Abstract
A number of trypsin inhibitor (TI) genes have been used to generate insect-resistant plants. Here we report a novel trypsin inhibitor from Indian mustard Brassica juncea (BjTI) that is unique in being the precursor of a 2S seed storage protein. The inhibitory activity is lost upon processing. The predicted amino acid sequence of the precursor based on the B. juncea 2S albumin (Bj2S) gene cloned and sequenced in this laboratory (Bj2Sc; GenBank(TM) accession number ) showed a soybean-TI active site-like motif GPFRI at the expected processing site. The BjTI was found to be a thermostable Kunitz type TI that inhibits trypsin at a molar ratio of 1:1. The 20-kDa BjTI was purified from midmature seeds and found to be processed in vitro to 9- and 4-kDa subunits upon incubation with seed extract. The Bj2Sc sequence was expressed in Escherichia coli pET systems as the inhibitor precursor. The radiolabeled gene product was expressed in vitro in a coupled transcription-translation system and showed the expected processing into subunits. Two in vitro expressed pre-2S proteins, mutated at Gly and Asp residues, were processed normally to mature subunits, showing thereby no absolute requirement of Gly and Asp residues for processing. Finally, the 2S gene was introduced into tobacco and tomato plants. Third generation transgenics expressing BjTI at 0.28-0.83% of soluble leaf proteins showed remarkable resistance against the tobacco cutworm, Spodoptera litura. This novel TI can be used in transforming seed crops for protection to their vegetative parts and early seed stages, when insect damage is maximal; as the seeds mature, the TI will be naturally processed to the inactive storage protein that is safe for consumption.
Highlights
Seed proteins play important roles in plants’ survival e.g. maintaining viability of seeds, providing nutrition during the early seedling stage, and protecting the seeds from microbes and insects [1]
Third generation transgenics expressing BjTI at 0.28–0.83% of soluble leaf proteins showed remarkable resistance against the tobacco cutworm, Spodoptera litura. This novel trypsin inhibitor (TI) can be used in transforming seed crops for protection to their vegetative parts and early seed stages, when insect damage is maximum; as the seeds mature, the TI will be naturally processed to the inactive storage protein that is safe for consumption
Isolation and characterization of the new trypsin inhibitor - The trypsin inhibitor was purified from mid-mature (28 DAF) B. juncea seeds by a combination of gel filtration and ion exchange chromatography
Summary
Seed proteins play important roles in plants’ survival e.g. maintaining viability of seeds, providing nutrition during the early seedling stage, and protecting the seeds from microbes and insects [1]. Seed proteins play an important role in human and animal nutrition by providing the major share of dietary protein These proteins may be classified as storage, structural and biologically active proteins [2]. TIs have been ascribed other functional roles, such as regulating endogenous plant proteinases to prevent precocious germination, inhibiting trypsin during passage through an animal’s gut, helping in seed dispersal, and protecting plants against pests and diseases [3,4,5,6] Their presence in major grain crops like cereals, legumes and oil seeds, has nutritional and clinical implications and importance of their structure-function relationships has made TIs the subject of extensive studies [3,7]
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