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Plant Cell Rep. 2016 Jan;35(1):43-51. doi: 10.1007/s00299-015-1865-y. Epub 2015 Sep 16.

CaMV-35S promoter sequence-specific DNA methylation in lettuce.

Author information

1
Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen, Nakaku, Sakai, Osaka, 599-8531, Japan.
2
Miyoshi Co., Ltd., 187 Kamisasao, Kobuchizawa, Hokuto, Yamanashi, 408-0041, Japan.
3
Tempstaff People Co., Ltd, 1-5 Shinsakae, Naka-ku, Nagoya, Aichi, 460-8482, Japan.
4
Iwate Biotechnology Research Center, 22-174-4 Narita, Kitami, Iwate, 024-0003, Japan.
5
Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen, Nakaku, Sakai, Osaka, 599-8531, Japan. mishiba@plant.osakafu-u.ac.jp.

Abstract

We found 35S promoter sequence-specific DNA methylation in lettuce. Additionally, transgenic lettuce plants having a modified 35S promoter lost methylation, suggesting the modified sequence is subjected to the methylation machinery. We previously reported that cauliflower mosaic virus 35S promoter-specific DNA methylation in transgenic gentian (Gentiana triflora × G. scabra) plants occurs irrespective of the copy number and the genomic location of T-DNA, and causes strong gene silencing. To confirm whether 35S-specific methylation can occur in other plant species, transgenic lettuce (Lactuca sativa L.) plants with a single copy of the 35S promoter-driven sGFP gene were produced and analyzed. Among 10 lines of transgenic plants, 3, 4, and 3 lines showed strong, weak, and no expression of sGFP mRNA, respectively. Bisulfite genomic sequencing of the 35S promoter region showed hypermethylation at CpG and CpWpG (where W is A or T) sites in 9 of 10 lines. Gentian-type de novo methylation pattern, consisting of methylated cytosines at CpHpH (where H is A, C, or T) sites, was also observed in the transgenic lettuce lines, suggesting that lettuce and gentian share similar methylation machinery. Four of five transgenic lettuce lines having a single copy of a modified 35S promoter, which was modified in the proposed core target of de novo methylation in gentian, exhibited 35S hypomethylation, indicating that the modified sequence may be the target of the 35S-specific methylation machinery.

KEYWORDS:

35S promoter; DNA methylation; De novo methylation; Gentian; Lettuce; Transgene silencing

PMID:
26373653
DOI:
10.1007/s00299-015-1865-y
[Indexed for MEDLINE]

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