Regulatory sequences for expressing gene products in plant reproductive tissue

a gene product and regulatory sequence technology, applied in the field of expression cassettes, can solve the problems of insufficient core promoter region to provide full promoter activity, inability to identify and specific regulatory regions, and difficulty in obtaining sufficient expression levels of gene products directed to expression in specific tissues, etc., to achieve the effect of enhancing the capacity for small molecule uptake, increasing the sink strength, and increasing the sink strength

Inactive Publication Date: 2010-08-12
SYNGENTA PARTICIPATIONS AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0020]“Exon” refers to a section of DNA which carries the coding sequence for a protein or part of it. Exons are separated by intervening, non-coding sequences (introns). For purposes of the present invention, the definition of the term “exon” includes modifications to the nucleotide sequence of an exon derived from a target gene, provided the modified exon does not significantly reduce the activity of its associated 5′ regulatory sequence.

Problems solved by technology

However, the core promoter region is insufficient to provide full promoter activity.
However, sufficient expression levels of gene products, especially those gene products directed to expression in specific tissues, is difficult to obtain.
However, to date, identifying and specific regulatory regions and incorporating them into a robust trait delivery platform has not been accomplished.

Method used

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  • Regulatory sequences for expressing gene products in plant reproductive tissue
  • Regulatory sequences for expressing gene products in plant reproductive tissue
  • Regulatory sequences for expressing gene products in plant reproductive tissue

Examples

Experimental program
Comparison scheme
Effect test

example 1

Method of Constructing Expression Cassettes Comprising Regulatory Sequences from the MADS Gene Family

[0052]1. Identifying target MADS genes.

2. Identifying high quality sequence for both the target's genomic DNA (gDNA) and cDNA.

3. Identifying the target gene's open reading frame on the cDNA. In general this is the longest open reading frame.

4. Using a candidate gene's cDNA sequence to annotate gDNA sequence and marking positions of the translation start codon, translation stop codon, introns, exons, the 5′-untranslated leader and the 3′-untranslated sequence. As is known in the art, marking the translation start codon and the translation stop codon identifies the 5′-regulatory sequence and the 3′-regulatory sequence of the gene. According to the present invention, the promoter, which includes the promoter regulatory sequence, is the sequence that extends approximately 1.5 to 2.5 kb upstream from the translation start codon, wherein the 3′-regulatory sequence of the present invention ...

example 2

1. Construction of the Assembly Vector pNOV6901 Containing the β-Glucuronidase (GUS) Coding Sequence

[0085]A. Preparation of GUS Coding Sequence.

[0086]The β-glucuronidase (GUS) coding sequence Narasimhulu, et al 1996, Plant Cell, 8: 873-886, which includes an engineered intron, was amplified from pNOV5003 in a Pfuturbo polymerase (Stratagene, Cat. No. 600250) reaction. The reaction mixture consisted of 1 μL pNOV5003 miniprep DNA 200 μM dNTPs, 20 μM GUS5 oligonucleotide primer 5′-atggtacgtcctgtagaaacc-3′ (SEQ ID NO 498), 20 μM GUS3 oligonucleotide primer 5′-gatcgagctetcattgtttgcctccctg-3′ (SEQ ID NO 499), 5 μL 10× cloned Pfu buffer and 2.5 Units of Pfuturbo DNA polymerase (Stratagene, Cat. No. 600250) in a final volume of 50 μL. The thermocycling program was at 95° C. for 30 seconds then 10 cycles of (95° C. for 5 seconds, 55° C. for 10 seconds, 72° C. for 2.5 minutes) then 20 cycles of (95° C. for 5 seconds, 57° C. for 15 seconds, 72° C. for 2.5 minutes) then 72° C. for 2.5 minutes. ...

example 3

Construction of the OsMADS5 Expression Cassette

[0098]A. Cloning the OsMADS5 5′-Regulatory Sequence

[0099]High-fidelity PCR was used to amplify the OsMADS5 5′-regulatory sequence from rice genomic DNA (gDNA). The 50 μL reaction mixture consisted of 100 ng rice gDNA, 200 μM dNTPs, 1 μL 20 μM oligonucleotide primer OsMADS5-P3 5′-tgagcaggtagccggcgaccaatcgcgag-3′ (SEQ ID NO 460), 1 μL 20 μM oligonucleotide primer OsMADS#5-P2 5′-catactgttacaaaaaagaaaatcagtggaccac-3′ (SEQ ID NO 461), 1 μL 10× Expand High Fidelity buffer and 1 μL Expand High Fidelity polymerase. The thermocycling program was at 95° C. for 2 minutes followed by 40 cycles of (94° C. for 15 seconds, 68° C. for 7.5 minutes) followed by 68° C. for 10 minutes. The 5.4 kb DNA product, encoding the OsMADS5 5′-regulatory sequence, was cloned with the TOPO XL PCR cloning kit. The pCR-XL-TOPO-OsMADS5-5′-gDNA recombinants, containing the OsMADS5 5′-regulatory sequence, were identified by digesting 5 μL pCR-XL-TOPO-OsMADS5-5′-gDNA minipr...

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Abstract

Expression cassettes causing specific regulatory control of transgene expression in plants, wherein the expression cassettes include regulatory sequences from the MADS gene family for expression of recombinant gene products in the reproductive tissue of plants for the purpose of generating abiotic stress tolerant plants.

Description

FIELD OF THE INVENTION[0001]The present invention includes expression cassettes that contain regulatory sequences derived from a target gene, for example, regulatory sequences from the MADS gene family, for tissue specific expression of recombinant gene products in plants.BACKGROUND OF THE INVENTION[0002]In agricultural biotechnology, plants can be modified according to one's needs. One way to accomplish this is by using modern genetic engineering techniques. For example, by introducing a gene of interest into a plant, the plant can be specifically modified to express a desirable phenotypic trait. For this, plants are transformed most commonly with a heterologous gene comprising a promoter region, a coding region and a termination region. When genetically engineering a heterologous gene for expression in plants, the selection of a promoter is often a critical factor. While it may be desirable to express certain genes constitutively, i.e. throughout the plant at all times and in most...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/82C12N9/16A01H5/00A01H1/00
CPCC12N15/827C12N15/8271
Inventor NUCCIO, MICHAEL L.LAGRIMINI, L. MARKMEGHJI, MOEZ
Owner SYNGENTA PARTICIPATIONS AG
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