Check patentability & draft patents in minutes with Patsnap Eureka AI!

Transgenic system for improving content of semiterpene in chlamydomonas and application thereof

A transgenic, Chlamydomonas technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve problems such as low efficiency and low yield, and achieve the effects of convenient extraction, reduced production cost, and increased terpenoid yield

Pending Publication Date: 2022-04-29
SHENZHEN UNIV
View PDF10 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the methods for synthesizing terpenoids through Chlamydomonas in the prior art all have the disadvantages of low efficiency and low yield.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Transgenic system for improving content of semiterpene in chlamydomonas and application thereof
  • Transgenic system for improving content of semiterpene in chlamydomonas and application thereof
  • Transgenic system for improving content of semiterpene in chlamydomonas and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Embodiment 1 CK and the construction of the transformation of foreign gene corresponding to IPK gene and expression vector

[0091] In this example, Chlamydomonas reinhardtii CC4533 (Chlamydomonas reinhardtii CC4533, derived from the Chlamydomonas reinhardtii Center) was selected as the recipient of the transgenic operation. This algae strain is a cell wall-deficient Chlamydomonas reinhardtii strain and can express foreign genes efficiently.

[0092] The culture medium used for the cultivation of Chlamydomonas reinhardtii is TAP medium, and the formula of the medium is as follows: 2.42g Tris, 25mL 4 × Beijerinck salts (16g NH 4 Cl 2 , 2g CaCl 2 -2H 2 O and 4g MgSO 4 -7H 2 O dissolved in water, to 1L), 1M potassium phosphate buffer and 1 mL Trace trace element mixture (11.4g H 3BO 3 , 5.6g MnCl 2 -4H 2 O, 22gZnSO 4 -7H 2 O, 4.99g FeSO 4 -7H 2 O, 1.61g CoCl 2 -6H 2 O, 1.57g CuSO 4 -5H 2 O, 1.1g (NH 4 ) 6 Mo 7 o 24 -4H 2 O and 50g Na 2 Dissolve EDTA i...

Embodiment 2

[0119] Example 2: Genetic Transformation of Chlamydomonas reinhardtii

[0120] (1) "Bead milling method" genetic transformation

[0121] FastPure Plasmid Mini Kit (Vazyme, Nanjing, China) was used to extract recombinant plasmids pOpt2-CTP-CrCK and pOpt2-CTP-CrIPK, respectively.

[0122] The specific steps of the "bead grinding method" are as follows:

[0123] a. Cultivate the cell wall-defective Chlamydomonas reinhardtii CC4533 to the logarithmic phase in continuous light and TAP medium, and the number of cells is about (1~2)×10 6 cells / mL. Centrifuge at room temperature at 5000rpm for 5min, collect the algae cells, and discard the supernatant;

[0124] b. Resuspend the algae cell pellet with sterilized fresh TAP culture medium, and adjust the cell concentration to 2×10 8 cells / mL;

[0125] c. Pipette 300μL of algae cell suspension into a 1.5mL EP tube (containing sterilized glass beads), and then add 1-2μg (0.5-1μg of pOpt2-CTP-CrCK and pOpt2-CTP-CrIPK respectively) sing...

Embodiment 3

[0134] Example 3 Screening and identification of transgenic Chlamydomonas reinhardtii

[0135] The expression vector of Chlamydomycin reinhardtii contains hygromycin or paromomycin gene, so it has hygromycin or paromomycin resistance. grown on flat plates. The identification of transgenic algae included PCR sequencing, RT-PCR analysis and determination of the content of semiterpenes (IPP and DMAPP).

[0136] (1) RT-PCR

[0137] Extract green monoclonal Chlamydomonas reinhardtii total RNA, take 1 μg for reverse transcription (conditions are 42°C for 60 min, 72°C for 15 min) (TaKaRa reverse kit) to obtain cDNA, and amplify the target gene fragment from the cDNA by PCR ( CrCK and CrIPK) and an internal reference gene (Actin gene), PCR amplification products were sent to Shanghai Sangon Bioengineering Company for sequencing.

[0138] Actin gene amplification primers are: 5'ACCCCGTGCTGCTGACTG 3'(SEQ ID NO.9) and 5'ACGTTGAAGGTCTCGAACG 3'(SEQ ID NO.10), target gene CrCK amplificat...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a transgenic system for increasing the content of chlamydomonas hemiterpenes and application thereof. The transgenic system for increasing the content of chlamydomonas hemiterpenes comprises a recombinant vector containing a choline kinase gene and / or a recombinant vector containing an isopentenyl phosphokinase gene, the choline kinase gene comprises a nucleotide sequence as shown in SEQ ID NO. 1; the isopentenyl phosphokinase gene comprises a nucleotide sequence as shown in SEQ ID NO. 2. The invention further provides the transgenic chlamydomonas capable of increasing the content of the semi-terpene in the chlamydomonas and a construction method of the transgenic chlamydomonas, the content of the semi-terpene in the obtained transgenic chlamydomonas is greatly increased, a chlamydomonas reinhardtii engineering algal strain for producing limonene can be further constructed, and reference is provided for synthesizing other high-value terpene substances in the chlamydomonas reinhardtii.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering, and in particular relates to a transgenic system for increasing the content of chlamydomonas semiterpene and its application. Background technique [0002] Terpenoids are the largest class of chemical substances in nature, with more than 80,000 known terpenoids. Because of its important application value in the fields of medicine, agriculture, essence, fragrance, cosmetics, cancer treatment and drug preparation, it has attracted much attention. [0003] All naturally occurring terpenoids are composed of two C 5 It is derived from terpene precursors, namely isopentenyl diphosphate (IPP) and its isomer dimethylpropenyl diphosphate (DMAPP). The isoprene precursors DMAPP and IPP are naturally synthesized by one of two pathways: the mevalonate (MVA) pathway consists of six reactions to generate IPP from acetyl-CoA, which is then isomerized from isoprene pyrophosphate The enzy...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/79C12N15/54C12N15/60C12N15/61C12N1/13C12P5/00C12R1/89
CPCC12N15/79C12N9/1205C12N9/1229C12N9/90C12N9/88C12Y207/01032C12Y207/04026C12Y503/03002C12Y402/03016C12P5/002C12N2800/22
Inventor 胡章立赵美丽贾彬
Owner SHENZHEN UNIV
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com