Transcriptome and Metabolome Revealed the Mechanism of NtBRL3 Overexpression Tobacco(Nicotiana tabacum L.K326)in Response to Drought Stress

Drought has severely affected the yield and quality of commercial crops.The BRI1 family plays an important role in plant response to drought stress,and BRL3 gene plays an important role in the study of drought in Arabidopsis thaliana.In this study,NtBRL3 was constructed as a vector and genetically transformed to obtain‘N.Tobacco K326’overexpression of NtBRL3.The enzyme activities of transgenic tobacco and wild-type tobacco were measured and transcriptome and metabolome analyses were performed.The results showed that the antioxidant enzymes of transgenic tobacco were more active under drought conditions,and 85 significantly differentially metabolites and 106 significantly differentially expressed genes were identified in the metabolome and transcriptome analyses,respectively.Transgenic tobacco NtBRL3ox demonstrated an excessive accumulation of droughtrelated metabolites,sugars such as sucrose and maltotetraose,and amino acids such as proline,compared with WT.We discovered drought-related differential genes in the root transcriptome,among which LOX6,RD22,WSD1,CCD8,and UGT were key genes which play an important role in plant response to drought stress.Our results demonstrate that NtBRL3 overexpression in K326 enhances drought resistance in transgenic tobacco.

Cloning and Expression of Calcium-Dependent Protein Kinase (CDPK) Gene Family in Common Tobacco (Nicotiana tabacum)

To further study the function of calcium-dependent protein kinase （CDPK） gene family in common tobacco （Nicotiana tabacum）, it is necessary to isolate more CDPKs from common tobacco and describe the sequence characteristics, evolutionary relationship and gene expression. Reverse transcription-PCR （RT-PCR）, rapid amplification of cDNA （RACE） and bioinformatics methods were used to isolate CDPKs from common tobacco. A phylogenetic tree was created using the MEGA4.0 program and expression patterns of the three full-length CDPK genes were studied by RT-PCR. After all aforementioned efforts, we obtained eight additional common tobacco CDPK genes, of which three possessed complete open reading frames （ORFs）. Phylogenetic analysis divided 1 1 full-length Nicotiana CDPK genes into four subfamilies, and two putative common tobacco and Arabidopsis orthologous CDPK genes might correspond to well-conserved functions. Three full-length CDPK genes in common tobacco were detected in all tobacco organs tested, but their expression patterns were significantly different. Eight non-redundant common tobacco CDPK genes were isolated in this study. Along with the previously characterized CDPK genes, at least 15 members of the CDPK family exist in common tobacco. This work establishes a foundation for a genome-wide study of this important gene family in common tobacco.

以单细胞压片技术观察烟草(Nicotiana tabacum L.)合子细胞器DNA的分布

通过实验建立了单个合子的压片技术,尝试了将染色与固定分开或同时进行的两种压片方法,两种方法均可以用于不同发育阶段合子细胞器DNA分布的检测,但二者各具特色。同时也利用共聚焦激光扫描显微镜观察了烟草(NicotianatabacumL.)合子细胞器的分布,并与单细胞压片技术进行了比较。实验表明它们各有其优越性。

Nonphytotoxic copper oxide nanoparticles are powerful“nanoweapons”that trigger resistance in tobacco against the soil-borne fungal pathogen Phytophthora nicotianae

Investigations into the potential application of nanoparticles acting as nanofungicides in sustainable agriculture are rapidly expanding due to the high antimicrobial properties of these compounds,which do not risk inducing pathogen resistance to fungicides.A detailed understanding of the impact of copper oxide nanoparticles(CuO NPs)on soil-borne phytopathogenic fungi is yet to be obtained.This study aimed to explore the in vitro antifungal activity and control efficacy of CuO NPs applied via irrigation with respect to tobacco black shank(TBS)disease caused by Phytophthora nicotianae.The results revealed that CuO NPs greatly interfered with the reproductive growth process of this fungus,repressing hyphal growth,spore germination and sporangium production.Additionally,morphological damage,intracellular ROS accumulation and increased SOD enzyme activity in hyphae were the antifungicidal mechanisms of these NPs.In pot experiments,treatment with CuO NPs at 100 mg L^(–1)significantly suppressed TBS development,compared with the effect on control plants,and the control efficacy reached 33.69%without inducing phytotoxicity.Exposure to CuO NPs significantly activated a series of defense enzymes,and resistance genes in tobacco can further explain the mechanisms by which CuO NPs suppressed fungal infection.The Cu content in both the leaves and roots of P.nicotianae-infested plants increased by 50.03 and 27.25%,respectively,after treatment with 100 mg L^(–1)CuO NPs,compared with that of healthy plants.In particular,a higher Cu content was observed in infected roots than in leaves.Therefore,this study showed the potential of CuO NPs applied as nanofungicides and as nanoinducers of fungus resistance genes for the management of TBS through inhibition of pathogen infection and stimulation of plant defenses.

Development of a Method to Produce Chromosome Lacking Lines (CLLs) in <i>Nicotiana tabacum</i>L. “Red Russian”

Monosomic lines of Nicotiana tabacum are helpful to confirm the location of genes on specific chromosomes. In the cross N. nudicaulis and N. tabacum, hybrid seedlings express lethal symptoms, which are controlled by the S subgenome of N. tabacum. To identify the responsible chromosome, we needed to produce chromosome lacking lines (CLLs) of N. tabacum L. “Red Russian” and use them to cross with N. nudicaulis. From a cross of (N. tabacum × N. tomentosiformis) × N. tabacum, 380 BC1 individuals were obtained. Using a Haplo-Q line (a monosomic line lacking the single linkage group 11) and N. tabacum, we found that qPCR is a simple and reliable screening method for CLLs of N. tabacum. The marker PT30342 is located on linkage group 11, and the -Ct value (Ct Actin - Ct PT30342) was 2.0 for a disomic line and was 1.097 for a Haplo-Q line. By the use of flow cytometry, qPCR and chromosome counting together as a screening method, we identified 6 CLLs lacking 2 to 6 chromosomes. Compared with conventional methods, our method is a rapid technique for making and screening CLLs ofthe S or S/T subgenome of N. tabacum. Further, these CLLs will be useful to identify the location of two or more factors on chromosomes controlling a variety of genetic problems affecting breeding. Here, we only made CLLs of the S or S/T subgenome of N. tabacum. We will use the method established in this study to produce CLLs of the T subgenome of N. tabacum, and gather a full set of CLLs of N. tabacum. qPCR could also be applied to the identification of chromosome aberrations in other plants.

Interference of Quinolinate Phosphoribosyltransferase Gene QPT Affects Agronomic Traits and Leaf Quality in Nicotiana tabacum L.

Quinolinate phosphoribosyltransferase(QPRTase),a key enzyme in ensuring nicotinic acid is available for the synthesis of defensive pyridine alkaloids in Nicotiana species,also plays an important role in nicotinamide adenine dinucleotide(NAD)biosynthesis.In this study,the morphological traits,the quality characteristics and photosynthetic parameters in QPT-overexpressing/interfering tobacco plants were investigated,respectively.Results showed that the interference of QPT gene not only reduced significantly the morphological traits including plant height,stem girth,leaf number and leaf length,etc.at 20 days after transplanting(DAT),but the flowering period was delayed 10-15 d in interfered tobaccos compared with the overexpressed,control and wild-type counterparts.However,at 40 DAT and 60 DAT,only three indexes(plant height,stem girth and leaf number)in QPT-interfering plants appeared significant difference in comparison with other three types of tobacco lines.Meanwhile,the determination results from nicotine,sugar,K+and Cl-content showed the nicotine content in interfered plants was always significantly lower than that in overexpressed plants,control and the wild-type ones respectively whatever toppling or not.At the same time,the toppling treatment also caused the increasement of K+content among the four different tobacco lines,but the maximum increase amplitude of K+content was found in QPT-overexpressing tobaccos while the minimum appeared in QPT-interfering plants.Finally,QPT-interference in transgenic tobaccos likewise affected the photosynthesis by reducing net photosynthetic rate(Pn),stomata conductance(Gs)and transpiration rate(Tr),while there was no significant difference between QPT-overexpressing plants and the controls and the wild-types.

Quality Characteristics of Tobacco Leaves with Different Aromatic Styles from Guizhou Province,China

The relationships between chemical components and quality indexes were studied in the tobacco leaves with different aromatic styles. A total of 16 chemical components, 4 quality indexes, and 6 smoking quality indexes from 366 tobacco leaf samples with 4 different types of aroma from Guizhou Province, China, were subjected to principal component analysis and stepwise regression analysis. The tobacco leaves with different types of aroma showed remarkable difference in the contents of chemical components, quality indexes, and smoking quality indexes. The first principal factors （carbohydrates and nitrogen-containing compounds） of the chemical composition of the leaf were similar among different types of aroma, which showed that the quality of the leaf was mainly influenced by carbohydrates and nitrogen-containing compounds and their ratios. The factors for the second through the seventh principal components varied largely among varioUs aromatic types, suggesting the contribution of other chemical components to the leaf quality. In addition, the smoking quality of four different aromatic leaves showed significant correlation with the different chemical components. The quality of tobacco leaves with different types of aroma was influenced by multiple factors, especially ecological conditions and culture techniques, which may provide guidance for directive cultivation of high-quality tobacco leaves.

Changes of Polyphenols in Tobacco Leaves During the Flue-Curing Process and Correlation Analysis on Some Chemical Components

The changes of polyphenols in tobacco leaves during the flue-curing process and correlation analysis on some chemical components were studied. Leaf samples were taken from different tobacco-producing regions in Henan Province, China. The results indicated that the content of total phenols increased during the first 24 h of curing, and then decreased. It reached the lowest value at 72 h of curing and increased rapidly after that. The content of chlorogenic acid also increased during 0-24 h of curing. But the lowest point occurred at 60 or 72 h of curing and then it increased till the end of the curing process. The content of mtin generally increased with curing, and showed little fluctuations. The changes of PPO and POD activity were the opposite. Rutin was found to have a highly significant positive correlation with total sugar （r=0.822^＊＊）, but a highly significant negative correlation with starch, nicotine, and protein.

Research on Tobacco Transformation of Vacuolar H^+-ATPase Subunit c Gene from Iris lacteal

Vacuolar ATPase is the key enzyme in the aspect of plant stress, which contains 13 subunits. In this study, VHA-c which was cloned from the salt-tolerant plants Iris lacteal was transformed into tobacco, and then treated the transgenic tobacco with 0.7% NaC1 for 7 days. Detected the MDA content, the SOD activity and the POD activity. Moreover, the transgenic plants displayed obviously better growth than the normal plants under salt stress. These results suggested that VHA-c could confer the stress tolerance through enhancing the POD and the SOD activities, and by protecting membranes from damage by decreasing lipid peroxidation under salt stress.

Populational Fluctuation and Distribution of Epitrix spp.Foudras(Coleoptera:Chrysomelidae)in the Organic Tobacco Management in Santa Cruz do Sul,RS,Brazil

Among the chrysomelids, some species are considered agricultural pests, such as some representatives of Epitrix spp. Popularly known as tobacco flea, these beetles cause damage to tobacco (Nicotiana tabacum L.) by feeding on the leaf surface, causing injuries, reducing productivity and the final quality of the product to be marketed. However, studies of the biology and interactions of these individuals with the environment become essential in order to develop effective and sustainable control measures. The objective of this study was to analyze the distribution and population fluctuation of Epitrix spp., as well as to evaluate the possible influence of temperature, rainfall and adjacent vegetation on the population dynamics of this beetle in organic tobacco growing in the municipality of Santa Cruz of the South, RS. The collections were carried out weekly during the 2011/2012 harvest, using Malaise and Pit-fall traps, distributed in four points along three lines. A total of 1584 individuals belonging to the genus Epitrix were collected, and their greatest abundance was observed in late January and early February 2012 due to climatic factors, such as rainfall and temperature, favorable to the development of specimens and the tobacco leaf harvesting reduced the availability of food and conditioned a greater movement of these individuals in the crop. The results presented here are of great importance for deciding the best methodology for controlling the smoke flea, as well as knowing the influence of abiotic factors on the behavior of these organisms in the field.

烤烟6个农艺性状的QTL定位(英文)

由于烟草的分子标记开发和遗传图谱构建十分困难,迄今烟草中有关数量性状基因座（QTL）的定位研究仍非常有限。本研究利用一个由207个株系组成的烤烟DH群体及基于该群体所构建的含有24个连锁群、611个SSR标记,总长为1882.1cM的遗传图谱,采用复合区间作图方法,对株高（PH）、茎围（SG）、节距（IL）、叶片数（LN）、最大腰叶长（LWL）和最大腰叶宽（WWL）6个与叶片产量有关的农艺性状进行QTL定位分析。共检测到69个QTL,大部分QTL的效应值较小,仅有4个具有较大的效应值,可解释大约15%~20%的表型变异。6个性状之间大多彼此相关。与此相符,在基因组中发现存在许多小区域,每个区域包含2个或2个以上紧密连锁的不同性状的QTL。

基于电子鼻技术的烟丝受虫害前后挥发性成分差异分析

针对烟草(Nicotiana tabacum L.)中烟草甲(Lasioderma serricorne)检测技术不完善,检测结果不可靠等问题,以电子鼻检测结果为基础,通过主成分分析法(PCA)和Loading分析法对数据结果进行分析。结果表明,4种烟丝的主要挥发物质为硫类物质、短链烷烃、醇醚醛酮、小分子氮氧化合物等,虫样及性激素的主要挥发性物质是小分子氮氧化合物、硫化物。受虫害后烟丝中硫类化合物明显降低,在对无机硫化物敏感的传感器(W1W)中浓香虫害烟丝的响应值比浓香烟丝下降48.33%,清香虫害烟丝比清香烟丝下降59.48%;在对有机硫化物敏感的传感器(W2W)中浓香虫害烟丝的响应值比浓香烟丝下降35.72%,清香虫害烟丝比清香烟丝下降48.55%。PCA结果显示,4种烟丝样品表现出非常好的区分度,雌虫、雄虫样品在占比高达99.70%的主成分1上几乎没有差别。Loading分析结果显示,烟丝样品中气味主要成分为硫类物质,烟草甲虫样气味主要成分是小分子氮氧化物,其次为硫类物质。

烟草六个重要性状的QTL定位

以烤烟台烟7号与白肋烟白肋21作为杂交亲本,后代自交衍生的127个F2和F2:3家系为材料,构建了全长为3483cM的包括26个连锁群、190个标记位点的烟草遗传连锁图谱。通过一年两地各3次重复的随机区组田间试验,测定烟叶烟碱、总氯、总钾、叶长、茎叶夹角、白粉病6个重要性状,采用混合线性模型的复合区间作图法定位QTL并分析其遗传互作效应。结果检测到2个烟碱相关QTL、2个总氯相关QTL、1个总钾相关QTL、4个叶长相关QTL、茎叶夹角和白粉病相关QTL各1个,其中6个加性效应QTL和4对加加上位性效应QTL。这11个基因位点遗传效应中除加性效应外,上位性效应也具有重要作用。

预喷施CaCl_2溶液对低温胁迫下烤烟幼苗生理生态的影响

温度是决定烤烟幼苗生长发育的重要因子之一,影响着烟叶最终的产量和品质.南方多数烟区育苗阶段早春低温影响着健壮烟苗的培育.该研究选用云烟87和Msk326 2个烤烟品种烟苗预喷施质量分数为0.5%,1.0%,1.5%及2.0%的CaCl2溶液后进行低温(8℃)处理72 h,然后测定烟苗光合参数、叶绿素相对质量分数、膜保护酶、渗透调节物质可溶性糖和游离脯氨酸等生理指标,研究预喷施CaCl2溶液对低温胁迫下烤烟幼苗的生理生态影响.结果表明:2个品种的低温对照CK2光合指标除云烟87的胞间二氧化碳质量分数较常温对照未达到极限著水平外,其他光合指标、过氧化氢酶(CAT)及过氧化物酶(POD)活性均显著低于常温对照CK1;预喷施1.0%CaCl2溶液可有效提高过氧化氢酶(CAT)及过氧化物酶(POD)活性,增加可溶性糖和游离脯氨酸质量分数,减少丙二醛(MDA)质量分数,提高烟苗耐低温胁迫的能力,从而维持烟苗正常光合作用.

UGPase和反义4CL基因对转基因烟草纤维素和木质素合成的调控

用根癌农杆菌介导法将源于紫穗槐的尿苷二磷酸葡萄糖焦磷酸化酶(UGPase)基因、反义4-香豆酸辅酶A连接酶(4CL)基因以及两者的双价基因分别转移至烟草中。PCR和Southern杂交检测证实外源基因已整合到转基因烟草基因组中。测定全纤维素和Klason木质素含量的结果显示,增强UGPase基因的表达可提高转基因植株的纤维素含量,但对木质素含量没有影响;抑制4CL基因的表达可显著降低转基因植株的木质素含量,但对纤维素含量没有影响;转移双价基因的转基因植株中纤维素含量增加而木质素含量降低。

超量表达 CsADH17 基因提高烟草抗旱性及香气成分分析

乙醇脱氢酶是植物香气物质合成的脂肪酸代谢等途径中的关键酶之一,对茶叶芳香物质的形成有着重要作用,因此探究CsADH17基因有利于茶树的生长发育与品质提升。以本课题组前期筛选的CsADH17基因为研究对象,克隆CsADH17基因并构建植物表达载体,遗传转化烟草,对转基因烟草非生物胁迫下CsADH17的表达量和相关生理指标进行测定与分析。结果表明,对转基因与野生型烟草进行模拟干旱胁迫后,转基因烟草植株在干旱胁迫处理下的CsADH17基因相对表达量比野生型植株高。野生型烟草植株和CsADH17转基因烟草植株在干旱胁迫处理21 d后,超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)的活性均高于野生型烟草;胁迫处理后,野生型烟草叶片中丙二醛(MDA)含量显著高于转基因烟草。香气成分分析,转基因烟草苯甲醛比野生型含量提高了44.4%,而烟碱含量比野生型提高不到5%。CsADH17基因在烟草干旱胁迫下具有抗旱作用,同时影响烟草的香气成分,为进一步揭示CsADH在茶树生长发育以及抗逆响应中提供参考。

一个烟草Mlo基因的电子克隆及其序列特性分析

电子克隆并探讨了一个烟草Mlo基因的结构和功能.以GenBank公布的水稻Mlo基因序列为信息探针对烟草的EST数据库进行同源搜索,并将获得的同源性高的EST序列进行序列拼接,最后得到了一个烟草Mlo基因的cDNA序列.采用生物信息学方法分析该基因编码蛋白的一级、二级结构,并对其功能进行预测.结果表明,此烟草Mlo基因编码一个由288个氨基酸组成的蛋白,相对分子质量为33kD,有7个跨膜区域,蛋白二级结构以α螺旋为主,有1个DUF1084超家族的保守结构域.蛋白功能分析表明,该烟草Mlo基因编码蛋白具有跨膜介导信号传递的功能,可能是一种类似于G蛋白偶联受体的膜结合转运蛋白.

超表达JcFT基因对烟草农艺性状的影响

FT(Flowering locus T)基因编码的成花素蛋白,也称蛋白激素,在植物中超表达该基因往往可以显著提早开花,是创制早熟植物种质资源的重要潜在基因之一,影响植物生长发育。本研究以烟草为材料,分析了FT对烟草多个农艺性状的影响。结果表明,JcFT(FT of Jatruopha curcas L.)超表达在提早烟草开花的同时,显著改变了烟草根、茎、叶的大小和形态,而花的结构没有显著改变。

烟草西柏烷二萜醇类突变体的理化特征及其突变机制解析

以烟草突变体及其野生型为材料,通过测定农艺性状、腺毛密度、西柏烷二萜醇及其代谢前体物含量、西柏烷二萜醇类代谢关键基因的表达量,分析了突变体的生理生化和代谢差异,并结合基因表达差异初步解析了其突变的生理与分子机制。结果表明,突变体西柏三烯一醇和西柏三烯二醇含量分别是野生型的2.63倍和4.03倍,均极显著高于野生型;突变体分泌型腺毛密度为757.38条/cm 2,极显著大于野生型;突变体西柏烷二萜醇类代谢前体物IPP和GGPP分别为849.83 ng/g、298.29 ng/g,与野生型间的差异不显著;突变体西柏三烯一醇合成酶基因NtCYC-1、西柏三烯二醇合成酶基因NtCYP71D16的表达量分别是野生型的17.77倍和20.89倍,均极显著高于野生型。研究表明,突变体是一个培育优质、抗虫烤烟新品种的优良材料;其发生突变的原因是NtCYC-1、NtCYP71D16基因发生表达上调,促进了西柏三烯一醇、西柏三烯二醇的合成反应,同时,突变体的分泌型腺毛密度更高,为西柏烷二萜醇类的合成提供了更多的场所,使得西柏烷二萜醇类的含量显著提高。