Molecular cloning, characterization and gene expression of a water deficiency and chilling induced proteinase inhibitor I gene family from sweet potato ( Ipomoea batatas Lam.) leaves

Abstract

A 7-kDa-proteinase inhibitor, designated SPLTI (S̱weet P̱otato Ḻeaf Ṯrypsin I̱nhibitor) was partially purified from sweet potato ( Ipomoea batatas Lam.) leaves under water deficiency. The N-terminal amino acid sequence was determined and used to design two overlap degenerate primers for isolation of the SPLTI gene. Two full-length cDNA clones encoding proteinase inhibitor I (PI-I), designated SPLTI-a and SPLTI-b, were isolated. Both SPLTI-a and SPLTI-b are 98% identical to each other in both levels of nucleotide and amino acid sequence. Different from most of PI-Is that exhibit chymotrypsin-inhibitory activity, SPLTI from sweet potato and recombinant SPLTI-a overexpressed in Escherichia coli showed mainly trypsin-inhibitory activity. Furthermore, site-directed mutagenesis analysis of an Arg 46–G1u 47 motif of SPLTI-a, based on amino acid sequence alignment with other PI-Is, indicated that Arg 46–G1u 47 of SPLTI is a novel reactive site for PI-I family conferring the trypsin-specific inhibitory activity. Using SPLTI-a as a probe, we found that SPLTI gene exhibited a leaf-specific expression pattern. Additionally, this was the first report that the SPLTI genes were up-regulated by water deficiency and chilling as well as osmoticant treatments in the PI-I family in plants. As other PIs, the SPLTI transcripts were induced by wounding and also by exogenous applications of abscisic acid and methyl jasmonate; however, accumulation of the wound-induced transcripts were restricted locally in the injured leaves, but not systemically. These distinct expression patterns provided a new insight to the regulation of PI-I gene family in response to environmental stresses. Our results suggested that SPLTI could participate in defense systems against invasions of insects or bacteria as other PI-Is. Moreover, it may play a role against environmental stresses through regulation of endogenous proteolytic activities during leaf development.