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Research Topics
Research Topics
I. Elucidation of molecular mechanisms of cold sensing, cold signaling, and hormonal signaling
II. Production of allergens or useful bio-materials from plant tissues by using "Tsukuba system"
III. Production of genome editing crops by transient expression system
I. Elucidation of molecular mechanisms of cold sensing and cold signaling
I. Elucidation of molecular mechanisms of cold sensing and cold signaling
ICE1 (Inducer of CBF/DREB1 expression 1) is a transcription factor that controls cold-regulated genes and cold tolerance in plants. However, it is not known how ICE1 is regulated and how cold stress is perceived. To elucidate these molecular mechanisms, we have isolated several proteins interacting with ICE1, including MYC transcription factors, kinases, and calcium-binding proteins. We have been characterizing these proteins in relation to cold signaling and how they regulate ICE1. And now we try to identify a cold sensor. According to our results, a calcium channel is a candidate. Functional mechanisms of a calcium channel as a sensor are to be elucidated.
II. Production of allergens or useful bio-materials from plant tissues by using "Tsukuba System"
II. Production of allergens or useful bio-materials from plant tissues by using "Tsukuba System"
As a definitive therapy of pollen allergy, sublingual desensitization therapy attracts attention. However, under the present circumstances, an extraordinary labor is required for extraction of allergen to use for this purpose. The aim of this study is production of allergen and extraction of large quantities by using other plants (collaboration with Dr. Noguchi, Faculty of Medicine) .
Now we are making allergens of cedar, Japanese cypress, birch, ragweed, and orchard grass. Allergens are proteins, thus proteins can be produced in any organism. But for allergic activity, plant-specific glycosylation is required. That means no allergic activity is observed, when the proteins are produced in E.coli or yeast. That is why the proteins are required to be produced in plants. For high expression of these proteins, we are now using transient expression system by agro-infiltration. Our project is to produce and purify allergens from plants. Not only quality, but also quantity is required.
High expression system in plant cells has been established. By using our system called as "Tsukuba System", about 4 mg/g fresh weight of protein can be produced in Nicotiana benthamiana. This amount is compatible to the other protein expression system in E.coli or yeast. Furthermore, this system is applicable to other plants (see below. Left: our system, right: previous system).
III. Production of genome editing crops by transient expression system
III. Production of genome editing crops by transient expression system
To produce transgenic plants, the foreign gene is required to be integrated into the host genome. The conventional way to produce genome-edited plants is the following; First, transgenic plants containing Cas9 and sgRNA are produced. Second, the target gene is edited by Cas9 and sgRNA. Third, the foreign gene (Cas9 and sgRNA) is removed by segregation. Several crops are difficult for making transgenic plants. Thus, transient expression system will be used for genome editing. After introducing the mutation in the target gene, Cas9 and sgRNA are not required any more. Thus, Cas9 and sgRNA will be transiently expressed and the genome-edited plants will be obtained. After cutting plants with shoot apical meristem, dediffentiation, cell division, and redifferentiation are happened. During these periods, Cas9 and sgRNA will be transiently expressed in the cells. For transient expression, Tsukuba system will be used. Tsukuba system is transient expression system in plants, resulting in high level of protein production. Expression level of GFP in several crops by Tsukuba system is very high.
Tomatoes with high sugar are valuable trait for tomatoes. In Univ. Tsukuba, the mutation, which enhanced sugar content but did not affect fruit size, has been identified from mutation population in cv. Micro-Tom. To introduce this mutation into other cultivars, we are now using genome editing methods. As we published recently (Shimatani et al., 2017 Nat Biotechnol), we are applying “Target-AID” system to tomato for introducing the mutation and making tomatoes with high sugar content.
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