A putative serine protease from larval midgut of red palm weevil Rhynchophorus ferrugineus : partial purification and biochemical characterization

INTRODUCTION

Red palm weevil (RPW), Rhynchophorus ferrugineus, invaded Qatif in Eastern region of Saudi Arabia on 1987. RPW has expanded its range very rapidly (1-3). It causes sever damage to date palms and threat the dates industry. In the region, RPW control management is based mainly on the use of synthetic insecticides, which besides increasing production cost, cause environmental hazards. Therefore, development of an environmental friendly control method is a major goal of researchers in pest control. The expression of anti-metabolic proteins in transgenic plants is a quite attractive strategy to protect date palm trees from RPW. One way to do this is to incorporate inhibitors into date palm genome. In order to do this, however, it is important to identify the target digestive enzyme from the midgut of the insect to ensure successful implication of such strategy. To achieve this objective, biochemical characterization of such enzyme should be conducted.

The proteinase showed different sensitivities to inhibition by plant protein protease inhibitors, which were effective in protecting exogenous proteins from digestion by gut extracts (4).

Plant-derived proteinase inhibitors are of a particular interest because they are part of the plant natural defense system against predation. Many insect species possess serine-type proteinases, such as trypsin-like enzyme, in their digestive system for digestion of food proteins (5). Previous studies of the effects of dietary proteinase inhibitor, either artificially introduced into defined diets or already present in plant tissues, have shown that these proteinase inhibitors can be detrimental to the growth and development of a wide range of insects (6-7). When foreign plant proteinase inhibitor genes were introduced into tobacco plants, the production of proteinase inhibitor at relatively high levels made the transgenic tobacco plants resistant to typical tobacco insect pests (8-9). Recent progress in gene transfer technology makes it possible to produce new rice cultivars by introducing agronomically useful genes into rice (10). Potato protease inhibitor II gene was successfully introduced into several rice varieties and was stably inherited in four successive generations of the transgenic rice plants. More importantly, transgenic rice plants showed increased resistance to a major rice insect pest (11-12). The present study is focused on isolation and characterization of TLE from RPW. This finding will provide an important information to pave the way for transferring specific insecticidal proteinase inhibitor gene in palm trees.

MATERIALS AND METHODS

Insects: Fourth instar larvae of R. ferrugineus were collected from infected palm trees at AL-Hass Oasis, Saudi Arabia. Chemicals and Reagents. Trypsin (type 1 from bovine pancreas), soybean trypsin inhibitor, casein and bovine serum albumin (BSA) were obtained from Sigma Co. Purified Agar from DIFCO, Trypan blue and Thiomersal from BDH. Ion exchange DE-52 was obtained from Whatman Co.

Preparation of midgut extract from R. ferrugineus larvae: Larvae were anesthetized by exposing to diethyl ether for few seconds, then dissected and midguts were taken out in 20 mM Tris-HCl, pH 7.4 followed by washing several times by the same buffer. The fat tissues were also removed by small brush, a total of 20-30 cleaned midguts of the fourth instar of R. ferrugineus were homogenized by electrical homogenizer in 20 mL ice-cold homogenization buffer containing 20 mM Tris-HCl pH 7.4, 5 mM EDTA, 5 mM EGTA and 2 mM 2-mercaptoethanol. The homogenate was filtrated through double layer of cheesecloth, then centrifuged at 10000 xg for 20 min at 4oC as described by (13). The clear supernatant was kept for the next step of the enzyme purification.

Partial purification of a TLE: Seventeen milliliters of the crude homogenate of larva midguts (10 mg [mL.sup.-1]) were applied onto 8 mL packed column chromatography of DE-52, anion exchange has been previously equilibrated by buffer A (20 mM Tris-HCl pH 7.4, 5 mM EDTA, 5 mM EGTA and 2 mM 2-mercaptoethanol). The column was washed by 10 mL of buffer A then, the bound proteins were eluted stepwise by 10 mL of 0.3, 0.5 and 1.0 M NaCl in buffer A. Each fraction was tested for the activity of midgut trypsin-like enzyme as described by (13).

Preparation of the agar-substrate gel plates: Agar gels were prepared using 2% Noble agar in buffer containing (0.1M Tris-HCl, pH 7.4, 0.9% NaCl and 0.01 thiomersal as a microbial agent. The agar was mixed with 0.2 mL of 1% trypan blue, dissolved in water over a steam bath and cooled to 50-60 [degrees]C. Casein substrate was mixed thoroughly with 10 mL milted agar in the final concentration 0.1 mg [mL.sup.-1] or otherwise indicated. The agar-substrate were added in Petri dishes and allowed to cool and solidify. After solidification of the gel, 4-6 wells were punched in gels with 0.4 cm diameter and 0.3 depth. The center agar of the wells were removed by gentle suction using a Pasteur pipette. The wells were filled with 10-50 [micro]L of the tested enzyme alone or mixed with specific protease inhibitors.