Cloning of the Thaumatin Gene

A pyramidal-shaped fruit of Thaumatococcus daniellii kept at 80’C was powdered by mortar and
pestle in the presence of liquid nitrogen.

The mRNA was purified by using mRNA purification kit.

About 0.5 mg of the powder of the frozen tissue was dissolved in 0.4 mL of extraction buffer.

After the addition of 0.8 mL of elution

buffer, the mixture was centrifuged at 10,000 g for 1 min.

The supernatant was mixed with oligo (dT) cellulose suspension and centrifuged at 10,000 g for
10 s.

The precipitates were washed 5 times with 1 mL of high-salt buffer and then washed twice with
1 mL of low-salt buffer.

After washing, the supernatants were suspended in 0.3 mL of low-salt buffer and applied to the
MicroSpin column.

After centrifugation at 10,000 g for 5 s, the column was washed with 0.5 mL of low-salt buffer
three times and, finally, the mRNA was eluted with 0.4 mL of elution buffer that was preheated
to 65’C by centrifugation (10,000 g, 5 s).

Ethanol precipitation was performed by mixing of 400 AL ethanol, 10 AL glycogen solution, 40

AL potassium acetate solution, and 1 mL of 95% ethanol.

First-strand cDNA synthesis was performed using a First-strand cDNA Synthesis Kit
(Pharmacia, Uppsala, Sweden) as follows.

Precipitated mRNA was dissolved in 20 AL DEPC water and heated at 65’C for 10 min.

After being chilled on ice, isolated mRNA was reverse-transcribed using 1 AL of pd (N)6
primers, 1 AL of DTT solution, and 11 AL of bulk First Strand cDNA Reaction Mix at 37jC for 1 h.

The thaumatin gene was amplified by PCR using the first-strand cDNA as a template, and
5V-GCCACCTTCGAGATCGTCAAC-3V and 5V-CCTAGGGGCAGTAGGGCAGAA-3V as
primers (underlined Avr II site).

The PCR reaction was conducted using Taq polymerase for 1 cycle of 94jC for 1 min,
and 30 cycles of 96’C for 30 s, 67’C for 30 s, and 72’C for 1 min, and then 1 cycle of 72’C for 10
min.
The PCR product was analyzed by 1.2% agarose gel electrophoresis.

The fresh Taq polymerase-amplified PCR product (1 AL) was

immediately ligated into 1 AL of pCRR2.1-TOPOR vector.
After being chilled on ice for 5 min, the PCR product was
transformed into Top 10F competent cells (Invitrogen)
mixed with 2 A1 of ligation mixture. The reaction mixture
was stored on ice for 30 min, was subjected to heat shock at
42jC for 30 s, and then immediately transferred back to
the ice. Then 250 AL, of SOC medium was added. Blue/
white screening was performed on LB plates containing
50 AgmL1 carbenicilin, 40 AL of 40 mgmL1 X-gal, and
40 AL of 100 mM1 IPTG. After incubation overnight at
37jC, some white colonies were observed. Plasmid DNA
was isolated from E. coli by using the S.N.A.P. Miniprep Kit
(Invitrogen) and restriction analysis was performed by
digesting with EcoRI and Avr II at 37jC for 2 h. The DNA
sequence was analyzed by an ABI 310 DNA sequencer
(Applied Biosystems) by using Big Dye Terminator Cycle