载有磷酸甘油激酶基因启动子的新表达载体的构建以及在发酵性丝孢酵母中启动外源基因的表达研究

中国生物工程杂志

2012, Vol. 32

Issue (03): 39-46

研究报告

载有磷酸甘油激酶基因启动子的新表达载体的构建以及在发酵性丝孢酵母中启动外源基因的表达研究

王萍¹, 江木兰^1,2, 张银波^1,2, 万霞¹, 梁焯¹, 龚阳敏¹

1. 中国农业科学院油料作物研究所武汉 430062;
2. 农业部油料作物生物学与遗传育种重点实验室武汉 430062

Construction of a Novel Expression Vector with the Promoter of Phosphoglycerate Kinase Gene and Its Utilization of Heterogenous Gene Expression in Trichosporon fermentans

WANG Ping¹, JIANG Mu-lan^1,2, ZHANG Yin-bo^1,2, WAN Xia¹, LIANG Zhuo¹, GONG Yang-min¹

1. Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan 430062, China;
2. Key Laboratory of Biology and Genetic Improvement of Oil Crop, the Ministry of Agriculture, Wuhan 430063, China

全文: PDF(705 KB) HTML

摘要：

利用简并PCR技术从一株丝孢酵母(Trichosporon sp.)中克隆到磷酸甘油激酶基因的部分序列,然后利用染色体步移的方法克隆到了已知片段的上游序列约950bp。通过启动子序列分析软件分析,发现序列中含有启动子所需的必须元件如TATA BOX和CAAT BOX等,因此确定克隆到的基因片段含有启动子序列。将潮霉素基因置于该启动子下构建了丝孢酵母整合型表达载体pTFPH,并转化发酵性丝孢酵母(Trichosporon fermentans),转化后的酵母能够在含有潮霉素的抗性选择性平板长出,而未进行转化的对照菌株则不能生长。以上试验证明:丝孢酵母的磷酸甘油激酶基因启动子具有启动异源基因在发酵性丝孢酵母中表达的功能,这个结果为油脂酵母工程菌的构建和开发新的酵母表达宿主奠定了基础。

关键词： 磷酸甘油激酶启动子; 简并PCR; 染色体步移; 新的表达载体; 发酵性丝孢酵母

Abstract:

Upstream sequence and partial coding sequence (CDS) of the phosphoglycerate kinase (PGK) gene were cloned from oleaginous yeast Trichosporon sp. by degenerate PCR and chromosome walking. The computation analysis revealed that a promoter-like sequence in the upstream region of the PGK coding region including essential elements such as TATA BOX and CAAT BOX was cloned. The promoter-proximal region cloned was fused with the coding region of the hygromycin gene, and then cloned into the expression vector pTF-PHT. The recombinant vector was transformed into Trichosporon fermentans, and the transformed yeast was able to grow on the selective agar containing hygromycin. The results indicated that the promoter-proximal region of the phosphoglycerate kinase gene of Trichosporon sp was able to drive the expression of heterologous genes in the Trichosporon fermentans, which provides new avenues for developing novel engineering yeast or yeast expression system.

Key words: Phosphoglycerate kinase promoter Degenerate PCR Chromosome walking Novel expression vector Trichosporon fermentans

收稿日期: 2011-10-28 出版日期: 2012-03-25

ZTFLH:

Q78

基金资助:

Supported by the National Natural Science Foundation of China(30970262, 31000778),the 863 Program(2011AA100904) and the Major S&T Projects on the Cultivation of New Varieties of Genetically Modified Organisms (Grant 2009ZX08009-120B)

通讯作者: 江木兰,mljiang@oilcrops.cn E-mail: mljiang@oilcrops.cn

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引用本文:

王萍, 江木兰, 张银波, 万霞, 梁焯, 龚阳敏. 载有磷酸甘油激酶基因启动子的新表达载体的构建以及在发酵性丝孢酵母中启动外源基因的表达研究[J]. 中国生物工程杂志, 2012, 32(03): 39-46.

WANG Ping, JIANG Mu-lan, ZHANG Yin-bo, WAN Xia, LIANG Zhuo, GONG Yang-min. Construction of a Novel Expression Vector with the Promoter of Phosphoglycerate Kinase Gene and Its Utilization of Heterogenous Gene Expression in Trichosporon fermentans. China Biotechnology, 2012, 32(03): 39-46.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I03/39

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