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For the gene detection about N and Si uptake and assimilation, leaves or roots were collected from different treatments for analysis. For BPH treatment, leaf sheathes were collected for analysis of defense gene expression. There were 3 biological replicates in each treatment. All samples were immediately frozen in liquid nitrogen and stored at -80 ℃, and ground to fine powder in liquid nitrogen just before the total RNA extraction. Total RNA was extracted from 0.1 g leaf sample using TRIzol Reagent (Life Technologies) according to the manufacture’s instructions. First-strand cDNA was synthesized from 1 μg of total RNA using M-MLV Reverse Transcription Kit (Invitrogen) according to the manufacture’s instructions. Real-time PCR was performed using SYBR Premix Ex Taq II (Tli RNaseH Plus, Takara) with 7500 Fast Real-Time PCR Systems (Applied Biosystems). Reaction condition for thermal cycling were 90 ℃ for 30 s, 40 cysles of 95 ℃ for 15 s, 60 ℃ for 30 s, 72 ℃for 30 s (elongation and collected fluorescence data). Melting curve analysis and agarose gel electrophoresis were carried out to verify the amplicon specificity. Transcripts of targeted genes were calculated by the double-standard curves method, and the rice housekeeping gene OsActin was used for sample normalization as an endogenous control. All the specific primers in this study were listed in Table S1. There were three biological replicates for each treatment. 发自小木虫Android客户端 |
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