title: [Screening of high taxol producing fungi by mutagenesis and construction of subtracted cDNA library by suppression subtracted hybridization for differentially expressed genes].
abstract: OBJECTIVE
  To screen mutants with high yield of taxol, and construct cDNA subtractive library of obtained mutant and primary strain HD(1-3).
  
  
  METHODS
  The spores of taxol-producing fungus HD(1-3) 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 HD(1-3) driver was constructed by using suppression subtracted hybridization (SSH).
  
  
  RESULTS
  The optimal conditions for mutagenesis of strain HD(1-3) were as follows: the spore suspension was treated with 8% DES for 15 min, followed by UV irradiation (30 w, 30 cm distance) for 45 sec under magnetic stirring, a mutant UD(14-1) 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 microg/L (strain HD(1-3)) to 312.81 +/- 7.51 microg/L (strain UD(14-1)). The tilter of the constructed cDNA library was 1.2 x 10(7) cfu/mL, the recombinant rate reached to 75.3% and the length of the inserted fragments was mostly 300 bp-1.0 kb.
  
  
  CONCLUSIONS
  A mutant UD(14-11) with high yield was obtained, and cDNA subtractive library of the mutant UD(14-11) and strain HD(1-3) 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.
authors: Laboratory of Microbiology, College of Life Science, Heilongjiang University, Engineering Research Center of 150080, China. zk395@yahoo.com.cn
authors: Kai
authors: K
authors: Zhao
authors: Lixin
authors: L
authors: Sun
authors: Xuan
authors: X
authors: Wang
authors: Xiuliang
authors: X
authors: Li
authors: Xin
authors: X
authors: Wang
authors: Dongpo
authors: D
authors: Zhou
mesh_terms: D060026:Endophytes; D005658:Fungi / Q000235:genetics* / Q000378:metabolism*; D015723:Gene Library; D016296:Mutagenesis; D009693:Nucleic Acid Hybridization / Q000379:methods*; D017239:Paclitaxel / Q000096:biosynthesis*
chemical_list: D017239:Paclitaxel