The Transcriptome Analysis And Functional Genes Screening Of DBP Degradation Batrerium DNB-S1

Di-n-butyl phthalate(DBP), which belongs to the family of Phthalate acid esters(PAEs), is widely used in home furnishings and automotive interior, etc.. DBP tends to release from plastic products and then leach into the environment during use or after disposal since it is not chemically bound to the plastic structure. The toxicity of DBP has done great threaten to environmental organisms and human health. A substantial amount of researches have showed that DBP has carcinogenic, mutagenic, mutagenicity and reproductive toxicity. As a result, DBP was classified as priority control environmental pollutants by the United States Environmental Protection Agency(USEPA). Studies have shown that DBP is not easily degraded by hydrolysis and photodegradation, and biodegradation is the main way for degradation DBP.Recently, a large number of researchers have successfully screened DBP efficient degradation strains. And the metabolism pathways and degradation genes also have been studied by some scholars. The previous studies, researchers used traditional methods to screen functional genes by cloning, sequencing and comparing with known sequences, annotating their functions. These methods are often time-consuming to get very few functional genes. With the development of the Next-Generation Sequencing technology(NGS), RNA-sequencing(RNA-Seq) technology has become the most popular and powerful tool for species that lack reference genome information. RNA-Seq is less costly, more efficient, and allows faster gene discovery and more sensitive and accurate profiles of the transcriptome than microarray analysis or other techniques. RNA-seq has been widely used to study the expression profile, functional genes and differentially expressed genes.To gain detailed genetic information for degradation mechanism, three DBP treatments(500, 1 000, 1 500 mg/L) and one glucose treatment(CK) to Novosphingobium aromaticivorans DNB-S1 were sequenced using a high-throughput sequencing platform–Illumina/Solexa technology of Hi Seq2000 sequencing platform.Through RNA-seq sequencing, the transcriptome comprehensive information of DNB-S1 was obtained. Trinity software was used to splice and assemble of reads. And CK, 500, 1 000 and 1 500 obtained 23 287, 9 402, 9 209 and 9 576 unigenes, respectively. The different expressed genes(DEGs) of 500, 1 000 and 1 500 mg/L DBP treatments with CK was 8 166, 8 895 and 8 467, respecticely. The quantity of up-regulated genes was 751, 752 and 747, respectively, and the corresponding quantity of down-regulated genes was almost ten-times than that of the up-regulated genes. Through the GO functional enrichment analysis of DEGs, unigenes were clustered into “catalytic activity”, “metabolic process”, “cellular process” and “Binding”. The results of KEGG annotation showed that the dominant metabolic pathways were “metabolism pathway”, “biosynthesis of secondary metabolites” and “microbial metabolism in diverse environments”. 26 transcriptional regulators, 35 chemotaxis proteins, 47 degradation related genes and 2 detoxification enzyme significantly up-regulated in three DBP-treatments. This study showed that DBP was degraded by DNB-S1 by gentisate metabolic pathway. And microbial degradation of gentisate was initiated by gentisate 1, 2-dioxygenase through extradiol cleavage of the benzene ring. The results of q PCR showed the quantity of gene expression was consistent with the results level in RNA-seq.This study found a novel degradation pathway that was gentisate metabolic pathway in DBP degradation strain. The RNA-seq sequencing technology was used for the first time to sequence DBP degrading bacteria. This research could provide a substantial contribution to existing nucleotide sequence resources and molecular mechanism, and provided a new direction for bioremediation of DBP pollution.