Metabolism Study on Tobacco-specific N-Nitrosamines in Rabbit by Solid-phase Extraction and Liquid Chromatography-electrospray Ionization-mass Spectrometry Method

Metabolism of four tobacco-specific N-nitrosamines （TSNAs）, N＇-nitrosonornicotine （NNN）, N＇-nitrosoanatabine （NAT）, N＇-nitrosoanabasine （NAB） and 4-（methyl nitrosamino）-1- （3-pyridyl）-1-butanone （NNK） was studied by solid-phase extraction （SPE） and liquid chromato- graphy-electrospray ionization-mass spectrometry （LC-ESI-MS）. SPE and LC-ESI-MS method was evaluated to be rapid, simple, sensitive and selective for analysis of TSNAs in rabbit serum. The doses of TSNAs administrated were 4.666 μg/kg and 11.665 μg/kg according to different levels in cigarettes. Metabolic curves of four TSNAs and 4-（methyl nitrosamino）-1-（3-pyridyl）- 1-butanone （NNAL）, the metabolite of NNK, were obtained.

Construction of Marker-Free GFP Transgenic Tobacco by Cre/lox Site-Specific Recombination System

Marker-free GFP transgenic tobacco plants were constructed based on Cre/lox site-specific recombination system. A GFP gene was introduced into the tobacco genome using the Bar gene as a linked selectable marker flanked by recombination sites in a directed orientation. The Bar gene expression box was subsequently excised from the plant genome by a strategy of Cre gene retransformation. After removal of the Cre-NPT Ⅱ locus by genetic segregation through self-cross, plants that incorporated only the GFP transgene were obtained. Transgenic tobacco plants mediated by Agrobacterium tumefaciens were obtained, which resisted herbicide Basta and GFP expressed well, then the Cre gene was subsequently introduced into 5 plants of them, respectively, by retransformation. The leaf disks from Cre transgenic plants were used to test the resistance to Basta on the medium with 8 mg L-1 of PPT. The results showed that few discs were able to regenerate normally, and the excision at 76-100% efficiency depended on individual retransformation events. Evidence for a precise recombination event was confirmed by cloning the nucleotides sequence surrounding the lox sites of the Basta sensitive plants. The result indicated that the excision event in the recombination sites was precise and conservative, without loss or alteration of any submarginal nucleotides of the recombination sites. Bar gene excised plants were selfpollinated to allow segregation of the GFP gene from the Cre-NPT Ⅱ locus. The progenies from self-pollinated plants were scored for Kan senstivity, then the segregation of GFP gene from Cre-NPT Ⅱ locus in the Kan senstive plants were confirmed by PCR analysis subsequently. Hence, constructing marker-free transgenic tobacco plants by Cre/lox sitespecific recombination system was reliable, and the strategy presented here should be applicable to other plants for the construction of marker-free transgenic plants as well.

THE N-NITROSAMINES IN TOBACCO SMOKE AND ITS ADSORPTION AND DEGRADATION

N-nitrosamines are strong carcinogens for humans. This paper gives an overview of the nitrosmaines in cigarette smoke including the formation, the harmfulness, the analytical methods of the nitrosmaines and the adsorptions and degradations of N –nitrosamines.

Effects of Exogenous Amylases and Metal Ions on the Amylase Specific Activities and Starch Degradation of the Upper Leaves of ‘KRK_(26)' during Flue-curing

Objective] The aim of this study was to investigate the effects of exoge-nous amylases and Ca2＋, Mn2＋ and K＋ on the amylase specific activities and starch degradation of the upper leaves of ＇KRK26＇ planted in Yunnan Province during flue-curing. [Method] The amylase specific activities and starch degradation of the leaves were determined by using spectrophotometry. [Result] The 8 U/g exogenous α-amy-lase could improve the specific activity of the leaf α-amylase at yel owing and color-fixing stages, but could not at stem-drying stage, and similarly, the 80 U/g exoge-nous β-amylase could improved the specific activity of the leaf β-amylase at the yel owing stage and the early period of color-fixing stage. The leaf starch could be enhanced to degrade by the exogenous α- or β-amylases and the enhancing effect of the former was stronger than that of the later. 1.50 mg/ml Ca2＋ improved the specific activity of the leaf （α＋β）-amylase mainly due to its enhancing effect on the leaf α-amylase, and increased the starch degradation. 4 mmol/L Mn2＋ inhibited the leaf α-amylase from yel owing to the early period of color-fixing and the β- and （α＋β）-amylases from the yel owing to the later period of color-fixing, but enhanced the leafα-amylase from the later period of color-fixing to the later period of stem-drying and the β- and （α＋β）-amylases at the later period of stem-drying. Meanwhile, Mn2＋ ham-pered the starch degradation during yel owing, but promoted it from the early period of color-fixing to stem-drying. 1 mg/ml K＋ enhanced the leaf α-, β- and （α＋β）-amy-lases during the yel owing stage, but lowered them from the early period of color-fix-ing to the later period of stem-drying, and always inhibited the leaf starch degrada-tion. [Conclusion] The exogenous α-, β- amylases and Ca2＋ of suitable concentra-tions could be used to treat the tobacco leaves before flue-curing to improve the leaf starch degradation during the curing.

育苗盘规格对烟苗生长发育及烟叶产质量的影响

为筛选出既满足宜宾烟区井窖式小苗移栽又减少基质用量的育苗盘规格,采用湿润育苗的方式,研究了不同育苗盘规格对烟苗生长发育及烟叶产质量的影响。结果表明,128孔和162孔育苗盘所育烟苗生长较快,烟苗农艺性状、物质积累和合格烟苗率较高,大田烟株农艺性状、经济性状较高,但基质用量和所需育苗面积较大,育苗投入成本明显增加;200孔育苗盘所育烟苗素质、大田烟株农艺性状、经济性状等方面与128孔育苗盘相当,但其基质用量较128孔育苗盘降低39.88%,育苗盘面积较128孔育苗盘减少30.77%,育苗投入成本明显降低;288孔育苗盘虽然基质用量和所需育苗面积最低,但其所育烟苗素质,大田烟株农艺性状和经济性状显著低于其他处理。综合而言,200孔育苗盘是宜宾烟区比较理想的选择。

长效有机肥与烟草专用肥配施对烟草产量及品质的影响

长效有机肥可以在一定程度上解决化肥造成的环境压力,提高农作物的产量和品质。本研究探讨了长效有机肥与烟草专用肥配施对烟草产量和品质的影响。结果表明:长效有机肥与烟草专用肥配合施用,可以提高烟叶均价、上等烟比例和上中等烟比例,促进烟叶总植物碱、氧化钾等含量的增加,降低还原糖含量,烟叶化学成分更协调,但同时表现出从移栽期至团棵期的生长发育较缓慢,大田生育期增加。综合来看,以长效有机肥与烟草专用肥氮素配比为25%∶75%、总施氮量减少20%的模式最佳。

不同规格卷烟配方叶丝柔软性与物理质量的关联性研究

【目的】探究不同规格卷烟配方叶丝柔软性与物理质量的关系。【方法】对常规、中支、细支卷烟配方叶丝柔软性与物理质量进行方差分析、相关性分析、灰色关联度分析,建立不同规格卷烟配方叶丝柔软性XGBoost预测模型。【结果】(1)同等规格卷烟,随着价位的提升,叶丝柔软性值越小,叶丝柔软程度越好;同等价位,不同规格卷烟配方叶丝柔软程度排序为:细支卷烟>中支卷烟>常规卷烟;不同规格卷烟配方叶丝柔软性差异显著。(2)不同规格卷烟配方叶丝柔软性值均与长丝率、中丝率、整丝率、弹性呈极显著负相关关系,与填充值、含水率、碎丝率呈极显著正相关关系。(3)不同规格卷烟配方叶丝物理质量与其柔软性的灰色关联度大小依次为:第一类(中丝率、含水率)>第二类(填充值和碎丝率)>第三类(长丝率、整丝率、弹性)。(4)针对常规、中支、细支卷烟配方叶丝分别建立的XGBoost模型预测精度达到96.2%、92.4%、95.4%,XGBoost预测模型结果稳定可靠。【结论】可实现对叶丝柔软性的动态调控,改善叶丝质量,同时可为提高烟丝适用性提供技术支撑。

基于CP基因的云南烟草花叶病毒RT-LAMP快速检测体系的构建

为快速检测云南烟草花叶病毒(Tobacco mosaic virus,TMV),本研究根据来自云南烟草TMV分离物的外壳蛋白(CP)基因保守核苷酸序列,设计了5组引物进行筛选,并采用单一变量法对反应的温度、时间、甜菜碱浓度、dNTPs浓度、Mg2+浓度及内、外引物浓度比等进行逐一优化,建立了云南烟草TMV逆转录环介导等温扩增(RT-LAMP)检测体系。结果表明,最佳引物组为第4组,最适反应温度为60℃,甜菜碱、dNTPs、Mg^(2+)的最佳反应浓度分别为0.6、0.4、2.0 mmol/L,最佳内、外引物浓度比为4∶1,最佳反应时间40 min。优化后的RT-LAMP经SYBR Green I染色可肉眼判断结果,特异性高,灵敏度是常规RT-PCR的10倍。RT-LAMP检测体系的建立为云南烟草TMV的检测提供了一种便捷、高效、可靠的方法。

龙岩烟区烤烟磷钾养分配比优化研究

为探明龙岩烟区有效磷含量较丰富植烟土壤上适宜的磷钾施用比例,采用田间试验,在氮肥用量8.5 kg/666.7 m^(2)条件下,设计了磷肥3水平(P9、P7、P5,P_(2)O_(5)施用量分别为7.65、5.95、4.25 kg/666.7 m^(2))和钾肥3水平(K21、K18、K15,K2O施用量分别为17.85、15.30、12.75 kg/666.7 m^(2))的不同配比处理,以目前生产中配比P9K21为对照,研究了不同磷钾配比对烤烟生长发育和产质量的影响。结果表明,P7K18、P7K21处理综合农艺性状较好,株高显著高于P9K18、P7K15、P5K15处理,P7K21处理有效叶数显著高于P9K18、P5K18、P5K15。各处理间均价和中上等烟比例无明显差异,但P7K18、P7K21的产量、产值均最高,P9水平各处理烤烟产值显著低于施磷量为P5、P7的各处理,P5水平各处理上等烟比例均较高。各处理下部叶总氮含量及氮碱比偏高,上、中、下部烟叶总糖、总钾含量基本适宜。P7K18处理的上部烟叶烟碱、总糖、还原糖含量均在适宜范围,糖碱比、氮碱比等化学成分较协调。综上,烤烟专用肥N、P_(2)O_(5)、K2O比例由10∶9∶21调整为10∶7∶18,P_(2)O_(5)施用量由7.65 kg/666.7 m^(2)调整为5.95 kg/666.7 m^(2),K2O施用量由17.85 kg/666.7 m^(2)调整为15.30 kg/666.7 m^(2),烟株生长较好,化学成分较协调,经济性状最优。

烟草水热炭低温活化制备高性能电极材料的研究

水热炭是制备先进碳材料的重要前躯体,但是其不发达的孔隙结构极大地限制了水热炭的高值化利用。文章以烟草水热炭为原料,通过氨基钠低温活化合成多孔炭,并研究其作为超级电容器电极材料的电化学性能。研究结果表明:当氨基钠与水热炭的质量比为1时,合成的多孔炭具有分级孔隙结构,氮含量为3.51%,比表面积可达987.4 m2/g;以水热炭衍生多孔炭制备的碳电极材料在三电极体系下表现出了351 F/g的高比电容(电流密度为1 A/g),在二电极体系下实现了10.6 W·h/kg的高能量密度(功率密度为138.6 W/kg),循环10000次后仍能保持100%的库伦效率和97%的比电容保持率。

烟草种质基因分型核心SNP标记的开发

基于KASP(kompetitive allele specific PCR)技术平台,开发并验证可用于烟草核心种质基因分型的SNP(single nucleotide polymorphism)标记和对应检测引物,为烟草种质基因型鉴定评价、遗传多样性分析、核心种质筛选等提供技术和数据支持。利用Python和Perl脚本程序对覆盖烟草全基因组的1 179 154个SNP位点进行KASP引物设计和筛选,并通过试验验证其准确度和可用性。结果共有217 621个SNP位点完成了对应KASP引物设计,选择1 378个SNP位点进行试验验证,明确了732个可作为SNP标记,并确定48个SNP标记作为烟草种质资源基因分型的核心标记。这48个核心标记在烟草24条染色体上平均分布,平均PIC为0.36,平均MAF为0.39。利用确定的48个核心SNP标记,可以将各供试种质特别是将当前主栽烟草品种基因型进行区分,且标记具有极高的可靠性。

井窖式移栽打孔规格及填土封口时间对烤烟生长发育和产质量的影响

为筛选出与烤烟井窖式移栽技术配套的打孔规格及填土封口时间,在利川开展井窖式移栽打孔规格及填土封口时间对烤烟生长发育和产质量影响的研究。结果表明,井窖式移栽技术以直径10 cm打孔器打孔,栽后20 d填土半封井窖的处理烤烟农艺性状、经济性状表现较好,烤烟内在化学成分表现较协调。该研究对烤烟井窖式移栽技术的大面积推广应用及烟叶产质量提高提供技术支持。

烟草黑胫病不同发病程度与根际微生物间的响应关系

为探究烟草黑胫病不同发病程度与土壤生态环境影响因子之间的关系,明确与黑胫病发病程度相关的因素,为今后黑胫病的生物防疫工作提供理论基础。本研究通过田间调查,收集烟草黑胫病不同发病程度的根际土壤(病级:0、5、7、9级),测定了土壤理化指标和酶活性,并采用16S DNA和ITS基因测序技术分析了烟株发病与未发病根际土壤细菌及真菌群落结构的差异。结果表明:(1)黑胫病发病土壤的碱解氮(AN)、硝态氮(NO_(3)^(-)-N)、铵态氮(NH_(4)^(+)-N)、有效磷(AP)、速效钾(AK)、蔗糖酶(SUC)、过氧化氢酶(CAT)及酸性磷酸酶(ACP)与正常土壤相比最高分别增加了197.53 mg/kg、82.67 mg/kg、33.17 mg/kg、65.7 mg/kg、358.07 mg/kg、8.84 mg/(d·g)、56.80µmol/(h·g)、3938.69 nmol/(h·g),且各处理间达到显著差异(P<0.05);(2)与未发病土壤相比,发病土壤细菌、真菌群落结构多样性较低,且随着发病程度增加,多样性逐步降低;(3)发病土壤厚壁菌门、镰刀菌属、棘壳孢属相对丰度高于正常土壤,而变形杆菌门、放线菌门、酸杆菌门、绿弯菌门、浮霉菌门、被孢霉属、孢球托霉属和沙蜥属群落丰度则低于正常土壤;(4)指示物种分析表明,与正常土壤相比,发病土壤中放线菌门、酸杆菌门、厚壁菌门、孢球托霉属和棘壳孢属存在显著正响应,为病害发生的指示物种;(5)冗余分析(RDA)表明,放线菌门、酸杆菌门、孢球托霉属与病害发生呈负相关,而厚壁菌门、棘壳孢属则相反。综上所述,根际土壤中放线菌门、酸杆菌门、孢球托霉属相对丰度增加和厚壁菌门、棘壳孢属相对丰度降低是烟草黑胫病发病的重要微生物因素。

优化烟草专用基肥对烤烟生长发育及产质量的影响

近年来,化肥不合理施用及连作导致烟田土壤供肥能力下降,烟叶品质不同程度降低。为进一步提高肥料利用率,改善植烟土壤理化性状,提高烟叶产质量,对现有烟草专用基肥进行优化改进,研究其对烤烟品种云烟87生长发育及产质量的影响。结果表明:与现有烟草专用基肥相比,不同优化基肥配方处理烤烟有效叶片数、茎围、产量、产值、均价均有所提高,其中以优化基肥配方2号对改善烟株农艺性状、提高烤烟经济效益效果最好,可在烟叶生产过程中推广应用。

Intake of tobacco nitrosamines of smokers in various provinces of China and their cancer risk:A meta-analysis

Nitrosamines are a class of carcinogens which have been detected widely in food,water,some pharmaceuticals as well as tobacco.The objectives of this paper include reviewing the basic information on tobacco consumption and nitrosamine contents,and assessing the health risks of tobacco nitrosamines exposure to Chinese smokers.We searched the publications in English from“Web of Science”and those in Chinese from the“China National Knowledge Infrastructure”in 2022 and collected 151 literatures with valid information.The content of main nitrosamines in tobacco,including 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone(NNK),N-nitrosonornicotine(NNN),N-nitrosoanatabine(NAT),N-nitrosoanabasine(NAB),total tobacco-specific nitrosamines(TSNA),and N-nitrosodimethylamine(NDMA)were summarized.The information of daily tobacco consumption of smokers in 30 provinces of China was also collected.Then,the intakes of NNN,NNK,NAT,NAB,TSNAs,and NDMA via tobacco smoke were estimated as 1534ng/day,591 ng/day,685 ng/day,81 ng/day,2543 ng/day,and 484 ng/day by adult smokers in30 provinces,respectively.The cancer risk(CR)values for NNN and NNK inhalation intake were further calculated as 1.44×10^(-5)and 1.95×10^(-4).The CR value for NDMA intake via tobacco smoke(inhalation:1.66×10^(-4))indicates that NDMA is similarly dangerous in tobacco smoke when compared with the TSNAs.In China,the CR values caused by average nitrosamines intake via various exposures and their order can be estimated as the following:smoke(3.75×10^(-4))>food(1.74×10^(-4))>drinking water(1.38×10^(-5)).Smokers in China averagely suffer 200%of extra cancer risk caused by nitrosamines in tobacco when compared with non-smokers.

Effects of specific expression of iaaL gene in tobacco tapetum on pollen embryogenesis

The indoleacetic-acid-lysine synthetase (iaaL) gene from Pseudomonas syringae subsp. savastanoi was fused to tobacco tapetum-specific expression promoter TA29, and introduced into tobacco. The expression pattern of this chimeric gene was studied, and the endogenous indoleacetic acid (IAA) levels in different organs were assayed. The results demonstrated that TA29 promoter was only able to direct the specific expression of iaaL gene in transgenic tobacco anther, and resulted in the decrease of endogenous IAA levels in transgenic tobacco anther. No significant phe-notype variation was observed among the transgenic plants at the whole plant level. However, the percentage of pollen embryogenesis was reduced to 11 % when anthers of the transgenic plants were cultured on the modified hormone-free Nistch H (NH) medium, while those of both CK1 and CK2 (see sec. 1.2.2) were more than 50% ; when the an-thers were cultured on NH medium supplemented with 0. 2 mg/L IAA, the percentage of pollen embryogenesis re-stored to the same level of that of the wild type (up to 55. 7% ). This study demonstrates that the IAA metabolism in anther tapetum cells is of significant importance to the androgenic development in anther culture.

Co-transformation to tobacco of Cre/lox site-specific recombination system and its precise recombination

For the temporally and spatially regulated expression of the barnase gene in plant, two kinds of plasmids with ere gene and its directly repeat recognition sites lox from bacteriophage P1 were constructed and co-transformed into tobacco by agrobacterium mediated procedure. The transgenic plants were conformed by PCR analysis. The blocking fragment between the two lox directly repeat sites was excised by Cre protein in the transgenic plant genome. Cloning and seguencing the DNA fragment from the co-transformed plant DNA showed that the precise DNA excision occurred in transgenic tobacco genome directed by Cre//ox site-specif ic recombination .

毛细管气相色谱法测定烟草中的亚硝胺

N 亚硝胺是烟草和卷烟烟气中一类重要的具有毒性和致癌性的化学成分。卷烟主流烟气中的N 亚硝胺一半左右直接来源于烟草 ,而其余则来源于抽吸过程中的热合成作用。我们研究开发了一种配备氮 磷检测器的毛细管气相色谱方法来同时测定烟草中挥发性的亚硝胺 (VNA)和烟草特有的亚硝胺 (TSNA) ,主要过程包括将烟草样品在柠檬酸 磷酸氢二钠缓冲液中超声波萃取 ,然后将萃取液在硅藻土层析柱上进行固相萃取 ,浓缩液用DB 5毛细管柱进行气相色谱分析。该方法具有操作简便快速 ,重复性好 ,无需专用仪器等特点 。

烟草花药特异表达基因启动子的克隆及序列分析

通过PCR扩增,从烟草(Nicotiana tabacum cv.NC89)中克隆了花药绒毡层中特异表达基因的启动子,序列分析表明,该启动子含1303个核苷酸,与已报道的序列比较,核苷酸的同源性为99.4％。

不同产地和品种白肋烟烟草特有亚硝胺与前体物关系

收集了我国4个白肋烟产区33份白肋烟晾制后上部叶样品,并以美国、马拉维样品为对照,测定了烟草特有亚硝胺(TS-NA)、生物碱和硝酸盐含量,以揭示TSNA含量与前体物之间的关系。结果表明,四川达州和云南宾川白肋烟TSNA含量水平较低,分别与美国和马拉维相当,不同产区间NNN和总TSNA含量差异较大,NNK、NAT和NAB含量差异相对较小。烟碱转化导致降烟碱含量升高是造成国内一些产区NNN和总TSNA含量较高的主要原因,随着总TSNA含量的增加,NNN所占比例增大。烟叶降烟碱含量、烟碱转化率与NNN和总TSNA含量呈极显著的正相关关系。NNK、NAT、NAB含量与硝酸盐含量呈显著正相关,同一产区不同烟叶样品NNK含量与硝酸盐含量呈极显著的正相关。品种间比较可知,鄂烟1号和鄂烟3号烟碱转化率较高,是导致其TS-NA含量升高的主要原因。