Molecular Cloning and Characterization of Stored mRNA in Cotyledons of Vigna unguiculata

From a gt1O cDNA library constructed from the total polyadenylatedRNA (poly A⁺ RNA) of mature cowpea cotyledons, we selected recombinantphages that hybridized with a cDNA probe complementary to polyA⁺ RNA from cotyledons collected 24 h after the onset of imbibition(cDNA-B) but that did not hybridize with a probe from cotyledonsat developmental stage II (13 to 15 days after flowering; cDNA-A).cDNA inserts of two of these phages were subcloned in pUC18plasmids and the resultant plasmids were designated pSAS5 andpSAS1O, respectively. We also selected two recombinant phagesthat hybridized with cDNA-A but not with cDNA-B, and one ofthem was subcloned in pUC18 to generate pSASC1. We then characterizedthe pSASlO cDNA insert as the cDNA of a putative stored mRNAof cowpea seeds and the pSASC1 insert as a reference cDNA. TheRNA-blot hybridization after gel electrophoresis, with pSAS10as probe, indicated that an essentially full-length cDNA hadbeen cloned, and the hybrid-select translation of pSAS10 mRNAgave a 10-kDa product. pSASC1 mRNA was shown to code for a 46-kDapolypeptide, which corresponds in size to one of the major storageproteins of the cowpea. pSAS10 mRNA was detectable only in cotyledonsat developmental stage III (17 to 19 days after flowering) orlater, and the level of the mRNA began to decline when seedsgerminated. The mRNA was also present in cowpea embryonic axes.mRNA hybridizable to pSAS10 cDNA was found in extracts of someother plants. (Received July 17, 1989; Accepted October 17, 1989)