Enzymes involved in triterpene synthesis

A technology of amyrin synthase and methyltransferase, which is applied in the direction of transferase, cells modified by introducing foreign genetic material, angiosperms/flowering plants, etc., and can solve the problem of lack of peptidase activity and other problems

Inactive Publication Date: 2014-02-26
PLANT BIOSCI LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These enzymes - serine carboxypeptidase-like acyltransferases - have homology to peptidases but lack peptidase activity and are instead able to acylate natural products (Milkowski C & Strack D, (2003), Phytochemistry 65:517; Fraser CM et al. ( 2005) Plant Physiology 138: 1136)

Method used

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  • Enzymes involved in triterpene synthesis
  • Enzymes involved in triterpene synthesis
  • Enzymes involved in triterpene synthesis

Examples

Experimental program
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Effect test

Embodiment 1

[0161] Isolation of Genomic and cDNA Fragments of Serine Carboxypeptidase-Like Protein (AsSCPL1), Methyltransferase (AsMT1) and Glucosyltransferase (AsGT2)

[0162] Genomic polynucleotide fragments encoding genes affected by serine carboxypeptidase-like protein (AsSCPL1), methyltransferase (AsMT1) and glucosyltransferase (AsGT2) were isolated from the BAC library derived from the accession number S75 genomic DNA and cDNA libraries were prepared from oats as follows.

[0163] The BAC library was constructed from the black oat accession number S75 genomic DNA ((Qi X. et al., 2006, Proc.Natl.Acad.Sci.U.S.A103:18848-18853). The DNA probe was derived from Sad1 (Osbourn et al., 2007 On March 6th, US, 7,186,884B2) and Sad2 (people such as Osbourn, US2006-0112448A1), this probe is used for screening complete BAC library.Construction spans a gene cluster (Qi X. et al., 2004, Proc.Natl.Acad.Sci.U.S.A.101:8233-8238) BAC contig. BAC fingerprint and BAC end sequence analysis allowed us ...

Embodiment 2

[0172] Isolation and characterization of Sad7 oat mutants

[0173] Seeds of diploid oats (Oats black) were mutagenized with sodium azide and M2 seeds from individual M1 plants were germinated and root fluorescence assessed for primary screening to identify saponin-deficient or Sad oat mutants. Candidate avenantia root saponin-deficient mutants were identified based on reduced root fluorescence and confirmed by TLC and HPLC analysis of methanolic root extracts from homozygous M3 seedlings.

[0174] generate mutants

[0175] Seeds of diploid oats (Oats black) (accession number S75 from Institute of Grasslands and Environmental Research, Aberystwyth, Wales, UK) were mutagenized with sodium azide essentially as described (Rines, H.W., 1985, Env. Exp. Bot., 25:7-17). Briefly, mutagenesis was performed as follows. Seeds were presoaked in Erlenmeyer flasks sealed with rubber stoppers, using 0.5 mL of water per seed, and shaken at 120 cycles per minute in an orbital platform sh...

Embodiment 3

[0183] Isolation and characterization of Sad9 oat mutants

[0184] The original mutants #616, #825, #376 and #1243 (Papadopoulou et al. 1999. PNAS9612923-1928) did not contain any nucleotide changes in the AsMT1 and AsGT2 genes. The sequence analysis was extended to 82 new Sad mutants, which were isolated as described in Example 2. Three mutant M3 lines (#195, #961 and #1310) were identified as having point mutations in the AsMT1 gene (Table 2). No mutations were identified in the AsGT2 gene for any mutants in the pool. DNA sequence analysis confirmed a single nucleotide change in the coding sequence in three mutants (#195, #961 and #1310). Each of these nucleotide changes was predicted to result in an amino acid change (Table 2).

[0185]The roots of these three mutants lacked the bright blue fluorescence associated with avenantia root saponin A-1 and showed dark purple fluorescence under UV excitation. These mutants are called "purple mutants". A more purple mutant #8...

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Abstract

The invention discloses enzymes involved in triterpene synthesis, and particularly relates to isolated polynucleotides. The polynucleotides encode enzymes comprising a carboxypeptidase-like protein, a methyltransferase and a glucosyltransferase. The enzymes are involved in the biosynthesis of beta-amyrin-derived triterpenes in plants and seeds. The invention also relates to construction of recombinant DNA constructs comprising all or a portion of the isolated polynucleotides. The polynucleotides are, in sense or antisense orientation, operably linked to at least one regulatory sequence.

Description

[0001] This application is a divisional application of the Chinese patent application 200780053491.4 "Enzymes Involving Triterpene Synthesis" filed on June 25, 2007. technical field [0002] The invention relates to the field of plant molecular biology. More specifically, the present invention relates to polynucleotides encoding enzymes involved in the biosynthesis of β-amyrin-derived triterpenes in plants and seeds. The invention also includes transgenic plants in which altered expression levels of polynucleotides of the invention result in altered levels or structures of [beta]-amyrin-derived triterpenes, including saponins. Background technique [0003] Terpenoids, also known as isoprenoids, form the largest family of natural products, with more than 22,000 individual compounds described. Triterpenes or terpenoids (semiterpenes, monoterpenes, sesquiterpenes, diterpenes, triterpenes, tetraterpenes, polyprenols, etc.) perform various functions in plants such as hormones, p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/63C12N5/10C12N1/19C12N1/21A01H5/00A01H4/00
Inventor A.奥斯伯恩X.齐
Owner PLANT BIOSCI LTD
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