怀地黄叶片中总环烯醚萜苷及总苯乙醇苷含量快速分析模型的建立

本文旨在建立地黄叶片中总环烯醚萜苷及苯乙醇苷定量分析模型。利用紫外-可见分光光度法测定不同种质怀地黄生育期内的128份地黄叶片中总环烯醚萜苷及总苯乙醇苷的含量,并将其作为基础值,结合地黄叶片的近红外光谱图,利用TQ8.0分析软件结合偏最小二乘法(PLS),分别建立地黄叶片中总环烯醚萜苷及总苯乙醇苷的定量分析模型。地黄叶片中总苯乙醇苷定量校正模型决定系数(R2)为0.998 2,校正均方根偏差(RMSEC)为0.089 9,预测均方决定差(RMSEP)为0.142,交叉验证均方根偏差(RMSECV)为0.707 2;总环烯醚萜苷定量校正模型的内部交叉验证决定系数(R2)为0.972 1,校正均方差(RMSEC)为0.259,预测均方决定差(RMSEP)为0.095 4,交叉验证均方根偏差(RMSECV)为0.869 4。预测值与实测值差异无统计学意义。该定量模型可用于怀地黄叶片中总环烯醚萜苷及总苯乙醇苷含量的快速测定。     

Functional analysis of the 3'' control region of the potato wound-inducible proteinase inhibitor II gene.

Proteinase inhibitor genes are expressed strongly in specific plant tissues under both developmental and environmental regulation. We have studied the role of the 3' control region of the potato proteinase inhibitor II gene (PI-II) that is inducible in leaves in response to herbivore attacks or other severe wounding. Comparison of the terminator from the PI-II gene with two different terminators from the 6b and 7 genes, driven by a common PI-II promoter-cat fusion molecule, indicated that the PI-II terminator provided the most efficient expression of cat. The PI-II terminator also caused a significantly elevated cat gene expression driven by the cauliflower mosaic virus 35S promoter. The increase in the level of expression is probably not due to the presence of an enhancer element in the PI-II terminator region, but to cis-acting elements involved in mRNA processing or stability. Both transient and stable transformation analyses of the deletion mutants in the 3'-flanking sequence indicated that about a 100-base pair DNA fragment surrounding the polyadenylation site is essential for the efficient gene expression. This region seems to consist of several regulatory elements, including the conserved sequence, CGTGTCTT, which is located 9 bases downstream from the polyadenylation site. The elements appear to contribute to the increased stability of mRNAs containing the PI-II terminator.     

Differential expression of the potato proteinase inhibitor II promoter in transgenic tobacco

We studied temporal and spatial expression patterns of the potato proteinase inhibitor II (PI-II) promoter, using transgenic tobacco (Nkotiana tabacum L cv. Xanthi) plants that carried a fusion between the PI-II promoter and the chloramphenicol acetyltransferase (cat) gene. Pl-ll promoter activity was low when plants were young, but increased as plants grew. In 8-week-old plants, old leaves showed higher activity than young leaves. At flowering stage (ca. 15 weeks), the overall promoter activity was reduced to a lower level except in the petals. Compared with stems or petioles at the flowering stage, the roots and floral organs showed minimal activity for the Pl-ll promoter. We used several environmental stimuli to examine the induction of the Pl-ll promoter in different organs. Promoter induction was effected by wounding or methyl jasmonate in stems, petioles, sepals, and leaves. The induction was highest in leaves, as was sucrose-enhanced wound induction. These results suggest that the Pl-ll gene is temporally and spatially regulated. We also established a transient assay system in tobacco BY2 suspension cells to elucidate the upstream regulatory region of the Pl-ll promoter. A field strength of 0.75 kV/cm and 400 μF capacitance were optimal electroporation conditions for our transient assay.     

Wound-induced expression of a tobacco peroxidase is not enhanced by ethephon and suppressed by methyl jasmonate and coronatine

In tobacco plants, wounding induces production of a set of defense-related proteins such as basic pathogenesis-related (PR) proteins and proteinase inhibitors (PIs) via the jasmonate/ethylene pathway. Although class III plant peroxidase (POX) is also wound-inducible, the regulatory mechanism for its wound-induced expression is not fully understood. Here, we describe that a tobacco POX gene (tpoxN1), which is constitutively expressed in roots, is induced locally 30 min after wounding and then systemically in tobacco plants. Infection of necrotizing virus also induced tpoxN1 gene. The wound-induced expression was not enhanced by known wound-signal compounds such as methyl jasmonate (MeJA) and ethephon in contrast to other wound-inducible genes such as basic PR-1 and PI-II genes. And treatment with MeJA and coronatine, biological analogs of jasmonate, rather suppressed the tpoxN1 expression. Salicylic acid, an antagonist of jasmonate-based wound signaling, did not suppress the wound-induced expression of tpoxN1. Only spermine, which is reported as an endogenous inducer for acidic PR genes in tobacco mosaic virus-infected tobacco leaves, could induce tpoxN1 gene expression. These results suggest that wound-induced expression of the tpoxN1 gene is regulated differently from that of the basic PR and PI-II genes.     

PP333对怀地黄试管苗形态及生理特性的影响

研究PP333对怀地黄试管苗生长及一些生理指标的影响。通过单因子实验、比色法和愈创木酚法探讨PP333对试管苗的影响。结果表明,不同浓度PP333均促进试管苗芽的萌发,使根系粗壮,根数增加,低浓度PP333(0.01、0.05mg·L-1)促进试管苗茎的伸长生长,高浓度(0.1、2mg·L-1)抑制茎、叶生长,PP333浓度为2mg·L-1时壮苗效果最佳。PP333处理使试管苗生长中期叶片可溶性蛋白含量、POD活力提高。适宜浓度的PP333可以改变试管苗的生理特性,达到培育壮苗的目的。     

Crystal structures of the Nicotiana glutinosa ribonuclease NT in complex with nucleoside monophosphates

Ribonuclease NT (RNase NT), induced upon tobacco mosaic virus (TMV) infection in Nicotiana glutinosa leaves, has a broad base specificity. The crystal structures of RNase NT in complex with either 5'-AMP, 5'-GMP, or 2'-UMP were determined at 1.8 A resolutions by molecular replacement. RNase NT consists of seven helices and seven beta strands, and the structure is highly similar to that of RNase NW, a guanylic acid preferential RNase from the N. glutinosa leaves, showing root mean square deviation (rmsd) of 1.1 A over an entire length of two molecules for Calpha atoms. The complex structures revealed that Trp42, Asn44, and Trp50 are involved in interactions with bases at B1 site (primary site), whereas Gln12, Tyr17, Ser78, Leu79, and Phe89 participate in recognition of bases at B2 site (subsite). The 5'-GMP and 5'-AMP bind both B1 and B2 sites in RNase NT, while 2'-UMP predominantly binds B1 site in the complex. The nucleotide binding modes in these complexes would provide a clue to elucidation of structural basis for the broad base specificity for RNase NT.     

Selective loss of cysteine residues and disulphide bonds in a potato proteinase inhibitor II family

Disulphide bonds between cysteine residues in proteins play a key role in protein folding, stability, and function. Loss of a disulphide bond is often associated with functional differentiation of the protein. The evolution of disulphide bonds is still actively debated; analysis of naturally occurring variants can promote understanding of the protein evolutionary process. One of the disulphide bond-containing protein families is the potato proteinase inhibitor II (PI-II, or Pin2, for short) superfamily, which is found in most solanaceous plants and participates in plant development, stress response, and defence. Each PI-II domain contains eight cysteine residues (8C), and two similar PI-II domains form a functional protein that has eight disulphide bonds and two non-identical reaction centres. It is still unclear which patterns and processes affect cysteine residue loss in PI-II. Through cDNA sequencing and data mining, we found six natural variants missing cysteine residues involved in one or two disulphide bonds at the first reaction centre. We named these variants Pi7C and Pi6C for the proteins missing one or two pairs of cysteine residues, respectively. This PI-II-7C/6C family was found exclusively in potato. The missing cysteine residues were in bonding pairs but distant from one another at the nucleotide/protein sequence level. The non-synonymous/synonymous substitution (Ka/Ks) ratio analysis suggested a positive evolutionary gene selection for Pi6C and various Pi7C. The selective deletion of the first reaction centre cysteine residues that are structure-level-paired but sequence-level-distant in PI-II illustrates the flexibility of PI-II domains and suggests the functionality of their transient gene versions during evolution.     

Molecular cloning of cDNAs encoding ribonuclease-related proteins in Nicotiana glutinosa leaves, as induced in response to wounding or to TMV-infection

We earlier isolated a cDNA clone (NGR1) encoding a wound-inducible ribonuclease (RNase NW) from leaves of Nicotiana glutinosa [Kariu et al. Biosci. Biotechnol. Biochem., 62, 1144-1151 (1998)]. In this study, two distinct cDNA clones, NGR2 and NGR3, encoding proteins with a ribonuclease-related sequence in the N. glutinosa leaves, were amplified and sequenced. The nucleotide sequences of NGR2 and NGR3 consist of 1244 bp and 1069 bp, and have open reading frames encoding 277 (RNase NGR2) and 236 (RNase NGR3) amino acid residues, respectively. The deduced amino acid sequences of the putative RNases NGR2 and NGR3 showed 33% and 58% amino acid sequence identity, respectively, with that of RNase NW and 32% identity with each other. Sequence comparison showed that NGR2 is similar to RNase RNS2 (61%) from Arabidopsis thaliana, while NGR3 is related to RNase LX (84%) from tomato (Lycopersicon esculentum). RNA gel blot analysis showed that the RNase NGR2 gene is constitutively expressed to measurable levels; it is not increased by either wounding or TMV infection. In contrast, the expression of the NGR3 gene is induced after 48 h upon TMV infection.     

Comparison of transient protein expression in tobacco leaves and plant suspension culture

Transient gene expression is being developed to provide a more rapid means of assessing plant tissues as a protein production platform without the labor-intensive and time-consuming process of generating stably transformed transgenic plants. Transient expression of the gus-intron reporter gene was facilitated in three different tobacco species. Two different approaches to T-DNA delivery were compared: (1) infiltration of a prototrophic strain of Agrobacterium into leaves and (2) coculture of plant cell suspension cultures with an Agrobacterium auxotroph. Wounding of plant tissues with a wire brush prior to infiltration had a large positive impact on Nicotianabenthamiana leaves but not for Nicotiana tabacum or Nicotiana glutinosa. The best expression level achieved by leaf infiltration was in N. benthamiana (0.025% total soluble protein). A cell suspension culture line of N. glutinosa achieved an expression level greater than 0.04% TSP. The tissue culture-based technique therefore provides improved levels of transient expression under aseptic conditions to facilitate improvements in expression by control of the plant cell culture and Agrobacterium coculture environments.     

N2 fixation and cycling in Alnus glutinosa, Betula pendula and Fagus sylvatica woodland exposed to free air CO2 enrichment

We measured the effect of elevated atmospheric CO(2) on atmospheric nitrogen (N(2)) fixation in the tree species Alnus glutinosa growing in monoculture or in mixture with the non-N(2)-fixing tree species Betula pendula and Fagus sylvatica. We addressed the hypotheses that (1) N(2) fixation in A. glutinosa will increase in response to increased atmospheric CO(2) concentrations, when growing in monoculture, (2) the impact of elevated CO(2) on N(2) fixation in A. glutinosa is the same in mixture and in monoculture and (3) the impacts of elevated CO(2) on N cycling will be evident by a decrease in leaf δ(15)N and by the soil-leaf enrichment factor (EF), and that these impacts will not differ between mixed and single species stands. Trees were grown in a forest plantation on former agricultural fields for four growing seasons, after which the trees were on average 3.8 m tall and canopy closure had occurred. Atmospheric CO(2) concentrations were maintained at either ambient or elevated (by 200 ppm) concentrations using a free-air CO(2) enrichment (FACE) system. Leaf δ(15)N was measured and used to estimate the amount (N(dfa)) and proportion (%N(dfa)) of N derived from atmospheric fixation. On average, 62% of the N in A. glutinosa leaves was from fixation. The %N(dfa) and N(dfa) for A. glutinosa trees in monoculture did not increase under elevated CO(2), despite higher growth rates. However, N(2) fixation did increase for trees growing in mixture, despite the absence of significant growth stimulation. There was evidence that fixed N(2) was transferred from A. glutinosa to F. sylvatica and B. pendula, but no evidence that this affected their CO(2) response. The results of this study show that N(2) fixation in A. glutinosa may be higher in a future elevated CO(2) world, but that this effect will only occur where the trees are growing in mixed species stands.     

Reactive sites of an anticarcinogenic Bowman-Birk proteinase inhibitor are similar to other trypsin inhibitors.

The three-dimensional structure of the Bowman-Birk type proteinase inhibitor (PI-II) has been determined by x-ray crystallography and refined at 2.5-A resolution. This protein is a specific inhibitor of trypsin. Two reactive site loops, one at each end of the PI-II molecule, are structurally similar to each other and to reactive-site loops of pancreatic secretory trypsin inhibitor (Bolognesi, M., Gatti, G., Menegatti, E., Guarneri, M., Marquart, M., Papamokos, E., and Huber, R. (1982) J. Mol. Biol. 162, 839-869) and bovine pancreatic trypsin inhibitor (Deisenhofer, J., and Steigemann, W. (1975) Acta Crystallogr. B31, 238-250). PI-II is the first reported Bowman-Birk type inhibitor structure to be refined at high resolution, providing further insight into inhibitor mechanisms.     

地黄叶和茎的解剖学及组织化学研究

采用解剖学和组织化学方法对地黄叶和茎的显微结构以及梓醇、多糖的分布进行观察研究,以明确梓醇和多糖在地黄叶和茎中的分布特征。结果显示:(1)地黄叶的上、下表皮均分布有腺毛和非腺毛,腺毛都属于头状腺毛,包括长柄和短柄的头状腺毛,两类腺毛的分泌物化学成分主要是黄酮和多糖;叶的上、下表皮上都分布有无规则型气孔,下表皮的气孔密度比上表皮的大,但气孔指数相差不大;栅栏组织由2～3层薄壁细胞构成,排列紧密,海绵组织薄壁细胞形状无规则,细胞间隙大。(2)组织化学研究表明,海绵组织中黄斑样的薄壁细胞是梓醇和多糖的贮存场所,这类薄壁细胞在叶片边缘的齿末端处最为集中,茎的皮层、韧皮部和木质部的薄壁细胞也都是梓醇和多糖的贮存场所。     

Structural and functional characteristics of plant proteinase inhibitor-II (PI-II) family

Plant proteinase inhibitor-II (PI-II) proteins are one of the promising defensive proteins that helped the plants to resist against different kinds of unfavorable conditions. Different roles for PI-II have been suggested such as regulation of endogenous proteases, modulation of plant growth and developmental processes and mediating stress responses. The basic knowledge on genetic and molecular diversity of these proteins has provided significant insight into their gene structure and evolutionary relationships in various members of this family. Phylogenetic comparisons of these family genes in different plants suggested that the high rate of retention of gene duplication and inhibitory domain multiplication may have resulted in the expansion and functional diversification of these proteins. Currently, a large number of transgenic plants expressing PI-II genes are being developed for enhancing the defensive capabilities against insects, bacteria and pathogenic fungi. Much emphasis is yet to be given to exploit this ever expanding repertoire of genes for improving abiotic stress resistance in transgenic crops. This review presents an overview about the current knowledge on PI-II family genes, their multifunctional role in plant defense and physiology with their potential applications in biotechnology.     

The suppressor of transgene RNA silencing encoded by Cucumber mosaic virus interferes with salicylic acid-mediated virus resistance

The Cucumber mosaic virus (CMV)-encoded 2b protein (Cmv2b) is a nuclear protein that suppresses transgene RNA silencing in Nicotiana benthamiana. Cmv2b is an important virulence determinant but nonessential for systemic spread in N. glutinosa, in contrast to its indispensable role for systemic infections in cucumber. Here, we report that Cmv2b became essential for systemic infections in older N. glutinosa plants or in young seedlings pretreated with salicylic acid (SA). Expression of Cmv2b from the genome of either CMV or Tobacco mosaic virus significantly reduced the inhibitory effect of SA on virus accumulation in inoculated leaves and systemic leaves. A close correlation is demonstrated between Cmv2b expression and a reduced SA-dependent induction of the alternative oxidase gene, a component of the recently proposed SA-regulated antiviral defense. These results collectively reveal a novel activity of Cmv2b in the inhibition of SA-mediated virus resistance. We used a N. tabacum line expressing a bacterial nahG transgene that degrades SA to provide evidence for a Cmv2b-sensitive antiviral defense mechanism in tobacco in which SA acts as a positive modifier but not as an essential component. We propose that SA induces virus resistance by potentiating a RNA-silencing antiviral defense that is targeted by Cmv2b.     

Inhibition of Sucrose Enhancer Effect of the Potato Proteinase Inhibitor II Promoter by Salicylic Acid

Effect of salicylic acid (SA) on the expression of the potato proteinase inhibitor (PI) II promoter was studied with transgenic tobacco plants (Nicotiana tabacum) carrying a gene fusion between the PI-II promoter and the chloramphenicol acetyltransferase (cat) reporter. As previously observed, the PI-II promoter was inducible by wounding and the promoter activity was further enhanced by sucrose. Addition of SA did not influence the wound induction of the PI-II promoter but significantly inhibited the sucrose response. The 5′-deletion mutant −573 was unable to respond to wounding but did respond to sucrose and SA. The 3′-deletion analysis indicated the presence of a sucrose-responsive element between −574 and −520. A study of the insertion mutants revealed the function of another sucrose-responsive element between −522 and −500. Enhancer effects of these sucrose-responsive elements were inhibited by SA. These studies suggest that SA inhibits PI-II promoter activity by decreasing the sucrose response. Analysis of SA-related chemicals revealed that only acetyl-SA showed a similar inhibitory effect, and other hydroxybenzoic acids had little or no effect on the sucrose enhancer activity. Therefore, it seems that the interaction between SA and the receptor molecule is specific.     

Uncoupling resistance from cell death in the hypersensitive response of Nicotiana species to cauliflower mosaic virus infection

Cauliflower mosaic virus strain W260 elicits a hypersensitive response (HR) in leaves of Nicotiana edwardsonii, an interspecific hybrid derived from a cross between N. glutinosa and N. clevelandii. Interestingly, we found that N. glutinosa is resistant to W260, but responds with local chlorotic lesions rather than necrotic lesions. In contrast, N. clevelandii responds to W260 with systemic cell death. The reactions of the progenitors of N. edwardsonii to W260 infection indicated that each contributed a factor toward the development of HR. In this study, we present two lines of evidence to show that the resistance and cell death that comprise the HR elicited by W260 can indeed be uncoupled. First, we showed that the non-necrotic resistance response of N. glutinosa could be converted to HR when these plants were crossed with N. clevelandii. Second, we found that cell death and resistance segregated independently in the F2 population of a cross between N. edwardsonii and N. clevelandii. We concluded that the resistance of N. edwardsonii to W260 infection was conditioned by a gene derived from N. glutinosa, whereas cell death was conditioned by a gene derived from N. clevelandii. An analysis of pathogenesis-related (PR) protein expression in response to W260 infection revealed that elicitation of PR proteins was associated with resistance rather than with the onset of cell death.     

Purification of potato virus X and preparation of its antiserum

Potato virus X (PVX) isolated from the potato leaf and tuber samples which were collected from various fields in Damavand and Ardabil. The initial isolations of the virus were made from potato by mechanical inoculation on Gomphrena globosa L. and Chenopodium spp. that produce local lesion, and then it causes mosaic on Nicotiana spp. and Datura stramonium L. An isolate of the virus inoculated to Nicotiana glutinosa L. and it was maintained throughout the work. Sap from infected N. glutinosa was ineffective after dilution to 10-6, 10 minutes at 70 degrees and 10 weeks at room temperature. The virus was readily purified from infected leaves and the best protocol was Moreira & Jones 1980 than the other 2 methods of Fribourg 1975 and Shepard & Shalla 1972. Antisera were prepared against native, degraded proteins and micro precipitin test showed that both antisera had a 1/512 titer. Precipitin lines with D - Protein antiserum was better of the native protein antiserum in agar double diffusion test than treated with SDS. The isolate of the virus was not transmitted by none of 2 species of Cuscuta but transmitted from infected leaves to healthy plants with sap inoculation without using Carburandum. This isolate showed positive reaction with gamaglubulin in kate received from CIP centre.     

Dianthins, ribosome-damaging proteins with anti-viral properties from Dianthus caryophyllus L. (carnation).

1. Dianthin 30 and dianthin 32, two proteins isolated from the leaves of Diathus caryophyllus (carnation), were purified to homogeneity by chromatography on CM-cellulose. 2. The mol.wt. of dianthin 30 is 29 500 and that of dianthin 32 is 31 700. Both dianthins are glycoproteins containing mannose. 3. Dianthins inhibit protein synthesis in a lysate of rabbit reticulocytes, with an ID50 (concentration giving 50% inhibition) of 9.15 ng/ml (dianthin 30) and 3.6 ng/ml (dianthin 32). They act by damaging ribosomes in a less-than-equimolar ratio. Protein synthesis by intact cells is partially inhibited by dianthins at a concentration of 100 microgram/ml. 4. Dianthins mixed with tobacco-mosaic virus strongly decrease the number of local lesions on leaves of Nicotiana glutinosa.