Method of constructing an expression cassette comprising regulatory sequences of a target gene of a plant for expressing gene products
View Patent ↗The present invention provides a method of constructing an expression cassette that causes specific regulatory control of transgene expression in plants, wherein the expression cassette includes 5′ and 3′ regulatory sequences obtained from a plant target gene that is selected based on its expression data or on its encoded protein's similarity to a protein encoded by another gene of interest, and wherein a nucleic acid molecule that is operably linked to the 5′ and 3′ regulatory sequences of the expression cassette is expressed in a manner that mimics the expression of the target gene.
1. A method of constructing an expression cassette, comprising the steps of:
a) selecting a target gene of a plant based on its expression data or its encoded protein's similarity to a protein encoded by another gene of interest;
b) identifying the open reading frame on cDNA of said target gene;
c) identifying a promoter, a translation start codon, a translation stop codon, a first intron, a first exon, a second exon, a 3′-untranslated sequence and a 3′-nontranscribed sequence of gDNA of said target gene by using said cDNA of said target gene to annotate said gDNA of said target gene;
d) constructing an expression cassette that comprises a 5′-regulatory sequence comprising said promoter, said first exon, said first intron, and said second exon identified in (c) above, wherein said 5′-regulatory sequence has been engineered 1) to include a translational initiation codon at approximately the 3′ end of said 5′-regulatory sequence, 2) to eliminate restriction endonuclease sites that hinder manipulation by recombinant DNA methods, and 3) to modify any additional translation initiation codons upstream of said translation initiation codon to be inoperable so that the target gene's protein coding sequence is silent, while preserving the length of the 5′-regulatory sequence in the expression cassette,
and a 3′-regulatory sequence comprising said 3′-untranslated sequence and said 3′-nontranscribed sequence identified in (c) above; and
e) operably linking a nucleic acid molecule to said 5′-regulatory sequence and said 3′-regulatory sequence of said expression cassette of (d), wherein said nucleic acid molecule is expressed in a manner that mimics the expression of said target gene.
2. The method of constructing an expression cassette according to claim 1 , wherein said target gene is a MADS gene.