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棉 花 学 报 Cotton Science 2008，20(4)：274-280
Transgene Silencing Caused by 35S Promoter Methylation in Upland Cotton (Gossypium hirsutum) Abstract：In this report, we found that transgenes were inactived during the transformation of cotton mediated by Agrobacterium , and we studied the mechanism of inactivation. Cotton embryogenic calli (EC) were transformed by Agrobacterium strain LBA4404 possessing the pBI121 binary vector. Ninety-seven transgenic plantlets were identified by PCR amplification for gus reporter gene and npt-II selection marker gene. Among the 97 transgenic plantlets, 10 (about 10%) gus inactive individuals were detected. After grafting the transgenic plantlets in the greenhouse, only one GUS-negative plant survived. In an effort to study the silencing mechanism of the GUS-negative transgenic plant, a GUS-positive plant generated from a single calli line was chosen for comparisons. Southern analysis revealed that the two transgenic plants possessed the same insertion copy numbers, and they had the same transformation event. GUS assay and RT-PCR analysis indicated that gus was silenced in the GUS-negative plant but it was expressed in the GUS-positive plant; while RT-RCR detection showed that the npt-II gene expressed in both transgenic plants. Restriction endonuclease-PCR analysis of methylation in the 35S promoter was conducted by using of the methylation-sensitive enzymes HapII/MspI . The results demonstrated that the HapII site in the TATA box of the 35S promoter region was methylated in the gus inactive transgenic plant and not in the gus active plant, which indicated that methylation of the 35S promoter caused gus silencing in transgenic cotton plants. This study represents the first report of transgenic silencing mechanisms in cotton transformation mediated by Agrobacterium.
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