JXB Advance Access originally published online on June 18, 2003
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Journal of Experimental Botany, Vol. 54, No. 389, pp. 1995-1996,
August 1, 2003
© 2003 Oxford University Press
OsSET1, a novel SET-domain-containing gene from rice
Received 21 April 2003; Accepted 25 April 2003
PKU-Yale Joint Research Center of Agricultural and Plant Molecular Biology, National Key Laboratory of Protein Engineering and Plant Gene Engineering, College of Life Sciences, Peking University, 5 Yiheyuan Road, Beijing 100871, PR China
* To whom correspondence should be addressed. Fax: +86 10 6275 1526. E-mail: shunongb{at}pku.edu.cn
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Abstract |
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A novel SET-domain-containing gene OsSET1 was isolated from rice (Oryza sativa L.). Its deduced protein consists of 895 amino acids. OsSET1 has a high degree of structure similarity to other SET-domain-containing genes such as CLF in higher plants and E(z) in animals. RT-PCR showed that the gene expresses throughout the entire plant. A transient expression assay in onion epidermis revealed that the OsSET1 protein is localized in nuclei. Over-expression of the SET domain of OsSET1 in Arabidopsis resulted in altered shoot development at seedling stages.
Key words: Gene expression regulation, rice, OsSET1, SET-domain-containing genes.
The SET domain is a conserved sequence containing 115 amino acids, named from three fruit fly genes, PEV inhibitor Su (var) 3-9, Enhancer of zeste and Trithorax, which contain the domain (Tschiersch et al., 1994). Recently, a breakthrough was made in understanding the function of such a conserved domain by discovering its histone methyltransferase (HMT) activity (Rea et al., 2000; for a review see Kouzarides, 2002). Structural analysis further elucidated its catalytic mechanism (for a review see Marmorstein, 2003). Together with other evidence about some well characterized SET-domain-containing genes which play important roles in regulating development by interacting with transcriptional factors in fruit fly and other organisms including plants (Satijn and Otte, 1999; Kennison, 1995; Goodrich et al., 1997; Grossniklaus et al., 1998), this group of genes has become a proper target for further investigation in understanding developmental regulation. The total number of SET-domain-containing genes in the databases has increased dramatically from about 40 in 1995 to 589 at this moment (http://smart.embl-heidelberg.de/smart). Among these, 67 were identified in Arabidopsis. To understand more about the function of SET-domain-containing genes in plants, searches for such genes were undertaken in other plant species. Recently, SET-domain-containing genes have been identified from tobacco (NtSET1; Shen, 2001), maize (MEZ1-3; Springer et al., 2002), spinach and pea (Trievel et al., 2002). Here the isolation and a brief description of the function of a novel SET-domain-containing genes from rice (Oryza sativa L.), the model plant in cereal crops, were reported.
To isolate SET-domain genes from rice, a conserved SET-domain sequence was first isolated with RT-PCR using the degenerated primers determined by the published sequences of CLF, E(z), MEA (CEM1: 5'-TCTGA(TC)T(TC)(TCG)(AC)(TC)GG(TAC)TGGGG TGC-3'; CEM2: 5'-GC(AT)(TC)C(TAC)TCTGG(TC)(CT)C(AG) TA(GCT)C(AGT)GTA-3'). A 344 bp PCR product was cloned in pGEM-T easy vector (Promega). After the PCR product was confirmed as a SET domain by sequencing, it was used as a probe to screen a cDNA library constructed from young panicles. A full length cDNA containing the SET domain was obtained by conducting 5'-RACE after the library screening. This cDNA is 2957 bp, contains an ORF that encodes a putative protein of 895 amino acids with calculated molecular mass of 99.8 kDa. This gene was designated as OsSET1 (GenBank accession number AF407010 [GenBank] ). It localizes at chromosome three in rice genome at the contig 1300 (http://www.softberry.com/berry.phtml?topic=gfind&prg=FGENESH; GenBank accession number AAAA01003815). Interestingly, five genes were predicted at this contig and the OsSET1 cDNA sequence was predicted as gene four and five by FGENESH1.1. The OsSET1 sequence data now rectified the prediction. According to the rice genome sequence data, the OsSET1 contains 17 exons (data not shown).
So far, 14 rice SET-domain-containing genes can be found in the SMART database, but in contrast to the two putative OsCLFs (AP005813 [GenBank] ; AP003044 [GenBank] ), the other 11 putative SET-containing genes have low sequence similarity with OsSET1 even in the SET domain. The detailed sequence analysis revealed that the OsSET1 gene has all known conserved regions, e.g. SET-N and SET-C in the SET domain, but lacks post-SET (Fig. 1). Similar to other plant SET-domain-containing genes such as CLF and MEZ1-3, OsSET1 only has a cysteine-rich region, no pre-SET domain. Based on the sequence characteristics, the OsSET1 could be grouped into the SET1 family.
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helices. Regions putatively involved in binding to the cofactor product AdoHcy were indicated with green, and other three highly conserved sequence regions (the last two of which form the unusual knot structure) were indicated with a blue bar below the aligned sequences. The invariant tyrosine residue implicated to function as a general base for catalysis was indicated with a black triangle below the alignment. No CLC and CRC conservative sequences were found in Post-SET region of OsSET1. DDBJ/EMBL/GenBank accession numbers: DIM-5_Nc (AAL35215
The expression pattern of OsSET1 was similar to that of the SET-domain-containing genes investigated in Arabidopsis and maize in terms of lacking organ specificity (data not shown). A transient expression assay revealed that the fusion protein of OsSET1 and green fluorescent protein (GFP) was located in the nuclei (Fig. 2). This was also similar to other SET-containing proteins such as E(z) and CLF. To investigate the function of the OsSET1 gene, a series of transgenic Arabidopsis and rice lines were constructed. Among them, about 53.8% transgenic Arabidopsis that over-expressed the SET domain resulted in altered shoot development shown in Fig. 3B, as well as large cotyledons (Fig. 3A, B). No tunic-corpus structure was observed in the sections of the shoot apex of transgenic plants with abnormal shoots (Fig. 3D). Further investigation on the function of the OsSET1 is still being undertaken.
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Acknowledgements |
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The authors thank Professor Xian-Sheng Zhang of Shandong Agricultural University and Professor Ying-Tang Lu of Wuhan University for their help in our construction of rice cDNA library, and Mr Di Liu and Professor Jing-Chu Luo for their kind help in bioinformatical analysis. This work is supported by grants to SNB from MST (J00-A-005, G19990 [GenBank] 116) and NSFC (30070361), and to ZHX from MST.
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