关键词: 热不对称交错PCR, 6-磷酸葡萄糖酸脱氢酶, 黄瓜, 上游序列

Abstract: The upstream sequence of the 6-phosphogluconate dehydrogenase (6PGDH) gene from cucumber (C. sativus L.) was isolated using modified TAIL-PCR method. Compared with the original protocol, the main modifications of the TAIL-PCR were introduced here: (1)among the battery of random 10 bp primers originally developed for RAPD analysis, suitable primers were selected as short arbitrary upstream primers according to the Primer 5.0 software prediction prior to PCR, and the annealing temperatures of two different stringency circles were also adjusted to be optimal accordingly.(2)the asymmetric interlaced thermal cycle was also applied in tertiary PCR so that the target product could be further preferentially amplified over non-target products. A 517 bp sequence upstream to the start codon of the 6PGDH gene of cucumber was successfully isolated after three rounds of amplification. The final result demonstrated that the modified of TAIL-PCR was an instant and efficient method to clone the flanking sequences from known region．

Key words: Thermal asymmetric interlaced PCR (TAIL-PCR), 6-phosphogluconate dehydrogenase(6PGDH), Cucumis sativus L., upstream sequence