Cloning and Expression Analysis of PtFATB Gene Encoding the Acyl-acyl Carrier Protein Thioesterase in Populus tomentosa Carr

Acyl-ACP thioesterases （FATs） terminates the fatty acid synthesis and allow the transport of fatty acids out of the plastids, which are the important determinants of cellular metabolism. FATB is a member of FAT enzymes that has been described previously in most of the plants. In silico cloning is a new method that utilizes the bioinformatics on the complete genome and available EST database. In this study, a full-length cDNA clone of PtFATB gene was isolated from Populus tomentosa using this approach. It is 1,450 bp in length and the open reading frame encodes a peptide of 421 amino acids. The predicted amino acid sequence shows significant homology with those from other plant species, which contain typical domains owned by FATB proteins. The transcripts of PtFATB were abundant in leaves, and less in roots detected by using semiquantitative RT-PCR. When the shoots were subjected to the stress treatments （cold, dry, NaC1） and ABA （Abscisic acid）, the expression of PtFATB was only slightly reduced under the treatment of low temperature. This suggests that the expression of PtFATB is in a constitutive fashion. This study provides the basis not only for the identification and characterization of this gene but also for the improvement of cold tolerance by controlling the expression of the PtFATB gene in trees in near future.

Segregation of AFLP Markers in A ( Populus tomentosa× P. bolleana) ×P. tomentosa Carr. BC_1 Family

To investigate the levels of polymorphisms and Mendelian segregation ratio in clone “TB01” (P. tomentosa×P. bolleana) ×clone “LM50” (P. tomentosa) BC 1 population at the entire genome level, amplified fragment length polymorphisms (AFLPs) analysis was conducted for both parents and 120 progenies. Forty one pairs of selective primers were used to detect 2?707 bands, of which 712 (26.4%) were polymorphic. Chi\|square tests were performed to examine if the observed genotypic frequencies of AFLP loci deviated from expected 1∶1 Mendelian segregation ratio ( P <0.01) in BC 1 population. Among the 712 loci 571 (80.2%) fit to Mendelian 1∶1 segregation ratio, corresponding to DNA polymorphisms heterozygous in one parent and a null in the other. The result shows that the AFLP markers are very suitable for fingerprinting and genetic mapping in the Chinese white poplar (Populus tomentosa Carr.).

Genetic Control of Air-Dried Wood Density, Mechanical Properties and Its Implication for Veneer Timber Breeding of New Triploid Clones in Populus tomentosa Carr

The wood samples of 9 triploid clones of Populus tomentosa Carr. taken from a 9 year old clonal test site were analyzed in order to investigate the genetic variation of wood properties, including air dried wood density and some mechanical properties. The results showed that significant or extremely significant difference in air dried wood density and the mechanical properties existed among the clones, this means these wood properties were under moderate or strong genetic controls and could be improved by genetic manipulations. The radial and vertical variation patterns of air dried wood density were also studied and the results were found to coordinate with other previous research results. The vertical variation patterns of most mechanical properties within the individual tree also conformed to the general wood theories except the modulus of elasticity and cross section hardness. Among the mechanical properties, modulus of elasticity (MOE) and tangent section hardness were under strong genetic control, with the clonal repeatabilities being 0 90 and 0 80, respectively. However, the clonal repeatabilities of other mechanical properties under study were a little lower than above two indexes. Genetic correlation analysis indicated that super clonal selection and breeding for veneer timber could be realized through indirect selection of wood density and form indexes.

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.

Lignin Characterization of Triploid Clones of Populus tomentosa Carr.

In order to understand the structural characteristics of lignin in triploid clones ofPopulus 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 orAl270 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.

THE FIBER MORPHOLOGY VARIATION OF TRIPLOID CLONE OF POPULUS TOMENTOSA CARR.AND ITS SUPPOSED HARVESTING AGE FOR THE PAPER INDUSTRY

The fiber morphology variation of triploid clone of Populus tomentosa Carr. and the supposed harvesting age for the paper industry were investigated in this paper. The results shows that in the first few years, 2-4 years for example, the fiber length at breast high grows faster than it does in the later years. But the standard deviation of distribution in fiber length at breast high in mature wood is larger than in the young tree. The technologically harvestable age of triploid clone of Populus tomentosa Carr., BL304 could be 4-7 year old to meet the requirement for paper industry. The distribution of fiber length become concentrated within an annual ring as the tree grows. The test results also show that the cloned poplar has a small Runkel ratio value (less than 0.4, generally about 0.3), and this ratio increases slightly with the tree grows.

Incidence and molecular markers of 2n pollen in Populus tomentosa Carr

Microscopic examination and AFLP molecular markers were employed to determine the incidence and related markers of 2n pollen (unreduced pollen) in Populus tomentosa Carr. The parallel and tripolar spindle at metaphase Ⅱ and the absence of cytokinesis at telophase Ⅱ led to the formation of 2n pollen. A group of 298 clones came from their indigenous areas was investigated for the production of 2n pollen based on the pollen size differences, within a clone and between n and 2n pollen. Pollen grains of 224 clones were collected, six of them only produced normal pollen, and the rest produced 2n pollen at different frequencies (0.6% -21.9%). Clones producing six normal and 22 2n pollen were selected for AFLP analysis. Following an initial screening with 55 primer combinations, the E50-M38 (CAT/ACT) primer was identified, which generated a PCR fragment (246 bp) from the normal clones, but not from the 2n pollen producers. In addition, the E31-M50 (AAA/CAT) amplified DNA fragment (204 bp) that was present in 2n pollen producers, and absent in normal clones. Two polymorphic bands were found and they were distinguished between normal and 2n pollen clones. They are very useful as AFLP markers for molecular-assisted selection in triploid breeding of 2n gametes in P.tomentosa.

Model of leaf energy distribution and its experimental validation of Populus tomentosa Carr

Leaf temperature of a plant is the result of heat transfer between the plant and its environment. There are many factors that can affect leaf temperature, such as the solar radiation energy, environmental temperature, wind velocity, evaporation on the leaf surface, photosynthesis, respiration and so on, which have different effects on the temperature of leaves. In first instance, we analyzed the heat transfer on leaves of Populus tomentosa Carr. theoretically and constructed a model of energy distribution. We then validated the model by analyzing seven different kinds of one-year-old P. tomentosa leaves experimentally. The result shows that solar radiation is the main energy input and the dominant ways of thermal diffusion are heat transfer between the upper and lower leaf surfaces and evaporation from the leaf surface.

Interrelationships between leaf heat conductivity and tissue structures of different varieties of Populus tomentosa Carr

Plant heat conductivity largely depends on tissue structure. Different structures lead to different heat conductivity. As well, water transfer also plays a very important role in heat transfer in plants. We have studied leaf heat conductivity and tissue structure of 3- and 30-year-old Populus tomentosa Carr. trees using infrared thermal imaging, steady state heat conductivity surveys and paraffin section and investigated the relationship between leaf heat conductivity, tissue structure and water content of leaves. The results show that the temperature on leaf surfaces among the various varieties of trees was almost the same. Leaf heat conductivity, temperature and water content of leaves are positively correlated. The thicker the leaf tissue structures, the larger the heat resistance. That is, the tighter the cells and the smaller the interspaces, the smaller the heat conductivity, which is not conducive for heat transfer.

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 Cart. 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-Ⅰ were found. Three allozyme bands at locus Mdh-Ⅰ 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-Ⅰ 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-Ⅰ 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.

Improvement in salt tolerance of Populus tomentosa from transformation by a mtl-D gene

Transgenic lines were achieved by transforming the E. coli 1-phosphate mannitol dehydrogenase gene （mtl-D） into the Populus tomentosa Cart. genome. An Agrobacterium tumefaciens strain （AGL1）, constructed by cloning mtl-D into the disarmed plasmid pBin438, was used to infect leaves of the clone YW2. The infected leaf discs were cultured on a medium containing 30 mg.L 1 kanamycin and 500 mg.L 1 cefotaxime. Transgenic plantlets regenerated from the infected leaves, rooted on the medium con- taining 30 mg.L 1 kanamycin. PCR and a Southern blotting test verified that the exogenous mtl-D gene had integrated into the trans- formation plants of the P. tomentosa genome. The mannitol content in control plant was 69 gg.gl FW, and the mannitol contents of the transgenic lines T1 to T5 ranged between 103.7 and 289.5 μg·g^-1 FW. Of the shoots of the control plants 20% survived; on the medium containing 0.6% NaCl, 60% and 70% of two transgenic shoots survived on a medium containing 0.8% NaCI.

Comparison of G6PDH Activity and LT50 Between P. tomentosa and P. suaveolens During Freezing Acclimation

The differences of glucose-6P dehydrogenase (G6PDH) activity and freezing resistance induced by freezing acclimation between cuttings of freezing-sensitive P. tomentosa and freezing-resistant P. suaveolens were compared for exploring the role of G6PDH on the enhancement of freezing resistance induced by freezing acclimation. After 5 d of freezing acclimation at -3 ℃, the LT50 of P. tomentosa has decreased from -6.2 ℃ in control cuttings to -14.3 ℃ in freezing acclimated ones, and the increase of G6PDH activity was observed in freezing acclimated cuttings as compared with control ones. Whereas, when P. suaveolens was freezing acclimated at -20℃ for 5 d, the LT50 has decreased from -27.1℃ in control cuttings to -43.5 ℃ in freezing acclimated ones, and the activity of G6PDH increased considerably. In addition, the increase of LT50 and the decrease of G6PDH activity resulting from 2 d of deacclimation at 25 ℃ were found in two kinds of freezing acclimated cuttings. It is concluded that the increase in the activity of G6PDH may associate with the inherited freezing resistance of species and the enhancement of freezing resistance of cuttings, and may play an important role in the antifreeze process under freezing temperature, which would provide the basis for the study on the molecular mechanism of freezing resistance in P. suaveolens and the cloning of gene associated with freezing resistance.

Comparison and early selection of new clones in Populus tomentosa

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.

成年雄性毛白杨组培幼化技术研究

【目的】毛白杨以无性繁殖为主,而无性繁殖材料往往存在老化现象,严重影响苗木质量,研究繁殖材料的幼化技术对于改善苗木质量,促进无性林业发展有重要意义。【方法】以北京大兴区站上村的200年生雄性毛白杨为对象,取其根萌条为繁殖材料,探索2种灭菌试剂处理条件下,不同灭菌时间对外植体成活与生长的影响,同时在MS培养基中设置不同激素组合研究其增殖的最佳效果,筛选出适合其生长的最佳增殖培养基,通过继代培养研究此200年生成年雄性毛白杨的组培幼化技术。【结果】结果表明,0.1%氯化汞溶液的灭菌效果比10%次氯酸钠溶液效果好,最佳灭菌处理为0.1%氯化汞7 min,成活率为57%;最佳增殖培养基为MS+0.7 mg/L 6-BA+0.01 mg/L NAA,最高丛生芽分化系数为2.23,茎高生长量≥1 cm苗的数量比例为53.33%,显著高于其他处理;随着继代次数的增加,生长周期由63 d(1代)降至44 d(4代),继代培养增殖系数由0.93(1代)提升至4.85(4代),茎高生长量≥3 cm苗的数量比也从2.23%(1代)显著增至62.34%(4代)。【结论】此200年生毛白杨繁殖材料存在一定程度的老化现象,母株的年龄可能影响其根萌条的幼化程度,但随着继代次数的增加,生长势有了显著的提高,说明多次继代培养有较明显的幼化作用,且随着继代次数的增加,幼化效果也越好。

不同污染程度下毛白杨叶表面PM_(2.5)颗粒的数量及性质和叶片气孔形态的比较研究

选择了北京市环境PM_(2.5)浓度不同的两个采样点的毛白杨(Populus tomentosa Carr.)作为研究对象,利用环境扫描电镜及X-射线能谱仪对杨树叶片表面滞留的PM_(2.5)颗粒进行了观察、统计和成分分析,并研究了叶片气孔对环境颗粒物污染的适应性变化。结果表明:夏秋两季西直门叶片样品上下表面的PM_(2.5)数量均多于森林公园样品这说明环境PM_(2.5)浓度是影响叶片表面滞留颗粒物数量的主要原因;其中叶片上表面是滞留PM_(2.5)颗粒的主要区域。森林公园样品中PM_(2.5)颗粒性质比较单一,硅铝酸盐颗粒和石英颗粒占很大比例,二者的主要来源均为天然源,如土壤扬尘、矿物颗粒等;而西直门采样点叶片样品滞留的PM_(2.5)颗粒的元素组成更为复杂,其中50%以上的硅铝酸盐颗粒检测出了明显的铜、钾、氯、钠等元素的谱峰其来源主要是工业排放;西直门样品PM_(2.5)的含硫量高于森林公园样品,且夏季明显高于秋季。研究还发现有少数PM_(2.5)颗粒进入了毛白杨叶片的气孔而且不同污染程度下气孔的形态特征存在差异。与森林公园毛白杨叶片的气孔相比,西直门处的毛白杨叶片气孔的长度、宽度、面积和气孔密度均较小,说明较高的PM_(2.5)污染程度对毛白杨叶片的形态发育有一定影响。研究结果可以为揭示植物叶片阻滞、吸收大气颗粒污染物的机制、合理选择和优化城市绿化树种从而改善空气质量提供一定的科学理论依据。

毛白杨形成层的活动周期及其POD同工酶的变化

北京地区生长的毛白杨 (PopulustomentosaCarr .)在春季芽膨大前 ,形成层带已经进入了恢复活动期。在芽展开后一周形成层进入活动期 ,细胞开始分裂 ,细胞层数也有所增加 ,同时分化出未成熟的韧皮部细胞。未成熟韧皮部细胞的数量在形成层活动初期快速增长 ,随后是一个持续而缓慢的连续过程。木质部的产生比韧皮部要晚 3周 ,未成熟的木质部细胞层数在开始变化不大 ,5月底～ 6月底 ,增长速度明显高于初期 ,随后便急剧下降。成熟木质部细胞的出现比成熟的韧皮部细胞要晚约一个月。与其他研究结果不同 ,毛白杨在4月底到 6月底这段时间 ,形成层细胞层数迅速下降 ,未成熟韧皮部细胞也略有减少 ,而未成熟和成熟的木质部细胞则大量增加。木质部在 9月底停止分化 ,较韧皮部要晚约 2 0d。 9月底形成层进入休眠期后 ,形成层细胞层数基本保持不变 ,直到翌年春天恢复活动。毛白杨形成层区域的过氧化物酶 (POD)同工酶在形成层活动期其活性低、酶带少 ,而恢复活动期和休眠期其活性高且酶带多 ,组织化学反应还表明 ,不同时期POD在不同的细胞和组织中的活性不同。本文对POD同工酶在形成层活动周期中的变化与其组织分化的关系进行了讨论。

运用AFLP技术估计毛白杨及其杂种毛新杨的遗传杂合水平

首次报道以毛新杨 (Populustomentosa×P .bolleana)无性系TB0 1×毛白杨 (P .tomentosa)无性系LM5 0的回交子代 12 0株随机个体为材料 ,利用扩增性片断长度多态性 (AFLPs)标记技术研究毛白杨及其杂种毛新杨的遗传平均杂合水平。研究结果表明 :30对不同的AFLP引物组合能够检测到毛白杨的遗传平均杂合水平范围为 9 10 %～ 30 19% ,平均为 18 71% ;而毛新杨的遗传平均杂合范围为 1 39%～ 2 0 31% ,平均杂和水平约为 8 4 7%。因此 ,对于分离群体亲本杂合度来说 ,毛白杨平均杂合度为毛新杨的 2 2 1倍。

毛白杨NBS型基因PtDRG01原核表达研究

为了分析毛白杨NBS型抗病基因PtDRG01(EF157840)编码蛋白的特性,研究构建了该基因的原核表达载体pGEX-Pt01,并导入大肠杆菌XA90,经IPTG诱导表达后,SDS-PAGE电泳分析证明,该特异蛋白的分子量约为79kD。对原核表达体系以及融合蛋白的表达特性进行优化与分析表明:最佳诱导表达条件为1mmol/L IPTG在37℃下诱导4h,所表达的融合蛋白为胞内分泌的可溶性蛋白。利用谷胱苷肽-琼脂糖亲和层析柱纯化获得了电泳纯级的目标蛋白,为PtDRG01基因编码蛋白的功能鉴定研究奠定了基础。

毛白杨花粉败育过程中Ca^(2+)-ATPase的异常变化

为研究木本植物花粉败育过程中Ca2+-ATPase的影响,本文利用氯化铈沉淀和电镜细胞化学方法,对不同育性毛白杨花药在小孢子发生发育过程中Ca2+-ATPase进行了定位。结果表明:Ca2+-ATPase在可育花药小孢子母细胞时期大量分布于小孢子母细胞质膜、液泡膜及内膜系统,花药内壁细胞内膜系统和绒毡层细胞质膜,随后Ca2+-ATPase在上述细胞中均减少甚至消失,并于小孢子时期和成熟花粉时期分别在小孢子和花药内壁细胞中再次沉积;不育花药小孢子母细胞以及此时期绒毡层细胞质膜上并无明显Ca2+-ATPase,花药表皮、药室内壁以及中层的Ca2+-ATPase都高于同一时期的可育花药,其绒毡层细胞解体不完全。由上述结果推测:毛白杨不育花药内小孢子母细胞和药壁中Ca2+-ATPase分布异常,影响细胞内钙离子的主动转运,可能导致钙离子异常累积,进而通过影响小孢子母细胞和药室内壁细胞的正常代谢、绒毡层细胞的及时降解而导致花粉败育。

杨树形成层区域扩张蛋白PtEXP1基因的克隆与分析

以矮牵牛的cDNA核苷酸序列为基础,利用杨树EST数据库和毛果杨基因组测序结果,通过序列的拼接设计合成了基因特异引物,从毛白杨形成层cDNA中扩增出一个长为1 009 bp的cDNA片段,将该片段连接到pGEM-T载体中并测序。测序结果表明,该片段含有完整的开放阅读框,共编码258个氨基酸残基。蛋白序列系统进化分析结果表明,该基因属于exp ansin基因家族-αexp ansin中的A亚家族,编码的氨基酸序列与芒果、欧洲山杨×美洲山杨杂交种、拟南芥和矮牵牛的-αexp ansin基因编码的氨基酸序列同源性分别为91%,88%,86%和86%。