Genes of mevalonate pathway enzyme encoding eucommia bark

A technology of mevalonate and genes, applied in genetic engineering, plant genetic improvement, biochemical equipment and methods, etc.

Inactive Publication Date: 2008-12-24
HITACHI ZOSEN CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, other enzymes involved in the mevalonate pathway have not been reported

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] (Example 1: EST analysis in Eucommia ulmoides)

[0036] (Material)

[0037] As the Eucommia plant sample, the phloem (bark) and xylem of young shoots of the current year collected from a standard tree of Eucommia growing in Ikuna Village, Ehime Prefecture in late May were used.

[0038] As the yeast mutant strain library, commercially available YKO Heterozygous Essential StrainCollection-Glycerol Stocks (Open Biosystems) was used.

[0039] (RNA extracted from Eucommia ulmoides)

[0040] About 4g of Eucommia ulmoides plant sample (the phloem and xylem of the branch of the current year) was cooled with liquid nitrogen, crushed with a mortar and a grinding rod, and suspended in 2 × CTAB solution (w / v) of 10 times the amount (w / v) of the sample ( 2% (w / v) cetyltrimethylammonium bromide (CTAB), 1% (w / v) 2-mercaptoethanol, 0.1M Tris-HCl (pH 9.5), 1.4M NaCl, 2OmM EDTA. It was incubated at 65° C. for 10 minutes, followed by treatment (washing) with chloroform / isoamyl alcohol...

Embodiment 2

[0047] (Example 2: Gene 1 Encoding Acetyl-CoA C-acetyltransferase)

[0048] (Acquisition of full-length cDNA)

[0049] By 3'-RACE (rapid amplification of cDNA ends, RACE), the sequence on the 3' end side was determined from the sequence obtained by the analysis in Example 1, and a full-length cDNA was obtained.

[0050] For 3'-RACE, 3'-Full RACE Core Set (manufactured by Takara Bio Co., Ltd.) was used. The oligo-dT primer was used in the reverse transcription reaction. For PCR amplification, primers for oligo-dT and sense primers having the same sequence as a part of the known sequence were used. The sense primers were designed according to the EST sequence information presumed to encode acetyl-CoA C-acetyltransferase obtained in Example 1. Using the RNA obtained in Example 1 as a template, the first time was to use N192-82-tree 1968_3R_S1 (SEQ ID NO.21) as a sense primer, and the second time was to use N192-82-tree 1968_3R_s2 (SEQ IDNO.22) as For sense primers, perform PC...

Embodiment 3

[0060] (Example 3: Gene 2 Encoding Acetyl-CoA C-acetyltransferase)

[0061] N192-84-tree_11012_3R_s1 (SEQ ID NO.25) (first time) and N192-84-tree_11012_3R_s2 (SEQ Full-length cDNA was obtained in the same manner as in Example 2 except that ID NO. 26) (second time) was used as a sense primer for 3'-RACE.

[0062] The obtained full-length cDNA has the base sequence of SEQ ID NO.3. The base sequence of the region shown by the 173rd to 1305th nucleotides of the base sequence of SEQ ID NO.3 has 83% homology with Kutki's acetyl-CoAC-acetyltransferase (Accession No. DQ347964). The open reading frame is 128-1354. The deduced amino acid sequence encoded by this cDNA is shown in SEQ ID NO.4. The full-length amino acid sequence (position 1-position 408) of SEQ ID NO.4 has 87% homology with Kutki's acetyl-CoA C-acetyltransferase (Accession No. ABC74567).

[0063] In this example, N219-36-tree_11012_s (SEQ ID NO.27) was used as a sense primer, N-219-36-tree_11012_a (SEQ ID NO.28) was u...

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PUM

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Abstract

The invention provides a gene cluster concerned with the biosynthesis of isoprenoid compound of eucommia. The invention also provides a gene concerned with the synthesis of isopentenylpyrophosate using mevalonate of eucommia, The gene cluster of the invention encodes acetyl-CoA C-acetyl transferase, HMG-CoA synthetase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase or mevalonate-diphosphate decarboxylase .

Description

technical field [0001] The present invention relates to a group of genes involved in the biosynthesis of isoprenoid compounds of Eucommia ulmoides. Background technique [0002] Polyisoprene (rubber), which is one of isoprene compounds, is classified into cis-type and trans-type according to the polymerization method of isoprene units. Many plants such as dandelion and mountain lettuce are known as plants that produce long-chain cis-polyisoprene. The cis-polyisoprene produced by Hevea brasiliensis is widely used commercially as natural rubber. As for long-chain trans-polyisoprene, it is known that it is naturally produced by a few plants such as Bakelite, but it is not commercially used. At present, trans-polyisoprene is chemically synthesized and used in golf ball skins, bandages, and sports protection equipment. Trans-polyisoprene is a thermoplastic elastomer showing a low melting point and high elasticity, and can be used as an insulator. [0003] The isoprenoid compo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N15/54C12N15/53C12N15/60
Inventor 福崎英一郎小林昭雄马场健史中泽庆久西河贵史
Owner HITACHI ZOSEN CORP
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