Os07g0508500

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Annotated Information

Function

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Expression

Plant Materials The blk1-1 mutant was isolated from the indica rice (Oryza sativa) variety Zhongxian 3037 following 60 Co;g-ray radiation. Seeds of the Tos17 insertion line NE7019, which we called blk1-2, were kindly provided by the Rice Genome Resource Center of the National Institute of Agrobiological Sciences. The other meiotic mutant, sgo1-1, was isolated in our lab previously (Wang et al., 2011). All plant material was cultivated in paddy fields. Molecular Cloning of BRK1 and Tos17 Insertion Site Mapping To fine map BRK1, STS markers (P1 to P5) were developed based on sequence differences between indica variety 9311 and japonica variety Zhonghua 11 according to data published on the National Center for Biotechnology Information website. All primer sequences are listed in Supplemental Table 1 online. Primer pair TosP (59-ATTGTTAGGTTGCAAGTTAGTTAAGA-39) and 7019P (59-TGTATCGCCAGCTTGATACG- 39) was used to amplify the Tos17-inserted regions of brk1-2. The PCR products were cloned into the pMD18-T vector (TaKaRa) and sequenced. Cloning the Full-Length BRK1 cDNA Total RNA was extracted from rice panicle using TRIzol reagent (Invitrogen), and 3 mg RNA was reverse transcribed with the oligo-dT(18) primer using MMLV-RT (Invitrogen). Gene-specific primers forward (59- ATGGTTCTGCTCGAACGCAC-39) and reverse (59-GATGCGTATATGTCTCGGT-39) were designed to amplify the predicted coding region of BRK1. PCR products were cloned into the pMD18-T vector (TaKaRa) and sequenced. 39- and 59-RACE was performed according to the kit protocols (39-Full RACE Core Set and 59-Full RACE Core Set; TaKaRa). For 39-RACE, the first and second PCRs were performed using 39RACE-F1 (59-CTGCTCAACGCGACCTCCAA-39) and the 39 adaptor (59-AAGCAGTGGTATCAACGCAGA-39), and 39RACE-F2 (59-AGCCTCCGGATGTCCTTCCA-39) and the 39 adaptor, respectively. For 59-RACE, total RNA was reverse transcribed using the 59 phosphorylated primer 59RACE-RT (59- CCTTACTGAAGAGAAACAGT-39). First and second PCRs were performed using 59RACE-S1 (59-CTCAGGTCTATCAACAAGGCT-39) and 59RACE-R1 (59-TGCTGCCTCGGGCACCACCAA-39), and 59RACE-S2 (59-GATCGGCTCAAGATTTGTGT-39) and 59RACE-R2 (59-CGTGCGTTCGAGCAGAACCA-39), respectively. The products of 39-RACE-PCR and 59-RACE-PCR were cloned and sequenced. Real-Time PCR Expression Assay Real-time PCR assay was conducted as described (Hong et al., 2010). Total RNA was extracted from the leaf, root, and panicle of Zhongxian 3037 and panicles of brk1 mutants. Real-time PCR analysis was performed using the Bio-Rad CFX96 real-time PCR instrument and EvaGreen (BIOTIUM). Real-Time PCR was performed using primers BRKRT-F (59-CTGCTCAACGCGACCTCCAA-39) and BRKRT-R (59-GCTACGAAGTTACGGAAGGT-39) for BRK1 and UBQF (59-CAAGATGATCTGCCGCAAATGC-39) and UBQR (59-TTTAACCAGTCCATGAACCCG-39) for Ubiquitin. The real-time PCR results were analyzed using Bio-Rad CFX Manager analysis software. Each experiment had three replicates. Construction of BRK1 RNA Interference Cassette and Rice Transformation A 310-bp fragment of the BRK1 cDNA sequence was amplified with the primer pair RNAiF, 59-ATTCTCGAGGCCAGTGTCGATGGTAATACA-39 (adding an XhoI site), and RNAiR, 59-ACGAGATCTCAACGCTGTGCAGTGTTTCCA-39 (adding a BglII site). The fragment was cloned into the BamHI-SalI and BglII-XhoI sites of the pUCRNAi vector. Subsequently, the stem-loop fragment was cloned into the pCAMBIA2300-Actin vector. Then the RNAi construct was transformed into an Agrobacterium tumefaciens strain, which was used for further rice transformation. Antibody Production and Protein Gel Blot Analysis Antibodies were produced as described (Wang et al., 2010). To generate the antibody against BRK1, a 330-bp fragment of the BRK1 cDNA (amino acids 396 to 505) was amplified with primers BRKAbF, 59-GTGAATTCCCGAAATCACAGACGAAACCTT-39 (adding an EcoRI site), and BRKAbR, 59-AGTCTCGAGTCCACAGCTCCACGTTCCAGT-39 (adding an XhoI site). The fragment was cloned into the expression vector pGEX 4T-2. The BRKAb-GST peptides were purified using Glutathione Sepharose 4B (GE). Polyclonal antibodies against BRK1 were raised in rabbit and mouse.

Evolution

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Labs working on this gene

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References

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