| 研究実績の概要 |
For the period of September 2018 until March 2019, we have made a vector for expression of Venus-rCBP fusion protein by refurbishing pRGEB31 originally used for CRISPR/Cas9 editing in rice. pRGEB31 is originally for stable transformation of rice callus using Agrobacterium transformation method. Assembly of this refurbished pRGEB31 uses NEBuilder High-fidelity Assembly DNA Cloning technique (NEB). DNA fragments were made via PCR or synthesis followed by DNA purification. DNA fragments are the following: (1) pRGEB31 backbone, (2) Native promoter, (3) PreNLS (nuclear localization signal before the fusion protein), (4) Venus, (5) 6x_TEV (6xHis and TEV), (6) rCBP_CDS (rCBP coding sequence), (7) PostNLS (nuclear localization signal after the fusion protein). The order of the DNA fragments when cloned was also as the above.
Rice protoplast extraction and transfection protocol was also developed and tested using etiolated and green protoplast.
ChIP-sequencing, via our collaborator (Dr. H. Saze, OIST), was also commenced and currently waiting for the results. We performed ChIP-sequencing on segregated wild type and 9-12b rCBP mutant line. These lines were of Nipponbare background. Enriched DNA-samples for the following modifications were submitted for ChIP-sequencing: (1) H3, (2) H3K9ac, (3) H3K9me2, (4) H3K9me3, and (5) H3K27ac.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
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理由
The original plan is to immediately isolate a stable transformant of transgenic line expressing Marker-rCBP fusion protein. However, upon discussion, it was decided to first perform preliminary analysis of the above vector (N-terminal Venus) and other vectors to be made (e.g. C-terminal Venus, etc.) for testing in a protoplast system. The protoplast system can also substitute analyses (e.g. complementation testing, etc.) much more efficiently than stable transformation system. Stable transformation will follow after protoplast testing to cater to analyses that require more sample material.
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| 今後の研究の推進方策 |
Experimental testing of the vectors expressing fusion protein and for other purposes in the protoplast system will be performed to achieve the following:
A) Complementation testing. We have already performed RNA-seq on rCBP mutants and have identified putative targets of rCBP. These differentially expressed target genes will be evaluated in the protoplast system upon expression of the Venus-rCBP fusion protein for complementation purposes.
B) Native promoter analysis. At the moment, there is no study on rCBP promoter; therefore, the expression of the fusion protein will easily confirm whether the native promoter we utilized was working or not.
C)Stable transformation. Upon confirmation of complementation and the functionality of promoter, we will start isolating stable transformants of transgenic lines expressing fusion proteins and others.
D) Analysis of transgenic lines expressing Venus-rCBP fusion protein, etc. We will analyze these transgenic lines for rescue of acetylation and other phenotypes, pulldown tests, ChIP-seq and ChIP-MS.
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