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Rhodotorula glutinis acetyl coenzyme A carboxylase gene and clone and function verification method and application thereof

A technology of Rhodotorula viscosus and acetyl coenzyme, applied in the field of genetic engineering, can solve the problems of less final product, affecting sensitivity, short detection cycle, etc., and achieve the effect of overcoming many steps and overcoming environmental pollution

Inactive Publication Date: 2012-07-18
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This type of detection method has a short detection cycle and relatively cheap reagents, but too many reaction steps, complex intermediate products, and small amount of final products affect its sensitivity.

Method used

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  • Rhodotorula glutinis acetyl coenzyme A carboxylase gene and clone and function verification method and application thereof
  • Rhodotorula glutinis acetyl coenzyme A carboxylase gene and clone and function verification method and application thereof
  • Rhodotorula glutinis acetyl coenzyme A carboxylase gene and clone and function verification method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Cloning of Rhodotorula glutinosa acetyl-CoA carboxylase gene

[0053] (1) Genomic DNA was extracted from Rhodotorula glutinis as the experimental material.

[0054] (2) if figure 1 As shown, the amino acid sequences of acetyl-CoA carboxylases of different species of yeast and filamentous fungi were downloaded from the KEGG database, and after using the software Clustal X1.83 for multiple sequence alignment, three conserved regions IANNGA, QKIIEEA, WGYFSV designed two pairs of degenerate primers IN-F / FY-R, QK-F / WG-R for PCR amplification. Primers are as follows:

[0055] QK-F CAGAAGATYATYGAGGAGGC

[0056] WG-R ACVGAGAAGTAACCCCA

[0057] IN-F ATYGCBAACAACGGNATYGC

[0058] FY-R GAGCCTCCTCRATRATCTTCTG

[0059] The PCR system is as follows (50uL): 10×buffer 5uL, 2.5mM dNTPs 4uL, upstream and downstream primers 1uL, E×Taq enzyme 0.5uL (5U / uL), dd H2O 37.5uL. Drop-down PCR program: 94°C pre-denaturation for 5 minutes; then 94°C for 1min, 51°C for 1min, the tem...

Embodiment 2

[0074] Example 2 Cloning and co-expression of the three functional domains of Rhodotorula viscosus acetyl-CoA carboxylase gene

[0075] (1) if figure 2 As shown, use E.Z.N.A. TM Yeast RNA Kit was used to extract total RNA from Rhodotorula viscosus.

[0076] (2) Refer to RevertAid TM First Strand cDNA Synthesis Kit instructions first digest total RNA with DNase I, the system is as follows: RNA 1ug, 10×DNase I reaction buffer with MgCl2 1uL, DEPC Water added to 9uL, DNase I 1uL. Total volume 10 uL. Incubate at 37°C for 30min, add 1uL of 25mM EDTA at 65°C for 10min to terminate the reaction.

[0077] Take 1 ug of total RNA as a template, add oligo(dT)18 as a reverse transcription primer, add DEPC-treated water to 12uL, mix slightly, incubate at 65°C for 5min, and briefly bathe in ice. Add the following components in sequence: 5×Reaction Buffer 4uL, Ribolock RNase Inhibitor (200u / uL) 1uL, 10mM dNTP Mix 2uL, RevertAid M-MuL V Reverse Transcriptase (200u / uL) 1uL. Mix lightl...

Embodiment 3

[0088] Example 3 Determining the fatty acid content of the recombinant strain when the three functional domains are co-expressed and then verifying the function of the gene

[0089] A large number of recombinant strains were induced to express, and the bacterial cells were collected by centrifugation, frozen at -80°C for 3 hours, and then vacuumized and freeze-dried. Using water bath method (Li et al., 2006) to extract bacterial fatty acids, the steps are as follows:

[0090] (1) Reagent preparation: 5% (volume percent) concentrated sulfuric acid / methanol solution; 0.2% (mass percent) BHT / methanol solution; 2.5mg / mL carbon seventeen fatty acid methyl ester / petroleum ether (90°C-120°C) solution ; 0.9% (mass percentage) NaCl / water solution;

[0091] (2) Grind the dried bacteria with a mortar, weigh 50 mg with an analytical balance and add it to a 20 mL crimp headspace bottle, add 2 mL of 5% concentrated sulfuric acid methanol solution (add in two times, 1 mL each time), and put...

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Abstract

The invention discloses a rhodotorula glutinis acetyl coenzyme A carboxylase gene and coded protein thereof and a clone method and a function verification method of the gene. A nucleotide sequence of the rhodotorula glutinis acetyl coenzyme A carboxylase gene is indicated as SEQ ID NO:1, and an amino acid sequence of the coded protein is indicated as SEQ ID NO:2. By testing fatty acid content of recombination bacterial strains during expression and coexpression of three function fields of the rhodotorula glutinis acetyl coenzyme A carboxylase gene respectively, functions of the gene are verified. The nucleotide sequence of the rhodotorula glutinis acetyl coenzyme A carboxylase gene, the coded protein sequence of the gene, the clone method of the gene and a prokaryotic expression method can be widely applied to oil microorganism research and oil crop transgenosis engineering.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the gene of Rhodotorula viscosus acetyl-CoA carboxylase, and also relates to the protein encoded by the gene, the cloning method and function verification method of the gene, and the application of the gene in transgenic oil crops. Background technique [0002] Acetyl-CoA carboxylase (ACC) (EC 6.4.1.2) is the rate-limiting enzyme that catalyzes the first step in fatty acid synthesis and metabolism. It carboxylates acetyl-CoA to generate malonyl-CoA, which is Biotin-dependent enzyme. The enzyme is present in most biological tissues, including bacteria, archaea, fungi, yeast, plants, animals and humans. Can be divided into 4 categories. The first major group of acetyl-CoA carboxylases is the multi-subunit type ACC, that is, the heterogeneous type, which exists in the plastids of bacteria, dicotyledonous plants and non-gramineous monocotyledonous plants. The ACC of Escherichia ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N9/00C12N15/10C12Q1/68C12Q1/02G01N30/02A01H5/00
Inventor 郑世学张吉斌喻子牛李洁琼王利兵
Owner HUAZHONG AGRI UNIV
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