电击法锈导的欧白英原生质体转化和转化植株再生

本研究使用电击法成功地将带有标记基因ＮＰＴⅡ的质粒ｐＣａＭＶＮＥＯ转入欧白英的原生质体,并获得了再生转化植株。通过用ｐＤＷ２质粒进行的ＣＡＴ基因短暂表达研究,确定了欧白英原生质体转化的电击条件为：电容３０ｎＦ、电场强度１５００Ｖ／ｃｍ、时间衰变常数５９．４微秒;质粒ＤＮＡ浓度为２０μｇ／２×１０＾６原生质体。在以上条件下,欧白英原生质体的相对转化率为１２．４％,绝对转化率为２．４×１０－４。在大多     

电击法介导的紫孢侧耳原生质体转化

使用基因脉冲导入仪成功地将糙皮侧耳DNA导入紫孢侧耳单核原生质体内,获得了具有"锁状联合”特征的双核转化菌株T₁,和T₂。转化率为8．2×10^-5,转化比为3．6％。酯酶同I酶分析结果表明,转化菌株除具有受体菌的酶带外,还存在供体菌的酶带,由此证明转化菌株确为紫孢侧耳和糙皮侧耳DNA重组的产物。转化菌株子实体形态也发生了变化。两菌株子实体均不释放孢子;T₁。菌柄中生,T₂成熟子实体菌盖中部易长出菌丝。     

石防风原生质体遗传转化及抗除草剂植株的再生

以石防风（Ｐｅｕｃｅｄａｎｕｍｔｅｒｅｂｉｎｔｈａｃｅｕｍ（Ｆｉｓｃｈ．）Ｆｉｓｃｈ．ｅｘＴｕｒｃｚ．）叶柄愈伤组织为材料建立以致密细胞团为主的悬浮培养物,用酶解法获得原生质体。用ＰＥＧ法将拟南芥菜（Ａｒａｂｉｄｏｐｓｉｓｔｈａｌｉａｎａ（Ｌ．）Ｈｅｙｎｈ．）抗除草剂基因导入原生质体后进行液体浅层培养。转化细胞经除草剂ｃｈｌｏｒｓｕｌｆｕｒｏｎ筛选,通过胚状体途径产生抗性小植株。转化处理１０６个原生质体,以加入４０～５０μｇ／ｍＬ质粒ＤＮＡ,ＰＥＧ终浓度为１０％,于１ｍＬ转化介质中,２６℃黑暗下处理２０ｍｉｎ的效果最好。ｃｔＤＮＡ的加入对转化结果无明显影响。分子杂交试验表明,突变的ａｌｓ基因已整合进转化植株的基因组中。转基因植株的细胞在脱分化过程中仍具有抗ｃｈｌｏｒｓｕｌｆｕｒｏｎ的能力;移栽成活的转基因小苗仍表现出抗除草剂特性,而未转化的植株在低浓度除草剂下即逐渐死亡。表明转入的ａｌｓ基因在石防风细胞内能够稳定表达。     

植物体细胞原生质体遗传转化研究

重点介绍了植物体细胞原生质体遗传转化的方法和当前已经取得的成果,同时提出了目前原生质体遗传转化中存在的问题,展望了今后的工作重点。植物原生质体遗传转化的方法主要有：PEG介导转化法、电击穿孔转化法、脂质体介导转化法、农杆菌共培养转化法等。     

大白菜和黄瓜原生质体电击基因转移的研究

本文以钙黄素、FITC-IgG 和溴乙锭-质粒 DNA 为荧光标记物,研究了电脉冲引起大白菜和黄瓜原生质体质膜通透性的改变及其动力学过程以及脉冲电学参数(脉冲幅值、个数、宽度)对原生质体成活率和外源物质导入率的影响。通过电击法成功地将外源 CAT(氯霉素乙酰转移酶)基因导入黄瓜原生质体并实现瞬间表达。45℃热激预处理原生质体有效地促进了CAT 基因的电击转移。     

球形芽孢杆菌TS—1原生质体电诱导质粒转化研究

This report gave the best conditions of Bacillus sphaericus Ts-1 protoplast-plasmid pHV33 electroporation. The highest transformation frequency and transformation efficiency induced by three pulse of 21 KV/cm and 10 microseconds duration applied at an interval of one sec., was 2.44 x 10(2) transformants/micrograms DNA and 3.16 x 10(-6) respectively. The saturated concentration of DNA absorbed by the protoplast was 5 micrograms DNA/10(9) cells/ml. By means of this method, pJB417, a recombinant mosquito larvicide clone, was introduced into B. subtilis 168M and B. sphaericus Ts-1. The transformants of B. subtilis 168M with biocide activity were obtained, but the toxicity of B. sphaericus Ts-1 was not increased.     

水稻原生质体再生植株研究进展

水稻是重要粮食作物之一,水稻的基因工程引起科学工作者关注。一般对植物基因工程的要求,除外源DNA体外重组及重组DNA分子引入植物受体细胞,并使其正确表达外;受体细胞还应分化成植株,使外源基因遗传至植物的后代,使其获得外源基因的性状。     

应用电击法获得转MT基因平菇

将MT基因用电击法转化平菇 (Pleurotusostreatus) ,MT基因表达蛋白与金属离子结合而形成络合物 ,用Zn诱导 ,转基因平菇能富集Zn ,可为缺Zn的人群补充Zn ,使平菇成为一种保健和治疗的食品或蔬菜。原生质体制备浓度为 6 .74 5× 10 6个 /mL。原生质体电击转化率为 0 .0 1%。PCR检测 ,2 0 0bp处有MT基因条带。蛋白检测 :转基因MT平菇ELISA检测阳性 ,表达率为 0 .6 %～ 0 .8%。SDS_PAGE显示有表达条带。Westernblot显示有阳性条带。抗ZnSO4结果 :野生型平菇抗ZnSO4浓度为 1.0mmol/L ,1.2mmol/L开始受抑制 ,转基因平菇抗ZnSO4浓度为 1.5mmol/L ,2 .0mmol/L开始受抑制。出菇试验结果表明 ,在米糠与锯沫比为 1∶3的培养基上生长 ,在米糠与锯沫比为1∶4的培养基上不生长。 2 4d菌丝可在广口瓶中长满 ,用于子实体培养。     

茄子子叶原生质体再生可育植株

将茄子子叶原生质体放在0.75％纤维素酶R-10、0.2％半纤维素酶Rhozyme和0.2％果胶酶溶液中分离。原生质体在培养基中诱导出小愈伤组织。愈伤组织在Ms+2mg/l KT+0.005mg/l NAA+2％蔗糖的固体培养基中,一个月后分化出芽。芽生长至3—4厘米高,转接在Ms+0.1mg/l 1AA+1％活性炭+2％蔗糖的培养基上,一个星期后可长出根,继而形成完整植株。随后移栽至灭菌的混合土壤中长到开花结果。     

Transgenic Rice Plants Produced by PEG-Mediated Plasmid Uptake into Protoplasts

Embryogenic cell suspension cultures were obtained from calli developed from mature rice seeds of a Japonica type Itahan cultivar, Roncarolo. Protoplasts were isolated and transformed by PEG-treated method with plasmid pHP23 carrying the NPT Ⅱ gene which encodes resistance to antibiotic G-418. Protoplast-derived colonies were selected in presence of the inhibitor. Plants were regenerated and transplanted into soil in the green house. The presence of foreign gene in the regenerated plants was verified by PCR and Southern analysis.     

Regeneration of Transgenic Plants from Electroporated Protoplasts of Asparagus officinalis L

An effective method for consistent regeneration of transgenic asparagus (Asparagus officinalis L) plants from electroporated protoplasts is described. Transgenic plants containing β-glucuronidase (GUS) and neomycin-phosphotransferase (NPT II) genes were obtained by electroporating callus-derived protoplasts of Asparagus officinalis L. Embryogenic callus tissue and plants from four kanamycin resistant lines expressed P-glucuronidase activity, as revealed by histological staining. The amplification of genomic DNA by polymerase chain reaction revealed the presence of both GUS and NPT II genes in transformed callus tissue and plants. Southern hybridization confirmed the integration of these genes into the asparagus genome.     

无选择标记基因植物转化系统研究进展

在转基因植物中,将选择标记基因去掉,将提高转基因植物的食用安全性和对环境的安全性,更易为广大消费者所接受,也有利于对同一个植物品种进行多次转基因操作。科学工作者已经在建立无选择标记基因转化系统方面作了大量尝试,获得了无标记基因的转基因植物(MFTPs:MarkerFreeTransgenicPlants)。本文将这方面的研究进展介绍给大家,以推动植物生物技术产业化进程。     

Evaluation of DAPI as a Fluorescent Probe for DNA in Viable Petunia Protoplasts

4',6-Diamidino-2-phenyl-indole (DAPI), is a fluorescent probe that specifically and quantitatively stains DNA. Electroporation of viable Petunia protoplasts in the presence of DAPI revealed integral fluorescence that was similar for both the electroporated and fixed protoplasts. indicating quantitative staining of DNA. DAPI fluorescence was localized in the nuclei of viable protoplasts of Petunia. Protoplasts had a short term viability of 56-65% of the control (non-electroporated. unstained) protoplasts as determined by fluorescein diacetate staining 24 hr following electroporation in the presence of DAPI. The majority (84% of the number originally cultured) of these protoplasts subjected to electroporation were able to form a cell wall, but most did not form microcalli because they were blocked in cell division. The three week plating efficiency for protoplasts exposed to DAPI was 4% of the original number of protoplasts initially cultured compared to 30% for the control. DAPI should not be used as a fluorescent probe for plant protoplasts when the protoplasts are cultured for sustained growth because the levels of DAPI required to obtain quantitative staining of the DNA resulted in inhibition of the cell cycle. DAPI may, however, be used as a fluorescent DNA probe for short term (24 hr) studies.     

Regeneration of Transgenic Soybean (Glycine max) Plants from Electroporated Protoplasts

Transgenic soybean (Glycine max [L.] Merr.) plants were regenerated from calli derived from protoplasts electroporated with plasmid DNA-carrying genes for a selectable marker, neomycin phosphotransferase (NPTII), under the control of the cauliflower mosaic virus 35-Svedberg unit promoter, linked with a nonselectable mannityl opine synthesis marker. Following electroporation and culture, the protoplast-derived colonies were subjected to kanamycin selection (50 micrograms per milliliter) beginning on day 15 for 6 weeks. Approximately, 370 to 460 resistant colonies were recovered from 1 × 10⁶ electroporated protoplasts, giving an absolute transformation frequency of 3.7 to 4.6 × 10⁻⁴. More than 80% of the kanamycin-resistant colonies showed NPTII activity, and about 90% of these also synthesized opines. This indicates that the linked marker genes were co-introduced and co-expressed at a very high frequency. Plants were regenerated from the transformed cell lines. Southern blot analysis of the transformed callus and leaf DNA demonstrated the integration of both genes. Single-plant assays performed with different plant parts showed that both shoot and root tissues express NPTII activity and accumulate opines. Experiments with NPTII and mannityl opine synthesis marker genes on separate plasmids resulted in a co-expression rate of 66%. These results indicate that electroporation can be used to introduce both linked and unlinked genes into the soybean to produce transformed plants.     

Transformation and Characterization of Transgenic Plants of Solanum dulcamara L.--Incidence of Transgene Silencing

A transformation system is described for Solanum dulcamara usingthe supervirulentAgrobacterium tumefaciens strain 1065, carryingboth the ß-glucuronidase (gus) and neomycin phosphotransferaseII (npt II) genes adjacent to the right and left T-DNA borders,respectively. Leaf explants were more efficient for the productionof transformed plants compared to stem explants on medium containing50 mg l^-1of kanamycin sulphate. A 1:10 (v:v) dilution of anovernight culture ofAgrobacterium gave optimal transformationin terms of transgenic plant regeneration. From a total of 174kanamycin-resistant plants selected by their antibiotic resistance,16 failed to exhibit GUS activity. Southern analysis revealedthat these GUS-negative transformants originated from threeindependently transformed cell lines. Restriction enzyme analysesshowed that the GUS-negative plants had both the gus and nptII genes integrated into their genome (one plant had a singlecopy of each gene; the other two plants had multiple copies),with major rearrangement of the gus gene occurring in plantswith several copies of the transgene. GUS-negative plants showedleaf malformations, delayed flowering and a reduction in flower,fruit and seed production compared to GUS-positive and non-transformed(control) plants. Although gene silencing of the gus gene occurred,albeit at a low frequency (9.2%), the transformation systemdescribed generates large numbers of phenotypically normal,stably transformed plants. Copyright 2000 Annals of Botany Company Agrobacterium -mediated transformation, gene silencing, Solanum dulcamara L. (Bittersweet, Woody Nightshade), T-DNA truncation, transgene expression     

Plant Regeneration from Petiole Protoplasts of Brassica napus

Two cultivars of Brassica napus, Altex and Canadian twins, were used as materials. Protoplasts isolated from petioles of plants grown in vitro were cultured in Nitsch medium supplemented with 0.5mg/L BA, 0.5mg/L NAA, lmg/L 2,4-D, 100mg/L serine, 800mg/L glutamine, 4% sucrose and 0.4mol/L mannitol. After 2 days of culture, the first division was observed. The division frequency estimated after 10 days of culture was 30-60%. One week after transferring onto MS medium containing 6mg/L GA3. and 3mg/L BA, protoplast-derived calli regenerated into shoots. The regeneration frequency of the two cultivars was 24% and 31% respectively. It was found that the protoplasts isolated from petioles could float on the surface of the 3% sucrose contained solution which was very favourable both to purification, and culture of the protoplasts.     

根癌农杆菌介导转化诸葛菜获得转基因植株

以诸葛菜下胚轴和子叶为材料,在附加BA和NAA的MS培养基上诱导芽再生,在1／2MS培养基上诱导生根,获得完整再生植株,建立了诸葛菜组织培养高频再生体系,再用很癌农杆菌介导转化诸葛菜下胚轴和子叶,在附加一定量的氨苄青霉素、头孢霉素和卡那霉素的相应培养基上进行筛选,并培养再生成苗,获得完整抗性再生植株,移植到盛有土壤的花盆中均可存活,生长正常。将再生植株叶片,进行GUS、NPTⅡ酶活性测定和Southernblot分子杂交,证实外源基因已稳定整合到植物基因组中,并高效表达。