关键词: 水稻, 谷蛋白, 基因克隆

Abstract:

Rice seeds with rich reserve of starch and protein are a major food source in many countries. Unlike the seeds of other plants, which typically accumulate one major type of storage protein, rice seeds contain two major proteins, prolamines and globulin-like glutelins. Glutelin, which accounts for 70%–80% of the total proteins of rice seeds, is coded by a multi-gene family, and to date at least nine different cDNAs have been isolated. After transcription in the cell nucleus, rice glutelin mRNA is targeted to a specific subdomain of the cortical endoplasmic reticulum (ER) where it is translated to synthesize a larger precursor with a signal sequence which is cotranslationally processed during translocation to the ER lumen whereupon correct folding and disulfide bond formation occur. The glutelin precursor is then transported to vacuolar protein bodies (PB-Ⅱ) presumably by way of the Golgi complex. At PB-Ⅱ, glutelin is proteolytically processed into acidic (a) and basic (h) polypeptides. Accoding to the primary sequence comparisons, glutelin can be classified into A and B types. Rice seed proteins are deficient in the essential amino acid, lysine. Therefore, nutritional improvement in the amino acid composition of rice proteins is needed. B type glutelin is superior to A type in terms of nutritional value since B type has more of the first limiting amino acid, lysine. For this reason, B type glutelin should be a noteworthy genetic resource to improve rice protein quality.
Here we reported the cloning and characterization of cDNA for a new rice glutelin gene from a local cultivar (Oryza sativa L). After screening the rice endosperm cDNA library by 32P-labeled GluB-2 cDNA probes (1 389 bp), we cloned a new glutelin gene cDNA, named GluB-7 (GenBank accession number AY987390). DNA sequence analysis showed that the size of the cloned cDNA was 1 588 bp, and carried entire coding sequences, which encode a 495 amino acid protein, corresponding to the size of the glutelin protein family. Signal peptide prediction with software found that GluB-7 included a 24-residue signal peptide with the cleavage site between arginine and glutamine and a 471-residue mature protein. Homology analysis showed that the deduced amino acid sequence of GluB-7 shared 57.8%–97.8% identity with others of rice glutelin gene family. Southern blot analysis of the genomic DNA showed the presence of multiple copies in the rice genome. Northern blot analysis using GluB-7 cDNA partial sequence as a probe showed that the GluB-7 was expressed specifically in rice endosperm, and the largest accumulation of mRNA occurred in 12 DAA, while no corresponding band was found in roots, stems, and leaves. The cloning of GluB-7 cDNA provides the basis for future studies on glutelin gene expression, the identification of the molecular mechanism of rice seed storatge protein biosynthesis, and especially the improvement of protein quality in rice breeding.

Key words: Rice, Glutelin, Gene cloning