Arabidopsis derived promoters for regulation of plant expression

a technology of plant genes and promoters, applied in the field of plant molecular biology, to achieve the effect of less biased analysis

Inactive Publication Date: 2010-01-21
SYNGENTA PARTICIPATIONS AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0016]As described herein, GENECHIP® technology was utilized to discover genes that are preferentially (or exclusively) expressed in various tissues including root and leaf, as well as those that are constitutively expressed, using labeled cRNA probes, determining expression levels by laser scanning and generally selecting for expression levels that were >2 fold over the control. The Arabidopsis oligonucleotide probe array consists of probes from about 8,1

Problems solved by technology

Moreover, the increasing interest in cotransforming plants with multiple plant transcription units (PTU) and the potential probl

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

GENECHIP® Standard Protocol

Quantitation of Total RNA

[0368]Total RNA from plant tissue is extracted and quantified.[0369]1. Quantify total RNA using GeneQuant 1OD260=40 mg RNA / ml; A260 / A280=1.9 to about 2.1[0370]2. Run gel to check the integrity and purity of the extracted RNA

Synthesis of Double-Stranded cDNA

[0371]Gibco / BRL SuperScript Choice System for cDNA Synthesis (Cat#1B090-019) was employed to prepare cDNAs. T7-(dT)24 oligonucleotides were prepared and purified by HPLC. (5′-GGCCAGTGAATTGTAATACGACTCACTATAGGGA GGCGG-(dT)24-3′; SEQ ID NO:27).

[0372]Step 1. Primer Hybridization:[0373]Incubate at 70° C. for 10 minutes[0374]Quick spin and put on ice briefly

[0375]Step 2. Temperature Adjustment:[0376]Incubate at 42° C. for 2 minutes

[0377]Step 3. First Strand Synthesis:[0378]DEPC-water-1 μl[0379]RNA (10 μg final)-10 μl[0380]T7=(dT)24 Primer (100 pmol final)-1 μl pmol[0381]5×1st strand cDNA buffer-4 μl[0382]0.1M DTT (10 mM final)-2 μl[0383]10 mM dNTP mix (500 μM final)-1 μl[0384]Superscri...

example 2

Characterization of Gene Expression Profiles During Plant Development Using the GENECHIP®

[0416]The Arabidopsis GENECHIP® provides a method to simultaneously scan over 30% of the genome for the expression profile of each gene on chip. By using RNA extracted from different tissue and developmental stages of development, a scan of the entire Arabidopsis plant is achieved. The advantages of a gene chip in such an analysis include a global gene expression analysis, quantitative results, a highly reproducible system, and a higher sensitivity than Northern blot analyses. Moreover, a gene chip with Arabidopsis DNA has a further advantage in that the Arabidopsis genome is well characterized.

[0417]Using the recently designed Arabidopsis high density oligonucleotide probe array, a total of 8,100 Arabidopsis thaliana genes were surveyed for temporal and developmental expression profiling. The objective was to identify known and novel genes that are expressed in specific organs (spatial expressi...

example 3

Further Analysis of Constitutively Expressed Genes

[0476]A standard curve of 50, 10, 2, 0.4, and 0.08 ng total RNA was generated for each primer / probe set tested. In this case, the 50 ng sample yielded a Ct value of 24.5 and the ng sample yields a Ct value of 26.7. The Ct value is defined as the threshold cycle whereby amplification occurs at an exponential rate. A low Ct value correlates with high gene expression. The threshold is determined empirically from the standard curve. By raising or lowering the threshold, the data set is maximized to represent optimal exponential amplification. A correlation coefficient (R2 of the best-fit line from the standard curve) greater than 99% and a slope of −3.3 (most efficient amplification) is ideal. For accurate repeatable results, the previous criteria must be met and the unknowns must fall within the range of the curve. The expression levels of the unknown can be interpolated from the unknown Ct values using the standard curve.

[0477]TaqMan c...

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Abstract

The invention provides a method to identify a plurality of plant promoters having specified characteristics and promoters identified by the method. Also provided are transgenic plants comprising the genes identified by the methods of the invention.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation-in-part of U.S. patent application Ser. No. 11 / 334,085 filed Jan. 18, 2006, which is a continuation-in-part of Ser. No. 09 / 887,567 filed Jun. 22, 2001, which claims the benefit of the filing date of U.S. Provisional Application Ser. No. 60 / 214,087 filed Jun. 23, 2000, U.S. Provisional Application Ser. No. 60 / 213,848 filed Jun. 23, 2000, and U.S. Provisional Application Ser. No. 60 / 258,692, filed Dec. 29, 2000 under 35 U.S.C. §119(e), the entire disclosures of which are incorporated by reference herein.FIELD OF INVENTION[0002]The present invention relates generally to the field of plant molecular biology. More specifically, it relates to the regulation of gene expression in plants.BACKGROUND OF INVENTION[0003]Manipulation of crop plants to alter and / or improve phenotypic characteristics (such as productivity or quality) requires the expression of heterologous genes in plant tissues. Such genetic manipulat...

Claims

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Application Information

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IPC IPC(8): A01H5/00C07H21/00C12N15/74
CPCC07K14/415C12N15/8216C12N15/8241C12N15/8227C12N15/8225
Inventor BROWN, DEVON L.HIPSKIND, JOHN
Owner SYNGENTA PARTICIPATIONS AG
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