采用农杆菌介导转化法的泡盛曲霉表达载体的构建

doi:10.7506/spkx1002-6630-200901043

食品科学 ›› 2009, Vol. 30 ›› Issue (1): 181-185.doi: 10.7506/spkx1002-6630-200901043

采用农杆菌介导转化法的泡盛曲霉表达载体的构建

陈波^1,2，王熙²，贺新生²，张义正^1,*

1.四川大学生命科学学院 2.西南科技大学生命科学与工程学院

收稿日期:2007-12-12 修回日期:2008-03-18 出版日期:2009-01-01 发布日期:2010-12-29
通讯作者: 陈波 E-mail:qqchen36@gmail.com
基金资助:
西南科技大学重大科学研究项目(023113)

Construction of Agrobacterium-mediated Aspergillus awamori Expression Vector

CHEN Bo1,2，WANG Xi2，HE Xin-sheng2，ZHANG Yi-zheng1,*

(1.College of Life Science, Sichuan University, Chengdu 610064, China；
2.College of Life Science and Engineering, Southwest University of Science and Technology, Mianyang 621010, China)

Received:2007-12-12 Revised:2008-03-18 Online:2009-01-01 Published:2010-12-29
Contact: CHEN Bo1 E-mail:qqchen36@gmail.com

摘要/Abstract

摘要：

为在泡盛曲霉中表达外源基因、克服泡盛曲霉常规转化法效率低下的瓶颈，本研究拟构建采用农杆菌介导转化法的泡盛曲霉表达载体。通过PCR，从糖化酶基因(glaA)高效表达的泡盛曲霉菌株SG1 基因组DNA 扩增获得glaA 2.1 kb 启动子片段(包含信号肽编码序列)及1.1kb 终止子片段，构建了外源基因表达盒。以根瘤农杆菌双元载体pCAMBIA1302 为基础，删除非必需区段及多余限制酶位点，插入上述外源基因表达盒及来自丝状真菌标记载体pAN7-1 的潮霉素抗性标记，构建采用农杆菌介导转化法的泡盛曲霉分泌整合型表达载体pSUAA52am。转化结果表明：采用原生质体法的转化效率为21 个转化子/106 分生孢子，而农杆菌介导法可达1106 个转化子/106 分生孢子，是原生质体法的53 倍。构建载体成功利用glaA 表达盒与农杆菌介导转化法的高效特性，可为利用泡盛曲霉高效表达外源基因奠定基础。

关键词: 泡盛曲霉, glaA表达盒, 农杆菌介导转化法, 表达载体

Abstract:

To utilize the high efficiency of glaA expression cassette of Aspergillus awamori and Agrobacterium-mediated transformation in the construction of A.awamori expression vector, a 2.1-kb glaA promoter fragment including signal peptidecoding sequence and a 1.1-kb glaA terminator fragment were amplified from the genomic DNA of A.awamori strain SG1 by PCR and then fused to form a foreign gene expression cassette. The Agrobacterium tumefaciens binary vector pCAMBIA1302 was used as backbone and its T-borders and necessary elements for E.coli and Agrobacterium-mediated transformation were retained whereas other regions in the vector were deleted. The foreign gene expression cassette described above and the filamentous fungal functional hygromycin B resistance marker from pAN7-1 were inserted into the region between T-borders. Finally a secretory integration Agrobacterium-mediated A.awamori expression vector pSUAA52am was constructed. Transformation experiment showed that transformation efficiency of pSUAA52am into A. awamori with protoplast method was 21 transformants/106 spores, while that with Agrobacterium-mediated method reached 1106 transformants/106 spores, 53 folds of the former. This indicated that the construction of this expression vector is successful.

Key words: Aspergillus awamori, glaA expression cassette, Agrobacterium-mediated transformation, expression vector

中图分类号:

Q782

陈波1,2，王熙2，贺新生2，张义正1,*. 采用农杆菌介导转化法的泡盛曲霉表达载体的构建[J]. 食品科学, 2009, 30(1): 181-185.

CHEN Bo1,2，WANG Xi2，HE Xin-sheng2，ZHANG Yi-zheng1,*. Construction of Agrobacterium-mediated Aspergillus awamori Expression Vector[J]. FOOD SCIENCE, 2009, 30(1): 181-185.

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采用农杆菌介导转化法的泡盛曲霉表达载体的构建

Construction of Agrobacterium-mediated Aspergillus awamori Expression Vector

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