Methods for identifying novel pesticidal gene homologues

Abstract

Methods and compositions for identifying novel pesticidal gene homologues are provided. Specifically, the methods of the invention comprise systematically designing oligonucleotide primers that are specific for a pesticidal gene of interest and performing successive rounds of PCR amplification of nucleic acid material from a microorganism, particularly a Bacillus thuringiensis strain, to identify novel homologues of known pesticidal genes. Oligonucleotide primers that can be used to practice the present methods are further disclosed.

1 . A method for identifying novel homologues of a target group of pesticidal genes, said method comprising:
 a) designing a set of degenerate oligonucleotide primers that is specific for said target group of pesticidal genes of interest;   b) selecting at least two pairs of degenerate primers from said set of degenerate oligonucleotide primers, wherein each pair of degenerate oligonucleotide primers comprises a forward primer and a reverse primer;   c) obtaining a first sample of nucleic acid material from a microorganism of interest;   d) mixing said first sample of nucleic acid material with said at least two pairs of degenerate oligonucleotide primers specific for said target group of pesticidal genes and a thermostable DNA polymerase under conditions that are suitable for amplification by polymerase chain reaction (PCR);   e) performing a first round of PCR and detecting PCR amplification products, thereby determining if PCR products are produced in the first round of PCR;   f) obtaining a second sample of nucleic acid material from the microorganism if PCR amplification products are detected in the first round of PCR;   g) subjecting the second sample of nucleic acid material to a second round of amplification by PCR using pairs of oligonucleotide primers that are specific for all known pesticidal genes in the target group, wherein said pairs of oligonucleotide primers specific for known pesticidal genes in the target group comprise nucleotide sequences that are different from the nucleotide sequences for said oligonucleotide primers of (a);   
       h) detecting PCR amplification products from the second round of PCR, thereby determining if PCR products are produced in the second round of PCR;
 i) obtaining a third sample of nucleic acid material from the microorganism if PCR products are detected in the first round of PCR and PCR products are not detected in the second round of PCR, wherein a microorganism that comprises nucleic acid material that is amplified in the first round of PCR and is not amplified in the second round of PCR comprises a putative novel homologue of the target group of pesticidal genes; 
 j) subjecting the third sample of nucleic acid material to a third round of PCR using at least one pair of oligonucleotide primers to clone the putative novel homologue; and, 
 k) analyzing the putative novel homologue of the target group of pesticidal genes of interest. 
 
     
     
         2 . The method of  claim 1 , wherein designing a set of degenerate oligonucleotide primers that is specific for said pesticidal gene of interest comprises:
 a) preparing an alignment of nucleotide sequences for a target group of pesticidal genes;   b) selecting a primer length;   c) viewing a window of nucleotides within said alignment, wherein said window is equivalent in length to said primer length of (b);   d) identifying a nucleotide sequence within a window that is conserved among all nucleotide sequences from the target group;   e) designing a set of all possible degenerate oligonucleotide primers based on the conserved sequence; and,   f) selecting at least two oligonucleotide primers from said set of degenerate primers and using a mixture of said selected primers in the first round of PCR amplification.   
     
     
         3 . The method of  claim 2  further comprising identifying a putative novel homologue of the target group of pesticidal genes using non-degenerate primers and comparing the putative novel homologue identified using non-degenerate primers with the putative novel homologue identified by the method of  claim 2 . 
     
     
         4 . The method of  claim 2 , said method further comprising determining if the nucleotide sequence within said window has the features of (a)-(e) below:
 a) does not have four or more contiguous identical nucleotide residues;   b) has no more than two guanine or cytosine residues within the last five residues of the 3′ end of the nucleotide sequence;   c) has a melting temperature (T m ) of between about 50° C. and 65° C.;   d) does not form hairpin or dimer structures; and,   e) is not conserved among non-target group pesticidal genes,   
       wherein a nucleotide sequence that is not conserved among non-target group pesticidal genes differs from each of the non-target group pesticidal genes by at least two nucleotide residues. 
     
     
         5 . The method of  claim 1 , wherein the microorganism of interest comprises a  Bacillus thuringiensis  strain. 
     
     
         6 . The method of  claim 5 , wherein obtaining a first, second, and third sample of nucleic acid material comprises preparing plasmid DNA from the  Bacillus thuringiensis  strain. 
     
     
         7 . The method of  claim 1 , wherein said target group of pesticidal genes of interest comprises  Bacillus thuringiensis  Cry genes. 
     
     
         8 . The method of  claim 7 , wherein said  Bacillus thuringiensis  Cry genes are Cry2, Cry3, or Cry8 genes. 
     
     
         9 . The method of  claim 1 , wherein said first and said second round of PCR comprise performing quantitative real-time PCR (RT-PCR). 
     
     
         10 . The method of  claim 9 , wherein said first round of PCR is performed in the presence of a fluorescent entity, said fluorescent entity being capable of indicating the presence of PCR products and providing a signal related to the quantity of the PCR products. 
     
     
         11 . The method of  claim 10 , wherein said fluorescent entity is a dye. 
     
     
         12 . The method of  claim 11 , wherein said dye is SYBR® Green. 
     
     
         13 . The method of  claim 9 , wherein said second round of PCR is performed in the presence of a nucleic acid probe, said probe comprising a fluorescent dye and a quenching dye. 
     
     
         14 . The method of  claim 1 , wherein analyzing the putative novel homologue of the target group of pesticidal genes comprises nucleotide sequence analysis. 
     
     
         15 . The method of  claim 4 , wherein said T m  is calculated using Formula I below:
     T   m =( EH°/[ES °+( R ×ln( Ct ))]−273.15+16.6 log([ X ]))×1.1144−14.964   wherein,   EH° (enthalpy)=ΣΔH;   ES° (entropy)=ΣΔS+0.368×19×1.585;   R (molar gas constant)=1.987;   Ct (total primer concentration)=log(0.00000005/4)×1000; and,   X (salt concentration [K + ])=0.05.   
     
     
         16 . The method of  claim 15 , wherein said T m  is between about 57° C. and 61° C. 
     
     
         17 . The method of  claim 4 , wherein determining if a nucleotide sequence within a window is not conserved among non-target group pesticidal genes comprises searching the full-length sequence of each gene from the non-target group pesticidal genes.