Multigene Family for Bowman–Birk Type Proteinase Inhibitors of Wild Soja and Soybean: The Presence of Two BBI-A Genes and Pseudogenes

Abstract

Genes for Bowman-Birk type protease inhibitors (BBIs) of wild soja (Glycine soja) and soybean (Glycine max) comprise a multigene family. The organization of the genes for wild soja BBIs (wBBIs) was elucidated by an analysis of their cDNAs and the corresponding genomic sequences, and compared with the counterparts in the soybean. The cDNAs encoding three types of wild soja BBIs (wBBI-A, -C, and -D) were cloned. Two subtypes of cDNAs for wBBI-A, designated wBBI-A1 and -A2, were further identified. Similar subtypes (sBBI-A1 and -A2) were also found in the soybean genome. cDNA sequences for wBBIs were highly homologous to those for the respective soybean homologs. Phylogenetic analysis of these cDNAs demonstrated the evolutional proximity between these two leguminae strains.Key words: cDNA cloning; Bowman-Birk inhibitors; Glycine soja; isoinhibitors; pseudogene Proteinase inhibitors are found in various plants and have been studied in the Leguminae, Gramineae, and Solanaceae.1,2) Especially, proteinase inhibitors of the Bowman-Birk family (BBIs) and the Kunitz family were discovered first in soybean seeds and have been well studied. [3][4][5] A number of functions have been proposed for BBIs, including the regulation of endogenous proteinases during germination, the storage of sulfur amino acids during dormancy, and the protection of the plant from insects and microorganisms, [6][7][8] while recent interest has focused on its medicinal utility as an antitumor agent, for instance.

Endonuclease com incompatibilidade heteroduplex para detectar mutação e variações genéticas de inibidores da tripsina em soja

“…Based on sequence comparisons, Wang et al (2008) suggests that both wild and cultivated soybean had similar BBI genes. This is probably due to the close phylogenetic relation between the two species (Deshimaru et al, 2004).…”

“…Based on their structural features and inhibitory characteristics, BBIs are grouped in three main types -BBI-A, BBI-C, and BBI-D -and consist in a multigene family (Deshimaru et al, 2004). Using cultivated soybean and wild soybean genomic DNA as templates, fragments of 350 bp for BBI-A, 480 bp for BBI-C, and 550 bp for BBI-D were amplified (Figure 4).…”

“…BBI-A was further divided into two subtypes -A1 and A2 -according to small differences in their nucleotide sequences (Deshimaru et al, 2004). Amplified fragments for BBI-A were further subjected to digestion by HindIII restriction enzyme ( Figure 5), for which only the coding sequence for BBI-A1 contains a clevage site, resulting in two fragments (Deshimaru et al, 2004).…”

“…Amplified fragments for BBI-A were further subjected to digestion by HindIII restriction enzyme (Fermentas, Vilnius, Lithuania) (Deshimaru et al, 2004), while amplified fragments for KTI3 were digested with Mse I (Tru1 I) restriction enzyme (Fermentas, Vilnius, Lithuania) (Wang et al, 2008). Amplification and restriction fragments were determined using electrophoresis on 2% agarose gel containing ethidium bromide (0.5 g mL -1 ).…”

“…Post-translational proteolysis indicates that BBI-EI is originated from BBI-DII. Thus, BBIs are grouped in three types with distinct characteristics: BBI-A, BBI-C, and BBI-D (Deshimaru et al, 2004;Wang et al, 2008;Barros et al, 2012).…”