| Oxytropis glabra DC.is one of the most harmful poisonous weeds in the world.It produces a toxic secondary metabolite of indolinidine,swainsonine(SW),which inhibits the activities of alpha-mannosidase I and Golgi alpha-mannosidase II,and causes the cell vacuolar degeneration of animals to lose the normal function,and even severe poisoning may lead to death in serious cases.In the medical field,SW has been used as a tool for the study of glycoprotein N-oligosaccharide synthesis.It was found to have good immunomodulatory and anti-tumor effects in the 1980 s and has gradually become a focus of attention.Our previous studies showed that the SW was synthesized by Alternaria oxytropis,an endophytic fungus in Oxytropis glabra,and the saccharopine reductase/dehydrogenase plays an important role in the SW synthesis in this fungus.The saccharopine reductase gene(sac)of the fungus was knocked out by homologous recombination to obtain the deletion mutant M1,and the level of SW in M1 was decreased.Since many details were still unknown in SW biosynthesis pathways of the Alternaria oxytropis,high-throughput transcriptome sequencing(RNA-Seq)of the wild-strain OW7.8 and mutant M1 was performed.The Unigenes related to SW biosynthesis were screened based on the sequencing results,and the expression patterns of chosen Unigenes were analyzed by qRT-PCR.Then key gene probably involved in SW biosynthesis was cloned to provide basic data for studying the synthetic pathways and molecular mechanism of SW.In addition,the effects of different light and medium conditions on the growth rates of OW7.8 and M1,and the dynamic changes to SW levels were also investigated.The main results shown as follows:1.The OW7.8 and M1 transcriptomes were cultured for 20 days by a new generation of high-throughput sequencing technology Illumina HiSeqTM 4000.A total of 47.91 million clean reads were obtained and de novo assembled to obtain 45,634 Unigenes,and the credibility is higher.Among them,5 genes were up-regulated and 11213 genes were down-regulated.2.A total of 41 DEGs related to SW biosynthesis were identified by using GO,KEGG and other public database information for comparative analysis,and enzymes related to SW biosynthesis pathways were predicted,including three saccharopine reductase,three pipecolic acid oxidases,two pyrroline-5-carboxylate reductases,and several CoAs related to hydroxylation.3.The SW biosynthesis pathways were speculated including P6 C and P2 C.?1-piperidine-2-carboxylic acid reductase,lysine 6-dehydrogenase and saccharin oxidase/L-piperidine oxidase were involved in P6 C.1-pyridine-2-carboxylate/1-pyridine-2-carboxylic acid reductase and ?1-piperidine-2-carboxylic acid reductase were involved in P2 C.The saccharopine reductase was involved in both pathways.The 1-keto-indolinidine was considered as a direct precursor of SW synthesis,which is catalyzed by hydroxymethylglutaryl-CoA lyase to synthesize SW.4.The cDNA sequence of hydroxymethylglutaryl-CoA lyase gene(Alhmgcl-1)were cloned,and the bioinformatics of the gene was predicted which provided basic data for later function analysis.5.The colony growth rate in of OW7.8 was the fastest on CDA medium under dark culture conditions(2.57±0.17mm/d),while the colony growth rate in M1 was the fastest on grew fastest on dark culturing conditions on PDA medium(4.93±0.10mm/d).The time for different strains to reach the maximum SW level was different under the same culture conditions or the same strain under different culture conditions.In addition,the SW level of OW7.8 was always higher than M1 under the same culture conditions.The statistical software analysis showed that there was no significant difference in the level of swainsonine under different medium and light conditions in the same strain,either OW7.8 or M1(0.01<P<0.05).However,there was significant difference in the level of swainsonine between OW7.8 and M1 under the same culture conditions(P<0.05). |
PDF Full Text Request