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).