由根癌农杆菌介导向毛白杨导入激素合成基因建立遗传转化系统

用携带基因1,2的根癌农杆菌AG(84)转化毛白杨外植体,在无激素的MS0培养基上获得转化根。分离单根或切成根段在分化培养基上能分化芽而再生完整植株。由T－DNA上带有基因4的根癌农杆菌C58C1（PBZ6111）转化毛白杨外植体,在MS0培养基上能直接分化不定芽而再生植株．在转化中使用叶柄作外植体比使用叶片的转化率提高一倍以上。基因1,2引入毛白杨后,植株根系发达,生根率达100％。基因4引入毛白杨则使植株节间变短,植株矮化．纸电泳分析表明,带有基因1,2的转化植株能表达特异的农杆碱,带有基因4的转化植株能表达特异的胭脂碱。     

通过农杆菌介导法将免防御麦NP—1基因导入毛白杨（P.tomentosa）

采用叶盘法将兔将御素ＮＰ－１基因导入毛白杨后,经ＰＣＲ分析、Ｓｏｕｔｈｅｒｎ杂交检测与筛获得了转基因植株,并经体外抑菌实验表明,转ＮＰ－１基因毛白杨植株组织提取物对某些微生物的生长具有明显的抑制作用。     

外源突变基因AP1M2转化毛白杨的研究

为探究毛白杨开花调控分子机制并获得不飞絮毛白杨株系,利用农杆菌介导法将显性负突变结构基因爿Pm挖转入毛白杨中,经PCR检测,共获得阳性转化植株7株。通过RT_qPCR检测转基因植株中外源基因AP1M2表达量,发现各株系间的表达水平差异显著,最高为内参的5．41倍,最低为1．89倍。对转基因毛白杨中内源刚尸,和其他开花关键基因的表达量进行检测,发现内源AP1的表达受到抑制,表达量明显下调;其他开花关键基LFY、AP3、PI、SEP3和FTI等的表达量也有不同程度的降低,而FT2表达量并没有明显变化。这些研究结果表明转4尸m扭毛白杨中API、LFY、AP3、PI、SEP3和FT2的表达受到明显抑制．对毛白杨花发育将有一定影响。     

通过根瘤农杆菌介导法获得菊花转基因植株

以带叶茎段为外植体,通过根癌农杆菌介导法,将兔防御素NP－1基因导入菊花品种“001”中。经梯度卡那霉素（kanamycin,Km）筛选,获得了大量Km抗性植株,其中部分Km抗性植株经Southem杂交鉴定为转基因植株。从而成功地建立了菊花遗传转化系统,为菊花分子育种奠定了基础。     

毛白杨非转化组培根及Ri质粒转化植株的根切段在培养中分化的比较(简报)

毛白杨(Populus tomentosa)是我国特有的树种,适应性强,枝叶茂盛,防护性能好,且材质轻软,纹理细致。既是重要的速生用材树种,又是防护林和行道绿化的重要树种,它也是造纸、火柴、纤维工业的重要原料。虽然毛白杨能通过扦插等技术进行繁殖,但毛白杨扦插时生根比较困难。通过导入外源的与激素合成有关的基因来调节再生植株在组织培养中的分化在草本植物中已有不少报道,见许智宏等。本研究试图通过比较正常植株与发根农杆菌Ri质粒转化的再生植株的根切段在培养中分化的差异,以期能建立一种有效的毛白杨的遗传转化系统。     

毛白杨CCoAOMT cDNA片段的克隆与转基因杨木质素含量的调控

用RT_PCR方法从毛白杨 (PopulustomentosaCarr.)中克隆了CCoAOMT基因的cDNA片段 ,并对其序列进行了分析。Northern杂交表明该基因在毛白杨正在木质化的次生木质部高水平表达 ,且与木质化进程同步。构建了该基因反义表达载体 ,根癌土壤杆菌 (Agrobacteriumtumefaciens (SmithetTownsend)Conn)介导转化杂交杨 (欧洲山杨×银白杨 ,P .tremula×P .alba)。采用PCR、PCR_Southern及Southern杂交对获得的转基因植株进行分子检测 ,并测定移栽 5～ 6个月的转基因植株的Klason木质素含量 ,其中一个转基因株系的Klason木质素含量比未转基因对照杨树下降 17.9%。结果表明利用反义RNA技术对杨树CCoAOMT基因的表达可以降低转基因植株的木质素含量 ,达到改善其造纸性能的目的     

水稻转基因植株后代中外源基因异常分离的研究

以用农杆菌介导法育成的日本晴等3个品种（系）的转基因水稻为材料,对外源基因的分离情况进行了研究。这几份转基因水稻都携带串联排列的抗虫基因cry1Ab和报告基因gusA等基因,在自交后代中,cry1Ab和gusA协同分离,但阳性植株与阴性植株之比都显著小于3：1,阴性植株显著偏多,以杂合转基因植株（gusA1-)作母本植株,与常规品种（-1－）杂交所得测交F1,阳性植株（＋／－）和阴性植株（－／－）的比例基本符合1：1,在反交时,阴性植株与阳性植株之比显著大于1：1的比例,比较日本晴转基因后代中gusA阳性株与阴性株的结实率发现,前者的结实率显著低于后者,综合上述结果可以看出,携带crylAb等转基因的花粉存在竞争劣势,可能是导致外源基因异常分离的主要原因。     

转双价抗虫基因毛白杨无性系85号抗虫性研究

以优化的毛白杨无性系85号最适遗传转化体系为基础,通过农杆菌介导法将Bt(CryⅠAc)和API双价抗虫基因导入毛白杨无性系85号基因组.在高浓度卡那霉素的培养基上诱导不定芽和根,初步选择到200株转化植株,PCR检测显示,66株呈阳性反应.Southern杂交和ELISA检测进一步证明,Bt(CryⅠAc)和API双价抗虫基因已整合到毛白杨无性系85号基因组中,并得到了表达.对转化植株用舞毒蛾幼虫进行饲虫试验,结果显示昆虫幼虫的死亡率高达60.0%~77.8%,且存活幼虫的生长发育也受到了明显抑制.研究结果为杨树抗虫育种提供了新的种质资源.     

玉米Ubi－1启动子在可育转基因玉米植株中的表达活性

本工作将玉米泛素基因－1启动子（Ubi-1)与大肠杆菌β－葡萄糖苷酸酶基因（gus,uidA)的编码区融合,通过基因枪粒子轰击方法转化来自水成熟胚盾片组织的I－型愈伤组织,经PPT选择获得可育的玉米转基因植株,并采用组织化学方法分析了Ubi-1启动子驱动的gus基因在不同组织,细胞中的表达活性,发现gus基因在除花药壁以外的其它所试组织中均可以有效表达。Ubi：GUS在花粉,卵细胞中T1代转基因植株未成熟胚中的表达显示该启动子在植株发育的早期阶段即具有活性。对T0代转基因植株的花粉进行GUS组织化学染色,gus基因呈1：1分离,显示外源基因在转基因植株中以孟德尔方式遗传。同时发现,使用玉米本身的启动子Ubi-1可以降低外源基因在转基因玉米中的拷贝数,进而避免基因沉默现象的发生。目前已得到第二代转基因种子。     

中华根瘤菌NP1氨单加氧酶基因的克隆与功能鉴定

以中华根瘤菌NP1(Sinorhizobium sp.NP1)为原始菌株,通过同源克隆与Tail-PCR方法,获得1089bp的氨单加氧酶基因(amo)全长序列.该基因编码362个氨基酸,其二级结构与Sinorhizobium meliloti1021AMO的二级结构相似,该蛋白有9个跨膜区段.以自杀穿梭质粒pJQ200SK为原始载体,构建NP1amo基因敲除质粒pJQ200SK-amo-Tc.采用三亲本杂交的方法将该质粒转入原始菌株NP1中,获得amo基因敲除菌株NP1∷amo.通过本贝洛氏(Berthelot)法对氨氮进行测定,发现NP1∷amo的脱氮效率比原始菌株NP1下降约35%.该结果表明,本实验中所克隆的氨单加氧酶基因为脱氮关键酶基因.     

兔防御素NP-1基因在转基因番茄中表达的初步研究

兔防御素ＮＰ－１是α－防御素的一种,含３３个氨基酸残基。最初从兔子的多形核嗜中性细胞中分离出来。它对革兰氏阴性菌、革兰氏阳性菌、分枝杆菌、真菌、被膜病毒以及ＨＩＶ病毒都有不同程度的抑制作用。兔防御素ＮＰ－１所带阳离子较多,可抗不具代谢活性的靶细胞。实验中将兔防御素ＮＰ－１基因构建到植物表达载体中,通过根瘤农杆菌介导转入番茄,得到了转基因番茄植株。对转基因番茄植株进行了ＰＣＲ、Ｓｏｕｔｈｅｍ杂交、Ｎ     

Putative molecular mechanism of defensin interaction with the membrane of the sensory neuron

NP-1 defensin decreased effective charge transfer in the activation gating system of TTX-resistant (slow) sodium channels in a dose-dependent manner. The dissociation constant and Hill coefficient values were KD = 2 pM and X--0.9. Geometry of the NP-1 defensin molecule was built using its primary structure with three S-S briges and fully optimised in the framework of molecular mechanics method. The data obtained explain experimental results of stechiometry 1:1 due to ligand-receptor interaction by the only outward directed carboxyl group of Glu 14 which might form a hydrogen bond with a single binding site of non-identified defensin membrane receptor.     

Comparative analysis of the effect of endogenous antibiotic defensin NP-1 and aminoglycoside antibiotic gentamicin on synaptic transmission in receptors of the frog vestibular apparatus

The effect of human and rabbit neutrophilic defensins NP-1 and amonoglycoside antibiotic gentamicin on the synaptic transmission in the afferent synapse of isolated vestibular apparatus of the frog has been comparatively studied. Both defensins proved active in the concentration range of 0.0001 to 1 nM and efficiently decreased the impulse frequency in the afferent nerve fibers in a concentration-dependent manner. No significant differences in the efficiency of rabbit and human defensin NP-1 have been revealed in these experiments. Gentamicin also had an inhibitory effect on the afferent discharge in the concentration range of 10–500 μM (0.5–25 mg/kg). The inhibitory effect of gentamicin on the impulse activity of the vestibular nerve was observed at therapeutic doses. The excitatory effect of the putative neurotransmitter L-glutamate was considerably inhibited by defensin NP-1. These findings suggest that the mechanism of defensin action involves a modification of the synaptic transmission in the hair cell receptor and modulation of the effect of L-glutamate.     

抗虫的转AaIT基因杨树的获得

通过根癌农杆菌叶盘法将构建在双元载体上的昆虫特异性神经蝎毒素AaIT基因转化至中国南方杨树N106(小叶杨×美洲黑杨,P.deltoides×P.simonii)中,共获得了62株再生植株。PCR分析及PCR产物Southernblotting的分析结果表明,AaIT基因整合在再生植株的基因组上。对部分转AaIT基因植株进行了杀虫实验,转基因植株A5对一龄舞毒蛾(Lymantriadispar)幼虫有明显的抗性,饲喂转基因杨树叶片的幼虫死亡率显著高于未转基因对照植株,其取食面积小,存活幼虫体重明显小于对照。ELISA分析证明了AaIT蛋白的表达。     

Over-expression of a putative poplar glycosyltransferase gene, PtGT1, in tobacco increases lignin content and causes early flowering

Family 1 glycosyltransferases catalyse the glycosylation of small molecules and play an important role in maintaining cell homeostasis and regulating plant growth and development. In this study, a putative glycosyltransferase gene of family 1, PtGT1, was cloned from poplar (Populus tomentosa Carr.). Sequence analysis showed that this gene encodes a protein of 481 amino acid residues with a conserved PSPG box at its C-terminal, suggesting that it is active in the glycosylation of plant secondary products. The PtGT1 gene was expressed in poplar stems and leaves, with a particularly high expression level in elongating stems. Transgenic tobacco plants ectopically over-expressing PtGT1 were obtained and phenotypes were analysed. Wiesner and M?ule staining showed that stem xylem of transgenic tobacco plants stained more strongly than controls. Measurement of the Klason lignins showed much higher lignin content in the transgenic lines than in control plants. Furthermore, the ectopic over-expression of PtGT1 in tobacco resulted in an early flowering phenotype. These findings offer a possible starting point towards better understanding of the function of poplar PtGT1, and provide a novel strategy for lignin engineering and flowering control in plants through the genetic manipulation of a poplar glycosyltransferase gene.     

Transformation of Populus tomentosa with Insecticidal Cowpea Proteinase Inhibitor Gene

Cowpea trypsin inhibitor (CpTI) gene, an insecticidal gene, was introduced into poplar ( Populus tomentosa Carr. ) by gene transformation mediated by Agrobacterium tumefac/ens (Smith et Townsend) Conn. The influences on regeneration and transformation frequency of poplar by the concentration and addition of kanamycin were compared. Kanamycin resistant (Kmr) plantlets were obtained by 3 -4 cycles screening in selective condition. The ability of leaf regeneration and shoot subculture and rooting from the transformed and non-transformed plants in the presence of 50 mg/L kanamycin was examined. The presence of CpTI gene in the transgenic plants were confirmed by PCR and PCR-Southern blot. Assay on proteinase inhibition activity demonstrated that leaf protein extracts of the transgenic poplar showed higher inhibition activity against trypsin than that of control plants.     

Over-expression of the Arabidopsis Na+/H+ antiporter gene in Populus deltoides CL?×?P. euramericana CL “NL895” enhances its salt tolerance

Soil salinity is a serious problem worldwide. It is necessary to improve the salt tolerance of plants to avoid the progressive deterioration of saline soil. We showed that the over-expression of AtNHX1 improves salt tolerance in a transgenic poplar (Populus deltoides CL × P. euramericana CL “NL895”) under mannose selection. Four transgenic poplar plants were obtained. Southern blot analysis showed that the pmi gene had integrated into the genome of the poplar. RT-PCR confirmed that AtNHX1 could be expressed normally in the transgenic plants. When tested for salt tolerance by NaCl stress, we measured a 100% increase in Na⁺ content in the three transgenic lines (T18, T50, T98) significantly higher than the 33% increase seen in wild-type plants. The chlorophyll content of the transgenic plants was not altered significantly, while the chlorophyll content in the control plants showed a small decrease. MDA content was decreased in the transgenic plants. These results show that the AtNHX1 gene may enhance salt tolerance due to increased vacuolar compartmentalization of sodium ions.     

A Flow Cytometric Assay Reveals a Suppression of Phagocytosis by Rabbit Defensin NP-3A in Mouse Peritoneal Macrophages

Phagocytosis of fluorescein isothiocyanate (FITC)-labeled polystyrene microparticles by peritonea] macrophages from thioglycollate-elicited mice was examined by means of flow cytometry (FCM). This assay revealed that rabbit defensin NP-3A suppressed the phagocytosis in a dose-dependent manner. The present results suggest that NP-3A released from neutrophils is one of the mediators which modulates the activity of macrophages in response to infection.     

The chitinase gene (Bbchit1) from Beauveria bassiana enhances resistance to Cytospora chrysosperma in Populus tomentosa Carr.

The Chinese white poplar (Populus tomentosa Carr.) is susceptible to infection by plant diseases which severely affect its growth and substantially decrease its economic value. A chitinase gene (Bbchit1) from Beauveria bassiana was introduced into Chinese white poplar (Populus tomentosa Carr.) by Agrobacterium-mediated transformation. The T-DNA of plant transformation vector contained the β-glucuronidase reporter gene (GUS) under the control of CaMV 35S promoter and the neomycin phosphotransferase selection marker gene (NPTII) driven by the nos promoter. GUS activity was detected in most of the kanamycin-resistant plants tested. Stable integration of transgenes in the plant genome was confirmed using PCR. RT-PCR analysis showed that the Bbchit1 gene was transcribed in the transformed plants. When evaluated for resistance to poplar fungal pathogens with an in vitro assay, crude extracts from leaves and shoots of transgenic lines were inhibitory against the pathogenic fungus Cytospora chrysosperma (Pers.) Fr. Similarly, Bbchit1 overexpression enhanced disease resistance to C. chrysosperma in the transformed poplar plants, indicating that is gene is potentially useful to protect the trees against fungal diseases.     

Transgenic tobacco and peanut plants expressing a mustard defensin show resistance to fungal pathogens

Defensins are small positively charged, antimicrobial peptides (~5 kDa in size) and some of them exhibit potent antifungal activity. We have cloned the complete cDNA containing an ORF of 243 bp of a defensin of mustard. The deduced amino acid sequence of the peptide showed more than 90% identity to the amino acid sequence of the well-characterized defensins, RsAFP-1 and RsAFP-2 of Raphanus sativus. We have generated and characterized transgenic tobacco and peanut plants constitutively expressing the mustard defensin. Transgenic tobacco plants were resistant to the fungal pathogens, Fusarium moniliforme and Phytophthora parasitica pv. nicotianae. Transgenic peanut plants showed enhanced resistance against the pathogens, Pheaoisariopsis personata and Cercospora arachidicola, which jointly cause serious late leaf spot disease. These observations indicate that the mustard defensin gene can be deployed for deriving fungal disease resistance in transgenic crops.