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.

The Application of Nicotiana benthamiana as a Transient Expression Host to Clone the Coding Sequences of Plant Genes

Coding sequences (CDS) are commonly used for transient gene expression, in yeast two-hybrid screening, to verify protein interactions and in prokaryotic gene expression studies. CDS are most commonly obtained using complementary DNA (cDNA) derived from messenger RNA (mRNA) extracted from plant tissues and generated by reverse transcription. However, some CDS are difficult to acquire through this process as they are expressed at extremely low levels or have specific spatial and/or temporal expression patterns in vivo. These challenges require the development of alternative CDS cloning technologies. In this study, we found that the genomic intron-containing gene coding sequences (gDNA) from Arabidopsis thaliana, Oryza sativa, Brassica napus, and Glycine max can be correctly transcribed and spliced into mRNA in Nicotiana benthamiana. In contrast, gDNAs from Triticum aestivum and Sorghum bicolor did not function correctly. In transient expression experiments, the target DNA sequence is driven by a constitutive promoter. Theoretically, a sufficient amount of mRNA can be extracted from the N. benthamiana leaves, making it conducive to the cloning of CDS target genes. Our data demonstrate that N. benthamiana can be used as an effective host for the cloning CDS of plant genes.

Structural and Functional Insights into an Arabidopsis NBS-LRR Receptor in Nicotiana benthamiana

Nucleotide-binding site leucine-rich repeat receptors (NBS-LRR/NLRs) are crucial intracellular immune proteins in plants. Previous article reported a novel NLR protein SUT1 (SUPPRESSORS OF TOPP4-1, 1), which is involved in autoimmunity initiated by type one protein phosphatase 4 mutation (topp4-1) in Arabidopsis, however, its role in planta is still unclear. This study employed Nicotiana benthamiana, a model platform, to conduct an overall structural and functional analysis of SUT1 protein. The transient expression results revealed that SUT1 is a typical CNL (CC-NBS-LRR) receptor, both fluorescence data and biochemical results showed the protein is mainly anchored on the plasma membrane due to its N-terminal acylation site. Further truncation experiments announced that its CC (coiled-coil) domain possessed cell-death-inducing activity. The outcomes of point mutations analysis revealed that not only the CC domain, but also the full-length SUT1 protein, whose function and subcellular localization are influenced by highly conserved hydrophobic residues. These research outcomes provided favorable clues for elucidating the activation mechanism of SUT1.

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)%。

Nicotiana tabacum Lin.-N. plumbaginifolia V iv.杂种的鉴定及其育性和黑胫病抗性的初步分析

为明确一株云烟87八倍体(2n=8x=96)与N.plumbaginifolia Viv.(2n=2x=20)的杂交后代的部分生物学特性。对该后代进行了形态观测,细胞学遗传学分析,育性分析,并进行黑胫病抗性的离体鉴定。结果显示,该杂交后代(无性系)大部分外观形态指数介于云烟87四倍体与N.plumbaginifolia之间;花冠颜色与云烟87相近,花药颜色与N.plumbaginifolia相近。其染色体数目为58条,其中含有来自N.plumbaginifolia的10条染色体。花粉离体培养未见萌发;与云烟87四倍体杂交,做母本时坐果率为100%,平均每个蒴果获得102.60±25.12粒可萌发的种子,以其为父本及自交时未获得成熟蒴果。该杂交后代(无性系)对黑胫病抗性强于云烟87,与N.plumbaginifolia相近。综上所述,该杂交后代为云烟87与N.plumbaginifolia的真杂种(2n=5x=58);其雄性败育,但雌性可育;且具较强的黑胫病抗性。

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。

Effects of Ppc Gene Construction of Monocotyledon on Seedling Growth of Transgenic Nicotiana tabacum

To compare the transformation effects of two different forms （cDNA in monocotyledonous plant Echinochloa crusgalli, DNA in monocotyledonous plant Zea mays） of phosphoenolpyruvate carboxylase （PEPC） gene （Ppc） on the growth of transgenic dicotyledonous plant, Agrobacterium-mediated transformation of Ppc genes into Nicotiana tabacum were carried out. Transgenic leaf plates and differentiated seedling leaves were verified by GUS histochemistry, PCR, and RT-PCR. Results showed that transgenic N. tabacum with Ppc-cDNA of E. crusgalli had relatively strong differentiation ability. However, N. tabacum after transformation of complete DNA sequence of Ppc genes in Z mays had relatively poor ability of growth. The differentiated green seedlings had the phenomenon of yellowing; and photosynthesis ability of leaves was poor. This might be caused by the misidentification and wrong splicing in transcription. This indicated that the expression rate of monocotyledonous complete DNA might be reduced in the monocotyledonous cells with relatively far genetic distances. Detection results of showed that Pn in most transgenic N. tabacum with Ppc-cDNA of E. crusgalli was was higher than that in control, which preliminarily proved that PEPC of monocotyledonous plant E. crusgalli had certain regulatory effects on photosynthesis of N. tabacum.

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.

Identification of stably expressed QTL for resistance to black shank disease in tobacco(Nicotiana tabacum L.) line Beinhart 1000-1

Cigar line Beinhart 1000-1 has effective durable resistance to black shank(BS) and is considered one of the most resistant sources in tobacco(Nicotiana tabacum L.). To investigate the inheritance and identification of stable quantitative trait loci(QTL) for BS response, F2,BC1 F2 individuals and BC1 F2:3 lines were produced from a cross between Beinhart 1000-1 and Xiaohuangjin 1025. Two major quantitative trait loci(M-QTL) named qBS7 and qBS17 were repeatedly detected under different conditions. QTL qBS7 was mapped to the region between PT30174 and PT60621 and explained 17.40%–25.60% of the phenotypic variance under different conditions. The other QTL qBS17 in interval PT61564–PT61538 of linkage group 17 was detected in a BC1 F2 population in the field and in BC1 F2:3 in both the field and at the seedling stage, explaining 6.90% to 11.60% of the phenotypic variance. The results improve our understanding of the inheritance of resistance to BS and provide information that can be used in marker-assisted breeding.

Effect of Nitrogen Fertilization on Growth and Photosynthetic Nitrogen use Efficiency in Tobacco （Nicotiana tabacum L.）

Soil pot experiments were conducted in a greenhouse to examine the effects of different nitrogen （N） supply （low, 0.15 g N/kg; middle, 0.3 g N/kg; and high, 0.6 g N/kg dry soil） on the growth, photosynthetic characteristics and photosynthetic nitrogen use efficiency （PNUE） of tobacco seedlings （Nicotiana tabacum L. Yunyan 87）. The results showed middle and high N significantly enhanced seedling growth including plant stem and leaf dry weight comparing with low N. High N supply could lead to a dramatic increase in the photosynthetic capacity of tobacco seedlings under low N conditions. There were significant differences in leaf N content between nitrogen treatments. About a 76% increase in leafN content in plants fed by high N resulted in about 43% increase in Rubisco content and 27% in net photosynthetic rate. The non-corresponding increases in photosynthetic rate in tobacco seedlings fed by high N relative to low N resulted from Rubisco activity and/or carboxylation efficiency （CE）. These results indicated that tobacco seedlings under high N application can maintain high net photosynthetic rate （Pn） but lower PNUE, will finally result in a decline in N use efficiency.

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.

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.

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.

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).