Differential display-mediated rapid identification of different members of a multigene family, HSP 16.9 in wheat.
Isolation of cDNAs encoding individual members of a gene family is essential for assessing their role in a biological phenomenon. However, this process is often laborious and slow due to highly conserved protein-coding region that interferes with the isolation of the individual members. Identification of gene-specific probes from 3' non-coding regions of different members can assist in the fast retrieval and characterization of individual members of a multigene family. We used the recent technique of differential display for the same purpose. As an example of a multigene family in plants, we selected a heat shock protein gene family, HSP16.9 from wheat, with estimated 12 members. We modified the original differential display technique for selective amplification of the 3' non-coding regions of different wheat HSP16.9 genes by replacing the random 10-mer in the original method with a conserved HSP16.9 gene family-specific primer. Sixteen cDNA fragments from these experiments were sequenced and they represent 8 different members of a 12 member gene family. Our success can be attributed to shorter 3' non-coding regions that are typical of higher-plant genes and use of highly conserved gene family-specific primer in these experiments. This modified differential display technique can be of general application to other plant systems where cloning of the different members of a gene family is desired.
Joshi
C P
CP
Department of Plant and Soil Science, Texas Tech University, Lubbock 79409-2122, USA.
Nguyen
H T
HT
D000595:Amino Acid Sequence; D001483:Base Sequence; D017124:Conserved Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D017343:Genes, Plant; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D005810:Multigene Family; D010940:Plant Proteins; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014908:Triticum / Q000235:genetics*
D017931:DNA Primers; D018076:DNA, Complementary; C101303:HSP16.9 protein, Triticum aestivum; D006360:Heat-Shock Proteins; D010940:Plant Proteins