3.1 Successful construction of LAC1 knock-down strain and
its phenotypic stability
For gene knock-down, we used pSilencer 4.1-CMV neo plasmids to design
two related vectors directing the transcription of shRNA, which
contained the same 21–base pair double-stranded LAC1 target
sequence, and loops of nine (5’-TTCAAGAGA for group pS-LAC1-A) or six
(5’-CTCGAG for group pS-LAC1-B) nucleotides. After chemical
transformation and G418 selection in YPDA broth, a part cells of two
experimental groups and a negative control (pS) group were prepared for
qRT-PCR, and the other were plated on a DOPA agar for 7 days. We found
that colonies of group pS-LAC1-A and pS-LAC1-B produced much less
melanin than that of group pS (Fig. 1C), which was in accord with the
qRT-PCR results of mRNA of LAC1 (p<0.01, Fig. 1B).
Therefore, shRNA containing either the long or short loops still
conducted its interfering function. But we still observed a little brown
pigment in the center of colonies of group pS-LAC1-A and pS-LAC1-B (Fig.
1C). When all the white-like colonies were passaged on DOPA agar every 5
days, the color of some colonies at the 3rd passage
evenly changed to grey and even dark (Fig. 1D). There were 4 brown or
black colonies of 39 in group pS-LAC1-A and 9 of 22 in group pS-LAC1-B
(white arrows in Fig. 1D). Then we plated the whitest colony in Fig. 1D
(belonging to group pS-LAC1-A) to a new DOPA agar for another 5 days (at
the 4th passage), and we found its color also turned
lightly grey (Fig. 1E).