Figure 4. Heterologous expression of pri-OsamiR395h in Nicotiana tabacum.

(a) The Schematic diagram of rice pri-OsamiR395h overexpression construct. Rice pri-OsamiR395h sequence containing stem-loop structure of OsamiR395h was cloned from rice genomic DNA and put under the control of the CaMV35S promoter. The hptII gene driven by CaMV35S promoter was used as selectable maker. The pre-OsamiR395h sequence was underlined. Sequence emphasized with red color indicates the mature miR395h. LB, Left border; RB, right border. (b) RT-PCR analysis of pri-OsamiR395h expression in wild type and three transgenic tobacco lines. Total RNA samples were prepared from two weeks old wild type and transgenic tobacco plants grown in MS medium. NtaL25 was used as reference gene. (c) Small RNA northern blotting analysis of mature miR395 transcripts in wild type and three transgenic tobacco lines. Total RNA samples were prepared from two weeks old wild type and transgenic tobacco plants grown in MS medium. rRNA was used as loading control. WT: wild type plant. OE: overexpression line.