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投稿时间：2023-10-10
投稿时间：2023-10-10
中文摘要: 启动子对基因的表达起着关键的作用，为获得黑曲霉强启动子，该文以柠檬酸生产菌株黑曲霉（Aspergillus niger）CGMCC 10142 为出发菌株，运用启动子捕获探针和基因重组技术筛选菌株高强度启动子，并确定其核心启动区域。结果显示：以红色荧光蛋白基因（Rfp）为报告基因，潮霉素抗性基因（HygR）为筛选标记，构建黑曲霉启动子捕获探针质粒pN3，借助T-DNA 随机整合到黑曲霉基因组，筛选具有高潮霉素抗性和强红色荧光表型的突变株，进而通过交错热不对称链式聚合酶反应（thermal asymmetric interlaced polymerase chain reaction，Tail-PCR）获得整合位点上游核苷酸序列，测序确定该序列为糖苷水解酶基因启动子区（Pgh），通过分析其具有多个转录因子结合元件。对Pgh 启动子进行5′端缺失分析，获得6 个不同长度5′端缺失的启动子序列，并与强启动子PglaA 对比，根据报告基因Rfp 表达情况确定启动子Pgh 的-1 066 ～-766 bp 为其核心区域。
Abstract:Promoter plays an important role in gene expression. To obtain the strong promoters of Aspergillus niger，citric acid producing strain A. niger CGMCC 10142 was used as the starting strain，and the promoter capture probe and gene recombination technique were employed to screen the ideal promoter and determine the core region. The results showed that pN3 was constructed as the capture probe vector with Rfp as reporter gene and HygR as marker gene. The T-DNA cassette was integrated into the genome of A. niger to screen mutants through a strong red fluorescence intensity. Subsequently，thermal asymmetric interlaced polymerase chain reaction（Tail-PCR）was used to obtain the nucleotide sequence upstream of Rfp with initiating activity.The nucleotide sequence was identified as upstream promoter of glycoside hydrolase gene by sequencing，named Pgh，with multiple transcription factor binding elements.In this paper，by analyzing Pgh promoter，six 5′ deletion cassette of Pgh was constructed and compared with PglaA. According to the expression of Rfp，it demonstrated that the-1 066--766 bp of promoter Pgh was the core region.
keywords: Aspergillus niger promoter promoter capture probe promoter element Agrobacterium tumefaciens infection
文章编号：202409024 中图分类号： 文献标志码：
基金项目:国家自然科学基金青年科学基金项目（31902193）
| 作者 | 单位 |
| 彭亚彬1，徐泽宇1，韩天琦1，王丽娟1，薛鲜丽1，2，王德培1，3* | 1.天津科技大学生物工程学院，天津 300450；2.天津市微生物代谢与发酵过程控制技术工程中心，天津 300450；3.天津工业发酵微生物教育部重点实验室，天津 300457 |
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