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.

黄花烟草(Nicotiana rustica)、迪勃纳氏烟草(Nicotiana debneyi)与Nicotiana tabacum K326互交亲和性的荧光鉴定

利用荧光显微镜对烟草互交组合Nicotiana tabacum K326×Nicotiana rustica与Nicotiana tabacum K326×Nicotiana debneyi授粉后花粉管生长部位和形态进行观察比较。结果显示,在正交组合中,N.rustica、N.debneyi花粉管大量生长进入Nicotiana tabacum K326花柱离柱头3/4区间后停止生长,在停止生长部位弯曲、膨胀,形成大量不规则胼胝质颗粒;用蒙导法、切割花柱法、已开放花朵授粉及已开花朵重复涂抹授粉等方法均未能有效促进2个父本花粉管在K326花柱中生长,而蕾期授粉花粉管可以生长进入子房,但授粉后没有获得种子;在反交组合中,K326花粉管可以在N.rustica、N.debneyi花柱中大量生长并进入子房,N.debneyi授粉后获得种子,种子萌发率为(49.67±1.53)%。

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。

外源生长素对烟草(Nicotiana tabacum L.)小孢子早期胚胎发生的影响

烟草(N icotiana tabacum L.)小孢子胚胎发生系统,不仅可以提供大量可供处理的小孢子胚,还由于小孢子胚胎发生的不同步性,可同时提供从2-3细胞原胚到分化胚一系列胚胎以供研究。利用这一便利系统,探讨了外源生长素处理对小孢子胚胎发育的影响。使用3种浓度的IAA:1、3、10μm o l/L,分别对不同发育时期烟草小孢子胚进行了处理,结果发现,对不同发育时期的小孢子胚,生长素处理的效果明显不同。外源生长素对胚胎发生有促进作用,表现为2-3细胞比例与非处理组相比升高,而当小孢子发育到小球形胚后,加入外源生长素对小孢子胚的进一步发育却表现出明显抑制作用。这说明在小孢子胚胎发育过程中早期和晚期发育对生长素的需求是不同的,且对生长素的敏感程度亦不同。反映了生长素调控机制在两个不同发育时期的差异。

Cloning of a Calcium-Dependent Protein Kinase Gene NtCDPK12, and Its Induced Expression by High-Salt and Drought in Nicotiana tabacum

Calcium-dependent protein kinases （CDPKs, EC 2.7.1.37） comprise a large family of Ser/Thr kinases in plants and play an important role in plant Ca^2＋ signal transduction. A full-length CDPK gene, NtCDPK12 （GenBank accession number GQ337420）, was isolated from common tobacco （Nicotiana tabacum） leaves by rapid amplification of eDNA ends （RACE）. The NtCDPK12 eDNA is 1 816 bp length and contains an open reading frame （ORF） of 1 461 bp encoding 486 amino acids. Sequence alignments indicated that NtCDPK12 contains all conserved regions found in CDPKs and shows a high level of sequence similarity to many other plant CDPKs. The results of real-time quantitative reverse transcription-PCR （qRT- PCR） showed that NtCDPK12 was highly expressed in stems and increased in roots treated with high-salt or subjected to drought stress, which indicates that NtCDPK12 was induced by high-salt and drought stresses.

Distribution of solanesol in Nicotiana tabacum

Solanesol is an important secondary metabolite in Nicotiana tabacum. Distribution of solanesol in Nicotiana tabacum was investigated by High Performance Liquid Chromatography （HPLC） method. The quantitative distribution of solanesol in various organs and tissues of N. tabacum showed that solanesol content, obviously different in all organs, was 6.8, 18.3, 27.5, 45.8, and 68.0 times higher in leaves than that in the stalks, flowers, seeds, fruits and roots, respectively. The contents of solanesol in various parts of leaf, stalk and flower were determined. The content of solanesol in top leaf, middle leaf and bottom leaf gradually decreased （6.124, 5.813 and 5.687 mg.g^-1, respectively） and the content of solanesol in various leaf-parts （leaf apex, leaf middle and leaf base） also gradually decreased. The content of solanesol in top stalk was 1.19 times and 1.92 times higher than that in the middle stalk and the bottom stalk, respectively. The content of solanesol in various tissues of stalk （epidermis, cortex and stele） dramatically decreased. The sepal contained higher concentration of solanesol （1.192 mg·g^-1） compared to any other parts in flower. The study will provide the base data for the regulation and control of solanesol, moreover, it will provide the scientific evidences for the rational development and utilization of N. tabacum resources.

Isolation and expression analysis of NtCHS6, a new chalcone synthase gene from Nicotiana tabacum

Chalcone synthases （CHS, EC 2.3.1.74） are key enzymes that catalyze the first committed step in flavonoid biosynthesis. In this study, we isolated a chalcone synthase, named NtCHS6, from Nicotiana tabacum. This synthase shared high homology with the NSCHSL （Y14507） gene and contained most of the conserved active sites that are in CHS proteins. The phylogenetic analysis suggested that NtCHS6 protein shared a large genetic distance with other Solanaceae CHS proteins and was the most closely-related to an uncharacterized CHS from Solanum lycopersicum. The expression analysis indicated that NtCHS6 was abundantly expressed in leaves, especially in mature leaves. By scrutinizing its upstream promoter sequences, multiple cis-regulatory elements involved in light and drought responsive were detected. Furthermore, NtCHS6 expression decreased significantly under dark treatment and increased significantly under drought stress suggested that NtCHS6 expression exhibited both light responsiveness and drought responsiveness, and important roles in ultraviolet protection and drought tolerance. Our results might play

Preparation of Exine-detached Pollen in Nicotiana tabacum

A method of preparing exine-detached pollen in Nicotiana tabacum was established. Anthers containing early-middle binucleate pollen were cold-pretreated at 4～6℃ for 7～14 days,and were suspended in 0. 3 mol/L sucrose solution for 2 days. During this process,the exine of most pollep grains dehisced. Then they were transferred into an enzyme solution containing 1 % cellulase, 1 % pectinase,0. 1 % pectolyase, I mol/L mannitol, 0. 3 mol/L sorbitol .0. 5 % potassium dextran sulphate and K3 medium macro elemnts. After 15～20 min enzymatic maceration, the exine was detached resulting in the release of exine-detached pollen. Factors affecting preparation of exine-detached pollen were investigated,including cold-pretreatment .osmoticum concentration and enzymes used.

Effects of Initial Infestation Levels of Callosobruchus maculatus (F.)(Coleoptera: Chrysomelidae) on Cowpea and Use of Nicotiana tabacum L.Aqueous Extract as Grain Protectant

This study determined the effects of initial infestation of cowpea seeds （Ife brown variety） with different insect densities （0, 2, 4 and 6 pairs per 50 g seeds） of Callosobruchus maculatus （F.） and evaluated the effects of aqueous leaf extract of Nicotiana tabacum L. on C. maculatus in the laboratory. It was observed that adult beetle population increased significantly （p〈0.05） with increase in insect density. The increase in population of beetles and corresponding weight loss of the seeds in different levels of infestation showed that the cowpea variety was susceptible to beetle infestation, emergence and survival of progeny. Significantly more adults emerged on higher infestation compared to lower and no infestation. In Nigeria, Nicotiana tabacum L. is a locally available plant, with known insecticidal properties. The plant leaf extract was easily extracted with water and confirmed its effectiveness as a protective agent for stored cowpea seeds. Experiment was conducted to assess the effects of aqueous extracts ofN. tabacum at 0, 0.1, 0.2 and 0.3 mL＂ 50 g-1 cowpea seeds on C. maculatus. Data was recorded and showed varying levels of effectiveness against C. maculatus. Result showed that seed appearance was dependent on levels of insect population, while N. tabacum aqueous extract exerted effects on survival of C. maculatus. Aqueous leaf extract of N. tabacum probably contained some insecticidal properties which might have significantly conferred beetle mortality and reduced beetle emergence leading to a decrease in seed weight loss.

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.

Evaluation of Transgenic <i>Nicotiana tabacum</i>with <i>deh</i>E Gene Using Transposon Based IRAP Markers

In the present study, five genetically modified herbicide tolerant Nicotiana tabacum cv. TAPM24 plants with a constructed vector pCAMBIA1301a carrying dehalogenase E (dehE) gene were compared with three non-transgenic controls using Tto1 retrotransposon specific IRAP markers. dehE gene was originally characterized in Rhizobium sp. and it produced an enzyme which degraded the Dalapon herbicide. IRAP protocol was applied on transgenic and non-transgenic plants to investigate the retrotransposon based genetic variation which may appear during transformation. Polymorphism rates were calculated as 0%-20% from IRAP-PCR products among all plant samples. These results show that transformation of tobacco plant with the dehE gene may cause Tto1 retrotransposon alterations appearing as different band profiles. The findings are expected to contribute to genetic engineering studies to obtain better results and also to understand how transposons contribute to features such as transgenesis. In our knowledge, this is one of the first experimental data of transgenic N. tabacum engineered with dehE gene originated Rhizobium sp. in terms of retrotranposon based variation.

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.

A TOM1 homologue is required for multiplication of Tobacco mosaic virus in Nicotiana benthamiana

The AtTOM1 gene of Arabidopsis thaliana had been shown to be essential for the efficient multiplication of Tobacco mosaic virus(TMV) in A.thaliana.In this study,we cloned an AtTOM1-like gene from Nicotiana benthamiana named as NbTOM1.Sequence alignment showed that NbTOM1 is closely related to AtTOM1 homologues of N.tabacum and Lycopersicon esculentum with 97.2% and 92.6% nucleotide sequence identities,respectively.Silencing of NbTOM1 by a modified viral satellite DNA-based vector resulted in complete inhibition of the multiplication of TMV in N.benthamiana.The result suggests that inhibition of NbTOM1 via RNA silencing is a potentially useful method for generating TMV-resistant plants.

Determination of the Bromine, Manganese and Antimony in Nicotiana tabacum Solanaceae by Using the Neutron Activation Analysis Technique

Tobacco addiction has been mentioned as a leading cause of preventable illnesses and premature disability. Smoking is the main cause of lung cancer and one of the factors that most contribute to the occurrence of heart diseases, among others. The herbaceous species Nicotiana tabacum is a plant of the solanaceae family used for tobacco production. Some authors have conducted research about heavy metals and the toxicity of tobacco. It is, frequently, found in low concentrations in the ground, and superficial and underground waters, even though they do not have environmental anthropogenic contributions. However, with the increase of industrial activities and mining together with the agrochemical use of contaminated organic and inorganic fertilizers, an alteration of the geochemical cycle occurs. As a consequence, the natural flow of these materials increases and is released into the biosphere, where they are often accumulated in the superior layer of the ground, accessible to the roots of the plants. During planting and plant development, fertilizers and insecticides, including organochlorines and organophosphates, are used;consequently, the smoke from cigarette smoking presents various toxic substances, such as bromine (Br), manganese (Mn) and antimony (Sb), elements studied in this work. The procedures for the preparation of the samples were carried out in our laboratories and submitted to irradiation with thermal neutrons at Nuclear and Energy Research Institute (IPEN/CNEN-SP), in the Atomic Energy Institute IEA-R1 research reactor. The irradiated material was, then, analyzed by gamma spectrometry, using a high purity germanium detector (HPGe).

烤烟黑胫病抗性评价及生理响应特性分析

本研究旨在揭示不同烤烟品种对烟草黑胫病的抗性机理。通过对抗病性的鉴定,从24个烤烟品种(系)中鉴定出4个高抗品种、8个中抗品种、8个中感品种和4个高感品种。对这些品种在烟草疫霉(Phytophthora parasitica var.nicotianae)侵染后,从中选取2个高抗品种和2个高感品种测定其抗氧化酶系活性、水杨酸含量以及抗病相关基因的表达情况。结果表明,在受到病原菌侵染时,高抗品种的抗氧化酶活性显著高于高感品种,暗示着更强的氧化应激防御能力。此外,高抗品种中的内源水杨酸合成以及抗病相关基因的诱导表达均显著高于高感品种,表明这些品种能够更有效地激活其防御系统以应对烟草黑胫病菌的侵染。本研究结果为理解烤烟对黑胫病的抗性机制提供了重要信息,并为烟草抗病育种提供了理论依据。

烟草磷转运蛋白基因NtPHT2;1克隆和表达分析

【目的】植物对磷酸盐的吸收与利用主要依靠磷转运蛋白,其中PHT2家族编码的低亲和磷转运蛋白主要负责植物在正常供磷条件下磷酸盐的吸收、转运与再利用,为了探究低亲和磷转运蛋白基因NtPHT2;1在烟草转运磷酸盐中的作用和表达模式,通过NtPHT2;1培育烟草磷素高效利用新品种。【方法】以普通烟草K326的cDNA为模板,克隆得到NtPHT2;1,对该基因进行生物信息学分析和蛋白质的亚细胞定位,并通过荧光定量PCR技术对该基因在低磷等非生物胁迫下的基因表达模式进行分析。【结果】(1)NtPHT2;1基因全长1764 bp,编码587个氨基酸;(2)亚细胞定位结果显示,NtPHT2;1蛋白定位于叶绿体上;(3)同源性比对发现,NtPHT2;1蛋白与辣椒CaPHT2;1蛋白的同源性最高达到91.00%;(4)NtPHT2;1启动子含有参与调控植物衰老、逆境胁迫相关的顺式作用元件;(5)NtPHT2;1在叶片表达量最高,新叶中的表达量比老叶中高;在低磷诱导条件下,该基因表达量与正常条件相比差异不显著;(6)非生物胁迫下的表达模式分析显示,NtPHT2;1基因表达量在盐胁迫和干旱胁迫下显著降低。【结论】NtPHT2;1基因主要是负责烟株正常生长发育条件下磷酸盐的转运与利用。