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目的筛选具启动活性的新生隐球菌荚膜相关基因CAP10编码区上游5′侧翼序列(启动子序列),为进一步研究其调控机制打下基础。方法利用氯霉素乙酰基转移酶(CAT)的编码基因为报告基因,构建了含不同长度的新生隐球菌荚膜相关基因CAP10编码区上游5′侧翼序列和报告基因的重组体,转化酵母,ELISA方法检测各转化子的CAT表达活性,并通过比较找到CAP10启动序列。结果发现含CAP10编码区上游303bp、390bp、500bp、951bp的转化子具有明显的CAT表达活性,而含202bp、102bp的转化子无明显的CAT表达活性。结论CAP10上游5′侧翼端303bp～0bp是具完整启动活性的最小单位。CAP10上游5′侧翼端202bp～303bp内含CAP10启动所必需的序列。
Objective To screen the 5 ’flanking sequence (promoter sequence) upstream of CAP10 coding region of C. neoformans capsule with promoter activity, and lay the foundation for further study of its regulatory mechanism. Methods The gene encoding chloramphenicol acetyltransferase (CAT) was used as the reporter gene to construct a recombinant containing the 5 ’flanking sequence and reporter gene upstream of CAP10 coding region of Cryptococcus neoformans capsule with different length. The CAT activity of each transformant was tested by ELISA, and the CAP10 promoter sequence was found by comparison. The results showed that the transformants with 303bp, 390bp, 500bp and 951bp upstream of the CAP10 coding region had obvious CAT expression activity, while the transformants with 202bp and 102bp had no obvious CAT expression activity. Conclusions 303bp ~ 0bp of 5 ’flanking upstream of CAP10 is the smallest unit with complete priming activity. The upstream 202 bp to 303 bp of the 5 ’flank of CAP10 contain the sequence necessary for CAP10 activation.