白桦反义CCoAOMT基因调控木质素生物合成

白桦是我国北方重要的造林树种,但其中的高木质素含量严重制约了它作为造纸资源植物的开发利用。本文利用RACE技术获得了白桦咖啡酰辅酶A-3-O-甲基转移酶（CCoAOMT）基因全长ORF序列,并构建了白桦CCoAOMT基因的反义表达载体,通过农杆菌介导法将其导入到白桦中。PCR检测表明反义CCoAOMT基因已整合到白桦的基因组中。对转化植株的半定量PCR检测显示转基因株系的CCoAOMT基因表达量下降;Wiesner染色发现,与野生型相比,转基因植株木质素含量有所下降。对七年生的转基因白桦和野生型对照进行了化学成分分析,结果表明转基因白桦的苯醇抽提物和Klason木质素显著减少,聚戊糖含量升高。上述结果暗示BpCCoAOMT基因参与白桦木质素的合成,反义表达该基因后木质素含量减少,更易于去除。白桦CCoAOMT基因对木质素的合成起重要作用,这为培育低木质素含量的制浆新品种白桦奠定了基础。     

Karyotype morphology and cytogeography in Brunnera and Cynoglottis (Boraginaceae)

A comparative study of karyotype morphology and heterochromatin patterns in Brunnera and Cynoglottis (Boraginaceae) was carried out with traditional methods and Giemsa C-banding. Two polymorphic species of Cynoglottis , each with two subspecies, and two of Brunnera were investigated using native population samples from the central-eastern Mediterranean and Middle East. Pollen size of these samples was measured to investigate relationships with ploidy level. C. barrelieri subsp. barrelieri and subsp. serpentinicola are characterized by In = 18 and smaller pollen grains in contrast to C. chetikiana subsp. chetikiana and subsp. paphlagonica , which are fundamentally tetraploid with 2n = 36. The occurrence of cytotypes with 2n –/2 and 2n = 24 in both subspecies of C. chetikiana , however, would suggest x = 6 as the original haploid number and x = 9 as a derived one. Furthermore, the finding of a hypoploid cytotype with 2n = 16 in C. barrelieri ssp. barrelieri was consistent with previous reports and suggested relationships with Anchusa. Karyoevolutionary processes possibly associated with such a wide chromosome variation in Cynoglottis are discussed. Brunnera macrophylla and B. orientalis share a complement of 2n= 12 and an apparently identical karyotype, which differs from Cynoglottis in terms of asymmetry, chromosome size and morphology. A basic C-banding style was present in Brunnera and Cynoglottis , but heterochromatin content increased from the former to the latter. The parallel increase in chromosome number, heterochromatin content and size of the pollen from Brunnera to Cynoglottis may reflect an evolutionary progression, and is consistent with the supposed ancient origin of Brunnera.     

Distinguishing between metabolically active and inactive roots by combined staining with 2,3,5-triphenyltetrazolium chloride and image colour analysis

The objective of the present work was to develop a method to distinguish between metabolically inactive and active parts of plant roots. White clover (Trifolium repens L.) roots were stained with 2,3,5-triphenyltetrazolium chloride (TTC) followed by root colour classification with an interactive scanner-based image analysis programme (WinRHIZO). Roots inactivated by boiling were unstained and pale brown, whereas fresh samples with predominantly metabolically active roots turned dark red, red or pale red after staining. A small amount of very young, presumable active roots (0.8% of total active root length) failed to stain red with TTC. The colour analysis of inactive and active roots was based on four colour classes for boiled roots and seven classes for fresh roots, respectively, as defined upon visual examination of images. Pixel colours falling outside the defined classes were allocated to the nearest defined class – an option that increased objectivity and stability and reduced the required number of colour classes. For the fresh white clover roots, 75–86% of the total root length was determined as active, while 3–7% of the boiled roots fell into the same category. The percentage of total root length measured by WinRHIZO that was identified as metabolically active was linearly correlated with the percentage of fresh roots in mixtures of fresh and boiled roots (R²=0.99). Colour classes chosen à priori from one experiment could be used to distinguish fairly satisfactorily between active and inactive roots of another white clover cultivar grown under other conditions, but failed to classify activity in ryegrass (Lolium multiflorum Lam.) root samples. In the latter case, colour classes needed to be re-defined in order to produce reliable data. Our work shows that WinRHIZOs colour identification sub-module provides a new promising tool to classify root activity as identified after staining with TTC, but colour classes must be carefully evaluated on every new occasion.     

Structural insights into the elastin mimetic (LGGVG)6 using solid-state 13C NMR experiments and statistical analysis of the PDB

Elastin is a crosslinked hydrophobic protein found in abundance in vertebrate tissue and is the source of elasticity in connective tissues and blood vessels. The repeating polypeptide sequences found in the hydrophobic domains of elastin have been the focus of many studies that attempt to understand the function of the native protein on a molecular scale. In this study, the central residues of the (LGGVG)(6) elastin mimetic are targeted. Using a combination of a statistical analysis based on structures in the Brookhaven Protein Data Bank (PDB), 1D cross-polarization magic-angle-spinning (CPMAS) NMR spectroscopy, and 2D off-magic-angle-spinning (OMAS) spin-diffusion experiments, it is determined that none of the residues are found in a singular regular, highly ordered structure. Instead, like the poly(VPGVG) elastin mimetics, there are multiple conformations and significant disorder. Furthermore, the conformational ensembles are not reflective of proteins generally, as in the PDB, suggesting that the structure distributions in elastin mimetics are unique to these peptides and are a salient feature of the functional model of the native protein.     

Evaluation of Rhodamine 123 As A Probe For Monitoring Mitochondrial Function In Trypanosoma Brucei Spp.

ABSTRACT. Rhodamine 123, a membrane potential-specific dye, has been evaluated as a probe to monitor the function of the mitochondrion in long slender bloodstream and procyclic trypomastigotes of several Trypanosoma brucei spp. By epifluorescence microscopy, mitochondrial development has been followed in long slender bloodstream and procyclic organisms stained with rhodamine 123. to photograph stained long slender bloodstream forms, it was necessary to develop a method to completely immobilize viable organisms. In both parasite forms, as the cell cycle progressed, the mitochondrion developed from a thread-like structure to a highly branched organelle. A dramatic reorganization occurred preceding cytokinesis to produce two progeny thread-like structures which were partitioned into newly formed daughter cells. the organelle within the long slender trypomastigote was found to stain optimally at 0.3 μ/ml of rhodamine 123, while the procyclic form required 3.0 μ/ml. the results suggest that the plasma membrane potential is higher in the long slender parasite than in the procyclic form. the effects of inhibitors that disrupt mitochondrial function were examined in long slender and procyclic parasites, and some of these agents were shown to affect rhodamine 123 accumulation and retention. In long slender trypomastigotes the trypanosome alternative oxidase does not appear to be coupled to proton pumping, whereas in procyclic organisms the effects of inhibitors indicate that this oxidase may be coupled to a pathway that is branched preceding an antimycin A₁-sensitive site.     

A fungal metabolite masks the host plant odor for the pine weevil (Hylobius abietis)

The pine weevil is one of the most important pest insects of conifer reforestation areas in Europe. Female pine weevils cover their eggs with chewed bark and feces (frass) resulting in avoidance behavior of feeding conspecifics towards egg laying sites. It has been suggested that microorganisms present in the frass may be responsible for producing deterrent compounds for the pine weevil. The fungi Ophiostoma canum, O. pluriannulatum, and yeast Debaryomyces hansenii were isolated from aseptically collected pine-weevil frass. The isolated fungi were cultured on weevil frass broth and their volatiles were collected by SPME and identified by GC–MS. D. hansenii produced methyl salicylate (MeS) as a major compound, whereas, in addition, O. canum and O. pluriannulatum produced 6-protoilludene. In a multi-choice lab bioassay, MeS strongly reduced pine weevil's attraction to the Pinus sylvestris volatiles. Thus, a fungal metabolite was found that strongly affects the pine weevil host-odor search.     

Backbone motion in elastin's hydrophobic domains as detected by (2) H NMR spectroscopy

The elasticity of vertebrate tissue originates from the insoluble, cross-linked protein elastin. Here, the results of variable-temperature (2) H NMR spectra are reported for hydrated elastin that has been enriched at the Hα position in its abundant glycines. Typical powder patterns reflecting averaged quadrupolar parameters are observed for the frozen protein, as opposed to the two, inequivalent deuterons that are detected in a powder sample of enriched glycine. The spectra of the hydrated elastin at warmer temperatures are dominated by a strong central peak with features close to the baseline, reflective of both isotropic and very weakly anisotropic motions.