The Endophytic Fungus Paclitaxel Biosynthesis-related Genes Were Analyzed By High-throughput Sequencing

Taxol has special effects on a variety of malignant tumors such as breast cancer, ovarian cancer, and cervical cancer. It has become recognized as the most promising cancer drug. Taxol was first extracted from the bark of Taxus brevifolia, not only the yield is not high, but not conducive to environmental protection. Currently, researchers are trying to solve the shortage of taxol source and finding mass-produced promising methods. The biosynthesis ways of endophytic fungi is the most ideal one to produce unlimited potential. However, the way of endophytic fungi is not very satisfactory in taxol production, is difficult to achieve large-scale industrial production. Use the modern molecular biology way to select high-yielding strains of endophytic fungi could improve the yield of taxol. The first should figure out the Taxol biosynthesis pathway of endophytic fungi, as well as the key biosynthetic pathway genes. High-throughput sequencing technology is a new fast and accurate method of gene sequence determination in the rise of recent years, with high sensitivity, high accuracy and low cost.The first test was to analyze the yields of taxol in different fermentation times of taxol high-yield endophyte HDFS4.26 (Nodulisporium sylviforme). The results showed that in sample 3d, strain HDFS4.26 began to produce Taxol, the production of sample 9d reached the highest value 521.63 ± 10.14p.g/L, then the taxol production of HDFS4.26 began to decrease. Therefore, the test to determine sample 1d,3d,7d,9d, 11d mycelium as high-throughput sequencing samples in HDFS4-26 fermentation.Then extract the total RNA of sample 1d,3d,7d,9d,11d mycelium in fermentation. Then make the mRNA fragment processing, reverse transcription to form a double-stranded cDNA, constructed Illumina PE library to sequenced. Finally, the data obtained were analyzed by bioinformatics. The sequenced results showed that the high quality sequence ratio of all the samples higher than 97%, there are 12,648 genes after sequence splicing, make an ORF predicted and then compare with database. The results of the analysis of differential expression between samples showed that:there are 383 different expressed genes between sample 1d vs 3d, there are 969 different expressed genes between sample 1d vs 7d, there are 2313 different expressed genes between sample 1d vs 9d, there are 2819 different expressed genes between sample 1d vs 11d, there are 674 different expressed genes between sample 3d vs 7d, there are 2086 different expressed genes between sample 3d vs 9d, there are 2900 different expressed genes between sample 3d vs 11d, there are 702 different expressed genes between sample 7d vs 9d, there are 1563 different expressed genes between sample 7d vs 11d, there are 167 different expressed genes between sample 9d vs 11d. There are nine different expressed genes related taxol production through the analysis of metabolic KEGG pathways annotations. These nine sequences were:comp2568_c0_seql belong to glutathione S-transferase family protein, comp145452_c0_seql and comp30968_c0_seql belong to glutathione S-transferase, comp168754_c0_seql and comp176327_c0_seql belong to acetyl-CoA-acetyltransferase, comp2885_c0_seql belong to GGPPS, comp6245_c0_seq11, comp6245_c0_seq2 and comp6245_c0_seq7 belong to NADPH-P450 reductase. The test obtained Taxol biosynthesis genes of HDFS4-26 with high-throughput sequencing technology, lay the foundation for the study of taxol biosynthesis pathway of N. sylviform and construct high-yield taxol engineered strains.