Generation and molecular analysis of <i>Osa-miR319b</i> transgenic lines.

<p>(A) Schematic diagram of the <i>Osa-miR319b</i> overexpression gene construct, p35S-<i>Osa-miR319b</i>/p35S-<i>Bar</i>, in which the <i>Osa-miR319b</i> gene is under the control of the CaMV 35S promoter. The CaMV 35S promoter-driven <i>Bar</i> is included for glufosinate resistance. LB, Left border; RB, right border. (B) Southern blot analysis of transgenic rice. Rice DNA and vector DNA were digested by HindIII, run on a 0.8% agarose gel, transferred to a nylon membrane, and probed with digoxingenin-dUTP against the amplified bar gene sequence. The wild-type rice DNA was used as negative control. (C) Expression pattern of <i>Osa-miR319b</i> in wild-type and transgenic lines by semi-quantitaive RT-PCR. cDNAs were normalized using the <i>Ef1-α</i> gene. (D) Verification of the mature-<i>Osa-miR319b</i> overexpression in <i>Osa-miR319b</i>-overexpressing transgenic rice lines by real-time RT-PCR. RNA was extracted from the flag leaves of the second generation of the transgenic rice plants. snRNA U6 served as a reference gene for the detection of miRNAs.