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Jatropha curcas L. diacylglycerol transferase and coding gene and application thereof

A diacylglycerol and transferase technology, applied in the field of genetic engineering, can solve the problems of oil production capacity limitation, inability to realize commercial application, etc., and achieve the effects of high quality and improving oil content

Inactive Publication Date: 2011-09-28
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, natural microalgae that can be used for bioenergy cannot be commercially applied due to their limited oil production capacity.

Method used

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  • Jatropha curcas L. diacylglycerol transferase and coding gene and application thereof
  • Jatropha curcas L. diacylglycerol transferase and coding gene and application thereof
  • Jatropha curcas L. diacylglycerol transferase and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Molecular Cloning of Jatropha curcas Ptdagat Gene

[0025] 1) Cloning of the middle fragment of Jatropha curcas DAGAT enzyme gene cDNA: by comparing the amino acid sequences of Δ12 fatty acid desaturases of four known higher plants (Perilla frutescens, AF298815; Glycine max, AY652765; Ricinus communis, AY366496; Arabidopsis thaliana, NP_179535), determine the conserved region II and region III, that is, the corresponding amino acid sequences are LKLVSYAHTNYD and YWRMWNMPVHKWM respectively, and the positions of the upstream and downstream degenerate primer design regions are respectively designed degenerate primers Jcdagat1, 5'-CHTAYG CWC ATA CWA RCT ATG A-3' and Jcdagat2, 5'-CAY TTR TGV ACA GGC ATA TTC CA-3'; Jatropha curcas leaf total RNA was extracted and purified using plant RNA extraction kit (Hua Shun Company, Shanghai), after The RNA was removed from DNA contamination and stored at -80°C for later use; using the purified total RNA as a template and Olig(dT)20 as a...

Embodiment 2

[0034] Transient Expression of JcDAGAT Enzyme Gene in Various Tissue Cells of Jatropha curcas

[0035] See Table 3 for the experimental materials and the time they were collected:

[0036] Table 3: List of materials for transient expression analysis of Jatropha curcas diacylglycerol transferase gene

[0037]

[0038]

[0039] Use the specific primers JcdagatTE1, 5'-GCT AAC ATCACG TGA AGA ATC TG-3' and JcdagatTE2, 5'-CT TAA TTC AGC ATT GCC TTT CCG A-3' of Jatropha curcas diacylglycerol transferase gene PCR, while doing PCR with specific primers Jc18s1 and Jc18s2 of the 18s ribosomal RNA gene of Jatropha curcas as a control. In this way, the success and quality of cDNA synthesis can be identified, and the expression of each gene can be relatively quantified, because the 18s ribosomal RNA gene is a housekeeping gene, and the expression level is relatively stable.

[0040] The results of transient expression of Jatropha curcas DAGAT enzyme gene in various tissue cells of J...

Embodiment 3

[0042] Comparison of Total Oil Content in Jcdagat Transgenic Yeast by Wet Weight

[0043] Heterologous expression of this gene was performed in the DAGAT mutant and wild type of S. cerevisiae (EUROSCARF collection).

[0044] Construction of expression vector: use primer JcdagatNH 5'-GCG AAGCTT ACC ATG GCT ACG ATT TTGGAG ACC ACT-3' (wherein the base in bold is the start codon of ORF, and the base underlined is a restriction endonuclease HindIII Recognition site, the base in italics is a glycine codon added for optimal expression), and the C-terminal primer JcdagatCX 5'-GCG CTCGAG TCA TCT TAA TTC AGC ATT GCC TTT CCG A-3' (the base in bold is the stop codon of ORF, and the underlined base is the recognition site of a restriction endonuclease XhoI) for PCR, using the synthesized cDNA as a template, and the polymerase uses high-fidelity Vent polymerase (New England Biolabs, USA ), the PCR parameters are: 95°C for 5min, followed by 35 cycles: 95°C for 30s, 60°C for 30s, 72°C for 3m...

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Abstract

The invention discloses Jatropha curcas L. diacylglycerol transferase (Jcdagat) and a coding gene and application thereof and belongs to the technical field of genetic engineering. The amino acid residue sequence of the Jcdagat is shown in SEQ ID No:2, or the amino acid residue sequence has 95% of homology with the amino acid residue sequence shown in SEQ ID No:2 and has the protein which is derived from SEQ ID No:2 and has the same activity or the protein which is derived from SEQ ID No:2 by replacing, deleting or adding one or more of amino acids to the amino acid in SEQ ID No:2 and has the same function. The invention also discloses a gene coding diacylglycerol transferase. Experiments show that the transgene disclosed by the invention can be utilized to significantly increase the oil content of plants or algae, thus the high-quality gene can be provided for the improvement of the oil content of plants or algae and have wide application prospect and important application value.

Description

technical field [0001] The present invention relates to a kind of plant genetic technology, particularly relate to the genetic engineering construction technology of the energy plant of high fat content and algae; ) and its coding gene and application belong to the technical field of genetic engineering. Background technique [0002] Energy is the lifeblood of national economic and social development. With the depletion of fossil energy resources, the price of crude oil has been rising all the way, and the combustion of fossil energy has led to the CO in the atmosphere 2 As a result of the net increase in production and the deterioration of the environment and climate, countries around the world have to consider accelerating the pace of research and development of petroleum alternative raw materials, among which biodiesel is regarded as a renewable alternative energy source and is receiving more and more attention. [0003] Fats and oils, the raw material for biodiesel, ar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/63C12N5/10C12N15/82C12N15/79C12R1/91
Inventor 卿人韦陈放李湘徐莺郭亮
Owner SICHUAN UNIV
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