1、第12期胡云龙,胡影,陈文晶,等.邻苯二甲酸酯对黑土胶体微界面中DNA的影响J.环境科学与技术,2022,45(12):89-95.Hu Yunlong,Hu Ying,ChenWenjing,et al.Effects of phthalates on DNA in black soil colloid microinterfacesJ.Environmental Science&Technology,2022,45(12):89-95.Environmental Science&Technology第45卷 第12期2022年12月Vol.45 No.12Dec.2022环境科学与技术 编
2、辑部:(网址)http:/(电话)027-87643502(电子信箱)收稿日期:2022-06-08;修回2022-09-06基金项目:黑龙江省省属高等学校基本科研业务费(135409705)作者简介:胡云龙(1990-),男,硕士,研究方向为环境微生物,(电子信箱);*通讯作者,(电子信箱)。邻苯二甲酸酯对黑土胶体微界面中DNA的影响胡云龙1,2,胡影1,2,陈文晶1,2,徐伟慧1,2,孟庆欣1,2,王志刚1,2*(1.齐齐哈尔大学生命科学与农林学院,黑龙江齐齐哈尔161006;2.黑龙江省农业微生物制剂产业化技术创新中心,黑龙江齐齐哈尔161006)摘要:邻苯二甲酸酯(PAEs)作为增塑剂
3、已在全球范围内普遍应用,尤其是在农业生产中由于地膜的大量使用导致其在土壤中不断累积,检出率逐年升高,对粮食安全、生态环境以及人类健康均造成了严重威胁。该研究通过荧光光谱、荧光猝灭以及质粒转化等试验研究了环境污染物PAEs对黑土胶体微界面中DNA结构及其水平转移的影响。结果表明:当不同浓度的邻苯二甲酸二乙酯(DEP)与邻苯二甲酸二丁酯(DBP)加入到EB-DNA荧光体系中时,DEP-EB-DNA与DBP-EB-DNA反应体系中的荧光强度均呈现降低的趋势,荧光强度与DEP、DBP的增加量呈负相关,且在浓度为4.510-4mol/L时DEP与DBP的荧光抑制率达到最大,分别为50%与36%,DEP对
4、荧光的抑制率显著高于DBP,说明DEP与DBP能够与DNA相互作用影响DNA结构且DEP的影响更为显著;KI对DEP-DNA与DBP-DNA体系进行猝灭时其猝灭常数Ksv值分别为7.947(R2=0.995 9)、11.03(R2=0.980 3),相比于对照组DEP的Ksv值10.27(R2=0.996 0)与DBP的Ksv值13.52(R2=0.980 6)均发生了明显的下降,说明DEP和DBP与DNA是通过嵌插作用相互结合;随着DEP和DBP浓度的增加,游离态与固定态的质粒转化率与转化子数均明显降低,且DEP的抑制作用更明显。研究表明,DEP和DBP能够以嵌插方式与DNA结合,改变DNA
5、的结构,并抑制黑土胶体微界面中DNA的水平转移,为揭示环境污染物PAEs对黑土胶体微界面DNA遗传特征及基因转移机制的影响提供理论支持。关键词:邻苯二甲酸酯;黑土胶体;DNA;嵌插;水平转移中图分类号:X172文献标志码:Adoi:10.19672/ki.1003-6504.1354.22.338文章编号:1003-6504(2022)12-0089-07Effects of Phthalates on DNA in Black Soil Colloid MicrointerfacesHU Yunlong1,2,HU Ying1,2,CHEN Wenjing1,2,XU Weihui1,2,M
6、ENG Qingxin1,2,WANG Zhigang1,2*(1.College of Life Science,Agriculture and Forestry,Qiqihar University,Qiqihar 161006,China;2.Heilongjiang Provincial Technology Innovation Center of Agromicrobial Preparation Industrialization,Qiqihar 161006,China)Abstract:Phthalic acid esters(PAEs)have been widely us
7、ed as plasticizers around the world,especially in agriculturalproduction,due to the extensive use of plastic film,PAEs continue to accumulate in soil,and the detection rate is increasingyear by year.PAEs pose a serious threat to food security,ecological environment and human health.In this study,the
8、influence of environmental pollutants PAEs on the genetic characteristics of DNA and its gene transfer mechanism in thecolloidal micro-interface of black soil were evaluated by fluorescence spectrum,fluorescence quenching test and plasmidtransformation test.The result showed that when different conc
9、entrations of diethyl phthalate(DEP)and dibutyl phthalate(DBP)were added to the EB-DNA fluorescence system,the fluorescence intensity of DEP-EB-DNA and DBP-EB-DNAreaction systems all showed a decreasing trend,and the fluorescence intensity was negatively correlated with the increase ofDEP and DBP.At
10、 the concentration of 4.510-4mol/L,the fluorescence inhibition rates of DEP and DBP reached the maximum of 50%and 36%,respectively,and the fluorescence inhibition rate of DEP was significantly higher than that of DBP,which indicated that DEP and DBP could interact with DNA and affect DNA structure,t
11、he effect of DEP is more pronounced.Moreover,compared with DEP(10.27,R2=0.996 0)and DBP(13.52,R2=0.980 6),the quenching constant Ksvvalues ofDEP-DNA(7.947,R2=0.995 9)and DBP-DNA(11.03,R2=0.980 3)were significantly decreased,this indicates that DEP andDBP bind to DNA through intercalation.Additionall
12、y,the conversion rate of free or fixed plasmids as well as the number of第45卷transformant in the colloidal micro-interface of black soil were decreased with the increase of DEP and DBP concentrations,and the inhibitory effect of DEP was more obvious.Therefore,DEP and DBP could bind to DNA in an inter
13、calated manner tochange the structure of DNA,as well as the horizontal transfer of DNA was inhibited in the colloidal micro-interface of blacksoil,in order to provide theoretical support for revealing the influence of environmental pollutants PAEs on the DNA geneticcharacteristics and gene transfer
14、mechanism in the black soil colloidal micro-interface.Key words:phthalic acid esters;black soil colloid;DNA;intercalative mode;horizontal transfer邻苯二甲酸酯类化合物(phthalic acid esters,PAEs)常被用作增塑剂用以增加塑料的强度和柔韧性,应用范围十分广泛1。然而,由于PAEs在塑料中的稳定性较差,其已在空气、水、土壤、食品中被广泛检测2,3。此外,随着塑料制品中大量PAEs的释放,其在生物体与环境中的富集量也不断增加4。有研究
15、表明,PAEs作为一种环境污染物具有致畸、致癌、致突变的作用,同时还会破坏动物器官,干扰人类生殖系统5,因此,PAEs已被美国环境保护署和中国列为优先污染物,受到学者们的广泛关注6。随着塑料制品应用行业和范围的不断扩大,残留在环境中的废塑料量也随之增加7。尤其是在农业种植过程中,大量塑料薄膜的应用极大地加剧了PAEs向农业种植土壤的迁移与累积,近年来土壤中的PAEs污染爆发式的增长对环境安全造成了极其严重的威胁8,9。Niu等对我国31省125个区域的15种PAEs 在农田土壤中的残留情况进行了分析,其中PAEs污染较为严重的新疆地区15种PAEs总含量均值达到1 088 g/kg,其污染主要
16、原因是塑料地膜的大量使用10;甘肃省农业土壤中邻苯二甲酸二甲酯(DMP)、邻苯二甲酸二乙酯(DEP)、邻苯二甲酸二正丁酯(DnBP)、邻苯二甲酸二(2-乙基己基)酯(DEHP)和邻苯二甲酸二正辛酯(DNOP)检出率为100%,参照USEPA制定的土壤PAEs控制标准,其中DnBP、DMP和DEP超标率达到94%、28%和27%,且土壤 PAEs含量与农田地膜残留量呈显著的正相关关系(P0.05)11。东北地区作为世界四大黑土地区之一,是我国最为重要的粮食生产基地12,13。然而,高达5万t的塑料薄膜年使用量导致黑土地区的PAEs污染问题更为突出14,15,哈尔滨菜地土中DBP含量达到7.6 mg/kg,鸡西黑土中的DBP含量超过10 mg/kg16,17,PAEs在黑土中大量富集不但会对数量庞大微生物群落、土壤酶活等造成极大影响威胁土壤健康18,19,同时也会使微生物以及动植物细胞通过主动分泌或细胞裂解所释放的大量DNA处于PAEs的污染之下。这些DNA在土壤中大部分并不会被分解,而是会被土壤胶体保存20-22,通过重组、转化、转导等方式在微生物之间,甚至微生物与植物之间进行转移23,