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用带PBI121质粒的农杆菌LBA4404菌株转化泗棉3号胚性愈伤组织,获得的再生植株进一步对gus和nptⅡ基因进行PCR跟踪检测,共得到97株阳性转基因植株。GUS组织化学检测发现,97株转基因幼苗中有10株GUS检测阴性,嫁接后,只成活一株。利用来源于同一愈伤系的GUS检测阳性植株作为对照,对这一GUS检测阴性的植株进行gus基因沉默机理研究。Southern分析表明,该GUS检测阴性植株与GUS检测阳性植株有相同拷贝数。GUS组织化学检测和RT-PCR分析显示,gus基因在GUS检测阴性植株中没有表达,而nptⅡ基因在这两株转基因棉花中都表达。用限制性内切酶-PCR法分析35S启动子区甲基化发现:GUS检测阴性植株35S启动子区TATA box的HapII/MspI酶切位点发生甲基化,而GUS检测阳性植株该位点没有甲基化。以上研究表明,这株gus沉默的转基因棉花可能是由于其35S启动子区甲基化引起的。
The Agrobacterium strain LBA4404 with PBI121 was transformed into Simian 3 embryogenic callus, and the regenerated plants obtained were further tested by PCR with gus and npt Ⅱ gene. A total of 97 positive transgenic plants were obtained. GUS histochemical examination found that 10 out of 97 transgenic seedlings tested negative for GUS, and only one survived after grafting. Using GUS-positive plants derived from the same callus as a control, this GUS-negative plant was subjected to a gus gene silencing mechanism. Southern analysis showed that the GUS negative and GUS positive plants had the same copy number. GUS histochemical detection and RT-PCR analysis showed that gus gene was not expressed in GUS negative plants, whereas nptII gene was expressed in both transgenic cotton plants. Analysis of the 35S promoter region by restriction endonuclease-PCR revealed that the HapII / MspI restriction site of the TATA box in the 35S promoter region of the GUS negative-sense plant was methylated, whereas the positive site of the GUS- No methylation. The above studies show that this gus-silenced transgenic cotton may be due to its 35S promoter methylation caused.