[1]陈丽娜,朱艳冰,倪辉,等.法夫酵母整合型表达载体的构建[J].集美大学学报(自然版),2014,19(3):185-191.
 CHEN Li-na,ZHU Yan-bing,NI Hui,et al.Construction of Integrative Expression Vectors for Phaffia rhodozyma[J].Journal of Jimei University,2014,19(3):185-191.
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法夫酵母整合型表达载体的构建()
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《集美大学学报(自然版)》[ISSN:1007-7405/CN:35-1186/N]

卷:
第19卷
期数:
2014年第3期
页码:
185-191
栏目:
水产、食品与生物技术
出版日期:
2014-05-25

文章信息/Info

Title:
Construction of Integrative Expression Vectors for Phaffia rhodozyma
作者:
陈丽娜1朱艳冰1倪辉123李利君1
1.集美大学生物工程学院,福建 厦门 361021;2.福建省高校食品微生物与酶工程技术研究中心,福建 厦门 361021;3.厦门市食品与生物工程技术研究中心,福建 厦门 361021
Author(s):
CHEN Li-na1ZHU Yan-bing1NI Hui123LI Li-jun1
1.College of Biological Engineering,Jimei University,Xiamen 361021,China;2.Research Center of Food Microbiology and Enzyme Engineering Technology (Jimei University),Fujian Province,Xiamen 361021,China;3.Research Center of Food Biotechnology of Xiamen City,Xiamen 361021,China
关键词:
法夫酵母整合载体构建G418抗性
Keywords:
Phaffia rhodozymaintegrated vectorsconstructionG418 resistance
文献标志码:
A
摘要:
为了建立法夫酵母虾青素代谢途径研究和分子育种的技术体系,构建了法夫酵母的整合型表达载体.通过测定法夫酵母对G418的抗性来确定抗性筛选物的质量浓度,以来源于法夫酵母本身的rRNA基因为基因同源重组片段,利用法夫酵母肌动蛋白启动子和甘油醛-3-磷酸脱氢酶(gpd)启动子,构建了携带G418抗性基因的整合型载体pMD-18spakta和pMD-18spgktg.结果表明:法夫酵母对20 μg/mL质量浓度的G418敏感,将构建的载体转化法夫酵母菌株后,筛选得到了能够在含有G418的培养基上生长的转化子;利用PCR的方法检测到转化子中存在的抗性基因.将筛选得到的阳性菌株连续传代培养10次后,发现该菌株的G418抗性仍然存在;以总DNA为模板,用PCR的方法仍可检测到G418抗性基因.说明已经构建得到了遗传稳定性良好的法夫酵母整合型表达载体,构建的质粒pMD-18spakta和pMD-18spgktg可作为法夫酵母整合载体应用于法夫酵母的DNA重组实验.
Abstract:
In order to set up a technology system for the research of astaxanthin metabolic pathways in Phaffia rhodozyma and molecular breeding,integrative expression vectors for P.rhodozyma were constructed in this study.The sensitivity of P.rhodozyma to G418 was determined to decide the screening concentration of G418.Two integrated vectors named pMD-18spakta and pMD-18spgktg containing ribosomal RNA gene portion of P.rhodozyma for stable integration in the genome and also containing G418 resistance gene cassette were promoted by Phaffia actin promoter and glyceraldehyde-3-phosphate dehydrogenase(gpd) promoter,respectively for conferring G418 resistance to P.rhodozyma strains.The result indicated that P.rhodozyma strain was sensitive to 20 μg/mL G418.Transformants showed that G418 resistance was screened after pMD-18spakta and pMD-18spgktg were transformed into P.rhodozyma by electroporation.Using total DNA extracted from the transformants as the template,the G418 resistance gene was also detected.After subculturing for 10 generations,the transformants still maintained their resistance to G418,and the G418 resistance gene cassette could also be detected by PCR,which indicated the genetic stability of the plasmids.In conclusion,the constructed plasmids pMD-18spakta and pMD-18spgktg could be used as integrated vectors in DNA recombination of P.rhodozyma.

相似文献/References:

[1]付爽,沈宁燕,倪辉,等.乙醇补料对法夫酵母发酵产虾青素的促进效果[J].集美大学学报(自然版),2017,22(4):20.
 FU Shuang,SHEN Ningyan,NI Hui,et al.Promotion Effect of Ethanol on Astaxanthin Synthesis During Fed-batch by Phaffia rhodozyma[J].Journal of Jimei University,2017,22(3):20.
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更新日期/Last Update: 2014-12-01