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BZIP type transcripton factors regulating expression of rice storage protein

A protein and plant transformation technology, applied in anti-plant immunoglobulin, hydrolase, genetic engineering, etc., can solve the problems of expression mode change, promoter activity reduction, etc.

Inactive Publication Date: 2003-02-19
NAT INST OF AGROBIOLOGICAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recently, it was found that the polymer of the GCN4 motif of the rice plant glutelin gene reproduced (reproduce) endosperm-specific expression in transformed rice plants, and it was found that due to the substitution or deletion of nucleotides on the GCN4 motif, the promoter activity was significantly reduced and its expression changes

Method used

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  • BZIP type transcripton factors regulating expression of rice storage protein
  • BZIP type transcripton factors regulating expression of rice storage protein
  • BZIP type transcripton factors regulating expression of rice storage protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] [Example 1] Isolation of a cDNA clone encoding a bZIP transcription factor from a seed cDNA library

[0112] The leaves and roots of rice plants (Oryza sativa L.c.v. Mangetumochi) cultured in hydroponic culture for 14 days were frozen in liquid nitrogen and stored at -80°C until use. Mature rice seeds were collected from rice plants grown in the field.

[0113] Oligonucleotide primers were designed according to the highly conserved amino acid sequence (SNRESA and KVKMAED) in the bZIP region of Opaque2(O2)-like protein, and poly(A) extracted from rice seeds + mRNA was used as template for RT-PCR. Poly A RNA was extracted from seeds 6 to 16 days after anthesis (DAF) (Takaiwa F. et al., Mol. Gen. Genet. 208:15-22, 1987), using Superscript reverse transcriptase (Gibco BRL, Paisly, UK) synthesized single-stranded cDNA by reverse transcription, and oligo(dT) 20 for primers. The cDNA was then amplified using the following primer pairs: 5'-TCC AAC / TA / CGI GAA / GA / TCIGC-2'; SE...

Embodiment 2

[0115] [Example 2] Identification of RISBZ cDNA

[0116] The newly confirmed RISBZ cDNAs (RISBZ1, RISBZ4, RISBZ5) were identified in detail as follows. RISBZ1 cDNA is the longest, 1742bp excluding polyadenylic acid, and contains a reading frame encoding 436 amino acids (estimated molecular weight is 46,491Dal). RISBZ4 and RISBZ5 have reading frames encoding 278 and 295 amino acids; their predicted molecular weights are 29383 and 31925 Dal, respectively.

[0117] RISBZ1 mRNA has a leader sequence (245 bases long) that is longer than the average leader sequence. Interestingly, a small open reading frame encoding 31 amino acid residues was found within the leader sequence upstream of the actual start codon of the RISBZ1 protein. Previous studies on maize Opaque2(O2) (Hartings H. et al., EMBO J.8: 2795-2801, 1989), wheat SPA (Albani D. et al., Plant Cell 9: 171-184, 1997), barley BLZ1 and BLZ2 (Vincente -Carbojos J. et al., Plant J.13: 629-640, 1998; Onate L. et al., J. Biol....

Embodiment 3

[0123] [Example 3] Genome structure of RISBZ1 gene

[0124] Using primers designed from the nucleotide sequence of RISBZ1 cDNA, the genomic region encoding the promoter and RISBZ1 protein was isolated. A PCR reaction was performed using rice genomic DNA as a template and two pairs of oligonucleotides as primers (RIS1f: 5'-ATGGGTTGCGTAGCCGTAGCT-3' / SEQ ID NO: 18 and RELr5: 5'-TTGCTTGGCATGAGCATCTGT-3' / SEQ ID NO: 19) and (RELf2: 5'-GAGGATCAGGCCCATAT-3' / SEQ ID NO: 20) and RIS1r: 5'-TCGCTATATTAAGGGAGACCA-3' / SEQ ID NO: 21). TAKARALA Taq polymerase (TAKARA) was used to amplify DNA fragments in a thermal cycler, using 30 cycle reactions, 98°C for 10 seconds, 56°C for 30 seconds, and 68°C for 5 minutes. The promoter region of the RISBZ1 gene was also amplified by thermal asymmetric staggered (TAIL) PCR based on the method of Liu et al., in which three oligonucleotides were used as specific primers, tail1: 5'-TGCTCCATTGCGCTCTCGGACGAG-3' / SEQ ID NO:22, tail2:5'-ATGAATTCGCGAGGGGTTTTCGA-...

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Abstract

cDNAs (RISBZ1, RISBZ4, and RISBZ5) encoding bZIP transcription factors were isolated from a cDNA library originating in rice plant seed. The cDNAs encode novel proteins and have binding activity to the GCN4 motif. Among them, RISBZ1 activated transcription mediated by the GCN4 motif by 100-fold or more. Since the expression of RISBZ1 precedes the expression of a seed storage protein gene and is expressed only in maturing seeds, it is suggested that RISBZ1 controls the expression of rice seed storage proteins. In addition, by linking the recognition sequence of the transcription factor, the GCN4 motif, in tandem and introducing it into the promoter for a gene encoding seed storage protein to facilitate its binding to the transcription factor RISBZ1, expression of a foreign gene under the control of the modified promoters is greatly enhanced.

Description

technical field [0001] The present invention relates to a novel transcription factor and its use in connection with endosperm-specific expression of storage proteins in rice plant seeds. Background technique [0002] Seed storage proteins are only expressed in the mature stage of seeds, and the analysis of genes encoding these proteins can serve as a suitable model for studying the mechanism of plant gene transcription regulation (Goldberg, R.B. et al., Science 266:605-614, 1994). It is known that the expression of genes encoding seed storage proteins is regulated by the coordinated action of multiple cis-factors in the promoter. The association of transcription factors with specific cis-regulators is important in the initiation of transcription and in tissue- and temporal-specific expression. This could be explained by the fact that the expression of seed storage proteins is induced by several types of cis-regulators that are involved in the regulation of seed-specific exp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H5/00C07K14/415C07K16/16C12N1/15C12N1/19C12N1/21C12N5/10C12N9/22C12N15/09C12N15/29C12N15/82C12P21/02C12P21/08C12Q1/02C12R1/91
CPCC12N15/8217C12N15/8238C07K14/415C12N15/8261C12N15/8234C12N15/8251Y02A40/146C12N15/11
Inventor 高岩文雄小野寺康之
Owner NAT INST OF AGROBIOLOGICAL SCI
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