A plant-based glycosyltransferase and its coding gene and application
A technology of glycosyltransferase and encoding, applied in the direction of transferase, plant gene improvement, application, etc.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0050] Embodiment 1, the cloning of Rowan phytoalexin glycosyltransferase gene
[0051] Total RNA was extracted from suspension cells of Sorbus australis, and cDNA was obtained by reverse transcription. Using the obtained cDNA as a template, PCR reaction was carried out using primer 1 and primer 2.
[0052] Primer 1: 5'-GAG GGATCC ATGAAGAGACCAGTACAACT-3';
[0053] Primer 2: 5'-GAG GCGGCCGC TTAAATTTGATTAATAAAAC-3'.
[0054] The PCR reaction system is as follows: KOD-Plus-Neo 2μL; 2mM dNTPs 10μL; 25mM MgSO 4 6 μL; 10×PCRbuffer for KOD-Plus-Neo 10 μL; 3 μL each of primer 1 and primer 2; 2 μL of cDNA template; ddH 2 O to make up to 100 μL.
[0055] PCR reaction conditions: 94°C for 10min; 35 cycles of 94°C for 30s, 55°C for 30s, 72°C for 1min; 72°C for 10min; 4°C for maintenance.
[0056] After PCR, the obtained PCR products were detected by 1.5% agarose gel electrophoresis and sequenced. Sequencing results showed that the sequence of the PCR product was "5'-GAG GGATCC...
Embodiment 2
[0057] Example 2, Exogenous Expression of Rowan Phytoalexin Glycosyltransferase
[0058] 1. Construction of recombinant expression vector
[0059] The PCR product "5'-GAG of embodiment 1 GGATCC +sequence 2+ GCGGCCGC After CTC" is purified and recovered, it is double-digested with restriction endonucleases BamH I and Not I, and the digested product is recovered and connected to the large backbone fragment of the pET-28a(+) plasmid that has undergone the same double-digestion to obtain a recombinant plasmid .
[0060] Sequencing showed that the recombinant plasmid after inserting the DNA fragment shown in sequence 2 between the restriction sites BamH I and Not I of the pET-28a(+) plasmid was named pET-28a-UGT7.
[0061] 2. Construction of expression host system
[0062] Transform the recombinant expression vector pET-28a-UGT7 constructed in step 1 into Escherichia coli Transetta (DE3) competent cells, select positive clones for PCR verification, and send them for sequencin...
Embodiment 3
[0073] Example 3, Verification of the Activity of Rowan Phytoalexin Glycosyltransferase
[0074] 1. Experimental method
[0075] 1. Screening of catalytic activity of exogenously expressed glycosyltransferases
[0076] In the preliminary screening of glycosyltransferase activity, the crude enzyme solution of SaUGT7 was used for screening experiments. Rhaphiolepsin (compound 1, as shown in formula I), 2'-Hydroxyaucuparin (compound 2, as shown in formula II) and Noraucuparin (compound 3, as shown in formula III) are the sugar groups UDP-glucose is used as the donor for the enzymatic reaction.
[0077]
[0078] The reaction system for screening glycosyltransferase activity was: SaUGT7 crude enzyme solution (ie, the supernatant in step 3 (h) of Example 2) 194 μL; UDP-glucose (40 mM) 4 μL; receptor substrate (40 mM) 2 μL.
[0079] In the experiment, the empty control supernatant prepared in Example 2 was used as a control.
[0080] The reaction conditions for glycosyltransfe...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More