Application of ceramide synthase genes in regulating biosynthesis of ganoderma triterpenes

A technology for biosynthesis, Ganoderma lucidum triterpenes, applied in the field of molecular biology

Active Publication Date: 2019-05-21
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previous studies mainly focused on the isolation, purification and pharmacological analysis of the active components of Ganoderma lucidum, while there were less related works on the biosynthesis of secondary metabolism

Method used

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  • Application of ceramide synthase genes in regulating biosynthesis of ganoderma triterpenes
  • Application of ceramide synthase genes in regulating biosynthesis of ganoderma triterpenes
  • Application of ceramide synthase genes in regulating biosynthesis of ganoderma triterpenes

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1: Construction of Ganoderma lucidum LAG-silencing strain

[0036] 1. Ganoderma lucidum LAG sequence analysis

[0037] The present invention obtained the sequences of Saccharomyces cerevisiae ceramide synthase LAG1 and LAC1 (accession numbers NP_011860.1 and NP_012917.3) obtained by searching in NCBI (https: / / www.ncbi.nlm.nih.gov / ). The sequence was aligned in the known Ganoderma lucidum protein library (Chen et al., 2011), and the three possible ceramide synthases LAG1, LAG2 and LAG3 (accession numbers were MH145349, MH145350 and MH145351) in Ganoderma lucidum were obtained. cDNA sequence. The full length of ceramide synthase gene LAG1 in Ganoderma lucidum is 1089bp, encoding 362 amino acids, the full length of LAG2 is 1317bp, encoding 438 amino acids, and the full length of LAG3 cDNA is 1431bp, encoding 476 amino acids. The amino acid sequence alignment results showed that the three ceramide synthase LAG1, LAG2 and LAG3 proteins in Ganoderma lucidum all cont...

Embodiment 2

[0065] Example 2: Effects of silencing the ceramide synthase gene LAG gene on the homeostasis of intracellular sphingolipids in Ganoderma lucidum

[0066] Ceramide is the simplest part of sphingolipid composition, and it is also the center of sphingolipid synthesis. It can be catalyzed by glucosylceramide synthase to generate glucosylceramide, and it can also be catalyzed by inositol phosphatidylceramide synthase to generate inositol phosphatidylceramide. . The changes of sphingolipid homeostasis in different silenced strains were detected by silencing the ceramide synthase gene LAG gene.

[0067] Systematic analysis of wild-type (WT), control (SiControl), single-silenced strains of LAG1i, LAG2i and LAG3i as well as LAG23i double-silenced strain sheaths by high performance liquid chromatography tandem mass spectrometry electrospray method (LC-ESI-MS / MS) technique Changes in lipid profiles, it was found that silent strains of LAG2i-1 and LAG2i-8, LAG3i-22 and LAG3i-26 showed n...

Embodiment 3

[0068] Example 3: Effects of silencing of ceramide synthase gene LAG on triterpenoid biosynthesis of Ganoderma lucidum

[0069] 1. The important role of Ganoderma lucidum LAG on the biosynthesis of Ganoderma lucidum triterpenes

[0070] In this patent, we detected the contents of Ganoderma lucidum triterpenes, key intermediate metabolites squalene and lanosterol in LAG-silenced strains.

[0071] The content of total Ganoderma lucidum acid is detected by UPLC method, and the specific method is as follows:

[0072](1) Inoculate the outermost bacterial mass of the cultivated mycelium in PDA liquid medium, 150rpm, culture at 28°C for 7 days, and then aseptically smash it with a homogenizer, and inoculate according to 5% (v / v) Amounts were inoculated in CYM liquid medium, and fermented at 150 rpm at 28°C for 7 days. The fermented mycelium was collected and dried in an oven at 60°C and ground to powder, and the content of total ganocidic acid was detected by UPLC method.

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Abstract

The invention provides application of ceramide synthase genes in regulating biosynthesis of ganoderma triterpenes. Ganoderma LAG2 and LAG3 genes are co-silenced by means of a gene silencing technologyto increase the biosynthesis quantity of the ganoderma triterpenes. Through an RNA interference means, an important genetic operation means for improving the biosynthesis of the ganoderma triterpenoids by co-silencing the ceramide synthase genes LAG2 and LAG3 is provided, and a theoretical basis for improving the biosynthesis of the ganoderma triterpenes by means of a genetic engineering technique is provided.

Description

technical field [0001] The invention mainly takes the field of molecular biology as the background, and specifically relates to the application of ceramide synthase / Longevity assurance gene (LAG) in regulating the biosynthesis of Ganoderma lucidum triterpenes. Background technique [0002] Sphingolipids, sterols and phospholipids are the three major lipid components that make up cell biofilms. Among them, sphingolipids are widely present in animals, plants and fungi in the form of sphingosine, ceramide, glucosylceramide, inositol phosphatidylceramide, etc. Among them, ceramide is the simplest part of sphingolipid composition. The de novo synthesis pathway of ceramide includes: the condensation reaction of serine and palmitoyl-CoA to generate 3-hydroxysphingosine; 3-hydroxysphingosine can be further hydroxylated to generate 3,4-dihydroxysphingosine; ceramide synthase Different types of ceramides are synthesized by recruiting different types of sphingosine and fatty acid cha...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N15/113C12N15/80C12P33/00C12R1/645
Inventor 赵明文鲁笑笑王云晓朱静任昂师亮于汉寿
Owner NANJING AGRICULTURAL UNIVERSITY
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