Construction and screening of plant genomic libraries

Abstract

A library of pea (Pisum sativum L) genomic DNA in bacteriophage EMBL3 was screened for seed storage protein genes of the legumin and vicilin families. Three genomic clones were isolated. One of the clones was found to contain a gene in the Leg A sub-family which was designated Leg E. The nucleotide and predicted amino acid sequence of Leg E were compared to those of Leg A. The coding sequences of both genes are strongly homologous with only 9 bases difference out of 1560 bases. A second genomic clone contained two genes from the Leg J subfamily. Leg J and Leg K. The clone was shown to overlap with a genomic clone isolated previously, JC5 (Gatehouse et al. 1988). Strong homology was found between the Leg K and Leg J sequences. The Leg K gene is predicted to be pseudogene, due to the conversion of the ATG methionine start codon to a GTG valine codon and the presence of a stop codon in the 5' end of the coding sequence in the reading frame predicted by the first subsequent start codon. A genomic library was constructed for Arabidopsis thaliana, using EMBL3 as a vector to sub-clone Sau3AI partially digested Arabidopsis genomic DNA. About 8 x 10(^4) random clones were obtained when the ligated vector DNA and insert were in vitro packaged. The Arabidopsis gene library was screened for clones containing sequences encoding the cell wall protein extensin, using a rape (Brassica napus L extensin cDNA as a probe. Six clones were isolated, two of which were restriction mapped. One of them was partially sequenced. This clone did not contain an extensin gene homologous to the probe sequence, and only contained a short extensin-like sequence which was responsible for the observed hybridisation. The putative gene may represent another type of protein, since it was expressed in the root of Arabidopsis and Brassica napus L, as shown by "Northern" blots which were probed with labelled DNA from the clone.