Molecular Cloning and Biochemical Characterization of a Recombinant Sterol 3-O-Glucosyltransferase from Gymnema sylvestre R.Br. Catalyzing Biosynthesis of Steryl Glucosides
Table 1
Details of primers used for PCR amplification of GsSGT ORF, heterologous expression, and real-time expression profiling.
Primer details
Primer’s name
Nucleotide sequence of primer (5′-3′)
Length (mer)
Tm (°C)
GC content (%)
Size of the amplicon
Primers used for partial clone, full length cDNA and heterologous expression
Degenerate
WsSGTF2
TGGCTACATCTGGACTCCTCAT
22
57.3
50.0
370 bp
WsSGTR2
TGGTTCATCACGGTGGTGCT
20
59.4
55.0
3′RACE
GymGTF1
GGCACTGGAGAATACAGGACAGAG
24
58.8
54.2
684 bp
GymGTF2
CCTCGTTGATTGCCCTCATGACTG
24
59.9
54.2
5′RACE
GymGTR1
GGTTTAGGCCCCTTCTGTATCCAC
24
59.2
54.2
1.7 kb
GymGTR2
CCAACAACATCAACCAGAGGGCC
23
60.6
56.5
GymURD01
AGAGACTCGATGATGGCCTTGAGC
24
60.5
54.2
GymURD02
AATCACTGAAGTTAGCATGAGTAGCCAGCC
30
61.6
46.7
GymURD03
ACTCCTGTAATTTCTTAGCCATAGCC
26
56.5
42.3
Complete ORF
2.3GymFLF1
TTGGATTAACATATTTGAGCATCCAGGC
28
57.6
39.3
2.1 kb
2.3GymFLF2
TTGAGCATCCAGGCTTCTCTTGCATGG
27
62.8
51.9
2.3GymFLR1
ATTCTGTAATTCCTTGTTCCGTTGTAAACC
30
57.5
36.7
pET-28a expression
PTGGTF0N
CATATGGATTCTGATGGGACTGGC
24
57.4
50.0
2.1 kb
PTGGTR0B
GGATCCCTAAGAACAACAAGGCAGG
25
59.4
52.0
Primers used for semiquantitative and real-time PCR expression studies
RT-PCR
Actin3F
TTGCCGAGATAATGGCCTAC
20
54.6
50.0
250 bp
Actin3R
TACCGCGACTTCGATCTTTT
20
54.0
45.0
SGTF2
TGGCTACATCTGGACTCCTCAT
22
57.3
50.0
370 bp
SGTR2
TGGTTCATCACGGTGGTGCT
20
59.4
55.0
Real-time PCR
PTGymGTF2
CCTCGTTGATTGCCCTCATGACTG
24
59.9
54.2
350 bp
PT1.8GymFL1
GGATCCCTAAGAACAACAAGGCAGGCTG
28
62.7
53.6
*Restriction sites for pET-28a expression are highlighted in bold and underlined.
