【作者】 王丽华；

【导师】 张颖；

【作者基本信息】 东北农业大学 ， 资源环境生态学， 2016， 硕士

【摘要】 邻苯二甲酸二丁酯(DBP)是邻苯二甲酸酯类化合物(Phthalic acid esters,PAEs)中常见的一种的塑料增塑剂,被普遍用在家具和汽车内饰等的生产和使用中。由于DBP与PVC树脂间是以氢键或者范德华力连接,因此DBP在产品中很容易扩散到环境中。DBP具有生殖毒性,且具有致癌性、致畸性、致突变性,因此DBP已被美国环保局(USEPA)列为优先控制污染物。由于DBP的性质稳定,在自然环境中不易降解,而微生物降解是降解DBP的主要方式。目前,国内外已有大量研究者成功筛选到DBP高效降解菌株,并且也有学者对降解菌的代谢路径和降解基因展开了研究。但在以往的研究中,研究者通常选用传统的研究方法如基因文库、基因克隆等方法来挖掘功能基因,这些方法往往耗时长且仅能获取很少的功能基因。随着第二代测序技术的发展,RNA-seq测序技术逐渐得到了研究者的关注,该方法不仅运行成本低,通量高,精确性高,而且可以对无参考基因组信息的物种进行转录组测序,已被大量研究者使用来研究生物的转录表达谱、功能基因的挖掘及差异表达基因分析。本研究采用Hi Seq2000测序系统的Illumina/Solexa技术对500、1 000和1 500 mg/L的DBP处理分别与葡萄糖作为对照(CK)的DBP降解菌株Novosphingobium aromaticivorans DNB-S1进行RNA-seq测序和差异表达基因分析,对降解菌株DNB-S1中的功能基因进行深度挖掘。通过RNA-seq测序,我们得到了DBP降解菌DNB-S1转录组的全面丰富的Unigenes信息。采用Trinity短序列组装软件对reads进行序列组装和拼接,CK与不同浓度DBP处理分别获得不同数量的Unigene,分别是23 287(CK)、9 402(500 mg/L)、9 209(1 000 mg/L)、9 576(1 500 mg/L)。在500、1 000和1500 mg/L DBP处理与CK的差异表达基因中,差异基因的个数分别是8 166、8 895和8 467,其中上调基因个数分别是751,752和747个,而相应的下调基因个数分别为7 414、7 642和7 719。差异基因的GO功能显著性富集分析表明,Unigene占主导的是“催化活性”,“代谢过程”,“细胞生理过程”和“结合功能”。KEGG注释结果显示,Unigene富集的主要的路径是“代谢途径”,“次生代谢产物的生物合成”和“不同环境中的微生物代谢”。对功能基因的挖掘,发现了26个转录调控因子,35个趋化蛋白,47个降解相关基因和2个解毒酶基因显著上调表达。根据降解基因的功能,推断DNB-S1降解DBP是通过龙胆酸降解途径进行代谢,其中龙胆酸1,2-双加氧酶在降解中起到开环的作用。在实时荧光定量PCR的验证结果中,基因表达情况与转录组测序中的基因表达水平保持一致。本研究利用RNA-seq测序技术对DBP降解菌进行转录组学研究,发现了DBP代谢途径为龙胆酸代谢途径,本研究为降解菌的功能基因和降解机理提供了丰富的基因数据,同时也为DBP污染的生物修复提供了新的方向。更多还原

【Abstract】 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.更多还原