高产紫杉醇菌株的诱变选育及其差异表达基因消减cDNA文库的构建  

Screening of high taxol producing fungi by mutagenesis and construction of subtracted cDNA library by suppression subtracted hybridization for differentially expressed genes

作　　者：赵凯[1,2] 孙立新[3] 王旋[1] 李秀凉[1] 王歆[1] 周东坡[1] 

机构地区：[1]黑龙江大学生命科学学院微生物黑龙江省高校重点实验室农业微生物技术教育部工程研究中心,哈尔滨150080 [2]黑龙江省农业科学院生物技术研究所,哈尔滨150086 [3]黑龙江大学校医院,哈尔滨150080

出　　处：《微生物学报》2011年第7期923-933,共11页Acta Microbiologica Sinica

基　　金：国家自然科学基金项目(30970090);中国博士后科学基金项目(20090450136);哈尔滨市科技创新人才研究专项资金项目(2010RFQXS043);黑龙江省普通高等学校新世纪优秀人才培养计划(1251-NCET-005);黑龙江省教育厅科学技术研究项目(11551377);黑龙江大学高层次人才(创新团队)支持计划(hdtd2010-17)~~

摘　　要：【目的】选育高产紫杉醇菌株,并构建选育到的高产紫杉醇菌株与出发菌株HD1-3差异表达的cDNA消减文库。【方法】分别采用硫酸二乙酯和紫外线与硫酸二乙酯复合诱变处理菌株HD1-3孢子;以选育到的高产紫杉醇菌株为tester,菌株HD1-3为driver,应用抑制性消减杂交技术构建选育到的高产紫杉醇菌株与菌株HD1-3差异表达的cDNA消减文库。【结果】试验确定的Nodulisporium sylviforme紫杉醇产生菌HD1-3孢子复合诱变的适宜条件为:将106 cfu/mL孢子悬液经过8%硫酸二乙酯处理15 min后,在电磁搅拌下,用紫外灯(30 w,距离30 cm)照射处理45 s,获得了1株遗传性状稳定、高产紫杉醇的突变株——UD14-11,其紫杉醇产量从出发菌株HD1-3的232.73±4.61μg/L提高至312.81±7.51μg/L;构建的文库滴度为1.2×107 cfu/mL,阳性克隆率75.3%,片段大小主要集中在300 bp-1.0 kb。【结论】选育到了1株遗传性状稳定、高产紫杉醇突变株;成功地构建了高产紫杉醇菌株UD14-11与菌株HD1-3差异表达的cDNA消减文库,为寻找、分离微生物生物合成紫杉醇相关基因和利用基因工程或代谢工程手段定向设计改造菌株奠定基础。[Objective]To screen mutants with high yield of taxol,and construct cDNA subtractive library of obtained mutant and primary strain HD1-3.[Methods] The spores of taxol-producing fungus HD1-3 were treated by diethyl sulphate（DES）,ultraviolet radiation and diethyl sulphate（UV ＋ DES）.cDNA subtractive library of taxol producing fungi from the mRNA of obtained mutant with high yield of taxol tester and HD1-3 driver was constructed by using suppression subtracted hybridization（SSH）.[Results] The optimal conditions for mutagenesis of strain HD1-3 were as follows： the spore suspension was treated with 8% DES for 15 min,followed by UV irradiation（30w,30cm distance） for 45sec under magnetic stirring,a mutant UD14-11 which was able to produce taxol with high yield and could be stably passed on genetics was found.Its ability to produce taxol was improved from 232.73±4.61 μg/L（strain HD1-3） to 312.81±7.51 μg/L（strain UD14-11）.The tilter of the constructed cDNA library was 1.2×107 cfu/mL,the recombinant rate reached to 75.3% and the length of the inserted fragments was mostly 300 bp-1.0 kb.[Conclusion] A mutant UD14-11 with high yield was obtained,and cDNA subtractive library of the mutant UD14-11 and strain HD1-3 was constructed.The study laid solid foundation for isolation of taxol biosynthesis related genes and construction of engineering strains with high yield of taxol by genetic techniques.

关 键 词：紫杉醇 内生真菌 诱变育种 抑制性消减杂交 CDNA文库 

分 类 号：Q933[生物学—微生物学]