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. 2017 Nov 10;8:2208. doi: 10.3389/fmicb.2017.02208

Figure 4.

Figure 4

T-DNA insertion and the disrupted gene in C. minitans mutant ZS-1TN1812. (A) Southern blot analysis of the copy number of T-DNA; the genomic DNA of ZS-1TN1812 and the wild-type strain ZS-1 were digested with SacI. Lane M, DNA weight marker, λ DNA digested with Hind?; Lane 1, ZS-1; Lane 2, Plasmid DNA used for transformation; Lane 3, the mutant ZS-1TN1812. Hygromycin resistance gene (HPH) labeled with α-32P was used as the probe. (B) Amplification of the region flanking the left side of T-DNA in ZS-1TN1812 using iPCR; Lane 1 and lane 2 were the PCR products of the first round and the second round amplification. (C) The full length CmSIT1 DNA sequences obtained by utilizing iPCR; Lane 1-2, iPCR products of the left border genome DNA; Lane 3–4, iPCR products of the region flanking the right side of T-DNA; (D) CmSIT1 has only one copy in the genome of C. minitans as determined with Southern blot analysis. Lane M, DNA weight marker, λ DNA digested with Hind?; Lane 1, Mock; Lane 2-4, genomic DNA digested by EcoRI, PstI, and XbaI. (E) Diagram of T-DNA insertion site, T-DNA was inserted at the non-transcription region between two putative genes, CmSIT1 and CmPEX14. (F) RT-PCR analysis of the expression pattern of putative CmSIT1 and CmPEX14 in the wild-type strain ZS-1 and mutant ZS-1TN1812 cultured on PDA medium.