Difference between revisions of "Os07g0508500"
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===Expression=== | ===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. | ||
===Evolution=== | ===Evolution=== | ||
Revision as of 02:56, 2 June 2014
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Contents
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.
Evolution
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Labs working on this gene
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References
<references>
[1]- ↑ BRK1, a Bub1-Related Kinase, Is Essential for Generating Proper Tension between Homologous Kinetochores at Metaphase I of Rice Meiosis
