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

摘要:

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%

关键词:关键词:紫杉醇，内生真菌，诱变育种，抑制性消减杂交，cDNA 文库

Abstract:

Abstract:［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.

Key words:Keywords: Taxol，endophytic fungi，mutagenic breeding，suppression subtracted hybridization，cDNA library