转抗菌肽基因毛白杨的培育及其对溃疡病的抗性

以毛白杨为转基因受体材料,利用根癌农杆菌介导法转化来源于益母草的阳离子抗菌肽基因LJAMP2。经卡那霉素筛选,共获得50株抗性植株。GUS组织染色和PCR检测显示有30株抗性植株呈阳性,初步证明外源目的基因已整合到毛白杨基因组中。RT-PCR证实抗菌肽基因LJAMP2在转基因植株中能大量表达。离体抗病性试验表明:转基因毛白杨细胞粗提液的抑菌能力明显强于非转基因植株。进一步将溃疡病菌接种在转基因和野生型毛白杨茎段上培养30天,转基因植株的病级指数均低于非转化植株。上述抗性试验结果表明:在毛白杨中超量表达益母草抗菌肽基因LJAMP2能显著提高其溃疡病抗病性。     

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。     

东北红豆杉内生真菌的鉴定-----新种和已知种及其代谢产物

从长白山自然保护区原始森林200多年生的东北红豆杉(TasxuscuspidateSiebetZucc.)的树皮中分离出94株内生真菌,鉴定出19种。其中,除10个新记录属种外,还包括2个新种:长线孢拟茎点(PhomopsislongiscoleosporuY.XiangetLuAnGuo,WuWenFang)和大孢盾壳霉(ConiothyriummacrospoumY.Xiang,LuAnGou,WuWenFang);1个新变种为紫杉链格孢[Alternariaalternata(Fr.)Keisslervar.taxiY.XiangetLuAnGuo];还有6个国内已知种:阿姆斯特丹散囊菌(EurotiumamstelodomiMgngin),匐散囊菌原变(EurotiumrepensdeBary),葡萄孢(Botrytissp.),桔青霉(PenicilliumcitrinumThom),黑附球孢(EpicoccumnigriumLinK),镰刀孢(Fusariumsp.)。用薄层层析技术,测定了各菌种发酵代谢产物,经筛选发现紫杉链格孢[Alternaria(Fr.)Keisslervar.taxiY.XiangetJ.X.Cui],葡萄孢(Botrytissp.),阿玛姆斯丹散囊菌(EurotiumamsteloodomiMgngin),匍匐散囊菌原变种(EurotiumrepensdeBary)4种内生真菌的代谢产物可与紫杉烷类物质显色剂——香草试液发生较明显的颜色反应,其中紫杉链格孢[Alternariaalternata(Fr.)Keisslervar.taxiY.XiangetJ.X.Cui]菌种的代谢产物中有一种含量较大的组分,还可与稀碘化铋钾生物碱试剂发生反应,该组分化合物经过光谱分析,为紫     

金丝小枣果实病害病原菌研究

金丝小枣是中国北方栽培的主要果树品种之一 ,特别是河北沧州、山东乐陵等主产区 ,金丝小枣收入在农业总收入中占较大的比重。近年来金丝小枣果实病害发生日趋严重 ,蔓延迅速 ,由 2 0世纪 90年代初发病率不足 5 %上升到现在的 30 %左右 ,严重的达 80 %以上。目前关于金丝小枣果     

In vitro anther culture and Agrobacterium-mediated transformation of the AP1 gene from Salix integra Linn. in haploid poplar(Populus simonii × P. nigra)

A reliable,efficient anther culture system,the dominant technique for generating haploid plants in breeding programs,that can be used for generating transgenic poplar plants has been needed.In the present study,therefore,an anther culture system was developed using isolated mid-and late-uninucleate anthers of poplar(Populus simonii x P.nigra).From a combination of SSR and ploidy analyses,six double haploid and two haploid lines were characterized from 86 plants grown from 16 regenerated anther cultured lines.After 48 months of development,two plant lines from the regenerated plants maintained their haploid level in vitro for over 2 years.A number of haploid plants from the different lines weretransferred to soil.The leaves of these transplants were then used as explants for transformation with the APETALA1(AP1) gene using Agrobacterium tumefaciens.Overexpression of AP1 in haploid poplar induced early flowering with obvious petals when ectopically expressed.To our knowledge,this is the first report on changes in flowering time in AP1-trangenic poplar,which is important for elucidating the regulatory mechanism of tree flower development.     

转果聚糖蔗糖转移酶基因银腺杨的获得

采用农杆菌介导的遗传转化方法,将来自枯草杆菌的果聚糖蔗糖转移酶基因(SacB)导入银腺杨,以提高杨树对水分胁迫的抗性。以来自无菌培养的叶片为外植体,通过大约10 0 0个叶盘与农杆菌LBA4 4 0 4共培养,将植物双元表达载体pKP中SacB基因导入银腺杨基因组,经卡那霉素筛选后,共获得10 2株卡那霉素抗性植株。经PCR特异性扩增和Southern点杂交分析,证明其中97株再生植株基因组DNA中整合了SacB基因。对其中的6 2个无性系进行RT PCR分析,结果表明SacB基因在其中的5 0个无性系中获得表达。温室生长观察表明,转基因无性系外部形态与对照相比没有稳定的显著差异,少数部分转基因无性系的生长明显受到抑制,其他转基因无性系生长正常。这些转基因无性系的获得为培育抗旱转基因杨树奠定了基础。     

Green fluorescent protein as a tool for monitoring transgene expression in forest tree species

The gene coding for green fluorescent protein (GFP) from the jellyfish Aequorea victoria was successfully used as a vital marker for the transformation of three woody plant species, black spruce (Picea mariana (Mill.) BSP), white pine (Pinus strobus L.) and poplar (Populus spp.). The gfp gene and the gene conferring resistance to kanamycin (nptII) were introduced by microprojectile bombardment or Agrobacterium tumefaciens-mediated technology. Screening by fluorescence microscopy of the transformed plant material, under the selection of kanamycin, identified five to eight cell lines from each tree species that clearly expressed GFP. Expression of GFP was observed in somatic embryonal cells of the coniferous species and in stem sections of poplar. For all species, GFP transgene expression was stable over multiple subcultures. Stable integration of the gfp gene into plant genomes was confirmed by Southern hybridization or polymerase chain reaction (PCR) analysis. We conclude that GFP can be used as a vital marker and reporter protein in transformation experiments with gymnosperms and deciduous trees.     

Transgenic poplar characterized by ectopic expression of a pine cytosolic glutamine synthetase gene exhibits enhanced tolerance to water stress

Physiological responses to water stress in hybrid poplar (INRA 7171-B4, Populus tremula L. x P. alba L.) lines transformed to overexpress a pine cytosolic glutamine synthetase (GS1) gene were compared with those of non-transgenic plants. Before, during and after a drought treatment, net photosynthetic rates (Anet) were higher in transgenic than in non-transgenic plants. Stomatal conductance (gs) was higher in transgenic than in non-transgenic plants before, but not after exposure to drought. Before drought treatment, a sudden reduction in photosynthetic photon flux caused a greater burst of CO2 efflux in transgenic than non-transgenic plants, indicating greater photorespiratory activity. Drought caused greater reductions in photochemical quenching, photosystem II (PSII) antennae transfer efficiency (Fv'/Fm') and light-adapted PSII yield (PhiPSII) in non-transgenic than in transgenic plants, especially at low irradiances. Antennae-based thermal dissipation was higher in transgenic plants than in non-transgenic plants both during the imposition of drought and 1 or 3 days after the relief of drought. Under severe water stress and subsequently, transgenic plants maintained a higher expression of glutamine synthetase, glutamate synthase and Rubisco and higher concentrations of chlorophyll and glycine than non-transgenic plants. These findings indicate that overexpression of pine cytosolic GS1 enhanced sustained photosynthetic electron transport capacity during severe stomatal limitation. The data also suggest that ectopic expression of cytosolic GS increases photorespiratory activity, and that this serves as a protective sink for electrons from photosynthetic reaction centers.     

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.     

Over expression of TaFer gene from Tamarix androssowii improves iron and drought tolerance in transgenic Populus tomentosa

Ferritin, a universal intracellular protein, can store large amounts of iron and improve plant resistance to abiotic and biotic stress. In this study, a ferritin gene(TaFer) from Tamarix androssowii Litv. was transferred into Populus tomentosa Carr. cv 'BJR01' via Agrobacterium. Six independent transgenic lines were obtained with a tolerance to kanamycin and three were randomly selected for further analysis. The PCR and RT-PCR results indicate that the TaFer gene had been integrated into the poplar genome. The effect of the gene on abiotic stress tolerance was tested, and the results show that transgenic plants improve growth, had higher chlorophyll and lower MDA contents, and higher relative electrical conductivity,fewer changes of SOD and POD activities, higher iron content, higher root ferric reductase activity and lower levels of ROS accumulation and cell death in response to drought, Fe-insufficient or Fe-excess tolerance. These results indicate that the TaFer gene can improve abiotic stress tolerance in transgenic Populus tomentosa.     

Overexpression of mtlD gene in transgenic Populus tomentosa improves salt tolerance through accumulation of mannitol

The mtlD gene encoding mannitol-1-phosphate dehydrogenase, which catalyzes the biosynthesis of mannitol from fructose, was cloned from Escherichia coli and transferred to poplar (Populus tomentosa Carr.) through Agrobacterium-mediated transformation. The transgenic plants were screened and selected on Murashige and Skoog (MS) medium containing 30-50 mg l(-1) kanamycin and verified by polymerase chain reaction (PCR) and Southern blotting. Expression of the gene led to synthesis and accumulation of mannitol in the transgenic plants. Gas chromatography and mass spectrometry (GC/MS) and capillary gas chromatography (GC) showed that transgenic plants accumulated much more mannitol in their tissues than the wild-type plants, whether cultured in vitro, or grown hydroponically or in the field. Increased salt tolerance of transgenic plants was observed both in vitro and in hydroponic culture. The transgenic buds rooted normally on MS medium containing 50 mM NaCl, whereas wild-type buds did not. In the 40-day hydroponic experiments, transgenic poplar plants survived in a 75-mM NaCl treatment, whereas the wild-type poplar plants tolerated only 25 mM NaCl. Under the same NaCl stress, stomatal conductance, transpiration rates and photosynthetic rates were all higher in transgenic plants than in wild-type plants, whereas cellular relative conductivity was lower. We demonstrated that the mtlD gene was expressed in transgenic poplar plants, resulting either directly or indirectly in mannitol accumulation and improved salt tolerance. The constant mannitol concentrations in transgenic plants during the NaCl treatments indicated that mannitol accumulation caused by the mtlD gene was not a consequence of NaCl stress. Height growth was reduced by about 50% in the transgenic plants compared with the wild-type plants in the absence of salt; however, relative growth rate was much less influenced by salt stress in transgenic plants than in wild-type plants. The stunted growth of the transgenic plants may in part explain their improved salt tolerance.     

转多基因1年生库安托杨对溃疡病的抗性分析

目的 本研究对转多基因库安托杨株系的溃疡病抗病性检测及抗病相关基因的表达分析,为通过基因工程手段培育抗病林木新树种提供有价值的参考。 方法 以转JERF36等多个外源基因的1年生库安托杨株系D5-9、D5-20、D5-21及非转基因受体D5-0为材料,对主干人工接种溃疡病菌的各株系病情指数进行了测定,同时对接种5 d后树皮组织中5个抗病基因的表达情况进行了实时定量PCR（qPCR）分析。 结果 在溃疡病菌胁迫下,3个转基因株系对溃疡病菌的抗性显著优于非转基因受体株系,转基因株系间抗病性也具有一定差异,D5-21的病情指数显著低于其它2个转基因株系;肉桂醇脱氢酶基因在3个转基因株系树皮中表达量均高于对照,类甜蛋白基因仅在D5-19树皮中的表达量高于对照,其他3个基因在转基因株系中的表达量或与对照相当或低于对照。 结论 转多基因库安托杨株系的溃疡病抗性与非转基因对照相比均有显著提高,且转基因株系间差异显著;同时转基因株系中不同抗病基因的表达模式也有很大差异,说明溃疡病菌胁迫下转多基因库安托杨抗病调控的复杂性,其分子调控机制还有待深入研究。     

A Preliminary Study on Colletorichum from Horned Gallnut

A pathogenic fungus Colletorichum gloeosporioides(Penz.) Sacc isolated from horned gallnut,a Chinese gallnut from Yunnan Province,was reported for the first time.Its morphological and molecular biological characteristics were identified, and its damage status and mycelium characters on gall and mydium were described.The pathogenic fungi C.gloeosporioides Sacc belongs to Melanconiales(Deuteromycotina:Coelomycetes).     

植物基因工程及其在林木害虫防治上的应用

植物基因工程主要包括目的基因的分离、基因工程载体的构建、植物细胞的遗传转化、转化细胞的组织培养和植株再生、外源基因表达的检测等几个方面。利用该技术目前已转化了20多种林木树种,并成功地将抗虫的苏云金芽杆菌内毒素基因和蛋白酶抑制剂基因转入林木树种。     

杨树油菜素内酯合成基因DET2的克隆与功能分析

[目的]DET2基因编码一个5α-还原酶,是油菜素内酯(BRs)合成过程中的关键限速基因。研究DET2基因在杨树生长发育中的作用,对于进一步研究油菜素内酯在木本植物中的调控机制有重要意义。[方法]从银腺杨84K(Populus alba×P. glandulosa,‘84K’)克隆得到拟南芥AtDET2同源基因PagDET2,利用生物信息学对其进行序列比对、生化特征分析、构建系统发育进化树。通过RT-PCR分析其在杨树中的表达模式。构建由CaMV 35S强启动子驱动的过表达载体,通过农杆菌介导的叶盘转化法转化84K杨,得到PagDET2-OE转基因植株。分析过表达DET2基因对于转基因植株内源BRs含量、植株生长和抗逆的影响。[结果]克隆了包含全长编码区PagDET2基因全长,可编码一个长度为257个氨基酸的蛋白质。其蛋白序列与毛果杨、拟南芥、水稻、棉花、大豆、番茄DET2蛋白同源性较高,说明该基因在进化过程中相对保守。PagDET2在84K杨不同组织中均检测到表达,其在茎中的表达较高。通过ELISA检测植物BRs含量发现,过量表达DET2可以显著提高杨树内源BRs含量。过量表达DET2基因,可以导致转基因植株的高生长,但对盐胁迫更加敏感。[结论]DET2基因作为BRs合成的关键基因,在杨树中过量表达可以显著提高内源BRs含量,促进植株增高。DET2转基因植株的获得为进一步分析BR参与木本植物生长发育的调控机制奠定了基础。     

杨树皮储藏蛋白基因启动子的克隆和功能研究

杨树树皮储藏蛋白BSP是类似种子储藏蛋白的氮素储藏物 ,冬季在韧皮部薄壁细胞中大量积累 ,是落叶树氮代谢中的重要成分。为了研究BSP基因启动子在转基因植物中的表达特性 ,探索其在植物基因工程研究中潜在的应用价值 ,我们用PCR方法从美洲黑杨基因组中DNA扩增得到了BSA启动子片段。与GUS基因融合构建中间载体后 ,转化烟草 ,获得了一批PCR检测为阳性的转化再生植株。经GUS组织化学检测 ,发现若干转基因烟草的茎和叶柄韧皮部以及叶脉都呈GUS染色阳性 ,初步证明杨树BSP基因启动子确有韧皮部表达特性 ,可介导GUS基因在转基因烟草韧皮部特异表达。     

抗虫转基因杨树的培育及其抗虫性初步研究

以杨树优良新品种欧美杨107(Populus×euramericanacl."74/76")为试材,通过根癌农杆菌(Agrobacteriumtumefaciens)介导法,将Bt毒蛋白基因导入107杨的叶片及茎段外植体,经潮霉素抗性筛选,获得了转基因再生植株。提取转基因植株的总DNA,经PCR扩增,部分植株呈阳性反应,证明目的基因已经整合到杨树基因组中。以转基因杨树叶片饲喂天幕毛虫幼虫的杀虫试验结果表明,转基因杨树表现出一定的杀虫活性。     

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%。     

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。