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1.
从香蕉胚性细胞悬浮系获得再生植株   总被引:5,自引:0,他引:5  
2个主栽香蕉品种的未成熟雄花诱导产生的胚性愈伤组织接种至液体培养基中,经3~4个月的继代培养后长成质地均匀的胚性细胞悬浮系(ECS),悬浮系中60%~80%是胚性细胞团.ECS接种至体胚再生培养基上约4~5周后开始出现再生体胚,萌发的体胚以MS培养基培养后可获得再生植株.  相似文献   

2.
以香果树(Emmenopterys henry,)未成熟种子为试材,探讨不同的接种量、基本培养基、糖浓度及植物生长调节物质等对体细胞胚胎生长的影响,建立稳定的香果树胚性细胞悬浮培养与植株再生体系。结果表明:悬浮培养条件下,最适接种量为2%(鲜重):较适合的基本培养基为MS;蔗糖浓度为1%时容易使球形胚聚合体愈伤化,浓度为3%和6%适合球形胚聚合体增殖。浓度为9%则容易使球形胚聚合体褐化:添加0.5mg·L^-1 6-BA和0.5mg·L^-1NAA的MS液体培养基,当初始蔗糖浓度为3%。然后逐步提高到6%则有利于香果树各个发育阶段的同步化;子叶胚转到不含任何植物生长调节物质的MS固体培养基中可以长成正常植株。  相似文献   

3.
影响茎用芥菜愈伤组织诱导和植株再生的因素   总被引:3,自引:0,他引:3  
茎用芥菜子叶培养在MS 0.25 mg·L-1NAA 0.5 mg·L-16-BA 0.25 mg·L-12,4-D培养基上,可获得较高质量的愈伤组织.愈伤组织培养在MS 1~1 mg·L-16-BA 0.2 mg·L-1NAA培养基上分化频率为8.3%,而在加有羧苄青霉素和头孢霉素的培养基上,最高分化频率可达52.4%.将获得的再生植株转移到1/2MS 0.1 mg·L-1 NAA的生根培养基中,可获得完整植株.  相似文献   

4.
A culture of callus cells has been developed from a transgenicline of tobacco which contains an introduced phyA-cDNA encodingphytochrome A. Suspension cultures of the cells were shown toaccumulate a significant immunodetectable level of the heterologousphytochrome, but not of the native phyA-gene product. The red-irradiatedform (Pfr) of the heterologous phytochrome was specificallydegraded in vivo, and the red-irradiated (Pfr) and far-red-irradiated(Pr) forms demonstrated different patterns of in vitro proteolyticcleavage. These results strongly suggested that the phytochromeapoprotein was associated with a chromophore moiety which mediatedred/far-red sensitive conformational changes of the molecule.Exogenous application of 4-amino-5-hexynoic acid (AHA) to thetransgenic suspension cultures resulted in the accumulationof a population of phytochrome which was stable under red lightand gave identical patterns of in vitro digestion in the redand far-red irradiated forms, i.e. the spectral activity ofphytochrome was inhibited. Application of exogenous 5-aminolevulinicacid (ALA) or biliverdin overcame the inhibitory effects ofAHA to restore spectral sensitivity of the phytochrome pool.These results are consistent with the proposed pathway of phytochromechromophore biosynthesis in intact plant systems. Thus, thetransgenic suspension cultures provided a single-cell systemin which spectrally-active phytochrome, apparently indistinguishablefrom the native phytochrome synthesized in etiolated seedlings,was accumulated. Photoregulation of expression of the genesencoding the small subunit of ribulose-1,5-bisphosphate carboxylaseand chlorophyll a/b binding proteins demonstrated that the heterologousphytochrome population mediated rapid changes in gene expressionin the de-differentiated cells. It is therefore proposed thatsuch a suspension culture of transgenic cells offers a modelsystem for the study of phytochrome function. Key words: Cell cultures, transgenic tobacco, phytochrome, oat-phy A-cDNA, gene expression  相似文献   

5.
重组人BMP-2在烟草不同组织中的表达   总被引:1,自引:0,他引:1  
骨形态发生蛋白(BMPs)是一类调节骨组织发育的生长因子。BMP-2是BMP家族中诱骨活性最强的。在骨组织工程研究和临床应用中需要大量的BMP-2。因此,研究出一种能够有效地大量生产BMP-2的方法是十分必要的。随着植物分子生物学的进展,转基因植物被用作一种生物反应器来生产目的蛋白。以gus作为报告基因,研究了重组人bmp-2基因在烟草中的表达。通过GUS活性检测、半定量PCR和Western blotting分析了根、茎、叶组织中基因表达的水平,结果显示融合蛋白在根和茎组织中表达量显著高于叶组织。由于根和茎组织中蛋白组成与叶组织相比相对简单,提示其更易于进行目的蛋白的纯化。  相似文献   

6.
Expression of the Human Milk Protein sCD14 in Tobacco Plant Cell Culture   总被引:1,自引:0,他引:1  
The human milk protein sCD14 was expressed in tobacco plant cell cultures. Tobacco cells were transformed with a modified cd14 cDNA minus the GPI-tail and either the native human signal peptide (SP) or a plant SP, under the control of the CaMV-35S promoter. Transformants were screened using PCR and Southern blot analysis. The functionality of the inserted cDNA was checked by northern blot analysis for the presence of recombinant sCD14 mRNA. The detection of the protein has been observed by western blot analysis at an estimated level of 5 μg l−1 in a non-soluble fraction of the culture medium.  相似文献   

7.
Summary By use of a method for regenerating wheat plants (Triticum aestivum L.) from cells from long-term suspension culture, the chromosome complement and stability of cultured cells of cv. Mustang were examined. Massive chromosome restructuring and genomic rearrangements were detected by HCl−KOH-Giemsa banding techniques. Chromosome structural variations involved mainly heterochromatin and centromeric regions. These included B genome chromosome elimination; heterochromatin amplification; megachromosomes and extrachromosomal DNA particles; translocations and deletions; telocentric, dicentric, and multicentric chromosomes; and somatic pairing and crossing over. At least 65 break-fusion sites were identified. Most of the sites were located in the B genome chromosomes (42 sites, 64.6%); 36.9% (20 sites) were located in the A genome chromosomes; and the fewest (3 sites, 4.6%) were detected in the D genome. Most of the chromosome break-fusion is in the heterochromatin and centromeric regions. The B genome chromosomes appeared to be eliminated nonrandomly, and the stability of the genome may vary among the genotypes and depend on culture duration. We also checked chromosome number of 1-year-old shoot-competent cells. Only 20% of the cells still had 2n=42 chromosomes. Most of the cells (60%) were hyperploid. These observed variations describe the types of tissue-culture-induced variations and suggest the unsuitability of using wheat cells from long-term cultures for genetic transformation experiments.  相似文献   

8.
The bone morphogenetic proteins (BMPs) are a family of growth factors that regulate the development of bone. BMP-2 is the most effective in the induction of bone tissue. A large amount of BMP-2 is needed for both bone tissue engineering research and clinical application. Thus, an effective way is necessary to produce sufficient BMP-2 protein. With the advance in plant biotechnology, transgenic plants have been targeted as a bioreactor to produce desired recombinant proteins. Here, the expression of recombinant human bmp-2 gene (rhbmp-2) was studied in tobacco plants using gus as a reporter gene. The difference of expression levels in root, stem and leaf tissues was analyzed by GUS activity assay, semi-quantitive RT-PCR and western blotting. The results indicated that the expression levels of fusion protein in root and stem tissues were significantly higher than those in leaf tissue. For the protein compositions in root and stem tissues were simpler than those in leaf tissue, this suggested that the purification process with root and stem tissues would potentially be easier.  相似文献   

9.
在转瓶和2L搅拌反应器中,利用重组杆状病毒AcNPV感染sf9昆虫细胞表达尿激酶原。在转瓶中,细胞接毒密度12×106/mL、MOI=30时,尿激酶原活性达到1065IU/mL。研究了尿激酶原表达过程中葡萄糖、乳酸的代谢变化。实验结果表明细胞状态对尿激酶原的表达水平有显著影响。  相似文献   

10.
重组[B18Ile]人胰岛素的鉴定和特征   总被引:2,自引:2,他引:2  
突变体「B18Ile」猪胰岛素前体经分离纯化,转肽,得到重组「B18Ile」人胰素「B18Ile」人胰岛素能结晶,其与受体的结合能力为猪胰岛素的82%,保留了与猪胰岛素基本相同的体内活力,从本文结果和分析表明B18Val可能不是胰岛素表现生物功能所必需的。  相似文献   

11.
以‘莱芜大姜’为试材,研究了生姜离体叶片愈伤组织的诱导以及细胞悬浮系建立与植株再生。结果表明,以生姜试管苗叶片为外植体,接种到MS+1.0 mg/L 2,4-D+0.5 mg/L 6-BA+30 g/L蔗糖的培养基上,可有效诱导出生长迅速、质地疏松的愈伤组织。将获得的愈伤组织接种到MS+0.15 mg/L 2,4-D+6.0 mg/L 6-BA+30 g/L蔗糖的液体培养基上,25℃黑暗条件下震荡培养25-30 d,可建立分散性好、生长迅速的悬浮细胞系,细胞悬浮系培养的适宜参数为:初始接种量为1.0-1.5 g,继代培养的适宜间隔期为15 d,继代培养液体培养基更新比例为3/4。将悬浮细胞接种到固体培养基MS+0.2 mg/L NAA+10.0 mg/L 6-BA+30 g/L蔗糖上可获得再生植株。  相似文献   

12.
以芥菜型油菜(Brcssica juncea)下胚轴来源的愈伤组织为材料,进行悬浮细胞振荡培养,得到游离的单细胞。这些细胞作浅层液体静置培养,可获得高频率的愈伤组织。在附加有水解乳蛋白200毫克/升、BA 2—3毫克/升及GA,0.1—1毫克/升的MS培养基上,这些愈伤组织分化了芽。分化的芽在无激素或附加有0.01—0.1毫克/升IAA或NAA的MS培养基上长出根,从而发育成完整植株。当对游离细胞获得的再生植株的茎切段愈伤组织,再经上述程序进行细胞培养时,发现愈伤组织诱导率及愈伤组织分化频率均远远高于原供体,从而建立了B.juncea易再生的体细胞无性系。  相似文献   

13.
毛白杨悬浮细胞系的建立及再生植株的获得   总被引:1,自引:0,他引:1  
以毛白杨基因型TC152无菌苗为材料,研究毛白杨悬浮细胞系建立与植株再生,结果表明,通过悬浮培养和固体培养两种方法诱导毛白杨悬浮细胞分化不定芽,最终获得无菌生根苗。愈伤组织在MS+1.5mg·L-12,4-D+30g·L-1蔗糖的液体培养基中振荡培养,12d可建立悬浮细胞系;悬浮细胞系继代培养基为MS+0.8mg·L-12,4-D+30g·L-1蔗糖,继代周期为7d,悬浮细胞在MS+1.0mg·L-16-BA+0.1mg·L-1NAA+0.5~1.0mg·L-1ZT+30g·L-1蔗糖培养基中悬浮培养,可分化大量不定芽,每个培养瓶中可得到40~50个芽,个别不定芽玻璃化;不定芽在1/2MS+0.6mg·L-1IBA+20g·L-1蔗糖+5.5g·L-1琼脂培养基上可分化不定根。悬浮细胞通过固体平板培养增殖为愈伤组织块后,在MS+1.0mg·L-16-BA+0.1mg·L-1NAA+1.0mg·L-1ZT+30g·L-1蔗糖+5g·L-1琼脂的固体培养基上,不定芽分化率可达到70.00%。  相似文献   

14.
生长素结合蛋白cDNA的克隆及其在烟草中的表达   总被引:3,自引:0,他引:3  
基于拟南芥内质网生长素结合蛋白基因的cDNA序列,设计合成了Ap5和Ap3两个引物,应用RT-PCR技术扩增了拟南芥的ABP基因。将该基因克隆在植物表达载体p35SSIN的35S启动子和Nos3’端之间,得到植物表达载体p35SE。通过农杆菌个导的方法对烟草SR1进行了转化,由分子杂交等检测证明,生长素结合蛋白基因已在烟草中表达,同时转基因烟草后代对生长素的敏感性明显增加。  相似文献   

15.
提高榨菜离体培养植株再生频率   总被引:11,自引:0,他引:11  
采用榨菜“浙桐1号”品种为材料,以MS为基本培养基,通过对不同植物生长调节剂的组合和不同外植体等主要因素的筛选,大幅度提高了榨菜离体培养植株再生频率。结果表明,2mg/L6.BA 0.2mg/L2,4-D的组合较为适宜,其不定芽再生频率可达50%,且外植体以下胚轴为好:而CPPU和2,4-D的适宜组合为1.5mg/L 0.2mg/L,其不定芽再生频率高达66.67%,最适外植体为带柄子叶。同时,研究结果显示,添加0.25~1mg/L的GA,对榨菜已分化的不定芽的伸长有抑制作用;子叶柄和下胚轴外植体的分化具有极性现象。  相似文献   

16.
17.
水母雪莲愈伤组织和悬浮培养细胞的抗冻性研究   总被引:12,自引:0,他引:12  
水母雪莲(Saussurea meduasa Maxim)是典型的高山雪线植物。本文研究了其愈伤组织及悬浮细胞的培养过程,并对其抗寒性做了初步研究,研究结果表明,水母雪莲愈伤组织和悬浮培养细胞分别可抵抗-6.5℃、-7.5℃的冰冻低温胁迫,水母雪莲愈伤组织细胞内丰富的蛋白质和淀粉粒多糖构成其较强抗冻能力的物质基础,低温锻炼后,悬浮细胞分泌蛋白中有新的多肽(76,48,27.5,19.5kD)合成,而33,51kD两条多肽合成增强,悬浮细胞抗冻能力的提高同蛋白质合成的增强是一致的。  相似文献   

18.
A large-scale statistical experimental design was used to determine essential cultivation parameters that affect biomass accumulation and geraniol production in transgenic tobacco (Nicotiana tabacum cv. Samsun NN) cell suspension cultures. The carbohydrate source played a major role in determining the geraniol yield and factors such as filling volume, inoculum size and light were less important. Sucrose, filling volume and inoculum size had a positive effect on geraniol yield by boosting growth of plant cell cultures whereas illumination of the cultures stimulated the geraniol biosynthesis. We also found that the carbohydrates sucrose and mannitol showed polarizing effects on biomass and geraniol accumulation. Factors such as shaking frequency, the presence of conditioned medium and solubilizers had minor influence on both plant cell growth and geraniol content. When cells were cultivated under the screened conditions for all the investigated factors, the cultures produced ∼5.2 mg/l geraniol after 12 days of cultivation in shaking flasks which is comparable to the yield obtained in microbial expression systems. Our data suggest that industrial experimental designs based on orthogonal arrays are suitable for the selection of initial cultivation parameters prior to the essential medium optimization steps. Such designs are particularly beneficial in the early optimization steps when many factors must be screened, increasing the statistical power of the experiments without increasing the demand on time and resources.  相似文献   

19.
Biosynthesis of ent-kaurene was investigated in extracts of cell suspension cultures derived from tobacco callus (Nicotiana tabacum L.), tomato callus (Solanum lycopersicum L.), and in germinating tomato seeds. Incubation of extracts derived from the two cell cultures with either isopentenyl pyrophosphate-14C or with 14C-labeled mevalonate, followed by alkaline phosphatase hydrolysis, resulted in the formation of trans-geranylgeraniol-14C and trans-farnesol-14C. The corresponding pyrophosphates of trans-geranyl-geraniol-14C and trans-farnesol-14C were also detected. No detectable amount of ent-kaurene-14C was produced by these enzymatic preparations when trans-geranylgeranyl-14C pyrophosphate served as substrate. However, copalyl-14C pyrophosphate served as a substrate for the production of ent-kaurene. Cell-free extracts derived from germinating tomato seeds catalyzed the formation of ent-kaurene-14C from mevalonate-14C, isopentenyl-14C pyrophosphate, trans-geranylgeranyl-14C pyrophosphate, and copalyl-14C pyrophosphate.  相似文献   

20.
t-PA cDNA在CHO细胞中的高效稳定表达   总被引:1,自引:0,他引:1  
我们曾报道t-PA mRNA非翻译区序列对其表达有明显的抑制作用,在此基础上,通过对5′-UTR及3′-UTR的改造,使t-PA在COS-7细胞中的表达水平提高30倍左右。将t-PA表达质粒用电击法转染中国仓鼠卵巢细胞二氢叶酸还原酶缺陷株(CHO-dhfr),经过混合加压及筛选,在CHO细胞中高效表达了t-PA,表达水平达到5000~6000 IU/10~6细胞/24hr。重组t-PA具有与天然t-PA相同的分子量及酶活性。经过8个月连续传代,表达水平未下降,表明细胞株是稳定的,其主要指标均符合工程细胞株的要求。  相似文献   

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