[1]聂鑫怡,薛 杨,王银春,等.黄曲霉中pyrG筛选标记循环利用的方法[J].福建农林大学学报(自然科学版),2021,50(02):270-275.[doi:10.13323/j.cnki.j.fafu(nat.sci.).2021.02.018]
 NIE Xinyi,XUE Yang,WANG Yinchun,et al.Establishment of a novel pyrG selectable marker recycling system in Aspergillus flavus[J].,2021,50(02):270-275.[doi:10.13323/j.cnki.j.fafu(nat.sci.).2021.02.018]
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黄曲霉中pyrG筛选标记循环利用的方法()
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福建农林大学学报(自然科学版)[ISSN:1671-5470/CN:35-1255/S]

卷:
50卷
期数:
2021年02期
页码:
270-275
栏目:
资源与环境
出版日期:
2021-02-15

文章信息/Info

Title:
Establishment of a novel pyrG selectable marker recycling system in Aspergillus flavus
文章编号:
1671-5470(2021)02-0270-06
作者:
聂鑫怡12 薛 杨12 王银春12 丁霞飞12 汪世华12
1.福建农林大学生命科学学院; 2.福建省病原真菌与真菌毒素重点实验室,福建 福州 350002
Author(s):
NIE Xinyi12 XUE Yang12 WANG Yinchun12 DING Xiafei12 WANG Shihua12
1.School of Life Sciences, Fujian Agriculture and Forestry University; 2.Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Fuzhou, Fujian 350002, China
关键词:
pyrG遗传筛选标记 循环利用 不依赖同向重复序列 黄曲霉
Keywords:
pyrG selectable marker recycling system direct repeat-independent Aspergillus flavus
分类号:
Q784
DOI:
10.13323/j.cnki.j.fafu(nat.sci.).2021.02.018
文献标志码:
A
摘要:
为解决黄曲霉遗传转化操作过程中遗传筛选标记受限及传统URA3/pyrG环出系统存在重复序列残留的问题,以具有尿嘧啶营养缺陷的黄曲霉CA14PTs(Δku70ΔpyrG)菌株为出发菌,利用pyrG筛选标记构建表达HA-SumO的重组菌株GHS(pyrG+),再通过重叠PCR法构建包含GHS(pyrG+)基因组中pyrG整合位点上、下游片段的融合片段,导入GHS(pyrG+)菌株原生质体,利用DNA同源重组和5-氟乳清酸(5-FOA)的负筛选剔除pyrG筛选标记,重新获得具有尿嘧啶营养缺陷的重组菌株GHS(pyrG-).通过测序分析,确认GHS(pyrG-)重组菌株基因组中的pyrG筛选标记已被剔除且无其他序列残留.免疫杂交分析结果表明,剔除pyrG筛选标记不影响重组菌株的蛋白表达模式.
Abstract:
Due to limited number of selectable markers and residual repeatable sequences in traditional URA3/pyrG recycling system, a novel pyrG selectable marker recycling system was established for the genetic transformation of Aspergillus flavus. Using CA14PTs(Δku70ΔpyrG), a A. flavus strain with uracil auxotroph as the original strain, the recombinant strain GHS(pyrG+) that expressing HA-SumO was screened by pyrG selectable marker. Without direct repeats, the flanking sequences of pyrG locus in GHS(pyrG+) recombinant strain were fused by overlap extension PCR and transformed into protoplasts of the GHS(pyrG+)strain. The pyrG selectable marker was knocked out from the genome by homologous DNA recombination in the presence of 5-fluoroolactic acid using negative selection. The GHS(pyrG-)recombinant transformants were further identified by sequencing and auxotrophic analysis. Moreover, western blotting was carried out to confirm that the protein expression pattern of GHS(pyrG-)was not affected in the absence of the pyrG selectable marker.

参考文献/References:

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备注/Memo

备注/Memo:
收稿日期:2020-09-24 修回日期:2020-10-23
基金项目:国家自然科学基金项目(31700050); 福建农林大学杰出青年科研人才项目(xjq201724).
作者简介:聂鑫怡(1982-),女,助理研究员.研究方向:微生物学与分子生物学.Email:xinyi_nie@126.com.通信作者汪世华(1976-),男,教授.研究方向:功能基因与病原真菌.Email:wshyyl@sina.com.
更新日期/Last Update: 2021-02-15