Journal: Journal of Experimental Botany
Article Title: Arabidopsis seed-specific vacuolar aquaporins are involved in maintaining seed longevity under the control of ABSCISIC ACID INSENSITIVE 3
Figure Lengend Snippet: Identification of tip3;1 and tip3;2 T-DNA insertion mutants and three TIP3;1 -RNAi transgenic lines ( TIP3;1 -RNAi/ tip3;2 ) in the tip3;2 mutant background. (A) Schematic representation of the tip3;1 and tip3;2 T-DNA insertion mutant lines. A triangle indicates the position of the T-DNA insertion, and the arrow indicates its orientation. The genomic sequences corresponding to the coding region (black boxes), untranslated region (grey boxes), and introns (black lines) are indicated. The positions of the primers (31LP, 31RP, 32LP, and 32RP) used for PCR analysis of the tip3;1 and tip3;2 T-DNA insertion mutants, respectively, are also indicated. (B) PCR analysis of genomic DNA of Col, tip3;1 , tip3;2 , and tip3;1/tip3;2 . LP, left primer; RP, right primer; LB, T-DNA left border primer. (C) Schematic representation of the construct used for the suppression of TIP3;1 in Arabidopsis seeds. RNAi technology was used with a segment of the TIP3;1 gene driven by the seed-specific TIP3;1 promoter. (D) qRT-PCR analysis of TIP3;1 , TIP3;2 , and ACT7 transcript abundance in mature seeds of Col, mutants, and RNAi lines. PP2A was used as the endogenous control, and the transcript abundance of TIP3;1 , TIP3;2 , and ACT7 was quantified by comparisons with that of PP2A . Values are means ±SD, n =3. (E) Immunoblot analysis of TIP3s in mature seeds from Col, tip3 mutants, and three TIP3;1 -RNAi/ tip3;2 transgenic lines (R3, R7, and R8). HSP17.6, which is expressed in mature seeds, was used as a loading control. (This figure is available in colour at JXB online.)
Article Snippet: Quantitative RT-PCR (qRT-PCR) analyses were performed using the SYBR Green method (SYBR premix EX taq, TaKaRa) with the StepOnePlus™ Real-time PCR System (Applied Biosystems).
Techniques: Transgenic Assay, Mutagenesis, Genomic Sequencing, Polymerase Chain Reaction, Construct, Quantitative RT-PCR