Polyketide synthases EnPKS1 and EnPKS2 from Erythroxylum novogranatense, and gene and application of polyketide synthases EnPKS1 and EnPKS2
A technology of polyketide synthase and coca, applied in application, genetic engineering, plant gene improvement, etc., can solve the problem of no coding gene identification, etc., and achieve the effect of good industrialization prospects
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0045] Cloning of coca polyketide synthases (Polyketide synthases, PKS) gene.
[0046] (1) Extraction of total RNA from coca shoots and synthesis of first-strand cDNA.
[0047] Take an appropriate amount of coca leaf bud tissue, grind it in liquid nitrogen, and extract total RNA with the Biotek Polysaccharide and Polyphenol Plant Total RNA Rapid Extraction Kit according to the instructions. The concentration and quality of RNA were detected by Thermo Scientific NanoDrop spectrophotometer, and the quality of RNA was detected by agarose gel electrophoresis.
[0048] cDNA was synthesized using the HiScript III 1st Strand cDNA Synthesis Kit reverse transcription kit from Novizyme, following the instructions of its product manual, using total RNA as a template.
[0050] Design specific primers, the specific primer sequences are as follows:
[0051] EnPKS1-F: 5'-atgaacggaaccgtcaagaaaatgaatg-3' (SEQ ID NO.1)
[0052] EnPKS1-R: 5'-tcattctgtaaccacag...
Embodiment 2
[0057] Prokaryotic expression verification PKS 1 and PKS 2 Gene function.
[0058] Introduced at the enzyme cleavage site EnPKS1 and EnPKS2 Gene. for EnPKS1 and EnPKS2 Gene Design Primers, PKS 1 forward primer with Bam The HI restriction site is shown in SEQ ID NO.9, and the reverse primer contains Sal I restriction site is as shown in SEQ ID NO.10; PKS 2 forward primers with Bam The HI restriction site is shown in SEQ ID NO.11, and the reverse primer contains xho The I restriction site is shown in SEQ ID NO.12. Amplified by PCR to obtain enzyme cleavage sites at both ends EnPKS1 and EnPKS2 After the gene, use the cohesive ends generated by these two restriction sites to separate the PKS 1 and PKS The complete sequence of the coding region of 2 was connected to the plasmid pET28a to obtain the recombinant expression vector pET28a- PKS 1 and pET28a- PKS 2.
[0059] The primer sequences are as follows:
[0060] BamHI-EnPKS1-F: 5'-cgggatccatga...
PUM
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com