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Cloning of an [alpha]-Amylase cDNA from Aleurone Tissue of Germinating Maize Seed

A number of hydrolases are required for the conversion of stored carbohydrate reserves in cereal grains into metabolizable products during germination. a-Amylase (EC 3.2.1.1) plays a key role in this process by catalyzing the endoglycolytic cleavage of amylose and amylopectin, the principal components of starch granules in the endosperm (Bewley and Black, 1985). Studies of a-amylase characterization and expression in cereals have been conducted mainly in barley, wheat, and rice, with very limited information available for maize (Zea mays L.). Multiple isozymes of this enzyme are synthesized in two tissues, the aleurone and scutellum of the embryo, and can be separated on the basis of their pI. In barley and wheat, the isoforms fall into two distinct groups (low pI [ ~ 5 . 5 ] and high pI [?5.5]); however, in rice and maize, there is only one group of isozymes, between pI 3 and 6 (McGregor et al., 1988; Sanwo and DeMason, 1993). To facilitate a more detailed understanding of gene structure and regulation in barley, wheat, and rice, a number of a-amylase genes have been cloned and characterized, and these studies have revealed that the cereal a-amylases are encoded by multigene families (Baulcombe et al., 1987; Knox et al., 1987; Khursheed and Rogers, 1988; Huang et al., 1990; O’Neill et al., 1990). Furthermore, it has been shown that although some of the isozymes are derived from different genes, others differ as a result of posttranslational modifications (Fincher, 1989; Jones and Jacobsen, 1991; Sticher and Jones, 1992). The function and significance of the multiple isozymes are not known. To further advance our understanding of a-amylase expression in maize, we have isolated cDNAs from maize libraries and report here the cloning of a full-length aamylase cDNA (Table I). Three Xgtll cDNA libraries were constructed from aleurone and scutellar tissues of germinating seeds of the maize inbred OH43. To accomplish this, total RNA was extracted approximately 7 d after inhibition from isolated aleurones of both a dent (wild type) and a shrunken2 isogenic line and from scutella of the dent isoline. Poly(A)+ RNA was isolated from each using the PolyATtract mRNA isolation system (Promega) and was subsequently used to synthesize cDNAs according to the specifications of a cDNA synthesis kit (Boehringer Mannheim).