Compositions and methods for modulating lignin of a plant

A technology for plants and transgenic plants, applied in the field of compositions and methods for regulating plant lignin, capable of solving problems such as inability to distinguish genes of the same family

Inactive Publication Date: 2009-04-15
ARBORGEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Furthermore, microarrays containing cDNA or EST probes cannot distinguish genes of the same family because of the sequence similarity in these genes

Method used

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  • Compositions and methods for modulating lignin of a plant
  • Compositions and methods for modulating lignin of a plant
  • Compositions and methods for modulating lignin of a plant

Examples

Experimental program
Comparison scheme
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example 1

[0474] Example 1 illustrates how monolignol synthesis, monolignol transport, and lignin polymerization genes and lignin-like monolignol synthesis, monolignol transport, and lignin polymerization genes were isolated and characterized in Eucalyptus grandis and radiata pine.

[0475] Total RNA was extracted from plant tissues (using the protocol of Chang et al., Plant Mol. Biol. Rep. 11:113-116 (1993)). Plant tissue samples were obtained from phloem (P), cambium (C), extended xylem (X1) and differentiated and lignified xylem (X2).

[0476] Use Poly(A)Quik mRNA Isolation Kit (Stratagene, La Jolla, CA) or Dynal Beads Oligo(dT) 25 (Dynal, Skogen, Norway) mRNA was isolated from total RNA preparations. A cDNA expression library was constructed from purified mRNA by reverse transcriptase synthesis, and the resulting cDNA clones were inserted into lambda ZAP using the ZAP Express cDNA Synthesis Kit (Stratagene) according to the manufacturer's protocol used. The resulting cDNA was pack...

example 2

[0484] To identify 5' or 3' extra sequences of partial cDNA sequences in the cDNA library, rapid 5' and 3' amplification of cDNA ends (RACE) was performed using the SMART RACE cDNA Amplification Kit (Clontech Laboratories, Palo Alto, Calif.). The required method typically first isolates poly(A) mRNA, performs first and second strand cDNA synthesis to generate double-stranded cDNA, truncates the cDNA ends, and then ligates SMART RACE adapters to the cDNA to form the adapter-adapted junctions. Sub-ds cDNA library. Gene-specific primers were designed for use in 5' and 3' RACE reactions with adapter-specific primers. Using 5' and 3' RACE reactions, 5' and 3' RACE fragments were obtained, sequenced and cloned. This method was repeated until the 5' and 3' ends of the full-length gene were identified. Full-length cDNA can be generated by PCR using primers specific for the 5' and 3' ends of the gene due to end-to-end PCR.

[0485] For example, to amplify a missing 5' region of a ge...

example 3

[0500] Example 3 illustrates a procedure for RNA extraction and purification, which is especially applicable to RNA obtained from conifer needles, xylem, cambium and phloem.

[0501] Tissues are obtained from conifer needles, xylem, cambium or phloem. Tissues were frozen in liquid nitrogen and pulverized. Total RNA was extracted using Concert Plant RNA Reagent (Invitrogen). The resulting RNA samples were extracted into phenol:chloroform and treated with DNase. The RNA was then incubated at 65°C for 2 minutes, followed by centrifugation at 4°C for 30 minutes. After centrifugation, RNA was extracted into phenol at least 10 times to remove contaminants.

[0502] RNA was further washed using RNeasy columns (Qiagen). Purified RNA was quantified using RiboGreen reagent (Molecular Probes) and the purity was assessed by gel electrophoresis.

[0503] RNA was then amplified using MessageAmp (Ambion). Aminoallyl UTP and free UTP were added to in vitro transcripts of purified RNA at...

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Abstract

Novel plant monolignol synthesis, monolignol transport, and lignin polymerization genes and polypeptides encoded by such genes are provided. These genes and polynucleotide sequences are useful regulating the lignification process and plant phenotype. Moreover, these genes are useful for expression profiling of plant monolignol synthesis, monolignol transport, and lignin polymerization genes. The invention specifically provides polynucleotide and polypeptide sequences isolated from Eucalyptus and Pinus.

Description

technical field [0001] The present invention generally relates to the fields of plant monolignol synthesis, monolignol transport and lignin polymerization genes and polypeptides encoded by said genes, and the use of said polynucleotide and polypeptide sequences for controlling plant phenotypes. Among other things, the invention provides polynucleotide and polypeptide sequences isolated from Eucalyptus and Pinus species and sequences related thereto. Background technique [0002] Lignin is a highly hydrophobic and cross-linked phenolic polymer that is the major constituent of the xylem of plants, especially of woody plants such as angiosperms and gymnosperms of the woody family. Lignin consists of three types of cinnamyl alcohols (p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol, which are in the C of the aromatic ring 3 and C 5 The degree of methoxylation at each position varies), although other phenols can be incorporated. See, eg, Sederoff et al. (1999) Curr. O...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/00C12N15/09C12N15/29C12N15/82C12N5/04C12N5/10C12N9/00C12N9/02C12N9/04C12N9/08C12N9/10C12N9/24C12N9/32C12N9/42C12N9/86C12N9/88
CPCC12N15/8255C07K14/415C12N9/00C12N9/0073
Inventor 威廉·H·罗特曼玛丽·B·康内特伦纳德·N·布洛克斯贝格理查德·L·福斯特
Owner ARBORGEN
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