【作者】 徐淑霞；

【导师】 刘华山；

【作者基本信息】 河南农业大学 ， 作物生理， 2013， 博士

【摘要】 二氯喹啉酸是防除稻田稗草的特效选择性除草剂,在稻田中普遍使用。在稻烟轮作地区,由于前茬稻田使用除草剂二氯喹啉酸,其土壤中的残留致使后茬烟草出现畸形生长现象,严重影响烟草产量和质量。生物降解是消除土壤中残留二氯喹啉酸的最主要途径。针对受二氯喹啉酸危害烟草的生长发育异常现象,本文从考察二氯喹啉酸危害烟草的主要生理机制着手,探讨了微生物对二氯喹啉酸危害烟草的修复作用及其主要机制。首先进行降解微生物的分离,将采集自农药厂的废水处理污泥样品,在二氯喹啉酸选择压力下进行富集培养驯化,获得一株二氯喹啉酸高效降解菌HN36,同时对菌株HN36从生理生化、蛋白质学角度进行比较系统的研究,探讨其降解特性、主要降解酶和中间代谢产物,并推测其对二氯喹啉酸的代谢途径。最后,通过盆栽试验研究了菌株HN36对二氯喹啉酸危害烟草的修复效应。本研究获得的主要结果如下:1.以盆栽烟草为试验材料,研究了不同浓度二氯喹啉酸对其农艺性状、生理特性及组织超微结构的影响。二氯喹啉酸对烟株的叶宽、叶长和株高均有显著抑制作用,对叶宽的抑制作用最强。二氯喹啉酸浓度为3.34×10-2mg/kg时,第40d的抑制率为叶宽(57.10%)>株高(53.52%)>叶长(22.25%)。二氯喹啉酸危害烟草的阈值为1.33×10-2 mg/kg干土。二氯喹啉酸处理后,烟草的保护酶SOD、POD及CAT活性与对照相比均下降,其中SOD和CAT响应土壤中二氯喹啉酸的灵敏度大于POD。·OH-、O2·-及MDA含量均随着处理时间的延长和二氯喹啉酸浓度的增大,含量升高。内源激素IAA、GA3及ZR的含量较对照显著降低,而ABA的含量则高于对照。透射电镜观察结果显示,受害烟株茎尖细胞的细胞壁、脂膜、细胞质、液泡等细胞器发育异常;叶片中叶绿体、基粒、外膜、片层结构等破坏严重,部分细胞发生病变。二氯喹啉酸对烟株的保护酶、氧自由基、内源激素、细胞超微结构等方面均产生危害,从而影响烟株的正常生长发育。2.采用富集驯化的方法,从生产二氯喹啉酸的农药厂废水处理污泥中,分离到两株二氯喹啉酸的高效降解菌株HN36和HN1,HN36菌株能以二氯喹啉酸为唯一碳源和能源生长,24 h对500 mg/L二氯喹啉酸的降解率达96.93%。菌株HN36的16S rDNA序列与Genbank数据库中荧光假单胞菌(P.aeruginosa)的同源性为100%。结合形态学和生理生化特征,将菌株HN36鉴定为假单胞菌Pseudomonassp.,HN1初步鉴定为土壤杆菌属(Agrobacterium sp.)。菌株HN36还能够利用邻苯二酚、喹啉、苯甲酸、苯酚和对苯二酚,但不能利用萘、菲和苯。菌株HN36对二氯喹啉酸降解的最适温度为35℃,最适pH为7。3.通过SDS-PAGE和双向电泳技术,研究了二氯喹啉酸胁迫下菌株HN36的蛋白表达差异,检测到两个可能与二氯喹啉酸降解相关的酶:邻苯二酚1,2-双加氧酶和邻苯二甲酸双加氧酶,并从菌株HN36中扩增到相关基因。通过邻苯二酚1,2-双加氧酶在含二氯喹啉酸液体培养基中,不同培养时间的酶活跟踪,发现该酶活变化与菌株的生长情况一致,证明菌株降解二氯喹啉酸的能力与该酶密切相关。利用RT-PCR技术进一步检测了邻苯二酚1,2-双加氧酶。将在含有以及不含二氯喹啉酸的培养液中生长的HN36菌株的RNA,通过反转录为cDNA,进行扩增,从RNA表达水平上,进一步证明了 HN36菌株的邻苯二酚1,2-双加氧酶是由二氯喹啉酸诱导产生的,并在降解二氯喹啉酸的过程中发挥着十分重要的作用。采用GC-MS技术共检测到三种二氯喹啉酸降解的中间代谢产物:4,7-二氯喹啉、2-氯对苯二甲酸以及2-氨基-4氯苯甲酸。结合分析鉴定的主要降解酶,初步推测了二氯喹啉酸的生物代谢途径:菌株HN36先通过脱羧作用把3,7-二氯-8-喹啉羧酸转化为3,7-二氯喹啉,再通过环的氧化裂解和基团转移作用,转化为2-氨基-4-氯苯甲酸,通过转氨基作用形成2-氯对苯二甲酸,在邻苯二甲酸双加氧酶的作用下形成氯邻苯二酚,再由邻苯二酚1,2-双加氧酶催化开环并脱氯,最后进入三羧酸循环。由于3,7-二氯喹啉极不稳定,试验中仅能检测到其同分异构体4,7-二氯喹啉。4.HN36对二氯喹啉酸危害烟株的盆栽修复试验结果表明:菌株HN36在60 d内能将土壤中0.085 mg/kg(干土)的二氯喹啉酸完全降解。一定范围内,二氯喹啉酸的降解率随降解菌接种量的增加而提高,最适接种量为5×109CFU/kg干土。二氯喹啉酸对土壤中过氧化氢酶、过氧化物酶、脲酶、蛋白酶、脱氢酶和蔗糖酶的活性有抑制作用,施加菌株HN36后,土壤酶活性升高,接种量为5×109 CFU/kg干土时,土壤酶活性最高。降解菌对二氯喹啉酸危害烟株的生理特性具有明显修复作用。降解菌能够提高二氯喹啉酸危害烟株的保护酶活性,其活性随接种量增加而增加,从而有效降低活性氧(O2·-和·OH和过氧化氢)的含量;接种量越大,根系的MDA含量越低,膜脂的过氧化作用就越弱,根系抗氧化能力越强,烟株的氨基酸含量越低,GSH含量越高,烟株修复效果越好。不同浓度二氯喹啉酸的影响规律基本相同,浓度越高根系保护酶活性低,活性氧含量高,MDA含量也越高,因此膜脂过氧化作用加剧。降解菌HN36通过加速二氯喹啉酸的降解,缓解其对叶宽、叶长和株高的抑制作用,接种量越大,烟株农艺性状的改善越显著,修复处理T3(接种量为5×1010CFU/kg干土)的农艺性状与健康烟株相比差异不显著,说明已得到有效修复。施加降解菌HN36后,受害严重的顶叶组织结构得到不同程度的修复,茎尖和顶叶细胞的超微结构的受害程度显著减轻。在烟叶主要化学成分上,修复烟叶的总糖、还原糖、淀粉和钾离子含量等均高于受害烟叶,总氮和烟碱含量均低于健康烟叶,还原糖/总糖、总氮/烟碱、总糖/烟碱的比例趋于合理,内在化学成分更协调。更多还原

【Abstract】 Quinclorac(3,7-dichloroquinoline-8-carboxylic acid)is a highly selective auxin herbicide.It is commonly used in rice production to control barnyard grass.Nevertheless,the widespread use of quinclorac(QNC)has led to large amount of residual in soil which do great harms to succeeding tobacco.The quality and yield of tobacco are greatly affected by the phytotoxicity of quinclorac.Microbial transformation and degradation are currently believed to be the most important strategy to remove the quinclorac in environment.This research aimed to screen bacteria capable of degradation quinclorac,study the degradation characteristics,identify the enzymes and metabolites involved in the biodegradation process,elucidate quinclorac biodegradation pathway and verify the bioremediation to damaged tobacco by quinclorac in soil.The main results were as follows:1.Effects of quinclorac to agronomic,physiological characteristics and ultrastructure of leaves of tobacco grown in pots were studied.leaf width,leaf length and plant height of were significantly inhibited by quinclorac applied in soil.The inhabitation rate of 3.343×10-2mg/kg quinclorac for leaf width in 40d was 57.10%,and 22.25%for leaf length.The results showed that threshold of quinclorac harm would be 1.33×10-2 mg/kg dried soil.The activity of SOD,POD and CAT of tobacco was decreased after quinclorac treatment.SOD and CAT were more sensitive to quinclorac than POD.Content of·OH-,O2·-and MDA in Leaves and roots increased with treatment time and increasing concentration of quinclorac.Compared with control,the level of endogenous hormones,IAA,GA3 and ZR decreased significantly,but the content of ABA was higher than the control group.Transmission electron microscope(TEM)observation showed that cell wall,lipid membrane,cytoplasm,vacuole and other organelles were abnormal in top stem cell of damaged tobacco.Chloroplasts in tobacco leaves were damaged seriously.Quinclorac does harm to protective enzyme,oxygen free radical,endogenous hormones and ultrastructure of tobacco.As a result,the growth of tobacco is inhibited.2.Strain HN36 and HN1 capable of removal quinclorac was obtained from a herbicide manufactory sewage sludge using gradient concentration of quinclorac enrichment strategy.Strain HN36 was considered to be Pseudomonas sp.,belonged to gram-negative bacteria,based on physiological and biochemical properties,morphology and a partial sequence of 16S rDNA.Strain HN1 was considered to be Agrobacterium sp.Strain HN36 could degrade 96.93%of quinclorac to undetectable level at the original concentration of 500 mg/L within 24 h.Strain HN36 was able to utilize a wide variety of aromatic compounds as carbon and energy source,including catechol,quinoline,benzoic acid,phenol and hydroquinone.Nevertheless naphthalene,phenanthrene and benzene could not be used to growth.The optimal temperature for quinclorac degradation was 35℃ and HN36 had the highest degradation rate at pH 7.3.The methods of SDS-PAGE and 2-DE were used to study the different proteins induced by quinclorac.Chlorocatechol 1,2-dioxygenas(1,2-CCD)and phthalate dioxygenase reductase(PDR)were identified and considered to be the key enzymes in the degradation of quinclorac.The activities change of 1,2-CCD and PDR in the culture process was accorded with the biodegradation of quinclorac and growth curve of HN36.It indicated that these enzymes were closely related to quinclorac degradation.1,2-CCD was verified by RT-PCR results.In addition,three metabolites,4,7-bichlorid quinolone,2-chlorine terephthalic acid and 2-amino-4 chlorobenzoic acid,were identified by GC-MS technique.The degradation pathway of quinclorac was proposed based on the obtained enzymes and degradation products.4.Strain HN36 was able to break down 0.085 mg/kg dried soil quinclorac in soil to undetectable level in 60d.To some extent,the degradation rate increased with increasing of inoculum size.The optimal inoculum amount was 5×109 CFU/kg dried soil.Quinclorac could inhibit the activity of catalase,peroxidase,urease,protease and dehydrogenase in soil.The activity of enzymes in soil increased with the application of strain HN36.The physiological characteristics of damaged tobacco by quinclorac were improved by HN36.Protective enzyme activities of tobacco increased and the content of,O2·-·OH and H2O2 decreased with HN36 added to soil.The level of MDA would be lower with a larger inoculum size.At the same time,Antioxidation activity of roots was stronger and the GSH content was higher.Thus growth of damaged tobacco was well improved.A similar results were obtained for bioremediation of different quinclorac concentration in soil.The inhibitation of leaf width,leaf length and plant height by quinclorac stress reduced when HN36 was applied to soil.Agronomic characteristics of repaired tobacco were similar with those of normal tobacco with a inoculum size of 5×1010CFU/kg dried soil.Cellar structure of damaged top leaves was repaired and ultrastructure of top stem and leaves was alleviated with application of HN36.Total sugar,reducing sugar,starch,potassium levels were as follows:phytotoxic tobacco>repaired tobacco>normal tobacco.The ratio of reducing sugar/total sugar,total sugar/nicotine,total nitrogen/nicotine of the normal tobacco and the repaired tobacco are more suitable than the phytotoxic one.更多还原