Prokaryotic expression analysis of an NBS-type PtDRG01 gene isolated from Populus tomentosa Carr.

In order to investigate the protein features of an NBS gene (PtDRG01, EF157840) isolated from Populus tomentosa Carr., the full-length open reading frame was fused into a prokaryotic expression vector pGEX-KG. PCR analysis and double endonuclease digestion showed that the recombinant vector was successfully constructed and transferred into an expression host E. coli strain XA₉₀. It was indicated by SDS-PAGE analysis that IPTG treatment successfully induced the expression of a fusion protein of about 79 kD, which was consistent with the predicted value. In addition, the prokaryotic expression system was also optimized. The result suggests that 1 mmol/L IPTG treatment for 4 h at 37°C was most effective, and the product was predominately soluble and not extra-cellular secreting. Moreover, the fusion protein was purified with an affinity chromatography column using Glutathione Sepharose 4B. This work will lay a foundation for further studies on biological functions of the PtDRG01 gene. __________ Translated from Acta Botanica Boreali-Occidentalia Sinica, 2008, 28 (5): 0882–0888 [틫自: 컷놱횲컯톧놨]     

Single nucleotide polymorphism discovery of Pinus radiata with chromosome walking PCR method

In this paper, the basic principle of chromosome walking is presented and we used an actin gene of radiata pine (Pinus radiata) as an example to conduct upstream and downstream chromosome walking for EST sequences. The full genomic sequence (2154 bp) of the actin gene, including promoters 5′ UTR, CDS and 3′ UTR, was identified by chromosome walking. PCR amplification and DNA band sequencing from 200 unrelated radiata pine trees revealed a total of 21 SNPs for the actin gene, three in the promoter region, 15 in CDS and 4 in 3′ UTR. The results of this experiment provide a technical framework for SNPs discovery in none coding regions of candidate genes. __________ Translated from Acta Botanica Boreali-Occidentalia Sinica, 2007, 27 (8): 1571–1576 [译自: 西北植物学报]     

哈茨木霉天冬氨酸蛋白酶基因SA76的3＇RACE扩增和序列分析

由EST获得全长cDNA对于结构基因组学和功能基因组学都是至关重要的,cDNA末端快速扩增技术RACE是该领域中的重要研究方法.利用BD SMART RACE技术扩增编码分泌天冬氨酸蛋白酶SA76基因的3＇末端,将其与哈茨木霉cDNA文库中的SA76基因的EST序列进行序列拼接,获得2019bp的全长cDNA序列,其开放读码框长1593bp,5＇非编码区266bp,3＇非编码区201bp,编码530个氨基酸,有信号肽.哈茨木霉天冬氨酸蛋白酶基因与玉蜀黍赤霉、粗糙脉孢菌、球毛壳菌天冬氨酸蛋白酶基因的同源性分别为53%, 37%, 36%.利用BD SMART RACE技术首次从哈茨木霉中克隆天冬氨酸蛋白酶基因,为验证SA76基因的功能奠定基础,为进一步研究蛋白酶的作用机制及生物防治功能提供依据.     

RAPD markers related to sex locus in Populus tomentosa

By using the methods of random amplified polymorphic DNA (RAPD) and bulked segregate analysis (BSA), we identified markers that are linked to the sex determination in the dioecious Populus tomentosa. Male and female bulks were created through rough mixing equal amounts of its five individual DNA. A total of 88 primers were screened. Twelve primers produced clear patterns with at least one band that appeared to be polymorphic between the two bulks. Subsequently, five male and female individuals were analyzed with those 12 primers, and only S60 (ACCCGGTCAC) could generate a common 1800 bp DNA fragment in all five male individuals and male pool but not in any female individuals. It can be concluded that the gender of P. tomentosa is most likely connected to the S60-1800 bp DNA fragment and RAPD markers. S60, therefore, can be used for selecting the gender of P. tomentosa. __________ Translated from Journal of Central South University of Forestry & Technology, 2008, 28(3): 80–83 [译自: 中南林业科技大学学报]     

Duplication of locus coding of malate dehydrogenase in Populus tomentosa Carr.

Horizontal starch-gel electrophoresis was used to study crude enzyme extraction from young leaves of 234 clones of Populus tomentosa Carr. selected from nine provenances in North China. Ten enzyme systems were resolved. One hundred and fifty-six clones showing unusual allozyme band patterns at locus Mdh-1 were found. Three allozyme bands at locus Mdh-1 were 9:6:1 in concentration. Further studies on the electrophoretic patterns of ground mixed pollen extraction of 30 male clones selected at random from the 156 clones were conducted and it was found that allozyme bands at locus Mdh-1 were composed of two dark-stained bands and a weak band. Only one group of the malate dehydrogenase (MDH) zymogram composed of two bands was obtained from the electrophoretic segregation of pollen leachate of the same clones. A comparison of the electrophoretic patterns one another suggested that the locus Mdh-1 coding malate dehydrogenase in diploid species of P. tomentosa was duplicated. The duplicate gene locus possessed three same alleles and was located in mitochondria. The locus duplication of alleles coding malate dehydrogenase in P. tomentosa was discovered and reported for the first time. [Supported by the “Tenth Five-year Plan” National Key Project in Science and Technology (Grant No. 2002BA515B0303) and the National “863” Project (Grant No. 2002AA241071)]     

Cloning and sequence analysis of atom gene from Sclerotimia sclerotiorum

An atom gene was cloned from genomic DNA of Scleortinia sclerotiorum by inverse PCR. The evolutionary relationships of S. sclerotiorum and other fungi in atom gene were studied. Results showed that the atom gene from of S. sclerotiorum has a single open reading frame of 4 773 bp and does not include any introns. The derived amino acid sequence consists of 1 590 residues, and it is homologous to all fungal AROM proteins studied so far. The theoretical isoelectric point （pl） and molecular weight （Mw） is 6.5 and 172.66 kD, respectively GC percentage of the arom gene is 44.94. According to the results of searching from CDD and Prosite database, AROM protein of S. sclerotiorum contains five conserve domains： 3-dehydroquinate synthase domain, 3-dehydroquinate dehydratase （3-dehydroquinase） domain, shikimate 5-dehydrogenase domain, shikimate kinase domain, and -enolpyruvylshikimate-3-phosphate synthase （EPSP sythase） domain, and four motifs： two EPSP synthase signatures, dehydroquinase class I active site, shikimate kinase signature. According to the PIR Site Rule PIRSR000514-1, four functionally important amino acid residues are found by alignment. Putative TATA box and CAAT box locate separately in -23 and -77 loci in 5＇ un-translated region, and two loci found in downstream atom gene are likely polyadenylation signals. In addition, phylogeny of atom gene is analyzed.     

基于cpDNA rps16序列分析兰考泡桐与白花泡桐和毛泡桐的遗传关系

【目的】通过测序法分析兰考泡桐与白花泡桐和毛泡桐在叶绿体rps16序列上的遗传差异,旨在分析三者之间在叶绿体基因上的变化特点和规律,探讨其种间的遗传关系。【方法】选取兰考泡桐、白花泡桐和毛泡桐各15个样本,对其提取的DNA用PCR扩增获得特异片段,并将其纯化与测序。利用软件Clustal X 2.0对所得序列进行排序;运行MEGA 4软件,进行多序列比对,分析其序列特征,并计算出K2P遗传距离。【结果】(1)对获得的rps16序列进行测定分析,得兰考泡桐序列长度分别为932 933 bp;白花泡桐序列长度为932 bp;毛泡桐序列长度分别为916918 bp。对所得rps16序列进行排序后的长度为938 bp,平均GC含量为34.31%。3个种所代表的个体之间共有10个变异位点,占整个序列长度的1.07%。其中有9个变异位点属于碱基插入或缺失类型,占变异位点总数的90%,占整个序列长度的0.96%。有1个变异位点属于碱基替换类型,占整个变异位点总数的10%,占整个序列长度的0.11%。(2)整个rps16片段的序列共有10个变异位点,其中兰考泡桐与白花泡桐在总的变异位点上,具有一致的碱基位点9个,占总变异的90%。而兰考泡桐与毛泡桐相比,没有相同的碱基。【结论】根据三种泡桐的rps16序列的序列特征和变异位点的分析,表明在叶绿体遗传方面,兰考泡桐具有与白花泡桐更多相似的遗传物质,其亲缘关系较近。综上所述,推测兰考泡桐与白花泡桐可能来自同一母系遗传。     

Discrimination of Bursaphelenchus xylophilus and Bursaphelencus mucronatus by PCR-RFLP technique

A polymerase chain reaction-restriction fragment length polymorphism analysis was used to discriminate isolates of Bursaphelenchus xylophilus and B. mucronatus. The amplifications of B. xylophilus isolates yielded one fragment of approximately 890 bp and that of B. mucronatus was about 930 bp. Digestion of amplified products of each nematode isolate with five restriction endonucleases revealed the following results: 1) Dra I digestion of the internal transcribed spacer (ITS) products of B. xylophilus populations yielded two fragments of 510 and 380 bp. Dra I could not digest the ITS products of B. mucronatus populations; 2) Sal I could not digest the ITS products of all B. xylophilus populations, but it could digest those of B. mucronatus populations into two fragments, which were 720 and 220 bp; 3) digested products of four B. xylophilus populations by Msp I yielded two fragments of 530 and 360 bp, except GZ02, which could not be digested. B. mucronatus populations yielded three fragments: 340, 290, and 180 bp; 4) all populations of B. xylophilus and B. mucronatus could not be digested by Apa I; 5) digestion of the ITS products of B. xylophilus and B. mucronatus yielded two fragments of 520 and 370 bp, and 530 and 400 bp respectively. The restriction endonucleases Dra I and Sal I could be used to identify B. xylophilus and B. mucronatus. Because the results of digestion of B. xylophilus and B. mucronatus were markedly different, they were very easy to be identified and applied; Msp I and Xho I were not suitable for identification of B. xylophilus and B. mucronatus and Apa I could not identify and distinguish between B. xylophilus and B. mucronatus. __________ Translated from Journal of Nanjing Forestry University, 2005, 30(4): 5–9 [译自: 南京林业大学学报]     

Variation of leaf characteristics in Populus tomentosa Carr.

An investigation was conducted to determine the extent of variations among nine provenances of Populus tomentosa Carr. in terms of leaf characteristics. A total of 263 accessions were studied under field conditions in the National Gene Bank of P. tomentosa in 2003. All of the accessions were characterized by 17 indices from 1 to 2-dimension constructions. Variance analysis of all characteristics showed that there were significant differences among the nine provenances and among individuals within each provenance. This study reveals that the evaluated germplasm appears to have a wide genetic base and high potential for further genetic improvements and it also indicates that abundant gene resources of P. tomentosa have been collected and preserved in the National Gene Bank. [Supported by the “Tenth Five-year Plan” National Key Project in Science and Technology (Grant No. 2002BA515B0303) and the National “863” Project (Grant No. 2002AA241071)]     

Molecular cloning of a cellulose synthase gene PtoCesA1 from Populus tomentosa and its genetic transformation in tobacco

A 3 125 bp cellulose synthase gene, PtoCesA1, which has a 98% identity to PtrCesA1 from Populus tremuloides, was cloned from cDNA prepared from secondary xylem of P. tomentosa. Four anti-expression vectors with different fragments of PtoCesA1, named as pBIPF, pBICC1, pBIPR and pBIBR, were constructed. Some traits of transformed tobacco of pBICC1, pBIPR and pBIBR differed from wild types, such as small leaves, “dwarf” phenotype and thinner xylem and fiber cell walls than wild plants consistent with a loss of cellulose. It indicated that the growth of transgenic tobacco was restrained by the expression of anti-PtoCesA1. Transgenic tobacco was obtained and the contents of cellulose and lignin were analyzed as well as the width and length of fiber cells, and xylem thickness for both transgenic and control plants. Transformed tobacco showed a different phenotype from control plants and it implied that PtoCesA1 was essential for the cellulose biosynthesis in poplar stems. [Supported by the Hi-Tech Research and Development Program of China (863) (2001AA244060 and 2003AA244020) and National Basic Research Program of China (973) (J1999016003)]     

Lignin characterization of triploid clones of Populus tomentosa Carr.

In order to understand the structural characteristics of lignin in triploid clones of Populus tomentosa and its changes in the processes of pulping and bleaching, milled wood lignin (MWL), lignin carbohydrate complex (LCC) and the residual lignin from kraft pulp (KP) and sulfite pulp (SP) were isolated and analyzed by Fourier transform infrared (FTIR) spectrum and ¹³C nuclear magnetic resonance (NMR). The most diagnostic peaks were assigned and the differences were discussed. The spectral patterns reveal that triploid P. tomentosa shows the specific features of hardwood from temperate areas, but in the spectrum of FTIR, the strength ratio of A 1270 cm⁻¹ to A1226 cm⁻¹ is 0.88, higher than the average of hardwood from temperate areas, which will make the lignin delignification more difficult during pulping and bleaching. The LCC from triploid P. tomentosa is mainly composed of xyloglucan and glucuronic acid, and other glucides have much lower ratio. In LCC FTIR, there are three peaks at 1 427, 1 329 and 1 046 cm⁻¹, indicating that both semi-cellulose and cellulose could exist in LCC, and that there might be relationships between cellulose and lignin. Compared with the residual lignin from KP and SP, the condensed structure in KP is more than that in SP.     

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.     

A novel cold-inducible promoter,P<Emphasis Type="Italic">ThCAP</Emphasis> from <Emphasis Type="Italic">Tamarix hispida</Emphasis>, confers cold tolerance in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Our previous studies have revealed that the ThCAP gene plays a vital role in transgenic Populus (P. davidiana × P. bolleana) in response to cold stress. However, the regulatory mechanism of ThCAP gene expression has been unclear. In this study, the 5′ flanking region of the ThCAP promoter (PThCAP) was cloned using a genome-walking method. By analyzing cis-acting regulatory elements of PThCAP, a DRE motif and MYC and MYB elements were found to be located in the promoter. To identify the regulatory elements that control the expression of the ThCAP gene promoter, a series of deletion derivatives of PThCAP, P1–P5, from the translation start code (?1538, ?1190, ?900, ?718 and ?375 bp), were fused to the GUS reporter gene, and then each deletion was stably introduced into Arabidopsis thaliana plants. Deletion analysis of the promoter suggested that only the P2 fragment had strong GUS expression in leaves and roots of A. thaliana exposed to low temperature stress. These results suggest that this 290-bp region (?1190 to ?900 bp), as an important part in PThCAP, was associated with cold tolerance of A. thaliana. Our results provide evidence for the regulatory mechanism of ThCAP gene involved in the response to cold stress, and that the gene is promising candidate gene for genetic improvement of crops.     

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.     

Biomass of fine roots and its relationship with water-stable aggregates in a composite ecosystem of triploid Populus tomentosa in the conversion of farmland to forest

A study on the biomass of fine roots and its relationship with water-stable aggregates (WSA) was conducted in two herbaceous models, triploid Populus tomentosa + Lolium multiflorum (TL) and triploid P. tomentosa + natural grass (TN). Both of the model triploid P. tomentosa stands were four years old converted from agriculture. Unconverted steep slope farmland was used as a control site. Results showed that the biomass of fine roots (⩽ 1 mm) in different layers varied in the following descending order: upper layer, middle layer and lower layer, at approximate ratios of 50:30:20. The average annual biomass of fine roots in ryegrass was twice that of the mixed natural grass-forest land. The total amount of natural grass roots was 4.4 times that of the ryegrass model. Water-stable aggregates of the upper, middle and lower layers and the unconverted farmland did not show any significant differences, whereas the amounts of water-stable aggregates of big-particles in the upper and middle layers were much larger than those of unconverted lands. The amounts of water-stable aggregates of natural grass-forest lands (TN model) were higher than those of managed grass-forest lands (TL model). Two-way analysis of variance indicated that fine roots (≤ 1 mm) could significantly enhance water-stable aggregates and total water-stable aggregates. We conclude that the program of converting agricultural lands to forest-grass lands is an effective way in improving soil anti-erosion capability. __________ Translated from Scientia Silvae Sinicae, 2007, 43(5): 24–29 [译自:林业科学]     

Chemical constituents from Gmelina arborea bark and their antioxidant activity

Gmelina arborea Roxb. is a fast-growing species and is known to have been used in traditional Indian medicine. Chemical constituents from the bark have not been reported, although some chemical constituents from part of this plant (heartwood, leaf, and root) are known. In this study, the bark meal was successively extracted with acetone and methanol. Fractionation of the acetone extract with n-hexane, diethyl ether, and ethyl acetate and subsequent chromatographic separation of the fractions led to the isolation of four compounds. The diethyl ether-soluble fraction yielded tyrosol [2-(4-hydroxyphenyl)ethanol] (1); (+)-balanophonin (2), an 8-5′ neolignan, with opposite optical rotation to known (−)-balanophonin; and gmelinol (3), a known lignan. The ethyl acetate-soluble fraction afforded a new phenylethanoid glycoside to the best of our knowledge, which was identified as (−)-p-hydroxyphenylethyl[5′″-O-(3,4-dimethoxycinnamoyl)-β-d-apiofuranosyl(1′" → 6′)]-β-d-glucopyranoside (4). From the methanol extract, two known compounds, 2,6-dimethoxy-p-benzoquinone (5) and 3,4,5-trimethoxyphenol (6), were isolated and identified. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay of the identifi ed compounds indicated that 3,4,5-trimethoxyphenol (6) exhibited moderate activity. Part of this report was presented at the 57th and 58th Annual Meetings of the Japan Wood Research Society, Hiroshima and Tsukuba, August 2007 and March 2008, respectively     

Artificial regeneration of Korean spruce in northeast China

Korean spruce (Picea koraiensis Nakai) has been included as a Chinese regeneration species since the 1960′s. In the 1970′s it became one of the four most important species in the northeast forest region of China. Korean spruce is a timber species with a wide range of adaptation, high survival rate, and few diseases or pests. Studies of 30-year trials show that the regeneration of korean spruce is successful and has a prospective future.     

Stereochemistry and biosynthesis of (+)-lyoniresinol, a syringyl tetrahydronaphthalene lignan in Lyonia ovalifolia var. elliptica I: isolation and stereochemistry of syringyl lignans and predicted precursors to (+)-lyoniresinol from wood

Steps leading to the biosynthesis of syringyl lignans and tetrahydronaphthalene and naphthalene lignans, especially the formation of the C2–C7′ linkage, have not been elucidated. Lyoniresinol is a typical syringyl lignan, as well as a tetrahydronaphthalene lignan found in Lyonia ovalifolia var. elliptica. To demonstrate the biosynthetic pathway for (+)-lyoniresinol, three putative biosynthetic intermediates of lyoniresinol, syringaresinol, 5,5′-dimethoxylariciresinol, and 5,5′-dimethoxysecoisolariciresinol, were isolated from wood. The identity of the putative intermediates was confirmed by spectroscopic analyses, as well as by comparison of spectral and chromatographic data with those of authentic samples previously synthesized. The stereochemistry (enantiomeric composition and absolute configuration) of the isolated lignans were determined as (±)-syringaresinol, (8S,8′S)-(−)-5,5′-dimethoxylariciresinol [46% enantiomeric excess (e.e.)], (8S,8′S)-(+)-5,5′-dimethoxysecoisolariciresinol (91% e.e.), and (8R,8′R)-(+)-lyoniresinol (42% e.e.). The absolute configurations of (+)-and (-)-5,5′-dimethoxylariciresinols, and (+)-and (-)-5,5′-dimethoxysecoisolariciresinols were determined by their synthesis (catalytic reduction) from (8R,8′R)-(+)-and (8S,8′S)-(-)-syringaresinols and by subsequent chiral high-performance liquid chromatography analysis. This report was presented at the 55th Annual Meeting of the Japan Wood Research Society, Kyoto, March 2005     

Molecular cloning and analysis of the partial sequence ofRhinopithecus roxellanae growth hormone gene

Growth hormone gene (GH) ofRhinopithecus roxellanae was amplified by PCR based on the sequences of the reported mammalian growth hormone gene for the first time. The amplified fragment was about 1.8 kb. It was cloned and its upper stream was sequenced. This sequencing region consists of a 5′flanking regulatory region, exon I and part of exon II, intron I of growth hormone gene. Comparing the corresponding sequences of growth hormone gene betweenRhinopithecus roxellanae and the porcine, we concluded that the homology reached 81% in the region, and there was high conservation in the 5′flanking sequence. The kinds of amino acids of exon I and exon II for about 90% were the same to those in pig. Many mutations occurred in the degenerate site of the triplet code. In the nucleotides of intron I, there were only 72% homologies with those in pig. It means that introns and 3′flanking sequence maybe play an important part in growth hormone gene regulation of the different animals. The project is supported by National Nature Science Foundation of china (No. 39970103). Xu Laixiang, male, born in March 1960, professor, Department of Biology, Qufu Normal University, Qufu 273165, P.R. China. Responsible editor: Zhu Hong