Optimized triterpenoid synthase coding gene and application thereof

A triterpene compound, coding technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve problems such as unsatisfactory expression and influence

Inactive Publication Date: 2013-05-08
INST OF BOTANY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the influence of codon bias, the expression of some OSCs in yeast is not ideal, thus affecting the identification of their functions

Method used

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  • Optimized triterpenoid synthase coding gene and application thereof
  • Optimized triterpenoid synthase coding gene and application thereof
  • Optimized triterpenoid synthase coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1, the acquisition of the SnOsOSC7 gene after codon optimization

[0026] According to the existing sequence information of Os06g28820 / OsOSC7 in the NCBI database (GenBank Accession Number: KC416147, sequence 3 in the sequence table, which is the gene sequence of unoptimized codons), according to the codon preference information of Pichia pastoris, without changing Based on the precursor of the deduced protein sequence, an optimized new SnOsOSC7 gene was designed according to the high-frequency codons of yeast. Its nucleotide sequence is sequence 1 in the sequence table (gene sequence after codon optimization). The encoded protein is OsOSC7, and its amino acid sequence is sequence 2 in the sequence list.

[0027] Sequence 1 in the sequence listing was artificially synthesized, and the DNA molecule indicated by the sequence was cloned into the multiple cloning site of the cloning vector pGEM-T-EASY to obtain pGEM-SnOsOSC7.

[0028] Sequence 3 in the sequence lis...

Embodiment 2

[0029] Embodiment 2, the application of the SnOsOSC7 gene after codon optimization

[0030] 1. Acquisition of recombinant yeast containing SnOsOSC7

[0031] 1. Construction of yeast expression vector

[0032] 1) The pGEM-SnOsOSC7 and pPICZA vectors (Invitrogen, V19020) obtained in Example 1 were completely digested with restriction endonuclease Not I, respectively. The digestion system is: 5 μg plasmid, 2.5 μL 10× digestion buffer, 1 μL Not I, plus ddH 2 O Supplement the reaction system to 50 μL. The enzyme digestion reaction conditions are: enzyme digestion at 37°C for 1 hour.

[0033] 2) Separate the digested products by agarose electrophoresis, recover about 2.2kb fragments containing SnOsOSC7 and about 3kb pPICZA vector fragments, and dissolve them in 20μL ddH 2 in o.

[0034] 3) Perform ligation reaction on the two fragments obtained in step 2). The ligation reaction system is: 1 μL T 4 DNA ligase, 2 μL 10× ligase buffer, 3 μL recovered DNA fragment containing the ...

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Abstract

The invention discloses an optimized triterpenoid synthase coding gene and application thereof. A DNA (Deoxyribonucleic Acid) molecule provided by the invention is any one of DNA molecules as follows: (1) the DNA molecule shown in a sequence I of a sequence table; (2) the DNA molecule which is hybridized with the DNA sequence defined in (1) and is coded and synthesized into a gap-associated protein of a triterpene compound under strict conditions; and (3) the DNA molecule which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with the DNA sequence defined in (1) and is used for synthesizing the gap-associated protein of the triterpene compound. Experiments prove that the sequence of rice SnOsOSC7 gene subjected to codon optimization, which is provided by the invention, can be used for preparing the triterpene compound alpha-amyrin and / or beta-amyrin.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an optimized triterpene synthase coding gene and its application. Background technique [0002] Oxidized squalene cyclases (OSCs) are a class of 2,3-oxidized squalene as a substrate, cyclized to produce a variety of triterpenoids with different structures (cycloartenol, lupeol, β-cinnamon It is a key enzyme in the synthesis pathway of phytosterols and triterpenes, and many products have important physiological and biological activities. For example, the triterpenoids in ginseng are its important medicinal ingredients, which have the function of improving human immunity (Shukla et al., Phytochemistry, 29:239-241). Cucurbitacin C in bitter gourd not only improves the plant's ability to resist diseases and insect pests, but also has anti-tumor and anti-diabetic effects (Tian et al., Chemistry & Biology 15: 263-273). The functions of many OSCs have been understood through heterologous ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/61C12N15/63C12N1/19C12N5/10C12P33/00
Inventor 薛哲勇漆小泉孙俊聪段礼新
Owner INST OF BOTANY CHINESE ACAD OF SCI
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