杨树PtRRI基因的组织特异性表达模式分析

[目的]模式植物在木本植物中鉴定的许多重要调控因子家族在木本植物中出现了基因家族成员扩张,但ARRs家族作为细胞分裂素响应调节因子在杨树基因组中成员数量反而减少,其在木本植物中如何行使功能需要进一步研究。[方法]本研究通过生物信息学构建PtRRI启动子与GUS融合表达载体,检测植物激素处理后PtRRI表达量和检测PPtRRI::GUS转基因植株在生根过程中GUS信号等方法,对杨树PtRRI基因的组织特异性表达模式进行分析。[结果]表明:PtRRI在杨树根部、形成层、木质部表达量相对较高,PtRRI转录受6-BA激素诱导,与其在不定根发育过程中激素调控下的表达相一致。[结论]PtRRI基因可能参与杨树的次级生长。     

Spatial distribution characteristics of fine roots of Populus euphratica in a desert riparian forest

The soil-plant system is a very important subsystem of the soil-plant-atmosphere continuum (SPAC). The water uptake by plant roots is an important subject in the research on water transport in this SPAC and is also the most active study direction in the fields of ecology, hydrology and environment. The study of the spatial distribution pattern of fine roots of plants is the basis of constructing a water absorption model of plant roots. Our study on the spatial distribution pattern of fine roots of Populus euphratica in a desert riparian forest shows that the density distribution of its root lengths can be expressed horizontally as a parabola. The fine roots are concentrated within the range of 0–350 cm from the tree trunk and their amount accounts for 91.9% of the total root mass within the space of 0–500 cm. In the vertical direction, the density distribution of the fine root lengths shows a negative exponential relation with soil depth. The fine roots are concentrated in the 0–80 cm soil layer, accounting for 96.8% of the total root mass in the 0–140 cm soil layer. __________ Translated from Chinese Journal of Ecology, 2007, 26(1): 1–4 [译自: 生态学杂志]     

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.     

Transgenic poplar with enhanced growth by introduction of the ugt and acb genes

The transgenic poplar (Populus tremula L.) was obtained by transfer of the ugt and acb genes via triparental mating, which was employed to deliver large fragments of TDNA as a cluster. Freshly harvested seeds of local poplar were placed on MS agar medium and plantlets were obtained. After 1 year of subcultivation, plantlets were infected with a transconjugant of triparental mating with target ugt and acb genes into axillary buds. The transformed sprouts so obtained were cut and subcultivated on agar medium with an addition of 0.6 mg/l indole-3-butyric acid as an auxin source. The transformed sprouts showed GUS activity and resistance to gentamycin and kanamycin. The integrity of the target ugt and acb genes into poplar genome was demonstrated via PCR and Southern blot hybridisation. The transgenic poplar plants revealed a higher growth energy, corresponding to a higher content of IAA as opposed to control plants. Both transgenic and non-transformed plants were potted into soil for outdoor acclimatisation and subsequently transferred to earth in beds. Growing outside during 3 years, the transgenic poplar demonstrated a higher growth rate with fast bud and branch development.     

农杆菌介导慈竹4CL基因遗传转化梁山慈竹

以梁山慈竹2种类型成熟胚的愈伤组织为材料,采用农杆菌遗传介导的方法,将已构建好的具有降低木质素含量的PBI121-4CL-RNAi表达载体导入愈伤组织,探讨愈伤组织预培养时间、菌液浓度、侵染时间、共培养时间和温度对遗传转化的影响。研究结果表明,淡黄色、颗粒状、疏松易碎的胚性愈伤组织是较好的遗传转化材料。以在愈伤组织培养基上预培养8天的淡黄色、颗粒状、疏松易碎的胚性愈伤组织为转化受体,在菌液浓度为OD600=0.05的EHA105中侵染20min后,在25℃、黑暗条件下共培养2天(共培养基表面加一层无菌滤纸),在含有卡那霉素为55mg.L-1的抗性筛选培养基上筛选30天,抗性愈伤组织率为90%,经PCR检测,慈竹4CL基因已导入梁山慈竹愈伤组织中。抗性愈伤组织在芽诱导培养基上诱导30天,可获得丛生芽,待丛生芽长至3～5cm后,在生根培养基上经过20～30天的诱导,可产生1～8条根,获得再生植株。经PCR检测,慈竹4CL基因已导入梁山慈竹再生植株中,获得了转基因植株,转化效率为9%。RT-PCR检测结果表明,转4CL基因的梁山慈竹愈伤组织和植株的内源4CL基因的表达受到抑制,且表达量比对照明显降低。     

Characterization of expressed genes in the establishment of arbuscular mycorrhiza between Amorpha fruticosa and Glomus mosseae

Arbuscular mycorrhiza （AM） formed between plant roots and fungi is one of the most widespread symbiotic associations in nature. To understand the molecular mechanisms of AM formation, we profiled 30 symbiosis-related genes expressed in Amorpha fruticosa roots colonized by Glomus mosseae and in non-mycorrhizal roots at different stages using differential-display RT-PCR （DDRT-PCR）. The expressed genes were confirmed by reverse Northern blotting. Eleven fragments were sequenced and putatively identified by homologous alignment. Of the eleven AM-related genes, five were obtained at the early-stage of plant-fungus interaction and six at the later stage. Three expressed se-quence tag （ESTs） sequences were found to originate from the fungi and eight from the host plant by use of PCR evaluation of gDNA of both plant and fungi. The target genes included an ATP-binding cassette sub-family transporter gene, a transposon-insertion display band, and a photosynthesis-related gene. The results provided information on the molecular mechanisms underlying the development of mycorrhizal sym-biosis between woody plants and AM fungi.     

Direct shoot organogenesis from leaf explants of Embelia ribes Burm. f.: a vulnerable medicinal plant

An efficient system was developed for direct plant regeneration from in vitro-derived leaf explants of Embelia ribes Burm. f., a vulnerable medicinal woody climber of the Western Ghats of India. The in vitro procedure involved three steps that included induction of shoot initials from leaf tissue, regeneration and elongation of shoots from the shoot initials, and rooting of shoots. The induction of shoot initials was achieved on Murashige and Skoog (MS) solid medium supplemented with different concentrations of thidiazuron (TDZ). The best medium for shoot induction was MS with 0.272 μM TDZ. Numerous shoot primordia developed within 2–3 weeks on the leaf margin as well as on the midrib region, without any callus phase. In the second step, the shoot clumps separated from the leaf explant on transfer to MS basal medium, resulting in the differentiation of 90% of the shoot initials into well-developed shoots. The 2- to 3-cm-long shoots rooted on half-strength MS basal medium supplemented with 4.90 μM indole-3-butyric acid (IBA) and 3% (w/v) sucrose in the third stage. The rooted plants could be established in soil with 70% success. This protocol could be utilized for in vitro propagation and conservation of this important threatened medicinal plant.     

蓝桉和尾叶桉混合菌根研究 Ⅱ.混合菌根的接种效应

接种苗木在生长量和生物量上均表现出极其显著的生长优势,ＥＣＭ真菌蜡蘑菌单接种及其与４种ＶＡ菌根菌剂混合接种,对苗木生长的促进作用尤为显著。在接种后１６周时,与对照苗相比,蓝桉和尾叶桉接种苗木的高生长量最大增幅分别为２８．８６％（ＬＳ）和８６．６５％（ＬＧ）;两种桉树地上部分平均干质量最大增幅分别为１２９．９３％（ＬＳ）和１３３．３４％（Ｌ）,地下部分分别为１１９．９３％（ＬＦ）和１７４．８３％（Ｌ     

大花蕙兰转齿兰环斑病毒外壳蛋白基因及检测

[目的]通过植物转基因技术获得抗病毒大花蕙兰种质资源,优化转化体系和鉴定方法.[方法]本研究克隆了齿兰环斑病毒外壳蛋白基因,并构建了该基因的pBI121表达载体,用根癌农杆菌介导法转化大花蕙兰,尝试以巢式PCR方法检测转基因再生植株.[结果]优化了大花蕙兰遗传转化体系,建立了利用巢式PCR技术检测转基因大花蕙兰植株的方法,获得了32株转基因株(系).[结论]优化了以类原球茎为外植体的农杆菌介导转化大花蕙兰的方法,确定以5%~10%类原球茎存活时的抗生素(卡那霉素)浓度为筛选浓度;获得了转ORSV CP基因大花蕙兰植株;对大花蕙兰转基因植株检测时,巢式PCR较普通PCR更灵敏、准确.     

In vitro regeneration of Salix nigra from adventitious shoots

Black willow (Salix nigra Marsh.) is the largest and only commercially important willow species in North America. It is a candidate for phytoremediation of polluted soils because it is fast-growing and thrives on floodplains throughout eastern USA. Our objective was to develop a protocol for the in vitro regeneration of black willow plants that could serve as target material for gene transformation. Unexpanded inflorescence explants were excised from dormant buds collected from three source trees and cultured on woody plant medium (WPM) supplemented with one of: (1) 0.1 mg l(-1) thidiazuron (TDZ); (2) 0.5 mg l(-1) 6-benzoaminopurine (BAP); or (3) 1 mg l(-1) BAP. All plant growth regulator (PGR) treatments induced direct adventitious bud formation from the genotypes. The percentage of explants producing buds ranged from 20 to 92%, depending on genotype and treatment. Although most of the TDZ-treated inflorescences produced buds, these buds failed to elongate into shoots. Buds on explants treated with BAP elongated into shoots that were easily rooted in vitro and further established in potting mix in high humidity. The PGR treatments significantly affected shoot regeneration frequency (P < 0.01). The highest shoot regeneration frequency (36%) was achieved with Genotype 3 cultured on 0.5 mg l(-1) BAP. Mean number of shoots per explant varied from one to five. The ability of black willow inflorescences to produce adventitious shoots makes them potential targets for Agrobacterium-mediated transformation with heavy-metal-resistant genes for phytoremediation.     

Reaction of manganese peroxidase ofBjerkandera adusta with synthetic lignin in acetone solution

The reaction of manganese peroxidase (MnP) of the white-rot fungusBjerkandera adusta with synthetic lignin dehydrogenation polymer, DHP) in acetone medium was investigated. Gel-permeation chromatography of the DHP treated by MnP demonstrated depolymerization of syringyl DHP in the reaction mixture containing 70% acetone; moreover, concomitant repolymerization occurred to give highly polymerized products. Guaiacyl DHP was only repolymerized by MnP in the same acetone solution without giving degradation products. Addition of ascorbic acid to reaction mixtures containing acetone resulted in preferential depolymerization of syringyl DHP.Part of this report was presented at the meeting of Kansai Branch, Japan Society for Bioscience, Biotechnology, and Agrochemistry in Kagawa, October 1996     

Overexpression of a fungal laccase gene induces nondehiscent anthers and morphological changes in flowers of transgenic tobacco

Laccases play important roles in the development of fruiting bodies and in lignin degradation by basidiomycetes. In this study, we present novel phenotypes of transgenic tobacco plants with a chimeric gene for fungal laccase under the control of the cauliflower mosaic virus 35S promoter. At the flowering stage, the transgenic plants that produced recombinant laccase had brownish anthers instead of the greenish anthers of wild-type plants. The brownish anthers exhibited male sterility with a nondehiscent phenotype at varying frequencies. The frequency of nondehiscence depended on the temperature at which plants were cultivated and it was higher at 24°C than at 29°C. The cell wall structures of transgenic anther tissues were almost the same as in the wild type, but the stomium was severely deformed, and abnormal components were apparent in cells of the endothecium and epidermis. Furthermore, the pattern of deposition of flavonoids in the transgenic anther epidermis differed from the wild-type pattern. The expression of laccase also induced other phenotypic changes in the flowers of transgenic plants, namely, increased petal number, fused and petaloid stamens, and doubling of floral organs. These results indicate that the ectopic expression of laccase influences various aspects of flower development.     

过量表达LAR3和Bbchit1以提高毛白杨对叶枯病的抗性

利用转基因技术将多种抗病基因共同转入毛白杨中以提高其抗性,从而获得毛白杨抗病新品种是目前解决杨树真菌病害的主要研究方向之一。本研究通过根癌农杆菌介导的二次遗传转化,将来源于球孢白僵菌几丁质酶基因Bbchit1转入过量表达无色花色素还原酶基因LAR3转基因毛白杨中,实时定量PCR显示Bbchit1与LAR3均能有效表达,离体抗病试验显示Bbchit1+LAR3共表达转基因毛白杨细胞粗提液对杨树叶枯病菌具有明显抑制作用,进一步将叶枯病菌接种在转基因和非转基因毛白杨叶片上培养30天,转基因植株的感病面积均低于非转基因植株且Bbchit1+LAR3共表达转基因株系抗病效果更明显。上述抗病试验结果表明:LAR3和Bbchit1在杨树中共表达可提高其对叶枯病的抗性。     

Over-expression of Tryptophan Decarboxylase Gene in Poplar and its Possible Role in Resistance Against Malacosoma disstria

Amines and their derivatives are known to influence insect behavior involved in feeding and reproduction. In order to examine the feasibility of improving the resistance of poplar to insect pests by the introduction of a plant-derived amine-generating transgene, explants from the hybrid poplar clone ‘INRA 717 1B4’ (P. tremula ×P. albo) were transformed with a Camptotheca acuminata tryptophan decarboxylase (TDC, EC 4.1.1.28) cDNA driven by the CaMV 35S promoter. The enzyme TDC catalyzes the decarboxylation of tryptophan to tryptamine, which, in addition to being a bioactive amine itself, is known to act as a precursor of various other indole derivatives. Putative transgenic lines were confirmed by PCR for the TDC1 gene sequence and by the expression analysis of the transgene mRNA and encoded protein. No visible phenotypic changes were associated with ectopic TDC1 expression. Chemical and radiotracer analyses of the transgenic plants revealed tryptamine accumulation as high as 4 mM in leaf tissue, and suggested that the tryptamine produced by ectopically expressed TDC was not further metabolized. Insect bioassays with the TDC transgenic plants showed that the tryptamine accumulation was consistently associated with adverse effects on feeding potential and physiology of Malacosoma disstria (forest tent caterpillar).     

Molecular cloning and expression analysis of a gene encoding a putative beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III) from Jatropha curcas

A cDNA clone encoding a putative beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III) was isolated from Jatropha curcas L., a woody oil plant. The cDNA clone (named JcKAS III) contained a 1203-bp open reading frame coding for 400 amino acids with a predicted molecular mass of about 42 kDa. The deduced amino acid sequence of the cDNA clone shares about 80% identity to KAS III from other plants, and contains a conserved Cys(176) in the active site and the amino acid motif G(355)NTSAAS(361) which is responsible for binding regulatory acyl-ACPs. Southern blotting analysis indicated that JcKAS III is a single copy gene in the J. curcas genome. Quantitative real-time PCR analysis showed that JcKAS III was expressed in all tissues examined with highest expression in roots, and that expression of JcKAS III increased as seeds developed.     

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.     

双价抗病基因植物表达载体构建及在烟草中表达的初步研究

多基因转化是基因工程研究热点之一。本研究应用DNA重组技术,将两个抗病机制不同,抗菌谱较广的抗病基因(天麻抗真菌蛋白GAFP和兔防御素NP1基因)构建在一个植物表达载体pBin35SGAFP-NP1上,两者具有各自的CaMV35S启动子和Nos终止子。通过根癌农杆菌介导,采用叶盘法转化烟草,PCR和PCR-Southern分析证明已将NP1和GAFP基因整合到烟草基因组中。离体抑菌实验表明转基因植株对真菌和细菌表现出一定的抗性。以上结果表明通过该表达载体进行遗传转化可获得含双价抗病基因植物,并能有效表达,提高转基因植物抗病能力。