著者

Takeshi Matsui Eiji Takita Seika Oiwa Asuka Yokoyama Ko Kato Kazutoshi Sawada

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

pp.21.0414a, (Released:2021-06-12)

参考文献数

47

被引用文献数

2

---

Plant-made oral vaccines can be a cost-effective method to control infectious diseases of humans and farm animals. Pig edema is a bacterial disease caused by enterohemorrhagic Escherichia coli producing the toxin Shiga toxin 2e (Stx2e). In our previous report, we chose the non-toxic B subunit of Stx2e (Stx2eB) as a vaccine antigen, and Stx2eB was expressed in lettuce (Lactuca sativa L., cv. Green wave). We found that a double repeated Stx2eB (2×Stx2eB) accumulates to higher levels than a single Stx2eB. In this study, we analyzed progeny plants introduced with 2×Stx2eB in which the gene was expressed under the control of conventional cauliflower mosaic virus 35S RNA (CaMV 35S) promoter, and found that the lettuce underwent transgene silencing and bore few seeds. We resolved these problems by using a transgene cassette which harbored a transcriptional promoter derived from the lettuce ubiquitin gene and a longer version of HSPT. The lettuce harboring this expression construct will be valuable in establishing the seed lot system on the basis that thousands of seeds can be obtained from one plant body and the resulting progeny plants accumulate 2×Stx2eB at high levels without the transgene silencing.

著者

Eiji Takita Kazuya Yoshida Shigeru Hanano Atsuhiko Shinmyo Daisuke Shibata

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.38, no.4, pp.391-400, 2021-12-25 (Released:2021-12-25)

参考文献数

41

---

Genetic modification in plants helps us to understand molecular mechanisms underlying on plant fitness and to improve profitable crops. However, in transgenic plants, the value of gene expression often varies among plant populations of distinct lines and among generations of identical individuals. This variation is caused by several reasons, such as differences in the chromosome position, repeated sequences, and copy number of the inserted transgene. Developing a state-of-art technology to avoid the variation of gene expression levels including gene silencing has been awaited. Here, we developed a novel binary plasmid (pTACAtg1) that is based on a transformation-competent artificial chromosome (TAC) vector, harboring long genomic DNA fragments on both sides of the cloning sites. As a case study, we cloned the cauliflower mosaic virus 35S promoter:β-glucuronidase (35S:GUS) gene cassettes into the pTACAtg1, and introduced it with long flanking sequences on the pTACAtg1 into the plants. In isolated transgenic plants, the copy number was reduced and the GUS expressions were detected more stably than those in the control plants carrying the insert without flanking regions. In our result, the reduced copy number of a transgene suppressed variation and silencing of its gene expression. The pTACAtg1 vector will be suitable for the production of stable transformants and for expression analyses of a transgene.

著者

Eiji Takita Kazuya Yoshida Shigeru Hanano Atsuhiko Shinmyo Daisuke Shibata

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

pp.21.0823a, (Released:2021-10-26)

参考文献数

41

---

Genetic modification in plants helps us to understand molecular mechanisms underlying on plant fitness and to improve profitable crops. However, in transgenic plants, the value of gene expression often varies among plant populations of distinct lines and among generations of identical individuals. This variation is caused by several reasons, such as differences in the chromosome position, repeated sequences, and copy number of the inserted transgene. Developing a state-of-art technology to avoid the variation of gene expression levels including gene silencing has been awaited. Here, we developed a novel binary plasmid (pTACAtg1) that is based on a transformation-competent artificial chromosome (TAC) vector, harboring long genomic DNA fragments on both sides of the cloning sites. As a case study, we cloned the cauliflower mosaic virus 35S promoter:β-glucuronidase (35S:GUS) gene cassettes into the pTACAtg1, and introduced it with long flanking sequences on the pTACAtg1 into the plants. In isolated transgenic plants, the copy number was reduced and the GUS expressions were detected more stably than those in the control plants carrying the insert without flanking regions. In our result, the reduced copy number of a transgene suppressed variation and silencing of its gene expression. The pTACAtg1 vector will be suitable for the production of stable transformants and for expression analyses of a transgene.

著者

Takeshi Matsui Eiji Takita Seika Oiwa Asuka Yokoyama Ko Kato Kazutoshi Sawada

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.38, no.2, pp.239-246, 2021-06-25 (Released:2021-06-25)

参考文献数

47

被引用文献数

2

---

Plant-made oral vaccines can be a cost-effective method to control infectious diseases of humans and farm animals. Pig edema is a bacterial disease caused by enterohemorrhagic Escherichia coli producing the toxin Shiga toxin 2e (Stx2e). In our previous report, we chose the non-toxic B subunit of Stx2e (Stx2eB) as a vaccine antigen, and Stx2eB was expressed in lettuce (Lactuca sativa L., cv. Green wave). We found that a double repeated Stx2eB (2×Stx2eB) accumulates to higher levels than a single Stx2eB. In this study, we analyzed progeny plants introduced with 2×Stx2eB in which the gene was expressed under the control of conventional cauliflower mosaic virus 35S RNA (CaMV 35S) promoter, and found that the lettuce underwent transgene silencing and bore few seeds. We resolved these problems by using a transgene cassette which harbored a transcriptional promoter derived from the lettuce ubiquitin gene and a longer version of HSPT. The lettuce harboring this expression construct will be valuable in establishing the seed lot system on the basis that thousands of seeds can be obtained from one plant body and the resulting progeny plants accumulate 2×Stx2eB at high levels without the transgene silencing.