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3-hydroxyl-3-mathylglutaryl-coa reductase albumen of barbadosnut and encoding gene thereof

A technology of methylglutaryl coenzyme and Jatropha curcas, which can be applied in the fields of molecular biology and genetic engineering, and can solve problems that have not yet been discovered.

Inactive Publication Date: 2010-06-23
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Prior art "Bioscience Biotechnology Biochemistry (biological science, biotechnology, biochemistry) 2008, 72 (8): 2049-2060" and "Wuhan Botany Research 2007, 25 (2): 123-126" have been reported from Brazil The 3-hydroxyl-3-methylglutaryl-CoA reductase gene has been cloned in the rubber tree and Euphorbia, and no literature report identical with the subject of the present invention has been found so far

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Cloning of JcHMGRs Protein Gene of Jatropha curcas

[0041] 1. Tissue separation (isolation)

[0042] The seeds of Jatropha curcas come from Panzhihua, Sichuan Province. After the seeds of Jatropha curcas are collected, they are stored in the laboratory.

[0043] 2. RNA isolation (RNA isolation)

[0044] Peel off the seed coat of Jatropha curcas seeds, take out the seed kernels, grind them with a mortar, add liquid nitrogen, grind them into powder, take 100mg and transfer them to 1.5mL EP tubes, and extract total RNA (two-step lysis method) . The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis, and then the RNA content was determined on a spectrophotometer.

[0045] 3. Cloning of Full-length cDNA

[0046] According to the amino acid conserved sequences of some plant HMGRs, design degenerate primers, use the principle of homologous gene cloning, and use SMART TM RACE cDNA amplification method (Clonetech kit) for full-length cDNA c...

Embodiment 2

[0058] Sequence information and homology analysis of JcHMGRs protein gene of Jatropha curcas

[0059] The length of the full-length cDNA of the new Jatropha curcas JcHMGRs protein of the present invention is 1950bp, and the detailed sequence is shown in SEQ ID NO.1, wherein the open reading frame is located at nucleotides 81-1832. The amino acid sequence of Jatropha curcas JcHMGRs protein was deduced according to the full-length cDNA, with a total of 584 amino acid residues, a molecular weight of 62410.72, and a pI of 7.63. See SEQ ID NO.2 for the detailed sequence.

[0060]The full-length cDNA sequence of Jatropha curcas JcHMGRs protein and its encoded protein were compared in the Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR databases using BLAST program. Protein homology retrieval, the result shows that it has 86% homology (Table 2) with N. brasiliensis HbHMGRs gene (AF429388) on nucleotide level; P29057) have 87...

Embodiment 3

[0192] Prokaryotic expression and purification of Jatropha curcas JcHMGRs protein in Escherichia coli

[0193] In this example, the full-length Jatropha curcas JcHMGRs protein coding sequence or fragment was constructed into a commercially available protein fusion expression vector to express and purify the recombinant protein.

[0194] Prokaryotic expression of Jatropha curcas JcHMGRs protein polypeptide in the form of fusion protein in Escherichia coli.

[0195] The construction of prokaryotic expression vector, and transformed escherichia coli: according to the aminoacid sequence of Jatropha curcas JcHMGRs protein, the primer of protein coding region is designed, and restriction endonuclease site is introduced respectively on forward and reverse primer (this according to the selected pQE30 carrier Depends), in order to construct the expression vector. Using the amplified product obtained in Example 1 as a template, after PCR amplification, the Jatropha curcas JcHMGRs prote...

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PUM

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Abstract

The invention relates to 3-hydroxyl-3-mathylglutaryl-CoA reductase albumen encoding sequence of barbadosnut, which belongs to the technical field of molecular biology and gene engineering. The DNA molecule separation comprises the following steps: the nucleotide sequence of polypeptide with barbadosnutJcHMGRs albumen activity is encoded, and the nucleotide sequence has a homology of at least 70 percent with the nucleotide sequence with nucleotide ranging from bit 81-1832 in SEQ ID NO.1; the nucleotide sequence can be hybridized with the nucleotide sequence with nucleotide ranging from bit 81-1832 in SEQ ID NO.2 at 40-55 DEG C. The invention is an enzyme, namely, 3-hydroxyl-3-mathylglutaryl-CoA generated by catalyzing acetyl-CoA and acetoacetyl coenzyme A. The enzyme is favorable for reducing the content of phorbol ester or the prosoma thereof in barbadosnut and greatly helpful for cultivating quality variety of aseptic barbadosnut.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and genetic engineering. Specifically, the present invention relates to a jc-Hmgrs protein expressed in Jatropha curcas (Jatrophacurcas 3-hydroxy-3-methylglutaryl-CoA reductase protein, Jatrophacurcas 3-hydroxy-3-methylglutaryl-coenzyme A reductase, JCHMGRs) and its nucleic acid sequence. Background technique [0002] The current research, development and application of Jatropha curcas seed oil as bio-fuel has become a hot spot in bio-energy research, and with the rapid development of Jatropha curcas as bio-fuel research, it is necessary to maximize its economic benefits and obtain good results. Eco-environmental benefits-the rational use of its by-products has become another hot spot in the research of Jatropha curcas. [0003] Jatropha curcas toxin-phorbol ester (12-deoxy-16-hydroxyphorbol, 12-deoxy-16-hydroxy-phorbol) is a diterpenoid compound, mainly present in Jatropha curcas pla...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/11C12N15/63C12N1/19C12N5/10C12Q1/68
Inventor 林娟金元杰侯嵘唐克轩
Owner FUDAN UNIV
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