48. Tnr1 -like element in the first intron of a type 2 metallothionein-like gene showing root specific expression in rice

Ikuo nakamura, Nanako kameya and Hiroyuki kanzaki

Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024, Japan.

 Many kinds of transposable elements have been identified from plant genomes as well as bacterial, fungal, insect and animal genomes. Transposable elements generally have common structural features such as terminal inverted repeats. Umeda et al. (1991) have found a short repetitive sequence, named Tnrl, in the 13th intron of starch synthase (waxy) gene in rice. Tnrl contains short inverted repeat sequence of about 75 bp and there are two base pairs (TA) target duplication at the both ends (Tenzen et al. 1994). Bureau and Wessler (1994) identified a new family of 47 repeat sequences, named Stowaway, after large-scale survey of DNA database, and found a much wide distribution of these elements in both monocotyledonous and dicotyledonous plants. Tnrl is considered a member of Stowaway family and non-autonomous transposon. Tnr1-like inverted repeats have been found along with the many genes of rice, such as alpha-amylase 3A (entry name in DDBJ DNA databank: OSRAMY3A), alpha-amylase C (RICAMYC), heat shock protein 82A (OSHSP82A), proliferating cell-nuclear antigen (OSPCNAGEN), and starch synthase (OSWAXY) (Bureau and Wessler 1994).

We identified three different cDNAs (SSK35, SSK171 and SSK211) encoding metallothionein-like proteins (MTs) from rice saline-cDNA library. The cDNA library

158 Rice Genetics Newsletter Vol. 13

was made from rice suspension cells cultured in 2% NaCl conatining liquid medium (Umeda et al. 1994) and the nucleotide sequences of three different MT-cDNAs were determined. SSK35 and SSK211 encoded a HVBZE40 homolog (71.4% / 84 aa.) from barley embryos (Smith et al. 1992) and ATATMT1 homolog (54.9% / 82 aa.) from Arabidopsis leaves, respectively. SSK171 encoded a homolog (66.2% / 74 aa.) with iron deficiency specific MT-like protein of barley roots (HVIDS, Okumura et al. 1991) and was identical to rice rgMT gene reported by Hsieh et al. (1995). The MTs are small ubiquitous proteins found in every organism and contain two Cys-rich domains both at the N- and C-terminal ends to form metal ion chelators. MT-like proteins of plant origin were classified into two types depending on the sequential differences of Cys residues of their Cys-rich domains (Robinson et al. 1993). As shown in Figure 1, SSK35 and SSK211 encoded type 2 MT-like proteins. On the other hand, SSK171 encoded a protein consistent with type 1 MT-like protein. Northern blot analysis showed that SSK35 transcript expressed in immature embryo as well as leaves, whereas SSK171 and SSK211 highly expressed in root tissues (Kanzaki et al. manuscript in preparation).

The genomic upstream sequences corresponding to each cDNA clone have been isolated from rice total DNA using the inverse PCR technique. One pair of inverted oriented primers were designed and synthesized based on coding sequence of each cDNA and total genomic DNA was digested by the restriction enzyme that could cut at single position between the primers. The digested DNA fragments were ligated to make circular molecules and subjected to PCR reaction to amplify unknown 5' flanking sequences. The amplified fragments were cloned in TA cloning vector (Invitrogen, Netherland) and the DNA sequences of the cloned fragments were determined by dye-terminator method.

Sequence analyses revealed that SSK35 and SSK171 contained a short intron of 141 bp and 117 bp separating protein coding region, whereas SSK211 contains a larger intron of 1046 bp (Fig. 2). We found that a short inverted repeat sequence resting in the intron
 

<N-terminal> Linker <C-terminal>
type 1 consensus CxCxxxCxCxxxCxC CxCxxxCxCxxCxC
HVIDS SSK171 MSCSCGSSCGCGSNCNC MSCSCGSSCSCGSNCSC *********. *****. * <43 aa.> <43 aa.> CSCGMICKCNPCNC CSCGSSCRCMPCNC **** * ******
type 2 consensus CCxxxCxCxxxCxC CxCxxxCx-CxxCxCx
ATATMTI SSK35 SSK211 MSCCGGNCGCGSGCKC MSCCGGNCGCGSGCQC MSCCGGNCGCGSGCQC ******************.* <50aa.> <52aa.> <50aa.> CKCGSDCK-CDPCTCK CKCGTSCSGCSCCSCN CKCGDNCT-CNPCNCGK ****.. *. *. *.*.
Fig. 1. Amino acid sequences of two Cys-rich domain both at N- and C- terminal ends of metallothionein-like proteins from rice. SSK171 belongs to type 1 whereas SSK35 and SSK211 encode type 2 metallothionein-like protein. I
Research Notes 159
of SSK211 was homologous to 73.1% with typical Tnr1 element described by Tenzen et al. (1994). The element of 239 bp in length contained an imperfect inverted repeats (76 bp) to form stem loop structure and TA found at both ends of the element might be foot-print as a result of target sequence duplication event (Fig. 3). We have also found two Tnr1-like sequences in the linked RFLP marker of rice blast resistance (RICINDICA) and promoter region of anther specific protein gene (RICASP2) from the DNA database (Fig. 3).

Robinson et al. (1993) indicated that type 2 MT-like genes primarily expressed in aerial tissues of plants and type 1 MT-like genes primarily expressed in root. SSK211, however, encoded a type 2 MT-like protein but expressed highly in root tissues of rice plant. As some members of Tnr1 and Stowaway family were transcribed to mRNA or served as cis-acting regulatory domains (Bureau and Wessler 994). We are interested whether Tnr1-like element in the first intron of SSK211 gene might be involved in the change of the tissue specificity from aerial tissues to root tissues in rice plants. And the presence or absence of Tnr1 -like element at these three loci could be checked by PCR among Oryza species and the distribution of Tnrl -like element at these three loci could give us the phylogenic implications about the genus Oryza as described by Tenzen et al. (1994). (Gene symbol: New system)

Fig. 2. Schematic representations of the position and length of the First intron of three different genes encoding rnetallothionein (MT)-like proteins of rice. Stripe and gray boxes show intron and exon of the MT genes, respectively. 160 Rice Genetics Newsletter Vol. 13 Fig. 3. A: Alignment of three different Tnr1-like elements found in this study. Arrows and box show inverted repeat sequences and target duplication (TA), respectively. Tnrl-like element in SSK211 gene (TnrI-21 1K), anther specific protein gene (Tnrl -asp) and rice blast resistance RFLP marker (Tnr1-blast). B: Secondary stem loop structure of Tnrl -like element in SSK211 gene. References

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Hsieh, H-M., W-K. Liu and P.C. Huang, 1995. A novel stress-inducible metallothionein-like gene from rice.
Research Notes 161
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