Preliminary Construction Of Genetic Operating System Based On Ku Gene Background In Blakeslea Trispora

Blakeslea trispora is a filamentous fungus which has been used in the industrial production of carotenoids and has very important economic value.The traditional mutagenesis strategy was used to select excellent strains of B.trispora with low efficiency,while the metabolic engineering technology was expected to improve the strain transformation effect.However,the non-homologous end joining repair?NHEJ?was the main part of DNA double-strand break repair in B.trispora,and the frequency of homologous recombination?HR?was very low,which caused the difficulty of genetic modification.Therefore,preventing the intracellular NHEJ repair of B.trispora is of great significance for the development of efficient genetic modification tools.In this paper,Btku70 and Btku80 gene were cloned?analyzed and knocked out.Finally strains?Btku70 and?Btku80 was gained,in the process,also explored the efficiency of the three methods of transformation mediated knockout mutant strains??Btku70 and?Btku80 phenotypic differences with wild type strains,as well as developed a reverse selection markers for B.trispora.The main research results of this paper are as follows:?1?Cloning and bioinformatics analysis of Btku70 and Btku80 genes.Btku70 and Btku80 gene were cloned by blastp based on the genome sequence information of B.trispora.The results showed that Btku70 gene was 2686 bp long,c DNA sequence was 1839 bp long,encoded 613 amino acids and there were two domains,v WFA and KU78 respectively.The full length of Btku80 gene was 2716 bp,and its c DNA sequence was 2202 bp long.It encoded 734 amino acids and had three domains,namely,v WFA,KU80 and Ku-PK-bind.Amino acid sequence comparison and phylogenetic tree analysis showed that ku gene of B.trispora had higher homology with Choanephora cucurbitarum.?2?The effects of three transformation methods in the knockout of Btku70 and Btku80 genes were compared,and a genetic transformation system mediated by Agrobacterium tumefaciens was constructed.Due to the low regeneration rate,the protoplast transformation mediated by Ca Cl₂/PEG did not have any transformants on medium.The spores transformation method mediated by agrobacterium had 11 transformants but all were false positive.The protoplast transformation method mediated by agrobacterium had 20-30 transformants,and they had been verified by PCR and sequencing,turned out that 18 to 21 transformants had knockout fragments,transformation rate was70%-90%,gained one?Btku70 and two?Btku80 strains,targeting rate was 3%to 10%.?3??Btku70 and?Btku80 phenotypic analysis with wild type strains.Compared with B.trispora wild-type strains,?Btku70 and?Btku80 two deficient mutants were slower in the growth and were less spore production,but had the same sensitivity on DNA damage agent?H₂O₂,MMS??temperature and osmotic pressure.But a test showed that?Btku70 and?Btku80 strains had an unstable hereditary.?4?To develop a new reverse screening marker for B.trispora.In the study of the lethal gene HSVtk,it was found that the growth of wild-type strain was not inhibited by the substrate F2d U,while the growth of transformants carrying the lethal gene HSVtk were gradually inhibited by the increase of the concentration of F2d U,and the transformants could not grow at the concentration of 50?mol·L^-1,indicating that the lethal gene HSVtk could be applied to the reverse screening of B.trispora.