中国白肋烟种质资源评价与土壤中有机氯农药残留分析
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摘要
本文旨在建立白肋烟种质资源的化学及分子生物学的评价方法，从白肋烟农艺学性状、化学成分、遗传关系以及中国植烟烟区土壤中有机氯农药残留及武汉地区土壤中有机氯农药残留各个方面对中国白肋烟种质资源的品质进行系统研究，最终为拓宽白肋烟种质遗传基础，找到优质白肋烟种质资源提供理论支持与应用基础。第一部分中国白肋烟种质资源的农艺性状及品质性状分析
为充分了解白肋烟种质资源之间的关系，根据测定的白肋烟19个农艺性状数据应用聚类分析软件NTSYS2.10对来自国内外149个白肋烟种质资源进行系统的聚类分析。结果表明，在相异性系数0.0375处可将149份白肋烟种质资源分为6个大类群及2个小亚群：种质类群Ⅰ含有25个品种，种质类群Ⅱ内含有22个品种，种质类群Ⅲ内含有23个品种，种质类群Ⅳ内含有18个品种，其中种质类群Ⅳ又可以被划分成2个小的亚群，种质类群Ⅴ内含有24个品种，种质类群Ⅵ内含有37个品种。白肋烟种质资源之间的相异性系数在0.01-0.53之间，表明了白肋烟种质资源之间存在着一定的差异。
第二部分白肋烟种质资源的化学评价
本章实验采用双列杂交及动态两种白肋烟材料，首先通过紫外分光光度法对36份双列杂交白肋烟样品叶面分泌物进行初步筛选，筛选出较高叶面分泌量的品种或杂交组合依次为：B37×KY8959，KY14×KY8959，BX2003×KY14，建选3号×B37，B37，B37×B21。
此外，采用气-质联用技术分析了双列杂交及动态组白肋烟烟叶中主要香气物质成分，共检出31种烟叶香气物质成分，其中新植二烯含量最高。
通过对36份双列杂交白肋烟种质资源上等烟率、亩产量、烟碱、钾含量、腺毛分泌物总量、香味物质总量、感官评析得分进行的综合分析，筛选具有较大利用价值的白肋烟品种或有性杂交组合：B37×BX2003（鄂烟101）、B21×KY14、KY8959×BX2003（鄂烟101）、KY8959×B21、B37×KY8959、建选3号×B37、建选3号×KY14、B21×BX2003（鄂烟101）、B37×B21。
分析白肋烟香气物质的动态变化，测定6个样品在移栽后56天，63天，70天，77天，84天时香气物质的含量，做出动态变化图，确定不同品种白肋烟的最佳采收期。
第三部分中国白肋烟种质资源的分子生物学评价
通过ISSR分子标记法对119份白肋烟种质资源的遗传多样性进行了研究。结果表明，从100个ISSR引物筛选出14条，总共扩增出了131条带，其中127个条带具有多态性，每个ISSR引物平均扩增出了9.1个条带。根据ISSR条带的数据，白肋烟品种之间的相似性系数在0.40-0.99之间。119份白肋烟样品的UPGMA系统树图中可以看出，白肋烟样品可以被划分成3个大组。其结果与利用农艺性状进行聚类研究的结果有一定的差异。联系白肋烟种质的遗传背景和农艺学/品质性状，本研究有助于利用分子标记的方法制定适当的策略，来协助白肋烟的育种工程。
第四部分土壤中有机氯农药残留状况分析
本章节以我国6大产烟区的植烟土壤及武汉地区土壤为研究对象，对土壤中有机氯农药残留状况进行了分析，主要研究结果如下：
1.建立了混合溶剂索氏提取法萃取，弗罗里硅土小柱净化，利用带微池电子捕获检测器的气相色谱仪同时分离检测植烟土壤中21种有机氯农药残留量的方法，该方法简便快速，准确灵敏。
2.全国主要烟区有机氯农药残留在6大烟区植烟土壤中普遍存在，其中滴滴涕和六氯苯的检出率最高，为90%。六六六和滴滴涕占有机氯农药残留的主要部分。其总的平均值ΣDDTs和ΣHCHs分别为6.69μg/kg、5.91μg/kg，与我国规定的滴滴涕和六六六的指标限值100μg/kg相比要小很多，可以保障正常的农业生产和人体健康。
3.武汉地区土壤样品中滴滴涕和六六六是土壤中有机氯农药的主要化合物，其中滴滴涕在总农药中比重为77.10%，其次是六六六（7.83%）、艾氏剂（4.21%），七氯（2.82%）和六氯苯（1.53%）。滴滴涕的浓度范围是nd至1198.0ng.g-1,滴滴涕的主要污染分布在武汉市汉南区和新洲区。六六六的浓度范围是n.d（未检出）至100.58ng.g1，黄陂区和汉南区土壤中检测到的六六六的含量较高。除七氯外，其他有机氯农药近期均未在土壤中使用。在武汉大部分农业土壤中滴滴涕的含量小于500ng.g-1，六六六也可以被归为无污染。
To explore the good germplasm of burley tobacco (Nicotiana tabacum L.) andestablish an effective evaluation method of the chemical and biologiacal, we analyzed withthe agronomy traits, chemical composition, genetic relationships, andorganochlorine pesticides residues in soil of tobacco ares in China and Wuhan, Hubeiprovince, China. It will provide theoretical support and application infrastructure to broadenthe genetic base and select quality germplasm of burley tobacco.
Part One Analysis of the agronomic/quality traits of the germplasmresources of burley tobacco
In order to access the similarity among burley tobacco germplasm,149burley tobaccocultivars from China and abroad were systematicall clustered by cluster software NTSYS2.10e based on data of19traits. Results showed that all burley tobacco cultivars can beclustered into6large cultivar groups and2samll cultivar subgroups at dissimilitudecoefficient of0.0375. Group1to group6included25,22,23,18,24and37cultivars,respectively. Total coefficient of all burley tobacco cultivars ranged from0.01to0.53,indicating that there existed some diversity among various germplasm.
Part Two Phytochemical evaluaion of the germplasm of burley tobacco
This chapter we use two kinds of burley tobacco leaves: diallel cross and dynamicmaterial, UV spectrophotometry was used for the secretions initial screening in36diallelburley leaf samples, screening a higher secretion of species or foliar crosses were: B37×KY8959, KY14×KY8959, BX2003×KY14, Jianxuan3×B37, B37, B37×B21.
In addition, gas chromatography-mass spectrometry technique was used to analysis thearoma components of the leaves of diallel and dynamic group.31kinds of tobacco aromacomponents were detected in the tobacco leaves, which neophytadiene was the highestcontent.
Through comprenshensive analysis of36diallel burley varieties resources, screening for potential usage value of burley varieties or hybrid varieties：B37×BX2003、B21×KY14、 KY8959×BX2003、KY8959×B21、B37×KY8959、Jianxuan3×B37、Jianxuan3号×KY14、B21×BX2003、B37×B21。
Analyses on the dynamic changes of aroma constituents in growth process of burleytobacco by GC/MS. six samples were measured56days after transplanting,63days,70days,77days,84days aroma substance content and make dynamic maps, determine thedifferent varieties of burley best harvest.
Part Three Molecular biological evaluation of the germplasm of burleytobacco
To assess the genetic diversity of germplasm resources of burley tobacco (Nicotianatabacum L.), we investigated genetic relationships among119burley tobacco accessions byusing inter simple sequences repeat (ISSR) markers. A total of14ISSR primers generated131bands, of which,127bands were polymorphic with an average of9.1bands per primerpair. Based on the ISSR band data, similarity indices between the accessions ranged from0.40to0.99. An unweighted pair group method using arithmetic averages algorithm(UPGMA) dendrogram was constructed and demonstrated that all119accessions could beassorted into3main groups. This result has certain differences with the clustering result,which was based on agronomic/quality traits. Accompanied with the genetic backgroundand agronomic/quality traits of germplasm resources of burley tobacco, these results willhelp in the formulation of appropriate strategies for assisting burley tobacco breedingprogramme by maker assisted selection.
Part Four Residues of organochlorin pestricides in agricultural soils
Soil samples collected from soils at six main tobacco growing regions in China werestudied, and the levels and distributions of OCPs were analyzed. The results were asfollowing.
1. The method for the determination of21organochlorine pesticides (OCPs) by gaschromatography with μ-electron capture detection was constructed, soil samples wereextracted by Soxhlet method by a mixed solvent, and purified by a florisil chromatographiccolumn.
2. Organochlorine pesticides residues in planting soils were widespread. DDTs and chlorobenzene had the highest detection rate of90%. The main residues of organochlorinepesticides in the soil were HCHs and DDTs, and the average content of ΣDDTs and ΣHCHswas6.69μg/kg,5.91μg/kg respectively, which were lower than the1imitation of nationalstandard (HJ332-2006). Risk assessment indicated that the soil could guarantee the normalagricultural production and human health.
3. According to the measured concentrations and detection frequencies in agriculturalsoils of Wuhan, dichlorodiphenyltrichloroethanes(DDTs), hexachlorocyclohexanes(HCHs),hepta chlor (HEPT), hexachlorobenzene(HCB) and aldrin were the predominantcompounds in soil. DDTs accounted for77.10%of total OCPs, followed by HCHs(7.83%),aldrin(4.21%), HEPTs(2.82%) and HCB (1.53%). The total DDT concentration srangedfrom nd to1198.0ng.g-1and the main contaminated areas were distributed in Hannan andXinzhou districts of Wuhan. The total HCH concentration sranged from nd to100.58ng.g1in soil and relatively higher levels were observed in soil samples from Huangpi and Hannandistricts. Source analysis showed that OCPs residues except heptachlor originated mainlyfrom historical application, besides slight recent introduction at some sites. Based on theChina National Soil Quality Standard, DDT pollution in most samples of Wuhanagricultural soils could be considered as no and low contamination, while the level ofHCHs was classified as no pollution. Our study indicated that there existed potentialexposure risk of OCPs in Wuhan agricultural soils although the use of OCPs has beenbanned.
为充分了解白肋烟种质资源之间的关系，根据测定的白肋烟19个农艺性状数据应用聚类分析软件NTSYS2.10对来自国内外149个白肋烟种质资源进行系统的聚类分析。结果表明，在相异性系数0.0375处可将149份白肋烟种质资源分为6个大类群及2个小亚群：种质类群Ⅰ含有25个品种，种质类群Ⅱ内含有22个品种，种质类群Ⅲ内含有23个品种，种质类群Ⅳ内含有18个品种，其中种质类群Ⅳ又可以被划分成2个小的亚群，种质类群Ⅴ内含有24个品种，种质类群Ⅵ内含有37个品种。白肋烟种质资源之间的相异性系数在0.01-0.53之间，表明了白肋烟种质资源之间存在着一定的差异。
第二部分白肋烟种质资源的化学评价
本章实验采用双列杂交及动态两种白肋烟材料，首先通过紫外分光光度法对36份双列杂交白肋烟样品叶面分泌物进行初步筛选，筛选出较高叶面分泌量的品种或杂交组合依次为：B37×KY8959，KY14×KY8959，BX2003×KY14，建选3号×B37，B37，B37×B21。
此外，采用气-质联用技术分析了双列杂交及动态组白肋烟烟叶中主要香气物质成分，共检出31种烟叶香气物质成分，其中新植二烯含量最高。
通过对36份双列杂交白肋烟种质资源上等烟率、亩产量、烟碱、钾含量、腺毛分泌物总量、香味物质总量、感官评析得分进行的综合分析，筛选具有较大利用价值的白肋烟品种或有性杂交组合：B37×BX2003（鄂烟101）、B21×KY14、KY8959×BX2003（鄂烟101）、KY8959×B21、B37×KY8959、建选3号×B37、建选3号×KY14、B21×BX2003（鄂烟101）、B37×B21。
分析白肋烟香气物质的动态变化，测定6个样品在移栽后56天，63天，70天，77天，84天时香气物质的含量，做出动态变化图，确定不同品种白肋烟的最佳采收期。
第三部分中国白肋烟种质资源的分子生物学评价
通过ISSR分子标记法对119份白肋烟种质资源的遗传多样性进行了研究。结果表明，从100个ISSR引物筛选出14条，总共扩增出了131条带，其中127个条带具有多态性，每个ISSR引物平均扩增出了9.1个条带。根据ISSR条带的数据，白肋烟品种之间的相似性系数在0.40-0.99之间。119份白肋烟样品的UPGMA系统树图中可以看出，白肋烟样品可以被划分成3个大组。其结果与利用农艺性状进行聚类研究的结果有一定的差异。联系白肋烟种质的遗传背景和农艺学/品质性状，本研究有助于利用分子标记的方法制定适当的策略，来协助白肋烟的育种工程。
第四部分土壤中有机氯农药残留状况分析
本章节以我国6大产烟区的植烟土壤及武汉地区土壤为研究对象，对土壤中有机氯农药残留状况进行了分析，主要研究结果如下：
1.建立了混合溶剂索氏提取法萃取，弗罗里硅土小柱净化，利用带微池电子捕获检测器的气相色谱仪同时分离检测植烟土壤中21种有机氯农药残留量的方法，该方法简便快速，准确灵敏。
2.全国主要烟区有机氯农药残留在6大烟区植烟土壤中普遍存在，其中滴滴涕和六氯苯的检出率最高，为90%。六六六和滴滴涕占有机氯农药残留的主要部分。其总的平均值ΣDDTs和ΣHCHs分别为6.69μg/kg、5.91μg/kg，与我国规定的滴滴涕和六六六的指标限值100μg/kg相比要小很多，可以保障正常的农业生产和人体健康。
3.武汉地区土壤样品中滴滴涕和六六六是土壤中有机氯农药的主要化合物，其中滴滴涕在总农药中比重为77.10%，其次是六六六（7.83%）、艾氏剂（4.21%），七氯（2.82%）和六氯苯（1.53%）。滴滴涕的浓度范围是nd至1198.0ng.g-1,滴滴涕的主要污染分布在武汉市汉南区和新洲区。六六六的浓度范围是n.d（未检出）至100.58ng.g1，黄陂区和汉南区土壤中检测到的六六六的含量较高。除七氯外，其他有机氯农药近期均未在土壤中使用。在武汉大部分农业土壤中滴滴涕的含量小于500ng.g-1，六六六也可以被归为无污染。
To explore the good germplasm of burley tobacco (Nicotiana tabacum L.) andestablish an effective evaluation method of the chemical and biologiacal, we analyzed withthe agronomy traits, chemical composition, genetic relationships, andorganochlorine pesticides residues in soil of tobacco ares in China and Wuhan, Hubeiprovince, China. It will provide theoretical support and application infrastructure to broadenthe genetic base and select quality germplasm of burley tobacco.
Part One Analysis of the agronomic/quality traits of the germplasmresources of burley tobacco
In order to access the similarity among burley tobacco germplasm,149burley tobaccocultivars from China and abroad were systematicall clustered by cluster software NTSYS2.10e based on data of19traits. Results showed that all burley tobacco cultivars can beclustered into6large cultivar groups and2samll cultivar subgroups at dissimilitudecoefficient of0.0375. Group1to group6included25,22,23,18,24and37cultivars,respectively. Total coefficient of all burley tobacco cultivars ranged from0.01to0.53,indicating that there existed some diversity among various germplasm.
Part Two Phytochemical evaluaion of the germplasm of burley tobacco
This chapter we use two kinds of burley tobacco leaves: diallel cross and dynamicmaterial, UV spectrophotometry was used for the secretions initial screening in36diallelburley leaf samples, screening a higher secretion of species or foliar crosses were: B37×KY8959, KY14×KY8959, BX2003×KY14, Jianxuan3×B37, B37, B37×B21.
In addition, gas chromatography-mass spectrometry technique was used to analysis thearoma components of the leaves of diallel and dynamic group.31kinds of tobacco aromacomponents were detected in the tobacco leaves, which neophytadiene was the highestcontent.
Through comprenshensive analysis of36diallel burley varieties resources, screening for potential usage value of burley varieties or hybrid varieties：B37×BX2003、B21×KY14、 KY8959×BX2003、KY8959×B21、B37×KY8959、Jianxuan3×B37、Jianxuan3号×KY14、B21×BX2003、B37×B21。
Analyses on the dynamic changes of aroma constituents in growth process of burleytobacco by GC/MS. six samples were measured56days after transplanting,63days,70days,77days,84days aroma substance content and make dynamic maps, determine thedifferent varieties of burley best harvest.
Part Three Molecular biological evaluation of the germplasm of burleytobacco
To assess the genetic diversity of germplasm resources of burley tobacco (Nicotianatabacum L.), we investigated genetic relationships among119burley tobacco accessions byusing inter simple sequences repeat (ISSR) markers. A total of14ISSR primers generated131bands, of which,127bands were polymorphic with an average of9.1bands per primerpair. Based on the ISSR band data, similarity indices between the accessions ranged from0.40to0.99. An unweighted pair group method using arithmetic averages algorithm(UPGMA) dendrogram was constructed and demonstrated that all119accessions could beassorted into3main groups. This result has certain differences with the clustering result,which was based on agronomic/quality traits. Accompanied with the genetic backgroundand agronomic/quality traits of germplasm resources of burley tobacco, these results willhelp in the formulation of appropriate strategies for assisting burley tobacco breedingprogramme by maker assisted selection.
Part Four Residues of organochlorin pestricides in agricultural soils
Soil samples collected from soils at six main tobacco growing regions in China werestudied, and the levels and distributions of OCPs were analyzed. The results were asfollowing.
1. The method for the determination of21organochlorine pesticides (OCPs) by gaschromatography with μ-electron capture detection was constructed, soil samples wereextracted by Soxhlet method by a mixed solvent, and purified by a florisil chromatographiccolumn.
2. Organochlorine pesticides residues in planting soils were widespread. DDTs and chlorobenzene had the highest detection rate of90%. The main residues of organochlorinepesticides in the soil were HCHs and DDTs, and the average content of ΣDDTs and ΣHCHswas6.69μg/kg,5.91μg/kg respectively, which were lower than the1imitation of nationalstandard (HJ332-2006). Risk assessment indicated that the soil could guarantee the normalagricultural production and human health.
3. According to the measured concentrations and detection frequencies in agriculturalsoils of Wuhan, dichlorodiphenyltrichloroethanes(DDTs), hexachlorocyclohexanes(HCHs),hepta chlor (HEPT), hexachlorobenzene(HCB) and aldrin were the predominantcompounds in soil. DDTs accounted for77.10%of total OCPs, followed by HCHs(7.83%),aldrin(4.21%), HEPTs(2.82%) and HCB (1.53%). The total DDT concentration srangedfrom nd to1198.0ng.g-1and the main contaminated areas were distributed in Hannan andXinzhou districts of Wuhan. The total HCH concentration sranged from nd to100.58ng.g1in soil and relatively higher levels were observed in soil samples from Huangpi and Hannandistricts. Source analysis showed that OCPs residues except heptachlor originated mainlyfrom historical application, besides slight recent introduction at some sites. Based on theChina National Soil Quality Standard, DDT pollution in most samples of Wuhanagricultural soils could be considered as no and low contamination, while the level ofHCHs was classified as no pollution. Our study indicated that there existed potentialexposure risk of OCPs in Wuhan agricultural soils although the use of OCPs has beenbanned.
引文
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