Tobacco (Nicotiana tobaccum) nuclear transgenics with high copy number can express NPTII driven by the chloroplast psbA promoter

A chloroplast expression vector containing the NPTII gene under the control of apsbA promoter (psbA-NPTII) was constructed, and was biolistically delivered into both suspension cells and leaf strips of tobacco (Nicotiana tabaccum). Analyses of subsequently recovered kanamycin-resistant transgenic plants indicate that the psbA-NPTII gene was not located in the chloroplast, but was in the nucleus in very high copy number. This conclusion was based upon results from: (1) Southern hybridization analyses of chloroplast and nuclear DNAs using NPTII, chloroplast-marker, and nuclear-marker probes; (2) pulse-field gel electrophoresis; and (3) kanamycin screening of sexual progenies. This study suggests that the nuclear expression of the NPTII gene may have been associated with many copies of the psbA-NPTII construction. Very high copy number in the nucleus might either allow NPTII expression from the otherwise inadequate psbA promoter, or might increase the chance of recombining with upstream tobacco regulatory sequences.     

A morphometric study of the geographic variation in Pinus contorta (Pinaceae)

This morphometric study of the geographic variation in Pinus contorta is based on 93 provenances cultivated in northern Jutland, Hjardemål Klitplantage, 57'04"08'48'E (Arboretum trial no. F275). The numerical methods employed were common principal components analysis (CPCA), non-metric multidimensional scaling (MDSCALE), minimum spanning trees (MST), and the neighbour-joining method (NJOIN). Although it is not always possible to draw a very clear line between the coastal var. contorta and the inland var. latifolia of P. contorta subsp. contorta, these varieties are distinct in their extreme forms. Critical provenances of both varieties seem to originate from the transition zone between the two taxa. The Californian or Sierra Nevada provenances of f contorta subsp. murrayana are very distinct from the Oregon provenances. Apparently most of the inland Oregon provenances of P. contorta are more or less intermediate between var. latifolia and subsp. murmyana s.str. The NJOIN method of phylogenetic reconstruction supports the division of P. contorta into two major groups or subspecies: 1. subsp. contorta, divided into var. contorta and var. latifolia, and 2. subsp. murrayana.     

A phylogeny of cycads (Cycadales) inferred from chloroplast matK gene, trnK intron, and nuclear rDNA ITS region

Phylogenetic relationships among the three families and 12 living genera of cycads were reconstructed by distance and parsimony criteria using three markers: the chloroplast matK gene, the chloroplast trnK intron and the nuclear ITS/5.8S rDNA sequence. All datasets indicate that Cycadaceae (including only the genus Cycas) is remotely related to other cycads, in which Dioon was resolved as the basal-most clade, followed by Bowenia and a clade containing the remaining nine genera. Encephalartos and Lepidozamia are closer to each other than to Macrozamia. The African genus Stangeria is embedded within the New World subfamily Zamiodeae. Therefore, Bowenia is an unlikely sister to Stangeria, contrary to the view that they form the Stangeriaceae. The generic status of Dyerocycas and Chigua is unsupportable as they are paraphyletic with Cycas and the Zamia, respectively. Nonsense mutations in the matK gene and indels in the other two datasets lend evidence to reinforce the above conclusions. According to the phylogenies, the past geography of the genera of cycads and the evolution of character states are hypothesized and discussed. Within the suborder Zamiieae, Stangeria, and the tribe Zamieae evolved significantly faster than other genera. The matK gene and ITS/5.8S region contain more useful information than the trnK intron in addressing phylogeny. Redelimitations of Zamiaceae, Stangeriaceae, subfamily Encephalartoideae and subtribe Macrozamiineae are necessary.     

The chloroplast psbA promoter is more efficient in Escherichia coli than the T7 promoter for hyperexpression of a foreign protein

The T7 promoter is commonly used in E. coli expression studies but has several disadvantages due to induction with isopropylthio--D-galactoside (IPTG) including the prohibitive cost of IPTG, reduction in cell growth, and the poisonous nature of IPTG prohibiting its use in pharmaceutical products. The chloroplast psbA promoter produces an 18.5 fold increase in expression in E. coli compared to the T7 promoter. Also, the psbA promoter does not have the disadvantages of induction with IPTG.     

Insertion analysis of putative functional elements in the promoter region of the Aspergillus oryzae Taka-amylase A gene (amyB) using a heterologous Aspergillus nidulans amdS-lacZ fusion gene system

Expression of the Taka-amylase A gene (amyB) of Aspergillus oryzae is induced by starch or maltose. The A. oryzae amyB gene promoter contains three highly conserved sequences, designated Regions I, II, and III, compared with promoter regions of the A. oryzae glaA encoding glucoamylase and the agdA encoding alpha-glucosidase. To identify the function of these sequences within the amyB promoter, various fragments containing conserved sequences in the amyB promoter were introduced into the upstream region of the heterologous A. nidulans amdS gene (encoding acetamidase) fused to the Escherichia coli lacZ gene as a reporter. Introduction of the sequence between -290 to -233 (the number indicates the distance in base pairs from the translation initiation point (+1)) containing Region III significantly increased the expression of the lacZ reporter gene in the presence of maltose. The sequence between -377 to -290 containing Region I also increased the lacZ activity, but its maltose inducibility was less than that of Region III. The sequence between -233 to -181 containing Region II had no effect on the expression. These results indicated that Region III is most likely involved in the maltose induction of the amyB gene expression.     

Spatially variable natural selection and the divergence between parapatric subspecies of lodgepole pine (Pinus contorta, Pinaceae)

? Premise of the study: Plant populations arrayed across sharp environmental gradients are ideal systems for identifying the genetic basis of ecologically relevant phenotypes. A series of five uplifted marine terraces along the northern coast of California represents one such system where morphologically distinct populations of lodgepole pine (Pinus contorta) are distributed across sharp soil gradients ranging from fertile soils near the coast to podzolic soils ca. 5 km inland. ? Methods: A total of 92 trees was sampled across four coastal marine terraces (N = 10-46 trees/terrace) located in Mendocino County, California and sequenced for a set of 24 candidate genes for growth and responses to various soil chemistry variables. Statistical analyses relying on patterns of nucleotide diversity were employed to identify genes whose diversity patterns were inconsistent with three null models. ? Key results: Most genes displayed patterns of nucleotide diversity that were consistent with null models (N = 19) or with the presence of paralogs (N = 3). Two genes, however, were exceptional: an aluminum responsive ABC-transporter with F(ST) = 0.664 and an inorganic phosphate transporter characterized by divergent haplotypes segregating at intermediate frequencies in most populations. ? Conclusions: Spatially variable natural selection along gradients of aluminum and phosphate ion concentrations likely accounted for both outliers. These results shed light on some of the genetic components comprising the extended phenotype of this ecosystem, as well as highlight ecotones as fruitful study systems for the detection of adaptive genetic variants.     

Origin and evolution of the chloroplast trnK (matK) intron: a model for evolution of group II intron RNA structures

The trnK intron of plants encodes the matK open reading frame (ORF), which has been used extensively as a phylogenetic marker for classification of plants. Here we examined the evolution of the trnK intron itself as a model for group II intron evolution in plants. Representative trnK intron sequences were compiled from species spanning algae to angiosperms, and four introns were newly sequenced. Phylogenetic analyses showed that the matK ORFs belong to the ML (mitochondrial-like) subclass of group II intron ORFs, indicating that they were derived from a mobile group II intron of the class. RNA structures of the introns were folded and analyzed, which revealed progressive RNA structural deviations and degenerations throughout plant evolution. The data support a model in which plant organellar group II introns were derived from bacterial-like introns that had "standard" RNA structures and were competent for self-splicing and mobility and that subsequently the ribozyme structures degenerated to ultimately become dependent upon host-splicing factors. We propose that the patterns of RNA structure evolution seen for the trnK intron will apply to the other group II introns in plants.     

Chloroplast phylogeography and evolution of highly polymorphic microsatellites in lodgepole pine ( Pinus contorta)

We employed a novel set of six highly polymophic chloroplastic simple sequence repeat (cpSSR) loci to investigate the phylogeography of lodgepole pine (Pinus contorta Dougl. Ex. Loud.), and to examine aspects of the evolutionary process operating on these repetitive DNA sequences. Chloroplast haplotypes of 500 trees, sampled throughout the range of lodgepole pine, were determined. We found a marked association of genetic distance with physical distance within the scale of 0 to 1,000 km, but no association beyond that range. Likewise, geographic clustering was observed only among recent clades in a dendrogram. These phylogeographic patterns are consistant with a rapid rangewide expansion (”big-bang”) followed by recent, local population differentiation (”galaxy formation”). In support of this expansion, coalescent simulations of the genealogical process gave a long-term effective population size in the low thousands, and a time to common ancestry of about 1,500 generations (12,000 years), consistent with a post-Pleistocene population expansion as documented by previous pollen-sediment analyses. Two lines of evidence (mapping mutational events onto a phylogeny, and evaluation of observed versus expected gene diversity) suggest that five of the cpSSR loci evolve primarily by a stepwise model of evolution of single repeat changes (but with a small proportion of changes involving two or more repeats), and the coalescent simulations point to a mutation rate of about 10^–3. Received: 7 December 2000 / Accepted: 17 May 2001     

小菜蛾NanosO基因启动子的克隆及功能验证

【目的】克隆小菜蛾Plutella xylostella NanosO基因PxnosO启动子,并验证其具有生殖腺特异性活性,以期应用于基因功能研究或转基因昆虫的构建,为小菜蛾等农业害虫的综合治理提供新的研究思路。【方法】根据小菜蛾基因组序列信息,利用PCR技术克隆NanosO的启动子并进行序列分析。构建PxnosO-EGFP表达质粒,利用脂质体细胞转染技术将PxnosO-EGFP和IE1-EGFP表达质粒转入到小菜蛾胚胎细胞系(Px-6)和草地贪夜蛾Spodoptera frugiperda卵巢细胞系(Sf9)中,通过激光共聚焦荧光显微镜观察和qRT-PCR技术分别定性和定量分析EGFP基因的表达,验证小菜蛾NanosO启动子的活性。【结果】克隆获得小菜蛾PxnosO (Px004767)启动子区序列,长1 743 bp。对启动子序列进行分析,发现该序列不仅包含启动子共有核心元件TATA box以及上游启动子成分CAAT box和GC box等,还包含有数十个转录因子结合位点。利用细胞转染技术,在PxnosO启动子驱动下成功地在Px-6和Sf9细胞系中表达外源基因EGFP。【结论】克隆了小菜蛾NanosO基因PxnosO启动子,在细胞水平上验证其能驱动外源EGFP基因的表达,为分析PxnosO在小菜蛾不同发育时期的表达模式和PxnosO启动子在体内的功能验证奠定基础。