequence having at least about 75% identity to SEQ ID NO:4 or SEQ ID NO:6 after optimal alignment or a nucleotide sequence capable of hybridizing to SEQ ID NO:4 or SEQ ID NO:6 or its complementary form under low stringency conditions. 
     
     
         4 . The isolated nucleic acid molecule of  claim 3 , comprising the sequence set forth in SEQ ID NO:4. 
     
     
         5 . The isolated nucleic acid molecule of  claim 1 , encoding a variant of the chymotrypsin wherein the variant is an N-terminal signal sequence comprising the amino acid sequence set forth in SEQ ID NO:3 or an amino acid sequence having at least about 75% similarity to SEQ ID NO:3 after optimal alignment. 
     
     
         6 . The isolated nucleic acid molecule of  claim 1 , wherein the sequence is set forth in SEQ ID NO:5 or a nucleotide sequence having at least 75% identity to SEQ ID NO:5 after optimal alignment or a nucleotide sequence capable of hybridizing to SEQ ID NO:5 or its complementary form under low stringency conditions. 
     
     
         7 . The isolated nucleic acid molecule of  claim 1  encoding a variant of the chymotrypsin wherein the variant comprises an amino acid residue other than arginine at position 192. 
     
     
         8 . The isolated nucleic acid molecule of  claim 7 , wherein the variant comprises a glutamine residue at position 192. 
     
     
         9 . A vector comprising a nucleic acid molecule of  claim 1 . 
     
     
         10 . The vector of  claim 9  wherein the vector is an expression vector. 
     
     
         11 . The vector of  claim 9 , wherein the expression vector is operable in a prokaryotic cell. 
     
     
         12 . The vector of  claim 1 , wherein the expression vector is operable in a eukaryotic cell. 
     
     
         13 . The vector of  claim 12 , wherein the eukaryotic cell is an insect cell. 
     
     
         14 . The vector of  claim 13 , wherein the vector is a baculovirus vector. 
     
     
         15 . A genetically modified cell comprising the nucleic acid molecule of  claim 1 . 
     
     
         16 . An isolated chymotrypsin from  Helicoverpa  spp., wherein said chymotrypsin exhibits resistance to a protease inhibitor from  Nicotiana alata  or a variant, derivative, homolog or analog of said chymotrypsin, wherein the isolated chymotrypsin comprises the amino acid sequence set forth in SEQ ID NO:2 or an amino acid sequence having at least about 75% similarity to SEQ ID NO:2 after optimal alignment. 
     
     
         17 . The isolated chymotrypsin of  claim 16  comprising the amino acid sequence set forth in SEQ ID NO:2. 
     
     
         18 . The isolated chymotrypsin of  claim 17  encoded by a nucleotide sequence having at least about 75% identity to SEQ ID NO:4 or SEQ ID NO:6 after optimal alignment or a nucleotide sequence capable of hybridizing to SEQ ID NO:4 or SEQ ID NO:6 or its complementary form under low stringency conditions. 
     
     
         19 . The isolated chymotrypsin of  claim 18  encoded by the nucleotide sequence set forth in SEQ ID NO:1. 
     
     
         20 . The isolated chymotrypsin of  claim 16 , wherein the variant comprises an N-terminal signal sequence. 
     
     
         21 . The isolated chymotrypsin of  claim 20  wherein the signal sequence comprises the amino acid sequence as set forth in SEQ ID NO:3 or an amino acid sequence having at least 75% similarity thereto after optimal alignment. 
     
     
         22 . The isolated chymotrypsin of  claim 16 , wherein the variant comprises an amino acid other than arginine at position 192. 
     
     
         23 . The isolated chymotrypsin of  claim 16 , wherein the variant comprises a glutamine at position 192. 
     
     
         24 . A plant comprising cells genetically modified to produce an antagonist of chymotrypsin-HpCh5, wherein said antagonist comprises the amino acid sequence of SEQ ID NO:81 or an antagonist of chymotrypsin-HpCh5 with at least about 75% sequence identity thereto. 
     
     
         25 . The genetically modified plant of  claim 24 , wherein the plant is a monocotyledonous plant. 
     
     
         26 . The genetically modified plant of  claim 24 , wherein the plant is a dicotyledonous plant. 
     
     
         27 . The genetically modified plant of  claim 24 , wherein the plant is cotton, sweet corn, tomato, tobacco, pimiento, potato, sunflower, citrus, plum, sorghum, leek, soybean, alfalfa, bean, pidgeon pea, chick pea, artichoke, curcurbit, lettuce,  Dianthus , geranium, cape gooseberry, maize, flax, linseed, alfalfa, lupin, broad bean, garden pea, peanut, canola, snapdragon, cherry, sunflower, marigold,  Helichrysum , wheat, barley, oat, triticale, carrot, onion, orchid, rose or petunia. 
     
     
         28 . The genetically modified plant of  claim 27 , wherein the plant is a cotton plant. 
     
     
         29 . The genetically modified plant of  claim 24  comprising a nucleic acid molecule encoding Pot1A and/or Pot1B. 
     
     
         30 . Seeds or other reproductive material from the plant of  claim 24 . 
     
     
         31 . A method for isolating and separating individual isoforms of chymotrypsin, said method consisting of:
 (i) affinity chromatography of insect gut extracts initially with benzamidine sepharose to bind trypsins;   (ii) further affinity chromatography of the unbound proteins using immobilized  N. alata  serine proteinase inhibitor C1 to bind all NaPI inhibitable chymotypsins followed by eluting bound proteins to produce an eluate; and   (iii) affinity chromatography of the eluate from (ii) with immobilized PotI and PotII or chymostatin to bind the remainder, followed by elution of bound NaPI-insensitive chymotrypsins using 8 M urea.   
     
     
         32 . A method for screening for an antagonist of a NaPI-insensitive chymotrypsin from  Helicoverpa  spp, said method comprising contacting said chymotrypsin with a potential antagonist and measuring chymotrypsin activity in the presence and absence of the potential antagonist, whereby an antagonist is identified when chymotrypsin activity is less in the presence than in the absence of the potential antagonist. 
     
     
         33 . An isolated antagonist identified by the method of  claim 32 . 
     
     
         34 . An isolated proteinase inhibitor comprising an amino acid sequence having at least 75% similarity to SEQ ID NO:81 after optimal alignment. 
     
     
         35 . An isolated antibody specific to the isolated proteinase inhibition of  claim 34 . 
     
     
         36 . A composition comprising the antagonist of  claim 34 . 
     
     
         37 . The composition of  claim 26 , wherein said composition comprises an insecticidal amount of the isolated proteinase inhibitor of  claim 34 .