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.     

Survival and development of immature Harmonia axyridis (Pallas) feeding on Chaitophorus populeti (Panzer) propagated on transgenic Populus alba × P. glandulosa

Laboratory feeding experiments with the poplar aphid, Chaitophorus populeti (Panzer), feeding on transgenic poplar (P. alba × P. glandulosa) varieties C13-5 and C013-5, were carried out to study the effect of transgenic poplar on the ladybird Harmonia axyridis (Pallas). The mortality and development time of the immature stages, the eclosion rate and body mass of H. axyridis were measured. The results indicated that C. populeti feeding on different varieties of transgenic plants had no statistically significant effect on the mortality of H. axyridis larvae. The development time of larval and pupal stages were not significantly different between the two transgenic poplars and a non-transgenic poplar. Furthermore, the body mass and eclosion rate did not show any difference between the H. axyridis feeding on aphids reared on transgenic plants and those from non-transgenic plants. It is suggested that transgenic plants have no deleterious effect on the predatory ladybird.     

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)]     

超量表达FBL1对84K杨根系和生长量影响研究

生长素及其信号转导系统对植物的生长发育具有重要的影响。本研究从银腺杨'84K'(Populus alba × P. glandulosa cl. '84K')中分离了生长素受体基因PtrFBL1,利用PMDC32构建了PMDC32-PtrFBL1超量表达载体,并通过遗传转化获得了超量表达植株17个。对温室定植的3个转基因株系和对照植株的根系、生长量和光合指标等性状分析结果显示:转基因株系总根长和总根面积达到显著或极显著差异,而根系干质量、平均不定根系长度、平均不定根直径差异不显著;株高、平均节间长、地径和高径比皆高于对照,且大多数转基因株系达到显著差异;除气孔限制值(Ls)低于对照外,气孔导度(Cd)、水分利用效率(WUE)、光能利用效率(LUE)和叶绿素相对含量皆高于对照,且大多数转基因株系达到显著或极显著差异。以上结果表明,可能是FBL1超表达增加了转基因株系根系面积,提高了水分和养分的吸收利用,进而导致转基因株系光能吸收和转化效率提高,引起转基因株系生长加快。     

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).     

Characteristics of senescent straw cell walls of dwarf, semidwarf, and normal strains of rice (Oryza sativa) plants

A normal variety of rice (Oryza sativa L.cv. Taichung 65, T65c), its isogenic dwarf line (T65d ₁), and a semidwarf variety of a different line (Oryza sativa L.cv. IR8, IR8) were studied. The results were compared with those of an isogenic dwarf line (Rh _i) of wheat straw, which was previously reported. Expression of the dwarf gene,d ₁, on the chemical composition and the structural features of lignin present in rice internodes differs from that in an isogenic dwarf line of wheat. The differences include the lignin content, total yield of alkaline nitrobenzene oxidation products, and distribution of wall-bound hydroxycinnamic acids. There was, however, no difference in the syringyl/ guaiacyl nuclei (S/V) molar ratio and neutral sugar composition. The lignin composition of rice straw cell walls, particularly that of the dwarf variety, contained more of the condensed structure and fewer syringyl nuclei than lignin in wheat straw cell walls. It is suggested that crosslinking between lignin and polysaccharides by ester-ether bridges via ferulic acid contributes to the mechanical properties of the cell walls of rice straw. Thus the chemical and structural characteristics of lignin in rice straw differ to some extent from those of other temperate grasses, such as wheat (Triticum aestivum) and phalaris (Phalaris aquatica), as reported previously. This can probably be attributed to the water environment of rapidly growing rice seedlings, but it also depends on the genetic variety of the rice plant.     

Treatment of poplar callus with ferulic and sinapic acids I: incorporation and enhancement of lignin biosynthesis

Ferulic acid (FA), tetradeuteroferulic acid (DFA), sinapic acid (SA), or heptadeuterosinapic acid (DSA) was exogenously supplied to poplar (Populus alba L.) callus. Administration of FA or SA increased the lignin content of the callus to about twice that of the control callus. Gas chromatographic analysis of the alkali hydrolysate of the cell wall residue revealed that only a trace amount of SA was bound to the cell wall, and the amount of FA was less than 2% of the total callus lignin. Thioacidolysis of the DFA-treated callus indicated that DFA is effectively converted to both coniferyl and sinapyl alcohols and then incorporated into the corresponding lignin. Incorporation of DSA into syringyl lignin or guaiacyl lignin was not observed, but yields of syringyl lignin thioacidolysis products were markedly increased by DSA treatment of the callus. These results suggest that SA may not be a precursor of sinapyl alcohol and syringyl lignin per se, but it may induce or enhance the biosynthesis of syringyl lignin in poplar callus.     

烟草和毛白杨次生维管发育相关MYB转录因子分析

[目的]为探讨额河杨和银灰杨天然杂种的起源机制,[方法]应用18对SSR标记,从分子水平上对新疆额尔齐斯河流域杨属植物的种间关系进行分析研究。[结果]表明:(1)SSR系统发育树将整个流域天然杨属植物分为两大类群,即黑杨派和青杨派为一类,白杨派为一类;(2)白杨派派内系统聚类图显示,银白杨、欧洲山杨、银灰杨三个树种均有较大的遗传分化,特别是杂种银灰杨似乎更大;(3)黑杨派和青杨派的UPGMA分类图显示,青杨派和黑杨派分属于2个分支,其中,青杨派内部分化相对简单,分为2支,均为典型的苦杨;黑杨派内部的分化较为复杂,可分为4类,包括典型的欧洲黑杨、额河杨和回交子代。[结论]杂种额河杨具有更多的欧洲黑杨的遗传成分,因此,将额河杨放到黑杨派是正确的。     

An improved transformation system for Lombardy poplar (Populus nigra var. italica)

We report an improved transformation system for Lombardy poplar (Populus nigra var. italica). A new binary vector, pBF2, with 11 unique restriction enzyme sites and the normal neomycin phosphotransferase II (NPTII) gene was constructed for the transformation of Lombardy poplar. Genetically transformed adventitious shoots were directly regenerated after cocultivation of stem segments with Agrobacterium tumefaciens EHA105 that harbored a binary vector with genes for the NPTII and enhanced green fluorescent protein. Successful transformation was confirmed by fluorescence microscopy, immunoblotting and Southern blotting analyses, and resistance to kanamycin and geneticin. This transformation system requires less time than our previous method for the regeneration of transgenic shoots. When explants were incubated on a smedium containing dithiothreitol, the transformation frequency increased to approximately 20%.     

Identification of <Emphasis Type="Italic">CpTI</Emphasis> gene integration for 2-year-old transgenic poplars at DNA level

The putative transgenic hybrid triploid poplars [(P. tomentosa × P. bolleana) × P. tomentosa] with CpTI gene have been outplanted in test field for 2 years. Although the authors’ previous studies have proved that they are highly resistant to 3 species of poplar-threatening insect pests and contain high content of CpTI protein in foliage, incorporation status of foreign CpTI gene in poplar genome is uncertain. In this present study, the incorporation of foreign CpTI gene in genome of 5 transgenic poplars was confirmed by PCR and Southern blotting analysis. DNA amplification showed that there were clear DNA bands of about 450bp specific to CpTI gene in transgenic lanes, while no corresponding band in non-transgenic lane was observed. Correspondingly, clear DNA hybridization signals and no signal were exhibited on film for DNA Southern blotting analysis in transgenic lanes and non-transgenic lane, respectively, which further confirmed the stable integration of foreign CpTI gene in genome of 2-year-old transgenic poplar.     

Assessment of rhizospheric microorganisms of transgenic Populus tomentosa with cowpea trypsin inhibitor ( CpTI ) gene

To have a preliminary insight into biosafety of genetically transformed hybrid triploid poplars (Populus tomentosa × P. bolleana) × P. tomentosa with the cowpea trypsin inhibitor (CpTI) gene, two layers of rhizospheric soil (from 0 to 20 cm deep and from 20 to 40 cm deep, respectively) were collected for microorganism culture, counting assay and PCR analysis to assess the potential impact of transgenic poplars on non-target microorganism population and transgene dispersal. When the same soil layer of suspension stock solution was diluted at both 1:1 000 and 1:10 000 rates, there were no significant differences in bacterium colony numbers between the inoculation plates of both transgenic and non-transgenic poplars. The uniform results were revealed for both soil layer suspension solutions of identical poplars at both dilution rates except for non-transgenic poplars at 1:10000 dilution rates from the same type of soil. No significant variation in morphology of both Gram-positive and Gram-negative bacteria was observed under the microscope. The potential transgene dispersal from root exudates or fallen leaves to non-target microbes was repudiated by PCR analysis, in which no CpTI gene specific DNA band was amplified for 15 sites of transgenic rhizospheric soil samples. It can be concluded that transgenic poplar with the CpTI gene has no severe impact on rhizospheric microorganisms and is tentatively safe to surrounding soil micro-ecosystem. [Supported by the National Project in Transgenic Plant and Application (Grant No. J2002-2003)]     

杨树转基因研究进展及展望

杨树是重要的栽培树种,也是研究林木基因工程的重要模式植物。杨树转基因研究可以打破种属限制,具有高效性和专一性的特点,是对杨树进行遗传改良的重要手段。为了更好的进行该方面的工作,本文介绍了杨树转基因涉及到的抗虫、抗除草剂、木材材性改良、抗逆、抗病、激素调控、开花调控和植物修复等应用领域的研究进展及现状,分析了影响杨树农杆菌转化效率的主要因素,并探讨了杨树转基因研究存在的问题及发展方向,希望能为后期从事林木基因工程研究的科研工作者提供依据。     

Cloning and RNAi construction of a LEAFY homologous gene from Populus tomentosa and preliminary study in tobacco

PtLFY, a LEAFY (LFY) gene, was cloned from Populus tomentosa (LM50) by PCR. Sequencing analysis indicated that PtLFY was 2 629 bp long, composed of three exons and two introns and encoded 378 amino acids. The splice donor sites and the splice acceptor sites were in identical positions to the LFY and its homologues. The amino acid sequence inferred was 68%-99% homologous to those of LFY and its homologues by blast analysis in GenBank. The Southern blot analysis indicated that there was a single copy of the PtLFY gene in genomic DNA of male and female P. tomentosa (LM50 and 5082). The pBI121-Ptalfy (reverse)-intron-Ptlfy-GUS-nos was constructed using RNA interference (RNAi) technique and verified by PCR and digestion identification and transformed into tobacco. Some transgenic tobacco plants were obtained by PCR and PCR-Southern identification. The growth was generally repressed in transgenic tobacco plants compared with wild-type ones and some phenotypic differences were observed. [Supported by the National Natural Science Foundation of China (Grant No. 30371175) and Postdoctoral Foundation of China (Grant No. 2002032041)]     

通过RNAi技术抑制杨树c3h基因表达提高糖转化效率

利用克隆得到的毛白杨c3h1基因构建其RNAi抑制表达载体,通过根癌农杆菌介导的叶盘法转化银腺杨无性系84 K,Realtime PCR检测表明其转基因株系323、325和322中c3h1基因表达量较野生型植株分别下调89.04%、82.22%和68.38%;茎横切片组化染色和显微结构观察表明转基因植株木质部发育和木质素沉积方式发生了改变;木质素、纤维素含量测定及苯酚—硫酸法总糖含量与HPLC法可溶性总糖和单糖含量检测结果表明:转基因植株木质素含量平均降低23.00%,最高可达39.71%;酸前处理效率最高提高了41.39%;未经酸处理直接酶解的糖化效率是对照植株的2.34～2.72倍,322株系和323株系比对照植株经酸前处理后再酶解的糖化效率高出81.18%和375.53%。     

Lignification and peroxidase in tension wood ofEucalyptus viminalis seedlings

Seedlings ofEucalyptus viminalis were grown for 50 days with their stems bent so tension wood would form. Every 10 days the lignin content, monomeric composition, and peroxidase activity in the tension wood were compared with those in the lower side (opposite wood) and in vertically grown controls. The lignin content in the developing tension wood started to decrease after 10 days of bending and kept decreasing for 50 days, whereas those in control plants and opposite wood remained almost unchanged. The yields of syringaldehyde from tension wood by nitrobenzene oxidation increased, and consequently the syringyl/ guaiacyl ratio of the lignin was higher in tension wood than in opposite wood and control plants. The peroxidase ionically bound to the cell walls (IPO) catalyzed oxidation of guaiacol and syringaldazine. The syringaldazineoxidizing activity of IPO from tension wood increased, whereas the activities of IPO from opposite wood and control plants did not show any marked change. In tension wood the increase in syringaldazine-oxidizing activity of IPO was consistent with an increase in the syringaldehyde yield. This suggests that IPO contributes to syringyl lignin deposition as other enzymes involved in the monolignol biosynthesis do in tension wood formation.This study was presented at the 50th Annual Meeting of the Japan Wood Research Society, Kyoto, April 2000     

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.     

Agrobacterium-mediated transformation of lombardy poplar (Populus nigra L. var.italica Koehne) using stem segments

Genetically transformed lombardy poplar (Populus nigra L. var.italica Koehne) plants were regenerated by co-cultivation of stem segments withAgrobacterium tumefaciens strain LBA4404 that harbored a binary vector (pBI121) which included genes for β-glucuronidase (GUS) and neomycin phosphotransferase. Successful transformation was confirmed by the ability of stem segments to produce calli in the presence of kanamycin, histochemical and fluorometric assays of GUS activity in plant tissues, and Southern blot analysis.     

Hybrid aspen with a transgene for fungal manganese peroxidase is a potential contributor to phytoremediation of the environment contaminated with bisphenol A

To assess the possible utility of a fungal gene for manganese-dependent peroxidase (MnP) produced by a transgenic plant in phytoremediation, we transformed hybrid aspen with a chimeric gene for MnP. Our gene construct allowed expression of the gene for MnP in plants and relatively high MnP activity was detected in the hydroponic medium in which roots of plants that expressed the transgene had been cultured. Some of our transgenic plants were able to remove bisphenol A from the medium more efficiently than wild-type plants. Our results demonstrate that, without any modification of the coding sequence, a chimeric gene for fungal MnP can be expressed in a woody plant, with secretion of active MnP from roots into the rhizosphere. Our strategy suggests new options using woody plants for phytoremediation.     

Effect of alkali extraction on the lignin monomeric composition inEucalyptus

The effect of alkali extraction on the lignin monomeric composition was examined inEucalyptus camaldulensis andE. globulus by thioacidolysis using extractive-free samples as a control. Results showed that the effect onEucalyptus is different among species and among sample positions in the trunk, although a small amount of lignin is solubilized during the extraction in all samples. In addition, it was proved that lignin extracted by the alkali extraction is not always guaiacyl-rich, probably relating to the original lignin monomeric composition, which depends on the sample species or the sample position in the trunk.