农杆菌介导的雪花莲凝集素基因转入玉米骨干自交系

以农杆菌AGL0介导,将雪花莲凝集素基因转入玉米骨干自交系齐319和掖515胚性愈伤组织细胞,从筛选后的抗性愈伤组织获得再生植株。农杆菌浓度和共培养时间均能显著影响侵染后玉米愈伤组织的抗性频率。在农杆菌浓度OD600 0．2～0．3,共培养时间3d时,侵染后玉米愈伤组织的抗性频率最高,平均约4％。对再生植株及其子代基因组DNA的PCR及Southern杂交分析表明雪花莲凝集素基因已经整合到玉米基因组中,并遗传给后代。在蚜虫人工接种试验中,转基因植株上蚜虫的繁殖力为非转基因对照植株上的50％,这表明转基因植株抗蚜性显著增强。     

农杆菌介导的胀果甘草愈伤组织遗传转化

目的:旨在建立一个农杆菌介导的甘草愈伤组织遗传转化的可行方案,并对转化条件进行优化。方法:选择EHA105和LBA4404两种根癌农杆菌菌株,热激法转入含有绿色荧光蛋白GFP基因的植物表达载体pBI121-gfp,挑选转化的农杆菌用于侵染胀果甘草愈伤组织。设置不同培养时间的愈伤组织作为受体材料和农杆菌不同侵染时间两组条件,经共培养后的甘草愈伤组织进行荧光检测。结果:愈伤组织在含有100mg/l卡那霉素的继代培养基上进行筛选培养,得到了具有卡那霉素抗性的转化甘草愈伤组织,转化愈伤组织经继代培养基后在紫外光下仍可见绿色荧光,PCR检测转化愈伤组织基因组中含有gfp基因。结论:试验建立了农杆菌介导的甘草愈伤组织的遗传转化体系,为目的基因导入甘草细胞利用基因工程手段调控甘草次生代谢产物生物合成的研究奠定了基础。     

利用gfp报告基因优化农杆菌介导的水稻遗传转化

为提高农杆菌介导的水稻遗传转化效率,以晚粳97为转化材料,绿色荧光蛋白gfp基因为报告基因,采用正交试验L9(33)对影响农杆菌介导水稻的遗传转化因子进行优化。通过观察愈伤组织荧光表达情况,分析菌液浓度、共培养温度与共培养时间对农杆菌转化水稻的影响。结果表明,在OD660值为0.1、共培养21℃～23℃黑暗条件下,农杆菌与水稻愈伤共培养72 h,最有利于水稻的遗传转化,该条件下晚粳97愈伤组织荧光表达率达到70.9%。     

根癌农杆菌介导的金边狗牙根遗传转化条件的优化

为建立根癌农杆菌(Agrobacterium tumefaciens,菌(L.)Pers.]最佳遗传转化体系,以葡萄糖醛酸糖苷酶(GUS)基因的瞬间表达率为指标,从愈伤组织继代时间、根癌农杆菌侵染时间、负压条件及光照时间等方面进行了筛选.结果表明,根癌农杆菌介导的金边狗牙根最佳的转化体系是:以继代培养2周的愈伤组织为起始材料,根癌农杆菌介导感染10 min,经负压处理(抽拉20次)并在全光条件下共培养转化.     

影响根癌农杆菌介导水稻转化的因素分析

根癌农杆菌与来自水稻成熟种子盾片的愈伤组织共培养,将GUS基因导入水稻愈伤组织,并获得了转基因植株。通过比较影响根癌农杆菌转化频率的各种因素,表明激素配比为2,4-D1mg/L、TDZ0.5mg/L、NAA1mg/L时,可以大大促进籼稻愈伤组织的分化能力;酚类化合物的加入使农杆菌的转化频率提高8.9%-23.5%;共培养时农杆菌的稀释方式及适当调整潮霉素（hygB）的使用浓度影响到农杆菌的转化频率。     

根癌农杆菌ATC15和438T与唐菖蒲细胞结合的扫描电镜研究

通过扫描电镜,研究了含烟草花叶病毒外壳蛋白基因的根癌农杆菌ATC15和438T转化单子叶植物唐菖蒲愈伤组织时,菌体与植物细胞结合的最适条件.研究结果表明,乙酰丁香酮(AS)在农杆菌培养和农杆菌与植物组织共培养时的最适浓度为30μg/ml.如果愈伤组织切块在共培养之前,在液体MS培养基中浸泡两小时,农杆菌在共培养时可大量附着到唐菖蒲组织切块的外表面细胞.     

农杆菌介导类胡萝卜素合成酶基因LycB转化水稻的研究

以3个水稻品种的成熟胚诱导的良好胚性愈伤组织为受体,以LycB为目的基因,应用根癌农杆菌介导法对水稻进行遗传转化,同时以抗性愈伤率为依据,对影响转化的几个因素进行优化研究。结果表明：预培养4d、侵染5～10min、农杆菌菌液浓度OD600值0.7～1.0、共培养2d有利于提高转化率。经潮霉素筛选获得的抗性植株经PCR和PCR-Southern分析鉴定,初步证明外源基因LycB已整合到水稻的基因组中。     

提高农杆菌转化水稻频率的研究

以16种重要的籼稻和粳稻栽培品种为材料,研究了影响农杆菌转化水稻频率的有关因素,结果表明,CC培养基是绝大多数水稻全国组织的最适诱导与继代培养基;添加2．5－5mg/L ABA可以有效地改善水稻愈伤组织的质量,籼稻愈伤组织所需的筛选剂浓度低于粳稻愈伤组织所需的浓度,根癌农杆菌EHA105菌株对水稻的转化效果优于LBA4404和AGL1菌株的效果,头孢霉素对农杆菌的抑制效果优于羧苄青霉素的效果,共培养后进行适当的干燥处理既可增强脱菌效果,又可提高转化频率,应用我们所优化的农杆菌转化技术体系,获得了10个品种的水稻转基因植株。     

苏云金杆菌内毒素蛋白基因转入葡萄胚性愈伤组织细胞及转基因植株再生的研究

以葡萄的胚性愈伤组织作为农杆菌介导,Ti质粒转化材料,利用共培养法将苏云金杆菌内毒素蛋白基因转入葡萄胚性愈伤组织细胞,通过胚状体发生途径再生转基因植株。实验发现:80μmol/L的乙酰丁香酮诱导处理农杆菌和葡萄愈伤组织后可将转化效率提高50倍。OD值为0.8的农杆菌菌液稀释8—10信后与在G培养基预培养10天的胚性愈伤组织共培养2—3夭,Ti质粒对葡萄愈伤组织细胞的转化效率可达50%左右。筛选得到的转基因植株在含Km 30 mg/L的选择培养基上继代存活6个月,生长正常;提取叶片染色体DNA做Southern blot,杂交结果为阳性。将转基因植株各部分切段置于含Km 50 mg/L的选择培养基上,能够脱分化产生抗性愈伤组织并能增殖。     

农杆菌共培养条件对小麦种子萌发和幼苗生理生化特性的影响

以‘郑9023’、‘中13’和‘西农1376’3个小麦品种(系)为主区,再分别以农杆菌共培养时间、共培养温度以及乙酰丁香酮(AS)浓度为副区,对农杆菌浸种处理后小麦种子萌发及幼苗生理生化特性进行了研究。结果表明,各小麦品种(系)与共培养时间、共培养温度以及AS浓度的互作效应不显著;随共培养时间的延长,小麦种子发芽率、幼苗株高、鲜重、叶绿素含量呈下降趋势,MDA含量、白化苗率、卡那霉素抗性苗率则呈上升趋势,而POD活性则呈先升后降的趋势,农杆菌对小麦种子及幼苗的伤害随共培养时间的延长而增大,且当共培养时间超过2 d时其伤害作用更为明显;共培养温度为25℃时,小麦种子发芽率、幼苗株高、鲜重、叶绿素含量达到或接近最低值,POD活性、MDA含量、白化苗率则达到最大值,此时农杆菌对小麦种子及幼苗不利影响最为明显;加入AS能促进农杆菌对小麦的侵染效果,并以150μmol/L AS的促进作用最强,对小麦种子萌发及幼苗生理生化指标的影响也最大;小麦不同品种(系)对农杆菌的反应存在一定基因型差异。依据共培养条件下小麦种子萌发和幼苗生理生化特性及卡那霉素抗性苗率综合分析认为,农杆菌浸种法转化小麦时较适宜的条件为:共培养时间应控制在2～3 d、共培养温度22～25℃、AS浓度为150μmol/L。     

Transformation of LRP gene into Brassica napus mediated by Agrobacterium tumefaciens to enhance lysine content in seeds

Lysine rich protein (LRP) gene derived from the seed of Psophocarpus tetragonolobus was transformed into Brassica napus, employing cotyledon petiole as explants and by using the Agrobacterium tumefaciens strain LBA4404. Transformation efficiency was found to be closely related with phytohormone concentration, infection incubation, and co-cultured time. A medium containing 4 mg/l 6-benzyladenine (6-BA) and 0.3 mg/l naphthalene acetic acid (NAA) was used for plant regeneration. With infection incubation of A. tumefaciens (OD600 = 0.4) for 20 min and co-culture of infected cotyledon petiole for 3 days, the highest transformation efficiency of 8.5% was obtained. To confirm LRP gene expression, PCR and Southern blot analysis were performed on leaf-isolated DNA from regenerated plants resistant to kanamycin. All transgenic plants of the generation T0 formed fertile seeds, which were sowed for the inheritance study of generational T1 and amino acid analysis. It was found that the lysine content of seeds from T1 generation increased by 16.7% compared with non-transgenic lines.     

农杆菌介导将Bt杀虫蛋白基因导入优良玉米自交系的研究

以杂交育种中广泛使用的优良玉米自交系340、4112为材料,用带有质粒pGBIL04（Pactin-Bt-Tnos）的根癌农杆菌LBA4404转化其幼胚及其初始愈伤组织,共培养3天后,在含PPT的培养基上连续筛选培养3代,然后分化获得再生植株。PCR检测证明目的基因已整合到再生植株的基因组中。实验结果表明幼胚预培养后形成的新鲜的初始愈伤组织是比较适宜的转化受体,结果还发现将共培养温度降到22℃可以提高农杆菌介导的玉米遗传转化的筛选频率。 Abstract:Excellent inbred-lines of maize,340 and 4112,which were used largely in hybridized combination were transformed with Agrobacterium tumefaciens.The immature embryos and their original calli were infected by A.tumefaciens LBA4404 containing plasmid pGBIL04.After 3 days of co-cultivation,the immature embryos and calli were continuously selected on the medium containing phosphinothricin (PPT) for 3 generations,then plants were regenerated.It was proved by PCR analysis that the target Bt gene had been integrated into the genome of regenerated plants.The results showed that fresh original calli from the immature embryos after pre-culture were suitable acceptors.The results also showed that it could increase the frequency of selection by properly lowering the co-culture temperature to 22℃.     

农杆菌介导的河北杨遗传转化体系的建立

以兼具生态和能源植物功能的木本模式植物——杨树(河北杨)为材料,研究了携带促生长基因(35S-DAS5)的根癌农杆菌载体介导的河北杨遗传转化若干因素对转化效果的影响。结果显示,较适宜的转化系统为预培养2-4 d,农杆菌菌液(OD600值为0.4)侵染20 min,共培养4 d,在含30 mg/L卡那霉素(Km)的培养基上诱导不定芽,生根培养基中Km的适宜浓度为10 mg/L。     

串叶松香草高效再生体系的建立与兔出血症病毒YL株外壳蛋白基因VP60对其遗传转化

通过对串叶松香草(Silphium perfoliatumL.)不同激素浓度配比的诱导分化实验,建立了串叶松香草离体培养高效再生体系,结果表明MS 6-BA(2.0mg/L) NAA0.1(mg/L)培养基可高效诱导愈伤组织和芽的分化,1/2MS IBA(0.1mg/L)培养基可快速诱导根的生成,形成再生植株。构建了植物表达载体pBI121-VP60,利用根癌农杆菌(Agrobacterium tumefaciens)介导叶盘法转化串叶松香草以研究高效的串叶松香草转化体系,结果显示以农杆菌LBA4404为介导菌株、以叶片为转化外植体、3d预培养时间和3～4d共培养时间、400mg/L羧苄青霉素和40mg/L卡那霉素筛选浓度转化效果较好,并已筛选到两株拟转基因植株,为利用串叶松香草生产兔出血症病毒动物可食用疫苗建立了初步的技术基础。     

根癌农杆菌介导的日本曲霉转化体系的建立

【目的】通过根癌农杆菌介导的方法构建日本曲霉转化子库,从而筛选出高产甘没氧化酶的日本曲霉突变菌株。【方法】本文通过三亲杂交的方法将双元载体pBI-hphII转移至根癌农杆菌EHA105中并作为侵染菌株,以日本曲霉As5999为受体菌株,建立了农杆菌介导的日本曲霉转化体系,构建了突变体库,并对影响转化效率的根癌农杆菌浓度,乙酰丁香酮(As)加入与否,共培养时间,共培养温度等因素进行了分析。【结果】对转化子的PCR检测和Southern杂交分析表明,T-DNA已整合进日本曲霉基因组中,随机挑选的9个转化子连续转接10代后均能稳定遗传。【结论】该转化体系的建立为筛选出高产甘油氧化酶的日本曲霉突变菌株奠定了基础。     

农杆菌介导GUS基因对多年生黑麦草转化的研究

通过检测愈伤组织中GUS基因的瞬间表达,研究农杆菌LBA4404/pCAMBIA1301介导多年生黑麦草的转化体系。通过对多年生黑麦草瞬间表达率的比较,确立了其遗传转化的最佳优化条件。研究发现,多年生黑麦草不同品种的转化率在25%~45%之间变化。多年生黑麦草遗传转化最佳优化条件是预培养10d的胚性愈伤组织、浓度为0.5~0.8OD的农杆菌菌液以及2d共培养时间。在共培养基中添加100μmol/L乙酰丁香酮能有效地提高植物瞬间表达率。两种侵染处理方法比较结果为滤纸滴加法比浸泡法更优。转化后对愈伤组织的干燥处理能抑制农杆菌过度繁殖,能改善愈伤状态,有利于提高转化率。     

Biochemical characterization of a sterol mutant plant regenerated from a tobacco callus resistant to a triazole cytochrome-P-450-obtusifoliol-14-demethylase inhibitor

We report here, for the first time, the biochemical characterization of a plant mutant impaired in sterol biosynthesis. A fertile plant was regenerated from a tobacco callus resistant to LAB170250F, a potent inhibitor of the cytochrome-P450-obtusifoliol-14-demthylase. The resistant callus and the leaves from the regenerated plant are characterized by profound qualitative and quantitative changes in their sterol content. Self-fertilization of this plant yielded seeds with the same biochemical features, indicating that the new phenotype is of mutational origin.     

提取工艺和植物生长调节剂对罗布麻愈伤组织中黄酮含量的影响

以罗布麻愈伤组织粉末为材,在单因素实验的基础上,利用响应曲面法对罗布麻愈伤组织中黄酮的提取工艺进行优化。响应曲面分析结果表明,提取试剂和提取温度对提取的黄酮含量存在显著影响。通过响应曲面分析得到罗布麻愈伤组织中黄酮提取的最佳条件为:提取试剂为70%甲醇,物料比1∶40,提取时间为4 h,提取温度为70℃。培养并比较了30种不同植物生长调节剂浓度与配比诱导100 d生长的愈伤组织中黄酮的含量,结果得出MB+KT(1.0 mg/L)+NAA(0.2 mg/L)上培养约100 d的愈伤组织中黄酮含量最高,为73.90mg/g。测定愈伤组织培养30 d内黄酮的积累动态,探明从培养的愈伤组织提取黄酮的最佳时段。通过优化提取工艺和筛选最佳植物生长调节剂浓度与配比,运用组织培养技术提高了罗布麻愈伤组织中黄酮含量。     

Isoenzymes of peroxidase and esterase related to morphogenesis in Mammillaria gracillis Pfeiff. tissue culture

In vitro propagated plants of the cactus Mammillaria gracillis Pfeiff. (Cactaceae) spontaneously produced callus. The habituated callus regenerated normal and hyperhydric shoots without the addition of grown regulators. Tumours were obtained by infecting cactus explants with Agrobacterium tumefaciens; the wild strain B6S3 (tumour TW) or with the rooty mutant GV3101 (tumour TR). Both tumour lines grew vigorously, never expressing any morphogenic potential. In this study, cactus shoots, callus, normal and hyperhydric regenerants and TW and TR tumours were compared with regard to peroxidase (EC 1.11.1.7) and esterase activity, and isoenzyme patterns. Guaiacol peroxidase activity was the lowest in the cactus shoots and in the normal regenerants. Callus, hyperhydric regenerants and tumours had peroxidase activity of 6 to 7 times higher. Esterase activity was measured with 1- and 2-naphthylacetate as broad-spectrum substrates. The highest esterase activity was determined in tumours with both substrates. All tissues, except the TR tumour, had higher esterase activity for 2-compared to 1-naphtylacetate. Peroxidase and esterase isoenzyme patterns were not completely identical among the investigated tissues.     

An improved procedure for production of white spruce (Picea glauca) transgenic plants using Agrobacterium tumefaciens.

An efficient and reproducible procedure for the transformation of white spruce (Picea glauca [Moench] Voss) embryogenic tissues was developed using A. tumefaciens-mediated gene transfer. Rapidly dividing white spruce embryogenic tissues were co-cultivated with disarmed A. tumefaciens strains containing additional copies of the virulence regions from plasmid PToK47. The plasmid pBi121, containing the neomycin phosphotransferase II (nptII) gene providing kanamycin resistance as a selectable marker and the beta-glucuronidase (uidA) reporter gene, was used as binary vector. The highest frequency of transformation (15 transformed tissues g(-1) FW of treated embryogenic tissue) was obtained with 5-d-old tissues grown in liquid medium and co-cultivated with Agrobacterium for 2 d in the same medium but containing 50 microM acetosyringone. Recovery of kanamycin-resistant tissues was improved when tissues were first grown for 10 d on a timentin-containing medium (400 mg l(-1)), to prevent bacterial overgrowth, before application of the selection pressure. After 6 weeks on kanamycin-selection medium, resistant tissues were obtained and showed stable uidA expression. The presence of the transgenes was demonstrated by PCR analysis and their integration into the genome was confirmed by Southern hybridization. Transgenic plants were regenerated from transformed tissues within 4 months after co-culture.