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Isopentenyl transferase sequences and methods of use

a technology of isopentenyl transferase and sequence, applied in the field of gene activity modulation of plants, to achieve the effects of reducing or eliminating the activity of an ipt polypeptide, reducing or eliminating the level of cytokinin in the plant, and modulating the root development of the plan

Inactive Publication Date: 2006-03-23
PIONEER HI BRED INT INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0010] Compositions further include expression cassettes, plants, plant cells, and seeds having the IPT sequences of the invention. The plants, plant cells, and seeds of the invention may exhibit phenotypic changes, such as modulated (increased or decreased) cytokinin levels; modulated floral development; modulated root development; altered shoot to root ratio; increased seed size or an increased seed weight; increased plant yield or plant vigor; maintained or improved stress tolerance (e.g., increased or maintained size of the plant, minimized tip kernel abortion, increased or maintained seed set); decreased shoot growth; delayed senescence or an enhanced vegetative growth, all relative to a plant, plant cell, or seed not modified per the invention.
[0012] Methods are provided for reducing or eliminating the activity of an IPT polypeptide in a plant, comprising introducing into the plant a selected polynucleotide. In specific methods, providing the polynucleotide decreases the level of cytokinin in the plant and / or modulates root development of the plant.
[0013] Methods are also provided for increasing the level of an IPT polypeptide in a plant comprising introducing into the plant a selected polyn. In specific methods, expression of the IPT polynucleotide increases the level of a cytokinin in the plant; maintains or improves the stress tolerance of the plant; maintains or increases the size of the plant; minimizes seed abortion; increases or maintains seed set; increases shoot growth; increases seed size or seed weight; increases plant yield or plant vigor; modulates floral development; delays senescence; or increases leaf growth.

Problems solved by technology

However, this pathway is not considered to be the main route for cytokinin synthesis (Chen et al.

Method used

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  • Isopentenyl transferase sequences and methods of use
  • Isopentenyl transferase sequences and methods of use
  • Isopentenyl transferase sequences and methods of use

Examples

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example 1

Cloning and Gene Characterization of ZmIPT1

[0388] Below we describe the identification and characterization of an IPT polypeptide from maize designated ZmIPT1.

[0389] Material and methods: A Mo17 BAC library was screened using a 3′-end fragment of the ZmIPT1 cDNA from B73, identified by sequence similarity to Agrobacterium ipt. One of the positive clones was digested by HindIII, and subcloned into pBluescript. Recombinant plasmids were screened using colony screening and a 3′-end fragment as a probe. One positive clone was sequenced. Samples used for RT-PCR were harvested in the field from three individual plants. Five μg of total RNA was used for RT-PCR using ThermoScript RT-PCR System from Invitrogen. The reverse transcribed mixture for PCR used primers designed across an intron-exon-intron junction in order to avoid amplification of genomic DNA.

[0390] Results:

[0391] A. Deduced Protein Sequence:

[0392] Two putative maize ipt ESTs were identified whose deduced amino acid sequenc...

example 2

Gene Expression of ZmIPT1

[0399] One of the identified ZmIPT1 ESTs was from a B73 embryo library, the other one was from a developing root library. In order to gain an impression of the level of expression of ZmIPT1, a search of the Lynx database was performed. A perfect tag was found in the 3′-end of the gene, 231 bp from the poly A tail start. Tissue types, number of library hits, and average ppm are presented in Table 1. Expression was found to be very low in most organs, but higher in seedling and embryo libraries. In embryo libraries, expression was higher at 10 DAP than at later development stages (FIG. 4).

TABLE 1Number of Lynx libraries containing the ZmIPT1 tag and average ppmvalues.Tissue# of LynxAveragetypelibrariesppmSeedling129Ear54Endosperm28Embryo410.75Stalk19Leaf23Root22.5

[0400] Using RT-PCR, the expression pattern of ZmIPT in various maize organs and on a kernel developmental series was tested. No amplification product could be detected after 20 cycles, confirming ...

example 3

Isolation and Gene Characterization of ZmIPT2, ZmIPT4, ZmIPT5. ZmIPT6, ZmIPT7, ZmIPT8 and ZmIPT9

[0402] The AtIPT1 and AtIPT3 to AtIPT8 protein sequences were blasted against the six possible frames generated by the maize genomic sequences and searched for some degree of similarity. Because rice and maize genomes show a significant degree of synteny, the same method was used against rice genomic database to optimize this search. The rice sequences with an E-score of at least 200 were then used for an additional screen of the GSS maize database. Since at that time, the GSS database had not been assembled into contigs, the sequences obtained which had an E-score of at least 150 were pooled and aligned using Sequencher.

[0403] Using this method, eight maize contigs encoding putative CK biosynthetic enzymes were identified (ZmIPT2 to ZmIPT9), six of them showing an open reading frame without introns. The translated proteins corresponding to these putative genes contained 320 to 380 amin...

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Abstract

Methods and compositions for modulating plant development are provided. Polynucleotide sequences and amino acid sequences encoding isopentenyl transferase (IPT) polypeptides are provided. The sequences can be used in a variety of methods including modulating root development, modulating floral development, modulating leaf and / or shoot development, modulating senescence, modulating seed size and / or weight, and modulating tolerance of plants to abiotic stress. Polynucleotides comprising an IPT promoter are also provided. The promoter can be used to regulate expression of a sequence of interest. Transformed plants, plant cell, tissues, and seed are also provided.

Description

[0001] This application claims priority to, and hereby incorporates by reference, U.S. provisional patent application 60 / 610,656 filed Sep. 17, 2004; 60 / 637,230 filed Dec. 17, 2004; and 60 / 696,405 filed Jul. 1, 2005.FIELD OF THE INVENTION [0002] The invention relates to the field of genetic manipulation of plants, particularly the modulation of gene activity to affect plant development and growth. BACKGROUND OF THE INVENTION [0003] Cytokinins are a class of N6 substituted purine derivative plant hormones that regulate cell division and influence a large number of developmental events, such as shoot development, sink strength, root branching, control of apical dominance in the shoot, leaf development, chloroplast development, and leaf senescence (Mok et al. (1994) Cytokinins. Chemistry, Action and Function. CRC Press, Boca Raton, Fla., pp. 155-166; Horgan (1984) Advanced Plant Physiology ed. MB., Pitman, London, UK, pp53-75; and Letham (1994) Annual Review of Plant Physiol 34:163-197...

Claims

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Application Information

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IPC IPC(8): A01H1/00A01H5/00C12N15/82C12N5/04
CPCC12N9/1085C12N15/8295C12N15/8261C12N15/8216Y02A40/146C12Y205/01027
Inventor BRUGIERE, NORBERT
Owner PIONEER HI BRED INT INC
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