Method for screening genes related to synthesis of target compound and application

A compound and purpose technology, applied in the field of genes related to compound synthesis, can solve the problems of less chimeras, differentially expressed genes, and unobtainable genes, etc., and achieve the effect of improving accuracy and high accuracy

Pending Publication Date: 2021-09-03
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the previous research on maize, Wang B et al. used the data of the second generation to evaluate whether the second generation could assemble the third generation through two different assembly software, Cufflinks and Trinity. The evaluation standard was whether the third generation transcription could be repeated. Based on the position of the conservative cut point in this book, the final results show that the assembly effect of Cufflinks is slightly better than that of Trinity, but the assembled transcripts are not

Method used

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  • Method for screening genes related to synthesis of target compound and application
  • Method for screening genes related to synthesis of target compound and application
  • Method for screening genes related to synthesis of target compound and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] 1. Extraction of secondary metabolites in Cinnamomum burmanii

[0062] Weigh 1g of fresh Cinnamomum burmanii leaves and remove the veins and petioles and cut them into pieces and place them in 60mL of absolute ethanol. The ratio of fresh leaf samples to absolute ethanol is 1:60 (g / mL). At a temperature of 80°C, the extraction time was 6h, and then the volatile oil was dilute to 100mL.

[0063] 2. GC-MS analysis of compound composition in two chemical types of Cinnamomum burmanii

[0064] Gas chromatography analysis using Agilent 7890A gas chromatograph (column: Agilent 19091N-113, 30m × 320μm × 0.25μm). The carrier gas was nitrogen with a flow rate of 2 mL / min. GC program: 70°C / min, rising to 100°C at 3°C / min, rising to 250°C at 15°C / min, and maintaining for 1 min. The injection volume was 1.0 μL without splashing. The injection port temperature is 220°C, and the detection temperature is 230°C.

[0065] Gas chromatography-mass spectrometry uses Shimadzu QP2010 PLUS...

Embodiment 2

[0068] 1. Extraction of RNA

[0069] TaKaRa MiniBEST Plant RNA Extraction Kit was used.

[0070] (1) Quickly transfer fresh or cryopreserved plant tissue samples to a mortar pre-cooled with liquid nitrogen, and grind the tissue with a pestle, during which liquid nitrogen is continuously added until it is ground into powder (no obvious visible particles , if it is not ground thoroughly, it will affect the yield and quality of RNA). Add the powdered sample (20-50 mg) into a 1.5 mL sterilized tube containing 450 μL Buffer PE, and pipette repeatedly until there is no obvious precipitation in the lysate.

[0071] (2) Centrifuge the lysate at 12,000 rpm at 4°C for 5 minutes.

[0072] (3) Pipette the supernatant carefully into a new 1.5mL sterile tube. Add 1 / 10 volume of Buffer NB to the supernatant (precipitation will appear at this time), shake and mix.

[0073] (4) Centrifuge at 12,000 rpm at 4°C for 5 minutes.

[0074] (5) Pipette the supernatant carefully into a new 1.5mL s...

Embodiment 3

[0093] 1. Three-generation full-length transcriptome sequencing

[0094](1) Synthesis of first-strand cDNA: The qualified RNA (or poly A+RNA) is reverse-transcribed into first-strand cDNA using Clontech SMARTerPCR cDNA Synthesis Kit; PCR amplification is used to synthesize double-stranded cDNA; PCR product purification: use AMPure PB Bead purifies PCR amplification products; SMRTbell library construction: including DNA damage repair, end repair, ligation of aptamers, and library quality assessment; Sequencing on the machine: annealing of SMRTbell library combined with primers and polymerase, using MagBead Loading on the machine sequencing.

[0095] (2) Using SMRT Link v5.0.1 supported by Pacific Biosciences to pair the raw sequencing data (reads) of the cDNA library

[0096] Sorted and clustered into consensus transcripts. Simply put, CCS (circular consensus sequence) is extracted from the BAM file. Then according to the cDNA primers and polyA tail signal, the CCS data were...

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Abstract

The invention discloses a method for screening genes related to synthesis of a target compound and application. The method in the invention comprises the following steps of: taking a third-generation transcript as a reference transcript, and comparing second-generation sequencing data to the transcript to obtain the abundance of each transcript; and quantifying the second-generation assembled transcript by using RSEM software, and carrying out differential expression analysis between genes to obtain a key gene in the terpenoid synthesis process. Compared with a second-generation transcriptome sequencing technology and a third-generation full-length transcriptome sequencing technology which are independently used, the invention has the advantages that: the advantage that the full-length transcriptome sequence can be obtained without splicing the third-generation transcriptome can be sufficiently utilized, so that the accuracy is high; the advantage of quantitative expression detection of genes by second-generation transcriptome sequencing data can be fully considered; the accuracy of genome annotation is greatly improved; and related genes in a synthetic route of the target compound are obtained through screening. According to the method, four genes related to cinnamomum burmannii monoterpenoid synthetase are obtained for the first time.

Description

technical field [0001] The invention belongs to the technical field of biological genes, in particular to a method and application for screening genes related to the synthesis of target compounds. Background technique [0002] D-borneol, also known as natural borneol, is a bicyclic monoterpene compound, which has antibacterial, anti-inflammatory, analgesic, and promotes the passage of drugs through the blood-brain barrier, improves the absorption and utilization of other drugs, and protects the heart and brain. It has been used for the prevention and treatment of diseases for more than 2000 years. [0003] The natural d-borneol currently on the market mainly comes from the extraction of borneol camphor (Cinnamomum camphora) and borneol-type yinxiang (Cinnamomum burmanni) - plum tree. However, the excellent quality of borneol camphor is very few, and the emergence amount is low, the management is time-consuming and laborious, and the economic and time costs are high; in cont...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2535/122C12Q2537/165C12Q2539/103
Inventor 苏健裕郭思源傅明辉
Owner SOUTH CHINA UNIV OF TECH
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