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Nucleic Acid Molecules and Other Molecules Associated with Plants

a technology of nucleic acid molecules and plants, applied in the field of plant biochemistry, can solve the problems of 1-2% error or base ambiguity rate, not yielding the best results under all conditions, etc., and achieve the effect of reducing the expression of a protein, reducing or depressing the expression of the protein

Inactive Publication Date: 2010-01-28
MONSANTO TECH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a purified nucleic acid molecule that encodes a maize protein or fragment thereof, which can be used to produce genetically transformed plants with improved traits. The invention also provides a method for regulating the expression of a protein in a plant cell to produce a desirable effect. The invention also includes a method for isolating a genetic region that encodes a protein or fragment thereof, and a method for determining the level or pattern of a protein in a plant cell. Overall, the invention provides a way to improve the agricultural value of plants through genetic modification.

Problems solved by technology

Automated single run sequencing typically results in an approximately 2-3% error or base ambiguity rate.
BLOSUM62 is tailored for alignments of moderately diverged sequences and thus may not yield the best results under all conditions.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0257]The CMz030 (Lib143) cDNA library is generated from maize (DK604, Dekalb Genetics, Dekalb, Ill. U.S.A.) heat shocked seedling tissue two days post germination. Seeds are planted on a moist filter paper on a covered try that is keep in the dark until germination. The trays are then moved to the bench top at 15 hr daytime / 9 hr nighttime cycles for 2 days post-germination. The day time temperature is 80° F. and the nighttime temperature is 70° F. Tissue is collected when the seedlings are 2 days old. At this stage, the colehrhiza has pushed through the seed coat and the primary root (the radical) is just piercing the colehrhiza and is barely visible. The seedlings are placed at 42° C. for 1 hour. Following the heat shock treatment, the seedlings are immersed in liquid nitrogen and crushed. The harvested tissue is stored at −80° until RNA preparation. The RNA is purified from the stored tissue and the cDNA library is constructed as described in Example 2. SEQ ID NO: 1 through SEQ I...

example 2

[0272]The stored RNA is purified using Trizol reagent from Life Technologies (Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A.), essentially as recommended by the manufacturer. Poly A+ RNA (mRNA) is purified using magnetic oligo dT beads essentially as recommended by the manufacturer (Dynabeads, Dynal Corporation, Lake Success, N.Y., U.S.A.).

[0273]Construction of plant cDNA libraries is well-known in the art and a number of cloning strategies exist. A number of cDNA library construction kits are commercially available. The Superscript™ Plasmid System for cDNA synthesis and Plasmid Cloning (Gibco BRL, Life Technologies, Gaithersburg, Md., U.S.A.) is used, following the conditions suggested by the manufacturer.

example 3

[0274]The cDNA libraries are plated on LB agar containing the appropriate antibiotics for selection and incubated at 37° for a sufficient time to allow the growth of individual colonies. Single selective media colonies are individually placed in each well of a 96-well microtiter plates containing LB liquid including the selective antibiotics. The plates are incubated overnight at approximately 37° C. with gentle shaking to promote growth of the cultures. The plasmid DNA is isolated from each clone using Qiaprep plasmid isolation kits, using the conditions recommended by the manufacturer (Qiagen Inc., Santa Clara, Calif., U.S.A.).

[0275]Template plasmid DNA clones are used for subsequent sequencing. For sequencing, the ABI PRISM dRhodamine Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq® DNA Polymerase, FS, is used (PE Applied Biosystems, Foster City, Calif., U.S.A.).

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PUM

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Abstract

Expressed Sequence Tags (ESTs) isolated from maize are disclosed. The ESTs provide a unique molecular tool for the targeting and isolation of novel genes for plant protection and improvement. The disclosed ESTs have utility in the development of new strategies for understanding critical plant developmental and metabolic pathways. The disclosed ESTs have particular utility in isolating genes and promoters, identifying and mapping the genes involved in developmental and metabolic pathways, and determining gene function. Sequence homology analyses using the ESTs provided in the present invention, will result in more efficient gene screening for desirable agronomic traits. An expanding database of these select pieces of the plant genomics puzzle will quickly expand the knowledge necessary for subsequent functional validation, a key limitation in current plant biotechnology efforts.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. application Ser. No. 11 / 353,150 filed Feb. 14, 2006 (pending), which is a continuation of U.S. application Ser. No. 09 / 565,306 filed May 4, 2000 (abandoned), which claims the benefit of U.S. Provisional Application No. 60 / 132,860 filed May 7, 1999 (expired) and U.S. Provisional Application No. 60 / 101,508 filed Sep. 22, 1998 (expired), all of which applications are herein incorporated by reference in their entireties.INCORPORATION OF SEQUENCE LISTING[0002]This application contains an electronic equivalent paper copy of the sequence listing submitted herewith electronically via EFS web and a computer-readable form of the sequence listing submitted herewith electronically via EFS web and contains the file named “P30114US04_seqlist.txt”, which is 49,918,550 bytes in size (measured in MS-DOS) and which was also created on Jun. 11, 2009.FIELD OF THE INVENTION[0003]The present invention is in the field ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01H5/00C07H21/04C07K14/415
CPCC07K14/415C12N15/8242C12N15/8261Y02A40/146
Inventor ANDERSEN, SCOTT E.CONNER, TIMOTHY W.VISHWANATH, RAGHU LALGUDI
Owner MONSANTO TECH LLC
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