Genome-wide search for segregation distortion loci associated with the expression of complex traits in Populus tomentosa

Segregation distortion of molecular markers has been reported in a broad range of organisms. It has been detected in an interspecific BC1 Populus pedigree established by controlled crossing between clone “LM50” (Populus tomentosa) and its hybrid clone “TB01” (P. tomentosa×P. bolleana). The study with a total of 150 AFLP markers (approximately 18.9% of the total loci) exhibited significant deviation from the Mendelian ratio (1:1) (p<0.01). Twenty-five percent of the markers were mapped on the pa-rental specific genetic linkage maps of clones “LM50” and “TB01” with a pseudo-test-cross mapping strategy. Twelve linkage groups had markers with skewed segregation ratios, but the major regions were on linkage groups TLG2, TLG4 and TLG6 in the linkage map of clone “LM50”. We also analyzed the association between distorted loci and expression of complex traits with Map-maker/QTL software. A total of 16 putative QTLs affecting 12 traits were identified in the distorted regions on seven linkage groups. Therefore we could detect the distribution of skewed loci along the entire genome and identify the association between quantitative traits and segregation loci via genetic mapping in an interspecific BC1 P. tomentosa family. Furthermore, the genetic nature and pos-sible causes of these segregation distortions for differentiation between female and male parents were also discussed.     

Cloning of <Emphasis Type="Italic">XET</Emphasis> gene from <Emphasis Type="Italic">Anthocephalus chinensis</Emphasis> and its plant expression vector construction

A full-length cDNA sequence of xyloglucan endotransglycosylase gene (XET), abundantly expressed in the cambium of Anthocepha-lus chinensis was cloned by conserved PCR, rapid-amplification of cDNA ends and by chromosome walking. Analytical results of the DNA sequence show that a 912 bp complete open reading frame (ORF) encoded a 303-amino acid protein was in the 1205 bp full cDNA sequence. The deduced amino acid sequence of AcXET, which contained the conserved specific EIDFE catalytic site sequence to XETs was homologous to the other known XET proteins. In order to study the gene function of AcXET and obtain transgenic plants, a plant expression vector pBIAcXET was constructed by recombinating the AcXET fragment from the cloning vector pMD19AcXET and the binary vector pBI121 between the XbaI and SmaI sites. The fragment of AcXET gene was inserted between the CaMV 35S promotor and the coding region of the GUS gene in pBI121. The identification results show that the plant expression binary vector pBIAcXET was constructed successfully. These results lay the foundation for studying the molecular mechanism of AcXET gene during wood formation.     

质壁分离处理刺五加合子胚促进了胼胝质合成酶基因的表达

在以前的研究中我们报道了经质壁分离预处理刺五加合子胚下胚轴,促进了体细胞胚形成。在本研究中,我们利用RT-PCR方法分析了刺五加合子胚经2,4-D,蔗糖,甘露醇处理后胼胝质合成酶基因的表达水平。结果显示:蔗糖和甘露醇处理明显促进了胼胝质合成酶基因的表达水平。而且我们观察到,体细胞胚发生过程中,外植体经质壁分离处理后细胞壁明显加厚。因此我们推测:胼胝质可能使表皮层细胞相互之间产生分离从而使其转变为具有胚性潜能的细胞。图2参20。     

Mathematical expression for the relationship between internode number and internode length for bamboo, <Emphasis Type="Italic">Phyllostachys pubescens</Emphasis>

We analyzed the relationship between internode number and internode length for one of the largest bamboo, Phyllostachys pubescens Mazel ex Houz. For 50 sample culms with various sizes felled in a purest and of P. pubescens, the internode number was assigned from base totip and the length for each internode was directly measured. The result indicated that the internode length should be cumulated from base to tip,and then the cumulated internode length should be relativized by the total culm length. It was inappropriate to relativize the internode length by themaximum intenode length. In addition, the relationship between therelative internode number (the internode number relativized by the total number of internodes) and the relative cumulated internode length should be described not by a power function but by a sigmoid function such as the third-order function. The determined function enabled us to estimate the actual internode length, with the root mean squared error being 4 cm.In conclusion, the mathematical expression presented here, i.e., there lativization of the cumulated internode length by the total culm length and the application of the sigmoid function, will be useful in describing the relationship between internode number and internode length for P.pubescens.     

Cloning and expression analysis of a homologous expansin gene <Emphasis Type="Italic">EXP2</Emphasis> in <Emphasis Type="Italic">Picea wilsonii</Emphasis>

Expansins are cell-wall-loosening proteins that have multiple roles during plant development and stress-related processes. In this study, a novel expansin gene PwEXP2 was cloned by rapid amplification of cDNA ends based on the cDNA library of Picea wilsonii and EST fragment of PwEXP2. It was found that PwEXP2 coded 253 amino acids, and putative signal peptides exist at the N-terminal, followed by 8 cysteines, a HFD (His-Phe-Asp) conserved domain, and 4 tryptophan residues at the C-terminal. PwEXP2 was located in cytoplasm and nucleus when transformed in an onion epidermal cell. Quantitative real-time PCR assays showed that PwEXP2 was expressed in various tissues with a relatively high level in needles and low level in mature pollen. The expression level of PwEXP2 dramatically increased after seed germination. Gene expression profiles in abiotic stresses showed that PwEXP2 was induced by high temperature and osmotic stress but not involved in ABA-dependent signaling pathway. These results display the important roles of the PwEXP2 in plant development and multiple adversity stresses.     

High-level expression of 4-coumarate:coenzyme A ligase gene Pt4CL1 of Populus tomentosa in E. coli

In order to investigate the enzymatic properties of the 4CL1 of Populus tomentosa, the recombinant expression vector pQE31-4CL1 was constructed. The recombinant was identified by three restriction endonucleases, then the vector pQE31-4CL1 was transformed into expression host M15 (pREP4) and induced by isopropyl-α-D-thiogalactoside (IPTG) to express 60 kD fused protein Pt4CL1. The biologically active Pt4CL1, expressed as soluble protein, was achieved with 0.6 mmol·L-1 IPTG induction as the ex-pression temperature declined from 37 to 28°C. The 6×His tag facilitates affinity binding to Ni2 -nitrolotriacetic acid (NTA) and enables one-step purification to acquire the molecular SDS-PAGE electrophoresis purity of the active 4CL1 protein by agarose cou-pled with Ni2 -NTA affinity chromatography. The optimal substrate for Pt4CL1 was 4-coumarate.     

High level expression of glucose-6-phosphate dehydrogenase gene <Emphasis Type="Italic">PsG6PDH</Emphasis> from <Emphasis Type="Italic">Populus suaveolens</Emphasis> in <Emphasis Type="Italic">E. coli</Emphasis>

In order to investigate the functions of the gene PsG6PDH and the mechanisms underlying freezing tolerance of Populus suaveolens, the recombinant expression vector pET-G (pET30a-G6PDH), which contained full encoding region of PsG6PDH gene, was established. The recombinant was identified by lawn-PCR and double enzyme digestion and then transformed into expression host XA90 and induced by isopropyl-â-D-thiogalactoside (IPTG) to express 100 kD polypeptide of G6PDH fusion protein. The results showed that the expressed amount of the fusion protein culminated after 1 mmol&#8226;L^–1 IPTG treatment for 4 h and that pET-G product was predominately soluble and not extra-cellular secreting.     

Constitutive expression of sense &; antisense <Emphasis Type="Italic">PtAP3</Emphasis>, an <Emphasis Type="Italic">AP3</Emphasis> homologue gene of <Emphasis Type="Italic">Populus tomentosa</Emphasis>, affects growth and flowering time in transgenic tobacco

To analyze the function of PtAP3, an APETALA3 （AP3） homologue gene isolated from Populus tomentosa Carr., the full length sequence （1797 bp） and a fragment （870 bp） of PtAP3 were fused to a CaMV 35S promoter of pBI121 to generate the sense and antisense constructs of PtAP3. These constructs were transformed into tobacco by Agrobacterium infection of leaf disks and selection on kanamycin medium. Some sense and antisense transgenic tobacco plants were obtained by PCR and Southern blot analysis. Great phenotypic differences in transgenic tobacco plants were observed. Almost all of sense PtAP3 to transgenic tobaccos showed a higher growth rate than those of antisense transformants and a few developed pregnancy earlier than wild type seedlings and antisense transformants under the same conditions.     

油桐LACS4基因的克隆与原核及真核表达

长链酰基辅酶A合成酶(LACS)在油脂代谢过程中发挥着重要的作用。以油桐近成熟种子为材料,在已构建的转录组数据库基础上,克隆得到一个油桐长链酰基辅酶A合成酶基因,在此基因c DNA全长序列的基础上,成功构建了油桐LACS4基因的真核表达和原核表达载体,其中,酵母表达载体p YES2-LACS4成功转入酿酒酵母菌株INVSc1中,并进行相关生长趋势分析;在大肠杆菌BL21(DE3)宿主细胞中,原核表达载体p ET30a-LACS4也成功诱导表达,并获得大约为74k Da的表观分子量的相应目的蛋白。     

Agrobacterium tumefaciens‐mediated transformation for targeted disruption and over expression of genes in the poplar pathogen Sphaerulina musiva

Sphaerulina musiva causes both leaf spots and cankers on poplar. Leaf spots can lead to defoliation and cankers on branches and primary stems can lead to stem breakage and tree mortality. The recent availability of both the S. musiva and Populus trichocarpa genomes offers a great opportunity to study host–pathogen interactions. To better understand the factors involved in S. musiva pathology, we present a strategy for the transformation of this species using Agrobacterium tumefaciens. Binary plasmids were generated with hygromycin B phosphotransferase (hph) flanked by upstream and downstream sequences of polyketide synthase‐like (PKS‐L1) gene to generate targeted gene disruptants by homologous recombination. Plasmids were also constructed for constitutive expression reporter genes eGFP and mCherry to help with histological characterization of the pathogen during infection. Gene knockouts were identified by PCR and confirmed by sequencing and Southern blotting. No significant differences were observed in melanin production between PKS‐L1 disruptants and wild type isolates. Colonies expressing reporter genes were identified by fluorescent stereomicroscopy. This method is a promising tool for the characterization of pathogen genes through reverse and forward genetics and for introducing markers for histopathological study.     

Agrobacterium-mediated genetic transformation of Camptotheca acuminata

UGPase gene related with wood cellulose synthesis was transferred into C. acuminata using the method of Agrobacte- rium-mediated genetic transformation, and an efficient transformation system was developed for C. acuminata on the basis of evaluations of several factors affecting Agrobacterium-mediated DNA transfer rate. The highest transformation rate was achieved when pre-cultttred leaf explants were infected with an Agrobacterium culture corresponding to OD600 （0.5） for 10 min, and cultured on explant regeneration medium for three days. The results of Southern hybridization showed that genomic DNA of the kanamycin-resistant shoots to an UGPase gene probe substantiated the integration of the transgene. Transformation efficiency （6%） was achieved under the optimized transformation procedure, This system should facilitate the introduction of important useful genes into C, acuminata.     

石河子纪念性景观情感表达分析

纪念性景观不仅是城市特有人文精神的传承者,更是纪念主体情感的表达者。融合于景观中的情感加强了景观的营造效果,保留了城市固有的风情风貌。本文选取石河子市为研究对象,从崇高感、哀悼感、缓释感的情感角度出发,研究纪念性景观情感的空间节奏变化、传播机制、表达方式以及情感互动体验。主要阐述石河子纪念性景观以中心突出、体量反差、材质肌理的方式表达崇高感,以陵园形象表达哀悼感,以广袤沧桑、自然融合的形式表达缓释感。     

Antisense expression of a caffeic acidO-methyltransferase ofStylosanthes humilis in transgenic poplar: Effect of expression onO-methyltransferase activity and lignin composition

Poplar (Populus tremula) was transformed with a construct carrying an antisense caffeic acidO-methyltransferase (COMT) cDNA (pOMT8) from a tropical pasture legume,Stylosanthes humilis. pOMT8 shows 83% overall homology to the corresponding COMT gene (pPCLA) of poplar. Of the 200 putatively-transformed plants regenerated on selective media after co-cultivation of poplar stem explants withAgrobacterium tumefaciens harbouring a CaMV 35S-antisensepOMT8 construct, a subset of 20 plants were randomly chosen for further analysis. PCR and Southern blot analysis demonstrated the stable integration of T-DNA into the genome of these plants. Antisense expression ofpOMT8 resulted in reductions in total COMT activity in the majority of the transgenic plants with the lowest total COMT activities (61–70% of untransformed control plants) being observed in four transgenic plants. The composition of lignin in transgenic plants was also changed, as detected by reductions in the content of syringyl units using infrared spectroscopy. However, no changes were found in the amount of insoluble lignin in transgenic plants as compared to untransformed control plants. These results indicate the potential of thepOMT8 gene to partially suppress COMT activity and modify the composition of lignin in transgenic poplar. This work was partly supported by General Management of Turkish Pulp and Paper Mills.     

Plant antifreeze proteins and their expression regulatory mechanism

Low temperature is one of the major limiting environmental factors which constitutes the growth, development,productivity and distribution of plants. Over the past several years, the proteins and genes associated with freezing resistance of plants have been widely studied. The recent progress of domestic and foreign research on plant antifreeze proteins and the identification and characterization of plant antifreeze protein genes, especially on expression regulatory mechanism of plant antifreeze proteins are reviewed in this paper. Finally, some unsolved problems and the trend of research in physiological functions and gene expression regulatory mechanism of plant antifreeze proteins are discussed.     

Problems of using pines in <Emphasis Type="Italic">Tuber melanosporum</Emphasis> culture: soils and truffle harvest associated with <Emphasis Type="Italic">Pinus nigra</Emphasis> and <Emphasis Type="Italic">P. sylvestris</Emphasis>

In Mediterranean pine forests, truffles and mushrooms generate greater profits than any other woodland products. However, there are no studies on Tuber melanosporum Vittad. associated with pines. For this reason, we have carried out a study of this truffle in mountain woods with Pinus sylvestris L. and P. nigra Arnold subsp. salzmannii (Dunal) Franco, in central Spain. Two hundred and eight Tuber melanosporum burns were monitored for 7 years in five different habitats within the same geographical area. An ANOVA test confirmed significant differences in carpophore production. In higher producing habitats, pines were less abundant. We also confirmed that in 433 burns, T. melanosporum was always unequivocally associated with the root base of Quercus or Corylus trees. Similarly, 14 truffle collectors confirmed that they had never found a single burn with carpophore production associated exclusively with pines. Nevertheless, soil analyses indicated that the soil of these pine woods was very favourable to Tuber melanosporum. We therefore conclude that at present Pinus nigra salzmannii and P. sylvestris are of little interest to Tuber melanosporum culture, as they hinder carpophore production. However, this study has also confirmed that Pinus nigra salzmannii and P. sylvestris mycorrhize easily with Tuber melanosporum, both in the laboratory and in natural environments. On this basis, we propose that pines may act as transmitters of T. melanosporum, although they do not induce fruiting. As a result, the commercial cultivation of Pinus nigra salzmannii and P. sylvestris seedlings mycorrhized with Tuber melanosporum is not recommended in truffle culture at the present time.