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| Line 12: |
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| | onset of anaphase I. In addition, the phosphorylation of inner centromeric histone H3 at Ser-10 (H3-pS10) during diakinesis | | onset of anaphase I. In addition, the phosphorylation of inner centromeric histone H3 at Ser-10 (H3-pS10) during diakinesis |
| | depends on BRK1. Therefore, we speculate that BRK1 may be required for normal localization of Aurora kinase before the | | depends on BRK1. Therefore, we speculate that BRK1 may be required for normal localization of Aurora kinase before the |
| − | onset of metaphase I, which is responsible for correcting the merotelic attachment.<ref name="ref1"> | + | onset of metaphase I, which is responsible for correcting the merotelic attachment.<ref name="ref1"></ref> |
| − | | |
| − | ===Expression===
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| − | '''Plant Materials'''
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| − | | |
| − | The blk1-1 mutant was isolated from the indica rice (Oryza sativa) variety
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| − | Zhongxian 3037 following
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| − | 60
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| − | Co;g-ray radiation. Seeds of the Tos17 insertion line NE7019, which we called blk1-2, were kindly provided by the
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| − | Rice Genome Resource Center of the National Institute of Agrobiological
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| − | 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.
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| − | | |
| − | '''Molecular Cloning of BRK1 and Tos17 Insertion Site Mapping'''
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| − | | |
| − | To fine map BRK1, STS markers (P1 to P5) were developed based on
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| − | sequence differences between indica variety 9311 and japonica variety
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| − | Zhonghua 11 according to data published on the National Center for
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| − | Biotechnology Information website. All primer sequences are listed in
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| − | Supplemental Table 1 online. Primer pair TosP (59-ATTGTTAGGTTGCAAGTTAGTTAAGA-39) and 7019P (59-TGTATCGCCAGCTTGATACG-
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| − | 39) was used to amplify the Tos17-inserted regions of brk1-2. The PCR
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| − | products were cloned into the pMD18-T vector (TaKaRa) and sequenced.
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| − | | |
| − | '''Cloning the Full-Length BRK1 cDNA'''
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| − | | |
| − | Total RNA was extracted from rice panicle using TRIzol reagent (Invitrogen), and 3 mg RNA was reverse transcribed with the oligo-dT(18)
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| − | primer using MMLV-RT (Invitrogen). Gene-specific primers forward (59-
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| − | ATGGTTCTGCTCGAACGCAC-39) and reverse (59-GATGCGTATATGTCTCGGT-39) were designed to amplify the predicted coding region of
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| − | BRK1. PCR products were cloned into the pMD18-T vector (TaKaRa) and
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| − | 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
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| − | 39-RACE, the first and second PCRs were performed using 39RACE-F1
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| − | (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
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| − | reverse transcribed using the 59 phosphorylated primer 59RACE-RT (59-
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| − | CCTTACTGAAGAGAAACAGT-39). First and second PCRs were performed using 59RACE-S1 (59-CTCAGGTCTATCAACAAGGCT-39) and
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| − | 59RACE-R1 (59-TGCTGCCTCGGGCACCACCAA-39), and 59RACE-S2
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| − | (59-GATCGGCTCAAGATTTGTGT-39) and 59RACE-R2 (59-CGTGCGTTCGAGCAGAACCA-39), respectively. The products of 39-RACE-PCR and
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| − | 59-RACE-PCR were cloned and sequenced.
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| − | | |
| − | '''Real-Time PCR Expression Assay'''
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| − | | |
| − | Real-time PCR assay was conducted as described (Hong et al., 2010).
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| − | Total RNA was extracted from the leaf, root, and panicle of Zhongxian
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| − | 3037 and panicles of brk1 mutants. Real-time PCR analysis was performed using the Bio-Rad CFX96 real-time PCR instrument and EvaGreen
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| − | (BIOTIUM). Real-Time PCR was performed using primers BRKRT-F
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| − | (59-CTGCTCAACGCGACCTCCAA-39) and BRKRT-R (59-GCTACGAAGTTACGGAAGGT-39) for BRK1 and UBQF (59-CAAGATGATCTGCCGCAAATGC-39) and UBQR (59-TTTAACCAGTCCATGAACCCG-39) for
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| − | Ubiquitin. The real-time PCR results were analyzed using Bio-Rad CFX
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| − | Manager analysis software. Each experiment had three replicates.
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| − | | |
| − | '''Construction of BRK1 RNA Interference Cassette and
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| − | Rice Transformation'''
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| − | | |
| − | A 310-bp fragment of the BRK1 cDNA sequence was amplified with the
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| − | primer pair RNAiF, 59-ATTCTCGAGGCCAGTGTCGATGGTAATACA-39
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| − | (adding an XhoI site), and RNAiR, 59-ACGAGATCTCAACGCTGTGCAGTGTTTCCA-39 (adding a BglII site). The fragment was cloned into the
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| − | BamHI-SalI and BglII-XhoI sites of the pUCRNAi vector. Subsequently,
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| − | the stem-loop fragment was cloned into the pCAMBIA2300-Actin vector.
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| − | Then the RNAi construct was transformed into an Agrobacterium tumefaciens strain, which was used for further rice transformation.
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| − | | |
| − | '''Antibody Production and Protein Gel Blot Analysis'''
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| − | | |
| − | Antibodies were produced as described (Wang et al., 2010). To generate
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| − | the antibody against BRK1, a 330-bp fragment of the BRK1 cDNA (amino
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| − | acids 396 to 505) was amplified with primers BRKAbF, 59-GTGAATTCCCGAAATCACAGACGAAACCTT-39 (adding an EcoRI site), and
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| − | BRKAbR, 59-AGTCTCGAGTCCACAGCTCCACGTTCCAGT-39 (adding an
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| − | XhoI site). The fragment was cloned into the expression vector pGEX 4T-2.
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| − | The BRKAb-GST peptides were purified using Glutathione Sepharose 4B
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| − | (GE). Polyclonal antibodies against BRK1 were raised in rabbit and mouse.
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| − | | |
| − | ===Evolution===
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| − | Please input evolution information here.
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| − | | |
| − | You can also add sub-section(s) at will.
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| − | | |
| − | ==Labs working on this gene==
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| − | Please input related labs here.
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| − | | |
| − | ==References==
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| − | <references>
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| − | <ref name="ref1">BRK1, a Bub1-Related Kinase, Is Essential for Generating Proper Tension between Homologous Kinetochores at Metaphase I of Rice Meiosis</ref>
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