Abi 7300

The upper three leaves of 15 tagged panicles were sampled from each replicant on 8 DPA of inferior spikelets. The leaves were first frozen in liquid nitrogen for 1 min before storing at minus 80 °C. Before RNA extraction, the samples were ground to fine powder and weighed nearly 100 mg per replicant for next step. Total RNA was extracted by using Plant RNA Kit (Omega Biotek, Inc., USA), and reversed-transcribed into the first-strand cDNA with the Prime-Script-TM RT Reagent Kit (Takara, Kyoto, Japan), oligo-dT. The qRT-PCR was performed by an ABI 7300 and SYBR Premix Ex Taq-TM (Takara, Kyoto, Japan) according to the manufacturer's protocol. All the experiments were analyzed by three biological replicants with three technical repeats per biological replicate. As shown in Additional file 1: Table S3, the cDNA was amplified by specific primers of 5′-UTR and 3′-UTR for the analysis of relative gene expression (OsSWEET11, OsSUT1, OsSUT2, OsSUT4, OsSPS1, OsSUS3, OsSUS4, OsAGPL1, OsAmy3, OsTPS1, OsTPP2, OsTPP6, OSK1, OSK24, OSK35, OsNCED1, OsABA3, and OsCYP707A6).

The gene expression level in brain tissues was determined by real-time PCR. Extracted total RNA was purified with 75% ethanol, and its concentration was determined by spectrophotometry. Then, the purified total RNA (200 ng) was retrotranscribed using a retrotranscription kit (DRR037A; Takara Bio, Inc.) and mixed to obtain the first-strand cDNA template. Subsequently, the expression levels of CD68, interleukin- (IL-)1β, and tumor necrosis factor- (TNF-) α were determined quantitatively by real-time PCR (ABI 7300) using the SYBR Premix Ex Taq kit (Takara Bio, Inc.).