High Level Expression of Glucose-6-phosphate Dehydrogenase Gene PsG6PDH from Populus suaveolens in E. coli

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-a-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·L–1 IPTG treatment for 4 h and that pET-G product was predominately soluble and not extra-cellular secreting.     

Cloning and sequence analysis of a glucose-6-phosphate dehydrogenase gene <Emphasis Type="Italic">PsG6PDH</Emphasis> from freezing-tolerant <Emphasis Type="Italic">Populus suaveolens</Emphasis>

A 1207 bp cDNA fragment (PsG6PDH) was amplified by RT-PCR from cold-induced total RNA of the freezing-tolerant P. Suaveolens, using primers based on the highly conserved region of published plant glucose-6-phosphate dehydrogenase (G6PDH) genes. The sequence analysis showed that PsG6PDH coding region had 1 101 bp and encoded 367 predicted amino acid residues. Moreover, the nucleotide sequence of PsG6PDH showed 83%, 82%, 79%, 79% and 78% identity, and the derived amino acid sequence shared 44.2%, 44.7%, 42.0%, 40.5% and 43.9% identity with those of the Solanum tuberosum, Nicotiana tabacum, Triticum aestivum, Oryza sativa and Arabidopsis thaliana, respectively. The results show that PsG6PDH is a new member of G6PDH gene family and belongs to the cytosolic G6PDH gene. This is the first report on cloning of the G6PDH gene from woody plants.     

Cloning and Sequence Analysis of a Glucose-6-Phosphate Dehydrogenase Gene PsG6PDH from Freezing-tolerant Populus suaveolens

A 1 207 bp cDNA fragment (PsG6PDH) was amplified by RT-PCR from cold-induced total RNA of the freez- ing-tolerant P. Suaveolens, using primers based on the highly conserved region of published plant glucose-6-phosphate dehydro- genase (G6PDH) genes. The sequence analysis showed that PsG6PDH coding region had 1 101 bp and encoded 367 predicted amino acid residues. Moreover, the nucleotide sequence of PsG6PDH showed 83%, 82%, 79%, 79% and 78% identity, and the derived amino acid sequence shared 44.2%, 44.7%, 42.0%, 40.5% and 43.9% identity with those of the Solanum tuberosum, Nicotiana ta- bacum, Triticum aestivum, Oryza sativa and Arabidopsis thaliana, respectively. The results show that PsG6PDH is a new member of G6PDH gene family and belongs to the cytosolic G6PDH gene. This is the first report on cloning of the G6PDH gene from woody plants.     

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.     

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.     

Successful <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Populus tomentosa</Emphasis> with apple <Emphasis Type="Italic">SPDS</Emphasis> gene

The problem of salinized soils has become one of the most serious constraints to agricultural and forest productivity. With the purpose of enhancing salt stress tolerance of Populus tomentosa, we transformed this tree species with spermidine synthase （SPDS） genes derived from an apple by an Agrobacterium-mediated method. Four transgenic clones were confu＇med by PCR and Southern blot analysis. As well, the expression of introduced SPDS genes was analyzed by real-time quantitative PCR.     

The role of antioxidant system in freezing acclimation-induced freezing resistance of Populus suaveolens cuttings

We investigated the changes in the contents of H2O2, malonaldehyde (MDA) and endogenous antioxidants, the activities of protective enzymes and some critical enzymes involved in the ascorbate-glutathione (ASA-GSH) cycle as well as freezing resistance (expressed as LT50) and correlations mentioned above, in detail using Populus suaveolens cuttings. The purpose was to explore the physiological mechanism of the enhancement of freezing resistance induced by freezing acclimation at –20°C, and to elucidate the physiological mechanisms by which trees adapt to freezing. The results showed that freezing acclimation enhanced the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), monodehydroascorbate reductase (MDAR), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and glutathione reductase (GR). And it increased the contents of reduced ascorbate (ASA), reduced glutathione (GSH), dehydroascorbate (DHA) and oxidized glutathione (GSSG). However, H2O2 and MDA contents and LT50 of cuttings were decreased. LT50 in cuttings was found to be closely correlated to the levels of SOD, POD, CAT, APX, DHAR, MDAR, GR, H2O2, MDA, ASA, GSH, DHA and GSSG during freezing acclimation. This suggested that the enhancement of freezing resistance of cuttings induced by freezing acclimation may relate to the distinct increase for the levels of SOD, POD, CAT, APX, DHAR, MDAR, GR, ASA, GSH, DHA, and GSSG. In addition, the observed levels of APX, DHAR, MDAR, GR, ASA, DHA, GSH and GSSG were higher than those of SOD, POD and CAT during freezing acclimation. It indicated that a higher capacity of the ASA-GSH cycle is required for H2O2 detoxification, and growth and development of cuttings. Based on the obtained results, it can be concluded that the ASA-GSH cycle plays an important role in enhancement of freezing resistance of P. suaveolens cuttings during freezing acclimation.     

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.     

Experimental study on characteristic curves of restoring forces of <Emphasis Type="Italic">Populus alba</Emphasis> var. <Emphasis Type="Italic">pyramidalis</Emphasis>

Static tests under cyclic loading were carried out on Populus alba var. pyramidalis to determine its characteristic curves of     

Pollen development and multi-nucleate microspores of <Emphasis Type="Italic">Populus bolleana</Emphasis> Lauche

Populus bolleana is a variety of P. alba, commonly used in poplar breeding programs in China. Developmental biology that involves staminate flowers, microsporogenesis and microgametogenesis ofP. bolleana is essential for Populus improvement in cross breeding for better characteristics in sexual reproduction. Flower morphology and pollen development were described and illustrated using anatomical, sectioning and stain-clearing techniques. The results show that microsporocytes undergo a regular meiotic process, but some multi-nucleate microspores occur at the microspore stage. It takes five days for microsporocytes to develop to mature pollen by forcing flower branches under greenhouse conditions. Additionally, an important relationship was found between stages of meiosis and anther colors. Microspore tetrads formed when the anther color turned yellow, whereas, when the pollen matured, the anther was red and the tapetum degenerated completely. When mature pollen grains are formed, flower buds develop into male catkins. In the end, filament elongated and pollen grains were released from dehisced anthers.     

Characterization and identification of leaf morphology of <Emphasis Type="Italic">Populus deltoides</Emphasis> Bartr. clones

Leaves are of fundamental importance to plants, representing their facility to generate power and are the sensing units of plants towards the environment. An attempt was made to characterize and compare the variations of leaf morphology of various Populus deltoides Bartr. clones by studying the winter buds and other leaf parameters of fully developed leaves. To achieve these objectives, forty-three exotic and indigenous clones of P. deltoides Bartr. were evaluated for different parameters. On the basis of various morphological characteristics the results reveal that each clone has a distinct color pattern of leaves. Different colors observed in these clones varied from light green through green to dark green. Two distinct lengths of the leaf apex were found, i.e., short and long; as well both acuminate and acute apex types were found. Erratic distribution of serration of leaves was also found. In this study, the morphological traits of leaves provided discriminatory grounds for separating various populations of P. deltoides Bartr. clones. Winter bud studies indicate that different clones vary considerably with regard to shape, color, shape of leaf scars and exudation.     

Establishment of cell suspension line of <Emphasis Type="Italic">Populus tomentosa</Emphasis> Carr.

Leaves of fine Populus tomentosa genotype TC152 were used as explants to establish cell suspension lines. The effects of plant growth regulators on callus induction and establishment of cell suspension lines were studied. The callus induction rate was the highest on a MS solid medium supplemented with 1.0 mg·L^-1 2,4-D. A cell suspension line could be obtained by inoculating calli which were not subcultured into a MS liquid medium supplemented with 1.5 mg·L^-1 2,4-D. The best subculture medium was MS ＋ 0.8 mg＇L-1 2,4-D ＋ 30 g·L^-1 sucrose with a subculture cycle of seven days.     

Comparison and early selection of new clones in <Emphasis Type="Italic">Populus tomentosa</Emphasis>

In our study, two experimental plantations, respectively, with 24 and 32 new clones of P tomentosa, were established in Weixian County, Hebei Province and Wuzhi County, Henan Province using a completely randomized block design. A comparative study was conducted on the continuous 5-year-old height and diameter at breast height （DBH） of new clones in the two plantations. As well, based on genetic correlation over the years of testing of these clones, a preliminary study of early selection was carried out. Results indicate that the growth traits of the new clones in Weixian were better than those in Wuzhi. The traits show weak correlation between the two plantations. In some stands, the height, DBH and seedling volume of 5-year-old clones presented statistically significant differences among clones. In both plantations, the new clones showed over 0.6 repeatability of height, DBH and volume, as well as larger coefficients of variation （CV）. The fact that these clones achieved the largest repeatability and CV in the second year suggests that these traits are highly controlled by heredity. Thus, based on the growth traits of the second year, the new clones B305, B307, B303, H75, BT18, BT17 and 21J-1 were considered suitable in Weixian. In Wuzhi, the new clones had variable repeatability and CVs in various years and their correlation of growth traits among different years was not high. We conclude that early selection of new clones was not feasible in Wuzhi.     

一个新的柽柳类萌芽素基因ThGLP的结构及其表达分析

萌芽素和类萌芽素蛋白在不同植物的各个生长阶段和胁迫相关过程中起到不同的作用。本研究首次从刚毛柽柳cDNA文库中获得类萌芽素蛋白全长基因ThGLP,该基因编码225个氨基酸,含有植物萌芽素和类萌芽素蛋白的功能序列。通过进化树分析发现该基因屑于真正萌芽素亚家族。利用实时定量PCR方法研究了该基因在PEG、NaCl、低温、CdCl2和ABA胁迫下不同时间的表达模式。结果显示PEG、NaCl、低温、CdCl2和ABA处理均能诱导ThGLP基因在柽柳的根和叶中的表达。结果表明ThGLP在柽柳根和叶中表达,参与非生物胁迫应答并由ABA依赖的信号传导途径调控。     

Structural characteristics and eco-adaptability of heteromorphic leaves of <Emphasis Type="Italic">Populus euphratica</Emphasis>

The microstructural and ultrastructural traits of three kinds of typical leaves ofPopulus euphratica Olive, including lanceolate, broad-ovate and dentate broad-ovate leaves, were studied by using electron microscope and optical microscope. The resuits showed that with the leaves changing from lanceolate shape to dentate broad-ovate shape, their structure obviously tended to be xeromorph: developed palisade tissue, undeveloped spongy tissue, thick cutin layer and sunken stomas. The amount of mitochondria tended to be increased, and the shape of chloroplasts varied from regular spindle to irregular rotundity or oval. The leaves were covered with wax without cilium, and the stomas on the upper and lower epidermis of the leaves opened unevenly. The stomas on the lower epidermis were deeper than those on the upper epidermis under the scanning electron microscope. The results implied that the structural characteristics of the diversiform-leaves ofP euphratica are related to its eco-adaptability.     

Impact of addition of soil amendments and microbial inoculants on nursery growth of <Emphasis Type="Italic">Populus deltoides</Emphasis> and <Emphasis Type="Italic">Toona ciliata</Emphasis>

Present study evaluated growth of Populus deltoides G48 and Toona ciliata over a period of 6 months, in nursery soil amended with 10% fly ash (v/v), 5% distillery waste (v/v), 20% farmyard manure (v/v) and microbial consortium of Pseudomonas striata and Azotobacter sp. @ 30 ml/pot in different combinations leading to 12 different treatments with 16 replicates in completely randomized block design. Biometric parameters such as plant height, collar diameter and total dry biomass were analyzed which indicated that the treatment (T8) comprising of fly ash @ 10% (v/v), farmyard manure @ 20% (v/v) and microbial consortium @ 30 ml/pot promoted growth of P. deltoides. The results indicated that combined addition of fly ash, farm yard manure and microbial inoculants can be used as a good potting mixture for improving survival rates and plant growth in forestry nurseries.     

Construction of a cDNA-AFLP reaction system of <Emphasis Type="Italic">Populus euphratica</Emphasis> for abiotic stress studies

Populus euphratica, after optimizing     

Stereochemistry of matairesinol formation by <Emphasis Type="Italic">Daphne</Emphasis> secoisolariciresinol dehydrogenase

Secoisolariciresinol dehydrogenase activity was detected for the first time from Daphne odora and Daphne genkwa (Thymelaeaceae), which are known to produce optically pure (+)-matairesinol. In sharp contrast, (–)-matairesinol was formed selectively over the (+)-antipode by the secoisolariciresinol dehydrogenase preparation from both D. odora callus and D. genkwa shoots.     

Feeding performance of <Emphasis Type="Italic">Clostera fulgurita</Emphasis> on three clones of <Emphasis Type="Italic">Populus deltoides</Emphasis>

Poplar leaf defoliator, Clostera fulgurita (Walker) larvae were reared on three Populus deltoides clones (PL1, PL5 and PL7) in the laboratory. The nutritional indices were computed for working out the relationship between food consumption and growth rate of 3rd, 4th and 5th instar larvae on three clones. The result showed that the consumption index (CI), approximate digestibility (AD), growth rate (GR), relative growth rate (RGR) and efficiency of conversion of ingested food (ECI) decreased with the increase in the age of the larvae. Efficiency of conversion of digested food (ECD) increased with increase in age of the larvae. GR and RGR varied significantly, indicating that larval development was enhanced on PL1 as compared to PL5 & PL7. The values of AD, ECI and ECD were not affected by the different clones. Feeding and growth indices could be useful to define a defoliation prediction model.     

The influence of Desert False Indigo, <Emphasis Type="Italic">Amorpha fruticosa</Emphasis>, and three arbuscular mycorrhizae fungi on the growth of <Emphasis Type="Italic">Populus ussuriensis</Emphasis> seedlings

The influence of Desert False Indigo, Amorpha fruticosa, on the growth of Populus ussuriensis seedlings inoculated with three arbuscular mycorrhizae (AM) fungi (Glomus mosseae, Glomus intraradices, Glomus sinuosa) was studied using the nylon net method. The results showed that all three AM fungi infected P. ussuriensis seedlings and G. intraradices and also G. mosseae infected A. fruticosa. The AM fungi promoted growth of P. ussuriensis and Desert False indigo seedlings. Moreover, under co-cultivation with A. fruticosa, the biomass of P. ussuriensis increased significantly. The concentration of nitrogen in P. ussuriensis grown with A. fruticosa and the concentration of soluble nitrogen in the rhizosphere were also higher than when grown alone. Hypha were found on the two plant seedlings inoculated with G. mosseae and G. intraradices, suggesting that AM fungi may transport nutrients from seedlings of A. fruticosa to the rhizosphere of P. ussuriensis seedlings, which may have promoted the growth of P. ussuriensis. The AM fungi played a critical role on the effect of A. fruticosa on growth of P. ussuriensis.