27. Expression analyses of glutelin genes in low glutelin rice mutant LGC-1
  S. M. JIANG1, T. IMBE2, N. HORISWE2, L. L. XU1 and J.M. WAN1,2*

1) State Key Laboratory of Crop Genetics & Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University; Nanjing 210095, China
2) Japanese Research Center of Agriculture, Japan
*) Corresponding author, Email: wanjm@mail.njau.edu.cn; Tel & Fax: +86-25-84396516

Glutelins, predominant storage and digestible proteins in rice grain (Cagampang, 1966; Tanaka,1975a; Tanaka, 1975b; Tanaka,1980), are encoded by a small multigene family consisting of two subfamily, GluA and GluB. So far, six glutelin genes has been isolated and sequenced (Takaiwa 1987 Takaiwa 1987b Masumura 1989 Okita 1989 Takaiwa 1991a Takaiwa 1991b). Mature glutelins consist of heterogenous acidic (37-39 kDa) and basic subunits (22-23 kDa) linked by a disulfide bond. Both subunits are derived from a common large 57 kDa precursor, which is post-translationally cleaved (Yamagata 1982).

LGC-1, a mutant rice with a low content of glutelin and a high content of prolamine, is a progeny from the backcross between Nihonmasari (NM) and its mutant, NM67, obtained by treating with 0.2% ethyleneimine. It has traits similar to its original cultivar NM except for low glutelin and high prolamine (Iida 1993). LGC-1 has been proved to be useful in the diet therapy of patients with chronic renal failure(Mochizuki 2000). Miyahara (1999) had studied the mutation mechanism of LGC-1 for long times. In Southern blot analysis, no difference, between LGC-1 and NM, was found in band pattern when both GluA and GluB genes were used as a probe, which indicated that there is no large deletion or duplication at glutelin genes in LGC-1. In Northern analysis, it was found that the mRNA level of GluA genes decreased slightly in LGC-1, on the other hand, that of GluB genes decreased dramatically and became lower than one tenth than that in Nihomasari. Two-dimensional gel electrophoresis showed that in LGC-1, the two spots of deletion, 1A and 4A, among several spots of the 37 kDa to 39 kDa glutelin acidic subunits, are encoded by different GluB subfamily genes (Miyahara 1999).

In this paper, expression analyses of all glutelin genes published were done by RT-PCR, no difference was found in mRNA level of each gene between LGC-1 and NM, which reached two conclusion: First, the trait of low glutelin content was not regulated at the level of transcription, but at the post-transcription. Second, low glutelin content was regulated at the level of transcription, but the suppressed glutelin genes were not among those published GluB families. The first conclusion didn't accord with the research of Miyahara, on the contrary, the second did. Therefore, according to the the second conclusion, in order to obtain the information about mutant glutelin genes, the cloning of new glutelin genes should be done, then the relationship between the new genes and the low glutelin content can be determined.

To obtain new glutelin genes, bioinformatics and rapid in silico cloning was applied. When Blastn searching was performed using the sequences of GluA-1 and GluB-1 as a probe, the prediction of gene with high homology by FGENESH program revealed, that in the seaching result of GluA-1, GluA-1 GluA-2 GluA-3 GluA-4 GluB-2 and GluB-4 existed in the first six contigs, respectively, and in that of GluB-1, GluB-1 GluB-2 GluB-3 GluB-4 and GluA-4 existed in the first five contigs, respectively, and that congtig23770, containing a new glutelin gene, appeared after the contigs described above in two groups of results. The gDNA and cDNA of new gene in Nihonmasari were obtained with the specific primers. When the deduced amino acid sequence of the new gene was compared with those of other glutelin genes, they showed about 80%, 70% and 60% between the new gene and GluB-4, GluB-1 or GluB-2, GluA, respectively, suggesting that the new glutelin gene belonged to the subfamily, GluB. Thus it wasdesignated as GluB-5 (Accession number AY196922, AY196923).

Comparison of the mRNA level of GluB-5 between LGC-1 and NM revealed that GluB-5 expressed normally and was under temporal control in NM, which was the same as other glutelin genes, and that its expression was dramatically suppressed and without temporal speciality in LGC-1(Fig. 1). The results suggested the suppression of GluB-5 might be related to the low content of glutelin in LGC-1, according with the research results by Miyahara, but not explaining all of characterestics in protein.

Contig23770 doesn't contain the 5'-UTR of GluB-5, for the gene lies in the end of the contig. To obtain more information about GluB-5, we are managing to search for the sequence of its 5'-UTR from other libraries of rice genomic data, and isolate its gDNA and cDNA in LGC-1. The identification of the results are underway.


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