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.

Antibacterial Activity and Mechanisms of Ethanol Extracts from Scutellaria baicalensis Georgi and Magnolia officinalis against Phytophthora nicotianae

Phytophthora nicotianae causes substantial economic losses in most countries where tobacco is produced.At present,the control of P.nicotianae mainly depends on chemical methods,with considerable environmental and health issues.We investigated the effects of ethanol extracts from Scutellaria baicalensis Georgi(SBG)and Magnolia officinalis(MO).On mycelial growth,sporangium formation,and zoospore release of P.nicotianae.Both extracts inhibited the growth of P.nicotianae,with mycelial growth inhibition rates of 88.92%and 93.92%,respectively,at 40 mg/mL,and EC50 values of 5.39 and 5.74 mg/mL,respectively.The underlying mechanisms were the inhibition of sporangium formation,the reduction of zoospore number,and the destruction of the mycelium structure.At an SBG extract concentration of 16.17 mg/mL,the inhibition rates for sporangia and zoospores were 98.66%and 99.39%,respectively.At an MO extract concentration of 2.87 mg/mL,the production of sporangia and zoospores was completely inhibited.The hyphae treated with the two plant extracts showed different degrees of deformation and damage.Hyphae treated with SBG extract showed adhesion and local swelling,whereas treatment with MO extract resulted in broken hyphae.Mixture of the extracts resulted in a good synergistic effect.

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.

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)%。

Transient Expression of BYDV-MP in Nicotiana benthamiana

[Objective]The aim of this study was to identify transient expression of movement protein （MP） gene in Nicotinana benthaminana rapidly and further investigate the function of this exogenous gene. [Method]The movement protein gene of barley yellow dwarf virus （BYDV） was cloned into potato virus X （PVX） viral vector of pGR107,and PVX-recombinant vector was obtained. After electroporation of Agrobacterium tumefaciens,PVX was inoculated into the lower leaves of tobacco by Agrobacterium infiltration assay to observe the infection of virus on tobacco. [Result]After infection for 7 days,upper non-inoculated leaves of tobacco infected by the PVX-recombinant vector showed the virus infection symptoms,while the control group had no viral infection phenomenon. Daily follow-up observations for two groups revealed that tobacco infected by PVX-recombinant vector had severe symptoms of virus infection and curling leaves,or even led to necrosis both in infiltrated and systemic leaves in late period. However,tobacco infected by PVX vector had only slight symptoms of virus infection and could recover from infection. RT-PCR of the infected tobacco indicated that exogenous gene BYDV-MP had a normal transcription and expression in tobacco. [Conclusion]As a determinant factor for viral disease,BYDV-MP promotes the systemic infection rate of PVX and its symptom. In addition,it is feasible to express exogenous MP gene in Nicotiana benthaminan via PVX expression vector.

Using Isolated Embryo Sacs and Early Proembryos for Localization of Calmodulin mRNA Before and After Fertilization in Nicotiana

An in situ hybridization technique for localization of calmodulin(CaM) mRNA in isolated entire embryo sacs and proembryos in Nicotiana tabacum L.cv.W38 has been developed. This technique can be applied to small amounts of materials in which a whole view of CaM mRNA distribution can be obtained. The authors revealed that CaM mRNA expression changes dramatically before and after fertilization. Especially interesting is that a prominent CaM mRNA band appears between the egg apparatus and polar nuclei temporarily during the period of pollination and fertilization. The band disappears just prior to fertilization and expands to a fan_shaped region that occupies the micropylar portion of the embryo sac. After fertilization, CaM mRNA accumulates in the elongated zygotes with higher concentration in their chalazal portion than in the micropylar portion. Such an asymmetrical pattern continues to manifest in the early proembryos. It is supposed that CaM mRNA may be involved in the early events and signaling steps associated with double fertilization and zygote polarization in higher plants.

烟草黑胫病原菌(Phytophthora parasitica Var·nicotianae)生理生化特性的研究

本文主要介绍了用人工合成培养基测定烟草黑胫病原菌的致死温度、最适生长的pH值和能够生长的pH值范围的方法及结果。对黄河等“适宜于马铃薯晚疫病菌的一种液体合成培养基”,在配方和实验方法上做了一些改进。通过实验,进一步证实了该菌在营养生长过程中钙素的重要性和三羧酸循环(TCA)中有机酸的重要性;该菌的营养生长并不需要较复杂的营养条件。

烟草Nicotiana glauca遗传瘤的诱导

以往的研究证明一些烟草杂种能自发形成遗传瘤,而正常条件下杂交亲本各自本身并不能自发形成遗传瘤。该研究首次从烟草Nicotiana glauca品种中诱导产生出遗传瘤,这些N.glauca遗传瘤与杂种N.glauca×N.langsdorffii遗传瘤具有相同的特征,即在无激素的培养基上能正常分化、对外源植物激素不敏感等特点。该研究结果同时还进一步证明种间杂交并不是诱导烟草产生遗传瘤的唯一因素。

Cloning and Gentic Transformation of NbDAD1 Gene from Nicotiana benthamiana

[Objective] The aim was to clone NbDAD1 gene from Nicotiana benthami- ana and study its genetic transformation. [Method] NbDAD1 gene was isolated from N. benthamiana by using RT-PCR technology and over-expression vectors were con- structed to obtain NbDADl-overexpression resistant plants and NbDADl-overexpres- sion resistant plants carrying HA tag. [Result] The 351 bp long NbDAD1 gene was cloned from N. benthamiana; recombinant plasmids pCAMBIA1301-NbDAD1 and pCAMBIA1301-NbDAD1HAtag were constructed successfully; 50T0-generation N. ben- thamiana Hyg-resistant transgenic lines of three genotypes were obtained, including 23 positive transgenic plants. [Conclusion] This study laid the foundation for investi- gating the specific functions of NbDAD1 gene in N. benthamiana and exploring the possible functional mechanism of DAD1 protein in programmed cell death of plants.

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.

Combined Toxicity Test of Complex Agents of Metalaxyl and Other Fungicides against Phytophthora parasitica var. nicotianae

[ Objective] To overcome the resistance of Phytophthora parasitica var. nicotianae against metalaxyl and effectively control its damage, new efficient complex agent of metalaxyl was studied and developed. [ Method] The toxieities of nine fungicides against P. parasitica were measured using growth rate method. On this basis, the fungicides with good effects were selected to compound with metalaxyl, and the optimum complex ratio was confirmed. [Result] The toxicity of metalaxyl was the strongest with EC50 of 2. 130 5μg/ml; followed by carbendazim, mancezeb and dimethomorph with EC50 of 2.357 9, 2.639 8 and 2. 778 8 μg/ml. The effect of cyazofamid was the poorest with EC50 of 6. 278 8 μg/ml. The optimum complex ratios of dimethomorph, carbendazim and mancezeb with metalaxyl were 40： 60, 30：70 and 20： 80, and their co-toxicity coefficients were 138.80,124.25 and 115.00, respectively. [ Conclusion] The complex agents had application and promotion value, which could be used to carry out further field trials.

基于SSR分子标记的Nicotiana tobacum–N.plumbaginifolia异源染色体植株的鉴定与筛选

以烟草(Nicotiana tabacum)品种云烟87的八倍体(2n=8x=96)和野生烟草N. plumbaginifolia (2n=2x=20)的基因组DNA为模板,对340对烟草SSR引物进行筛选以获得能扩增多态性条带的引物。利用多态性引物对种间杂交后代及190株回交后代的基因组DNA进行扩增,并对N.plumbaginifolia中的SSR标记的连锁情况进行简要分析。经筛选获得了多态性引物29对。结果显示,在190株后代中, 159株的基因组DNA能扩增出N. plumbaginifolia的特异SSR位点,可以判定该159株为N. tabacum的N. plumbaginifolia异源染色体植株,其余31株植株可能不含有N. plumbaginifolia的染色体。经UPGMA聚类分析,本群体中植株的遗传多样性较为丰富,部分分子标记在后代中的出现具有完全相关性。29个标记中14个可确定来源于5条不同染色体,N.plumbaginifolia的29个位点在回交后代中的扩增效率并不相同,且效率均较低(低于31.00%),说明该杂种中N. plumbaginifolia基因组的垂直传递效率较低。利用SSR分子标记可以判定云烟87八倍体与N.plumbaginifolia杂交获得的后代为真杂种,且自该远缘杂种回交后代中筛选获得大量异源染色体植株。这些结果和筛选获得异源染色体植株为进一步创制N.tabacum-N.plumbaginifolia抗黑胫病单体附加系以及易位系奠定了基础。

A Preliminary Study on the Resistance to Different Fungicides in Phytophthora parasitica var. nicotianae

[Objective] This study aimed to investigate the resistance to different fungicides in Phytophthora parasitica var. nicotianae. [Method] Under indoor incubation conditions, the resistance to dimethomorph, metalaxyl-mancozeb, propamocarb and ovraclostrobin.dimethomorph in P. parasitica strain isolated from Zhenyuan County in Qiandongnan State was analyzed with colony growth measurement method. [Result] P. parasitica exhibited different levels of sensitivity to four fungicides. To be specific, P. parasitica exhibited the highest resistance to dimethomorph, and ECho reached 1.19 μg/ml. [Conclusion] In Zhenyuan tobacco-growing area, long-term single use of dimethomorph possesses certain resistance risk in prevention and control of black shank disease.

枯草芽孢杆菌(Bacillus subtilis)cspB基因转化烟草(Nicotiana tabacuum)的研究

细菌冷休克蛋白cspB是原核生物RNA的分子伴侣,含有原始型的冷休克结构域,具有与核酸结合功能,可以防止RNase对mRNA的降解,也能纠正mRNA的折叠错误。为了寻找作物改良的潜在基因资源,从枯草芽孢杆菌XS-01基因组中克隆出cspB基因,并与pBI121构建成pBI121-cspB植物表达载体。利用叶圆盘转化法转化烟草,通过卡那霉素筛选和PCR、Southern杂交鉴定5个转化体株系。除TN010外,其他转基因株系在外观形态与野生亲本的没有差别,但TN001和TN011育性降低,TN010和TN012则完全丧失育性。干旱处理结果表明,转基因植株在土壤水分恢复后10 d,其平均单株干物质质量较之野生亲本的显著增加;叶片光合速率测定结果表明,在干旱处理时,转基因植株和对照亲本叶片光合速率均显著下降,在土壤水分恢复后,转基因植株的光合速率能快速恢复,但对照亲本的恢复缓慢。实验结果说明,cspB能够促进植物细胞从逆境伤害中快速恢复功能。

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.

Functional characterization of a potassium transporter gene NrHAK1 in Nicotiana rustica

The purpose of this study is to investigate the function of a novel potassium transporter gene (NrHAK1) isolated from Nicotiana rustica roots using yeast complement and real-time PCR technique. The complementary DNA (cDNA) of NrHAK1, 2 488 bp long, contains an open reading frame (ORF) of 2 334 bp encoding a protein of 777 amino acids (87.6 kDa) with 12 predicted transmembrane domains. The NrHAK1 protein shows a high sequence similarity to those of high-affinity potassium transporters in Mesembryanthemum, Phytolacca acinosa, Arabidopsis thaliana, and so on. We found that the NrHAK1 gene could complement the yeast-mutant defect in K+ uptake. Among several tissues surveyed, the expression level of NrHAK1 was most abundant in the root tip and was up-regulated when exposed to potassium starvation. Moreover, the transcript accumulation was significantly reduced by adding 5 mmol/L NH4+ to the solution. These results suggest that NrHAK1 plays an important role in potassium absorption in N. rustica.

Sugarcane mosaic virus infection of model plants Brachypodium distachyon and Nicotiana benthamiana

Sugarcane mosaic virus (SCMV;genus Potyvirus, family Potyviridae) is a causal pathogen of sugarcane mosaic disease, and it is widespread in regions where sugarcane (Saccharum spp. hybrids) is grown. It is difficult to investigate the molecular mechanism of pathogen infection in sugarcane because of limited genomic information. Here, we demonstrated that SCMV strain FZ1 can systemically infect Brachypodium distachyon inbred line Bd21 and Nicotiana benthamiana through inoculation, double antibody sandwich enzyme-linked immunosorbent, transmission electron microscopy, and reverse transcription PCR assays. The leaves of Bd21 developed mosaic symptoms, while the leaves of N. benthamiana showed no obvious symptoms under the challenge of SCMV-FZ1. We concluded that B. distachyon inbred line Bd21 is a promising experimental model plant compared with N. benthamiana for study on the infectivity of SCMV. This is the first report on the SCMV infection of model plants B. distachyon inbred line Bd21 and N. benthamiana, which will shed light on the mechanism of SCMV infection of sugarcane and benefit sugarcane breeding against sugarcane mosaic disease.

具翼烟草(Nicotiana alata)核型、C带与rDNA染色体定位研究

采用去壁低渗法、BSG法和双色荧光原位杂交技术,分别开展了具翼烟草(Nicotiana alata)的核型分析、C显带与45S和5S rDNA染色体定位研究。4个具翼烟草收集系根尖细胞有丝分裂中期染色体平均核型为2n=2x=12m+6st,染色体臂比的变异幅度为1.14~4.22,平均臂比值为2.17,最长与最短染色体之比为2.16,属2B型。C带研究结果显示,具翼烟草中期染色体C带丰富,多态性良好,共显带25条,包括7条着丝粒带、15条端带和3条中间带。45S和5S rDNA染色体定位研究结果显示,具翼烟草根尖细胞有丝分裂中期染色体细胞相分别显示了3对45S rDNA和2对5S rDNA基因位点信号,45S rDNA基因位点分别位于I S、III S、V L上,2对5S rDNA基因位点分别位于I L、IV S上(字母L和S分别代表染色体长臂和短臂,罗马数字代表同源染色体序号)。在I号同源染色体上同时存在45S和5S rDNA基因位点。研究结果对烟草属系统起源、遗传进化以及遗传育种等领域的进一步研究具有重要的参考价值。