Abstract
BackgroundThe chitin biosynthesis pathway starts with trehalose in insects and the main functions of trehalases are hydrolysis of trehalose to glucose. Although insects possess two types, soluble trehalase (Tre-1) and membrane-bound trehalase (Tre-2), very little is known about Tre-2 and the difference in function between Tre-1 and Tre-2.ResultsTo gain an insight into trehalase functions in insects, we investigated a putative membrane-bound trehalase from Spodoptera exigua (SeTre-2) cloned from the fat body. The deduced amino acid sequence of SeTre-2 contains 645 residues and has a predicted molecular weight of ~74 kDa and pI of 6.01. Alignment of SeTre-2 with other insect trehalases showed that it contains two trehalase signature motifs and a putative transmembrane domain, which is an important characteristic of Tre-2. Comparison of the genomic DNA and cDNA sequences demonstrated that SeTre-2 comprises 13 exons and 12 introns. Southern blot analysis revealed that S. exigua has two trehalase genes and that SeTre-2 is a single-copy gene. Northern blot analyses showed that the SeTre-2 transcript is expressed not only in the midgut, as previously reported for Bombyx mori, but also in the fat body and Malpighian tubules, although expression patterns differed between the midgut and fat body. SeTre-2 transcripts were detected in the midgut of feeding stage larvae, but not in pupae, whereas SeTre-2 mRNA was detected in the fat body of fifth instar larvae and pupae.ConclusionThese findings provide new data on the tissue distribution, expression patterns and potential function of membrane-bound trehalase. The results suggest that the SeTre-2 gene may have different functions in the midgut and fat body.
Highlights
The chitin biosynthesis pathway starts with trehalose in insects and the main functions of trehalases are hydrolysis of trehalose to glucose
A fragment of 690 bp was first obtained from pupal fat body cDNA by a second PCR using SeTre-F2 and SeTre-R
We performed 5' and 3' rapid amplification of cDNA ends (RACE) using several specific primers based on the sequence of the fragment and universal primers (Clontech)
Summary
The chitin biosynthesis pathway starts with trehalose in insects and the main functions of trehalases are hydrolysis of trehalose to glucose. The disaccharide trehalose consists of two α-glycosidically linked glucose units. It is a non-reducing sugar found in many organisms as diverse as bacteria, yeast, fungi, nematodes, plants, insects and some other invertebrates, but is absent in mammals [1,2,3,4]. Unlike in mammals, trehalose is the main blood sugar and is present in the hemolymph of larvae, pupae and adults [1,8,9,10,11]. It is the main reserve sugar in the hemolymph of flying insects and is indispensable for thermotolerance in larvae
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