33. Cloning and identification of rice prolamin 4a gene promoter

Laboratory of Molecular Biology, Biotechnology Research Center Chinese Academy of Agricultural Sciences, Beijing, 100081, China

Prolamin and glutelin are the major storage proteins of rice endosperm. Prolamines are alcohol-soluble, low molecular weight proteins ranging from 10 kd to 17 kd. They are encoded by a large gene family. So far, prolamin genes, 10 kd, Prol 14, Prol 4a etc. have been isolated from cDNA libraries and genomic libraries. (Masumura et al. 1989; Kim et al. 1988). No intron was found in their primary

Fig. 1. Two synthetic primers; Primer 1, Primer 2. * Changed base for creation of enzymatic cleavage site. V Signal peptide cleavage site.

Fig. 2. DNA pattern of the 5' region of prolamin 4a gene obtained by amplification in vitro. A: lambda Hind III molecular weight marker. B: Amplified 5' region of prolamin 4a gene in 2% agarose gel.

sequences. In general, they are initially synthesized around 10 DAF and are deposited in protein body-1 with the help of signal peptide. In vitro digestion experiments with bovine pepsin suggest that poor digestibility of PB-1 may be the reason for low nutritional value of prolamin.

Therefore, we are interested in cloning the 5'-flanking region of prolamin 4a gene containing signal peptide sequence to further investigate the relations among signal peptide, PB-1 and poor digestibility. So far, there is little information on prolamin gene expression and the interactions between cis-elements and trans-acting factors in the regulation of prolamin gene expression during the seed development, in comparison with the detailed analysis on the regulation of zein gene expression. More knowledge about the regulation of prolamin gene expression will help us to better understand the molecular biology of seed development for

Fig. 3. Cloning of the 5' region of prolamin 4a gene from PCR products. A: PCR products, H3: Hind III.

According to the prolamin 4a genomic sequence data (Kim et al. 1988), two primers were synthesized with a DNA Synthesizer (Fig. 1) and purified by FPLC. By using polymerase chain reaction, the promoter region of prolamin 4a gene of the Chinese rice variety Zhonghua 8 has been successfully amplified (Fig. 2). With the presence of different restriction enzyme cleavage sites in the primers, we have

Fig. 5. Autoradiography of DNA sequences of two putative promoters. G1 A1 T1 C1: Four lanes of pHB1. G2 A2 T2 C2: Four lanes of pHS31.

cloned these promoter fragments into pUC19 vector. Two different recombinant plasmids, pHB1 and pHS31 (Fig. 3), were selected for physical mapping (Fig. 4). There is no difference between the 5' region of prolamin 4a gene (Kim et al. 1988)

Fig. 6. Comparison of two putative promoter sequence with the 5'-flanking region of prolamin 4a gene (W.T. Kim, 1988).

Fig. 7. Construction of chimeric genes for transient expression. GUS1, GUS2, GUS3 from pBI1O1.1, pBI1O1.2, pBI1O1.3 respectively. H3: Hind III.

and the two promoter fragments of prolamin 4a gene from pHB1 and pHS31 in their physical map. Further analysis of these two fragments was made by cloning them into M13 mp 19 for sequencing by the dideoxynucleotide chain termination method (Fig. 5).

Two fragments have been partially sequenced and compared with the 5'-flanking region of prolamin 4a gene (Fig. 6). About 98% homology was found among them and no difference in signal peptide coding region. But in the long leader sequence, several base mutations were observed that may have resulted from using different rice varieties. Alternatively, the sequences may represent different members of the complex prolamin gene family. Several small ORFs were found in the leader sequence.

While studying their primary sequences, we have also constructed the chimeric genes. Three GUS reporter genes with different reading frame from pBI101.1, pBI101.2, pEI101.3 were translationally fused to the 5' region of prolamin 4a gene in pHS31 for transient expression analysis. We have also transcriptionally fused the GUS reporter gene with the 5' region of prolamin 4a gene in pHS31 without the signal peptide coding region. These four different constructs (Fig. 7) are being used in transient expression analysis for studying the function of the promoter of prolamin 4a gene.

Next, we will synthesize another primer to sequence the junction region between GUS reporter gene and prolamin 4a gene promoter to confirm the expected reading frame. By using binary vector system, we will transfer these chimeric genes into tobacco plants to investigate the tissue-specific and developmental stage-specific expression.

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Masumura, T. et al. 1989. cDNA cloning of an mRNA encoding a sulfur-rich 1OKDa prolamin polypeptide in rice seeds. Plant Mol. Biol. 12: 123-130.

Kim, W. T. and T. W. Okita, 1988. Nucleotide and primary sequence of a major rice prolamin. FEBS Letters 231(2): 308-310.

Kim, W. T. and T. W. Okita, 1988. Structure, expression, and heterogeneity of the rice seed prolamines. Plant Physiol. 88: 649-655.