Tobacco proteinase inhibitor I genes are locally,but not systemically induced by stress

A cDNA library of tobacco mosaic virus (TMV)-infected tobacco was screened with polymerase chain reaction products obtained using a degenerate primer corresponding to proteinase inhibitor I (PI-I) of tomato and potato. The resulting clones encoded two highly similar, putative tobacco PI-I proteins, indicating that both genes identified in tobacco are probably expressed. The tobacco PI-I's were approximately 50% identical to wound-inducible potato and tomato PI-I and 80% identical to an ethylene-regulated tomato PI-I. Northern blot analyses indicated that healthy tobacco leaf contains only minor amounts of PI-I mRNA, and that the inhibitor genes are induced by TMV infection, salicylate treatment, ethephon spraying, UV light irradiation and wounding. The results indicate that the tobacco PI-I genes are coordinately expressed with the genes for the basic pathogenesis-related proteins. Contrary to PI-I genes of tomato and potato, wound induction of the tobacco genes occurs only locally; the upper, unwounded leaves do not show any wound-induced PI-I gene expression.     

转双基因烟草对棉铃虫的杀虫活性评价

以含Ｂｔ杀虫蛋白基因（单基因）烟草和常规烟草为对照,系统测定了含Ｂｔ与豇豆胰蛋白酶抑制剂蛋白基因（双基因）的抗虫烟草对棉铃虫不同龄期幼虫的杀虫活性。结果表明：１￣３龄幼虫取食转双基因烟草３ｄ后死亡率为８０．５％￣９９．３％,取食６ｄ后死亡率达１００％,均显著高于转单基因烟草。２龄幼虫取食转基因烟草３ｄ后死亡率为８０．５％￣９９．３％,取食６ｄ后死亡率达１００％,均显著高于转单基因烟草。２龄幼虫取食     

烟草细胞色素P450的基因组学分析

细胞色素P450是一类含血红素的单加氧酶超基因家族, 在植物多种代谢途径中起着重要作用。为了解烟草中的P450的种类和数量, 文章将植物代表性P450蛋白质序列与烟草基因组序列比对, 在烟草基因组中鉴定了44个P450家族共263个成员。将这些烟草P450基因与烟草表达序列标签(EST)比对, 发现173个成员有EST证据。通过与拟南芥中已知的P450蛋白序列比较, 分析了部分烟草P450蛋白序列的特征和二级结构。根据烟草基因芯片数据和部分基因的RT-PCR结果, 发现73个烟草P450基因能够在不同的生长发育时期表达, 其中部分基因具有组织特异性。这些研究结果为烟草P450基因功能的深入分析奠定了基础。     

从烟草EST微卫星标记中筛选合子胚中优势表达的基因

为获得烟草合子胚中的优势表达基因,利用CAP3程序对来自烟草公共数据库的EST序列进行组装,利用MISA程序从组装后的EST中筛选SSR位点,将多态性的SSR位点在烟草合子文库中进行扩增并对等位基因进行分析。结果表明:具有多态性的16个SSR标记中,有9个基因能从烟草的合子库中成功扩增得到。该研究为筛选烟草合子胚中优势表达基因提供新的途径。     

Structure of tobacco genes encoding pathogenesis-related proteins from the PR-1 group.

Infection of Samsun NN tobacco with tobacco mosaic virus (TMV) was found to induce the synthesis of mRNA encoding a basic protein with a 67% amino acid sequence homology to the known acidic pathogenesis-related (PR) proteins 1a, 1b and 1c. By Southern blot hybridization it was shown that the tobacco genome contains at least eight genes for acidic PR-1 proteins and a similar number of genes encoding the basic homologues. Clones corresponding to three of the genes for acidic PR-1 proteins were isolated from a genomic library of Samsun NN tobacco. The nucleotide sequence of these genes and their flanking sequences were determined. One clone was found to correspond to the PR-1a gene; the two other clones do not correspond to known TMV-induced PR-1 mRNA's and may represent silent genes. Compared to the PR-1a gene, these genes contain an insertion or deletion in the putative promoter region and mutations affecting the PR-1 reading frame.     

烟草Trihelix转录因子家族鉴定及表达分析

运用生物信息学分析方法,通过对烟草(Nicotiana tabacum)全基因组数据中的Trihelix家族基因的理化性质、染色体分布、染色体共线性、基序组成、基因结构、顺式作用元件及其低温处理下的表达进行完整的鉴定分析,初步解析烟草Trihelix家族成员的结构与功能。从普通烟草中鉴定出32个Trihelix基因,分为GT-1、GT-2、GTγ、SH4、SIP1等5个亚家族;32个NtTH基因有着10种分布不均匀的基序,且有不同的基因结构,但在同一个亚家族内有相似性;烟草与番茄(Lycopersicon esculentum)、拟南芥(Arabidopsis thaliana)、水稻(Oryza sativa)存在同源的Trihelix基因,普通烟草Trihelix家族自身也存在共线性基因;鉴定出13种不同类型的顺式元件,主要与非生物胁迫、激素和生长发育有关,其中ABRE和MeJA是最多的两个顺式元件,表明NtTH基因的功能主要与非生物胁迫和生长发育响应调控有关;烟草NtTH3和NtTH16等11个基因在对低温胁迫的响应方面有着重要作用。本研究挖掘到与烟草抗冷有关的基因,为烟草抗冷性遗传改良提供了理论依据。     

烟草CONSTANS-like基因家族的鉴定与分析

烟草(Nicotiana tabacum)是基因功能分析的模式植物以及重要的经济作物之一, 适宜的生存环境对烟草的生长和繁殖至关重要。COL (CONSTANS-like)基因家族编码蛋白不仅调控植物开花, 而且在植物生物/非生物胁迫响应中发挥重要作用。该研究通过鉴定烟草COL基因家族成员, 分析其基因结构、进化关系、转录调控元件和表达模式, 探究其编码蛋白的生物学功能, 尤其是在烟草响应低温胁迫中的可能作用。结果显示, 在烟草中共鉴定出15个COL基因, 其编码的蛋白理化性质相近; 进化分析结果表明其包括3类, 每个类别的成员之间具有相似的外显子/内含子结构以及motif数量和类型; 烟草COL基因启动子区域含有大量与光、低温、干旱以及植物激素等响应相关的顺式作用元件; 基于二代高通量测序分析结果表明, 低温显著影响烟草COL基因的表达, 但对不同基因的影响存在差异, 不同COL基因的亲本(林烟草(N. sylvestris) (母本)和绒毛状烟草(N. tomentosiformis) (父本))具有表达偏好性, 且这种偏好性大部分会从6-7叶期保持到现蕾期。     

转基因抗虫烟草研究进展

烟草为模式植物,也是外源杀虫基因最早转化成功的植物。文章从转Bt内毒素基因,植物凝集素GNA,Plec,AHA基因,蛋白酶抑制剂PIⅠ,PIⅡ,MTI,SKTI基因,昆虫特异性神经毒素基因,几丁质酶基因,畸形细胞分泌蛋白基因以及双抗虫基因等方面综述了转基因抗虫烟草的抗虫性、转基因抗虫烟草的经济性状等,展望了转基因抗虫烟草的研究和应用前景,以期对烟草害虫的治理尤其是对其他转基因抗虫作物的培育和研究有借鉴作用。     

Engineering photoassimilate partitioning in tobacco plants improves growth and productivity and provides pathogen resistance

Expression of pathogenesis-related (PR) genes is part of the plant's natural defense response against pathogen attack. To study the in vivo role and function of the maize PRms protein, tobacco plants were transformed with the PRms cDNA under the control of the CaMV35S promoter. Transgenic tobacco plants grow faster and yield more leaf and seed biomass. By using immunoelectron microscopy, we found that PRms is associated with plasmodesmata in leaves of transgenic tobacco plants. Furthermore, we found that activation of sucrose efflux from photosynthetically active leaves and accumulation of higher levels of sucrose in leaf tissues are characteristic features of PRms tobacco plants. This, in turn, results in the constitutive expression of endogenous tobacco PR genes and resistance to phytopathogens. The expression of multiple plant defense genes can then be achieved by using a single transgene. These data provide a new approach for engineering disease-resistant plants while simultaneously improving plant yield and productivity through the modification of photoassimilate partitioning.     

An HR-induced tobacco peroxidase gene is responsive to spermine, but not to salicylate, methyl jasmonate, and ethephon

In Tobacco mosaic virus (TMV)-infected tobacco plants carrying the N resistance gene, a hypersensitive reaction or response (HR) occurs to enclose the virus in the infected tissue. Although a contribution of peroxidases to the resistance has been proposed, no evidence has been presented that tobacco peroxidase genes respond to HR. Here, we describe the HR-induced expression of a tobacco peroxidase gene (tpoxC1) whose induction kinetics were slightly different from those of acidic and basic tobacco pathogenesis-related (PR) protein genes. Interestingly, tpoxC1 was insensitive to the inducers of PR genes such as salicylic acid, methyl jasmonate, and ethephon. Spermine activated tpoxC1 gene expression at a low level and both acidic and basic PR gene expression at a considerably higher level. These results indicate that the induced expression of tpoxC1 is regulated differently from that of classical tobacco PR genes in the N gene-mediated self-defense system in tobacco plants.     

Introduction of bacterial metabolism into higher plants by polycistronic transgene expression

Multiple-gene transformation is required to improve or change plant metabolisms effectively; but this many-step procedure is time-consuming and costing. We succeeded in the metabolic engineering of tobacco plants by introducing multiple genes as a bacteria-type operon into a plastid genome. The tobacco plastid was transformed with a polycistron consisting of three bacterial genes for the biosynthesis of a biodegradable polyester, polyhydroxybutyrate (PHB). Accumulation of PHB in the leaves of the transgenic tobacco indicated that the introduced genes were polycistronically expressed. This "phyto-fermentation" system can be used in plant production of various chemical commodities and pharmaceuticals.     

Preliminary studies on differential defense responses induced during plant communication

We compared the expression patterns of three representative genes in undamaged tomato and tobacco plants in response to exposure to either tomato or tobacco fed on by Helicoverpa armigera (cotton bollworm). When tomato and tobacco, two species of one family, were incubated in the chambers with the tomato plants damaged by the cotton bollworm, the expression of the PR1, BGL2, and PAL genes was up-regulated in leaves of both plants. However, the levels of gene expression were significantly higher in the tomato than that in the tobacco. In addition, the activities of enzymes, peroxidase, polyphenol oxidase, and lipoxygenase were found to be higher in the tomato than those in the tobacco. Similar results were obtained when the damaged plants were replaced by the tobacco.     

Cloning and expression of two genes encoding auxin-binding proteins from tobacco

Two genes encoding the auxin-binding protein (ABP1) of tobacco (Nicotiana tabacum L.), both of which possess the characteristics of a luminal protein of the endoplasmic reticulum (ER), were isolated and sequenced. These genes were composed of at least five exons and four introns. The two coding exons showed 95% sequence homology and coded for two precursor proteins of 187 amino acid residues with molecular masses of 21 256 and 21 453 Da. The deduced amino acid sequences were 93% identical and both possessed an amino-terminal signal peptide, a hydrophilic mature protein region with two potential N-glycosylation sites and a carboxyl-terminal sorting signal, KDEL, for the ER. Restriction mapping of the cDNAs encoding tobacco ABP1, previously purified by amplification of tobacco cDNA libraries by polymerase chain reaction (PCR) using specific primers common to both genes, indicated that both genes were expressed, although one was expressed at a higher level than the other. Genomic Southern blot hybridization showed no other homologous genes except for these two in the tobacco genome. The apparent molecular mass of the mature form of tobacco ABP1 was revealed to be 25 kDa by SDS polyacrylamide gel electrophoresis using affinity-purified anti (tobacco ABP1) antibodies raised against a fusion protein with maltose-binding protein. Expression of the recombinant ABP1 gene in transgenic tobacco resulted in accumulation of the 25 kDa protein. A single point mutation of an amino acid residue at either of the two potential N-glycosylation sites resulted in a decrease in the apparent molecular mass and produced a 22 kDa protein. Mutations at both sites resulted in the formation of a 19.3 kDa protein, suggesting that tobacco ABP1 is glycosylated at two asparagine residues.     

Circadian expression and induction by wounding of tobacco genes for cysteine proteinase

Two sets of clones were isolated from a tobacco cDNA library, utilizing as a probe a PCR fragment obtained from tomato cDNA using a degenerate primer based on the sequence of tomato systemin. Contrary to expectation, the clones did not correspond to tobacco homologues of tomato pro-systemin. However, the cDNAs encoded two highly similar proteins with extensive structural homology to cysteine proteinases from a wide range of plant and animal species. Northern blot analyses showed that in unstressed tobacco leaf the genes for the putative proteinases are expressed according to a circadian rhythm. Furthermore, incision wounding enhances the expression approximately six-fold. Other forms of stress, such as infection with tobacco mosaic virus, treatment with ethephon or UV light do not result in induced expression of the tobacco cysteine proteinase genes.     

Expression of the ACC synthase and ACC oxidase coding genes after self-pollination and incongruous pollination of tobacco pistils

In tobacco, as in other species, ethylene is produced in response to pollination. Although tobacco is a self-compatible species, it displays unilateral incongruity with other Nicotianaplants. Incongruous pollination also results in ethylene production, but this production differs depending on the pollen used and is related to the extent to which pollen tubes grow in the tobacco style. In the investigation reported here we followed the expression of the ACC synthase- and ACC oxidase-coding genes upon pollination of tobacco pistils and compared self-pollination with incongruous pollination. The pattern of expression of these genes also correlated with pollen-tube growth, although wounding alone cannot explain the results obtained. We also examined the expression of these genes upon pollination of immature tobacco pistils, in which different pollen tubes grew indistinctly inside the tobacco style and reached the ovary at the same rate. In this situation no significant differences in gene expression could be observed between the different pollinations. Ethephon, a substance that produces ethylene, could, in some cases, minimize the arrest of incongruous pollen tubes inside the style.