Gene for catalyzing morella rubra UDP (Uridine Diphosphate)-rhamnose biosynthesis, encoding protein and application
A biosynthetic and protein-encoding technology, applied in plant gene improvement, biochemical equipment and methods, applications, etc., can solve the problems of low UDP-rhamnose content, difficult purification, and high synthesis cost
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Embodiment 1
[0010] Embodiment 1: Myrica rubra MrRHM1 gene cloning
[0011] The fruit of ‘Water Chestnut’ bayberry was used as the material, and the fruit pulp tissue was taken, quickly frozen with liquid nitrogen, and then stored in a -80°C refrigerator. RNA was extracted from bayberry pulp by CTAB method, according to PrimeScript TM RT reagent Kit with gDNAEraser (Takara) reagent instructions were used to synthesize cDNA.
[0012] Using the reverse transcription product cDNA as a template, use the primers shown in SEQ:NO.3 and SEQ:NO.4 to amplify MrRHM1 by PCR. The PCR reaction system is 50 μL, and the components are: 2×Phanta Max Buffer 25 μL, dNTP Mix (10mM each) 1μL, DNA polymerse (1U / μL) 1μL, upstream and downstream primers (10μM) each 2μL, cDNA 1μL, H 2 O 18 μL. The PCR program was: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 1 min and 40 s, and extension at 72°C for 5 min to obtain the amplified product.
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Embodiment 2
[0014] Example 2: Prokaryotic expression of MrRHM1
[0015] Design specific primers with multiple cloning restriction sites of the expression vector pET-28a vector, and the MrRHM1 primer sequences are shown in SEQ:NO.5 and SEQ:NO.6.
[0016] Using the plasmid returned correctly by sequencing as a template, MrRHM1 was amplified by PCR with the primers shown in SEQ:NO.5 and SEQ:NO.6. The PCR reaction system was 50 μL, and the components were: 2×Phanta Max Buffer 25 μL, dNTP Mix ( 10mMeach) 1μL, DNA polymerse (1U / μL) 1μL, upstream and downstream primers (10μM) each 2μL, cDNA 1μL, H 2 O 18 μL. The PCR program was: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 1 min and 40 s, and extension at 72°C for 5 min to obtain the amplified product.
[0017] The PCR amplified products were respectively connected to the pET-28a vector digested with SalI and HindIII to obtain the pET-28a-MrRHM1 recombinant plasmid.
[00...
Embodiment 3
[0021] Example 3: Detection and Analysis of Enzymatic Activity of MrRHM1 Recombinant Protein
[0022] For the enzyme activity detection of UDP-rhamnose substrate, the reaction system is 200 μL, containing 100 mM phosphate buffer (pH=9.0) containing 1 mM UDP-glucose as the reaction substrate, 2 mM NAD coenzyme and 2 mM NADPH coenzyme, 20 μL Purified recombinant protein.
[0023] All enzyme reaction systems were reacted at 37°C to obtain enzyme reaction products. The enzyme reaction product is detected and identified by product standard combined with HPLC. The HPLC detection conditions are as follows: Waters 2695-2996DAD detector, ODS C18 column (4.6×250mm) chromatographic column. 1.5% triethylamine aqueous solution (adjusted to pH=7.5 with formic acid) was used as mobile phase, eluted isocratically for 30 minutes, the detection wavelength was 260nm, the column temperature was 25°C, the flow rate was 1mL / min, and the injection volume was 10μL.
[0024] The result is as figur...
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