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培养基部分组份的一些功能

已有 4866 次阅读 2011-2-13 14:46 |系统分类:科研笔记| 培养基

经常问自己:Have I done something new and interesting?

低声说话;勤劳,踏实做事;心情平和,不让矛盾堆积,不计较得失;多吃水果蔬菜,植物油和不去麸的粮食,少吃肉类。要善于在不断总结中变得聪明起来。

 

NB基本培养基

KNO3                               2830 mg/L   

(NH4)2SO4                                      463 mg/L=374.8 mg/L NH4Cl

KH2PO4                                         400 mg/L

MgSO4.7H2O                       185 mg/L

CaCl2.2H2O                            166 mg/L

FeSO4.7H2O                        27.8mg/L 

Na2EDTA                         37. 5 mg/L  

MnSO4.4H2O                        10 mg/L   

H3BO3                                              3 mg/L    

ZnSO4.7H2O                          2 mg/L   

Na2MoO4.2H2O                    0.25 mg/L    

CuSO4.5H2O                      0.025 mg/L    

CoCl2.6H2O                       0.025 mg/L    

KI                                0.75 mg/L    

盐酸硫胺素 thiamine CHL VB1        10 mg/L   

盐酸吡哆醇 pyridoxine-CHL VB6      1 mg/L      

烟酸   nicotinic acid                  1 mg/L    

肌醇  myo-inositol                   100 mg/L   

水解酪蛋白                        300 mg/L

谷氨酰胺                          500 mg/L

甘氨酸                             2 mg/L

脯氨酸                            2878 mg/L

蔗糖                            30,000 mg/L

PHytagel                            2.6 mg/L

pH                                5.8

1.         硝酸盐是植物培养中重要的氮源,其代谢物亚硝酸盐是对植物有害的,所以亚硝酸盐还原酶NiR在组培中起重要作用。硝酸盐nitrateNR催化为亚硝酸盐nitrite,再经NiR催化为氨,再经GS合成谷氨酸;

2.         钙离子利于胚细胞的产生和维持;

3.         银离子阻止乙烯诱导的坏死,促进器官发生及体细胞胚胎发生;

4.         铜离子提高过氧化物酶的活性,诱导胚性愈伤的产生和分化;高浓度的铜离子作为重金属离子使得PPO蛋白变性,抑制其活性,阻止褐化。CuSO4.5H2O的分子量为250, 0.5mM1.25mg/L即可抑制PPO的活性到20%以下,培养基中0.6mg/L即可抑制PPO的活性到50%以下。另外,Vc、硝酸银、PVP等抗氧化剂也能抑制PPO的活性。

5.         缺乏锌离子利于小麦胚细胞的产生;

6.         19-22度低温利于共培养侵染;共培养的时间为55个小时侵染率最高;

7.         干燥和冷利于分化提高转化效率;

8.         硫代硫酸银和硝酸钙利于转化效率的提高;

9.         promotion or prevention of shoot regeneration by cupric sulphate and high EDTA

10.     侵染后亚精氨spermidine利于愈伤的恢复;

11.     硝酸铵利于分化和胚细胞的产生;

12.     The increase in ammonium nitrate concentration in regeneration medium resulted in substantial, concentration-dependent increase in homologous recombination frequency (HRF) (Boyko et al. 2009). ammonium-based salts induce conformational changes in human Rad51 leading to an increase in its activity and therefore promoting recombination. The transformation events we analyzed included better quality and more intact insertions of the transgene, achieved most probably due to the more frequent involvement of HR, an error-free repair mechanism rare earth elements during a tissue culture stage also holds much promise, as a positive effect of these elements on nitrogen metabolism has been documented

13.     氯化铈CeCl3 or 氯化镧 LaCl3 诱导活性氧的产生,激发抗病反应,chloride ions had a positive influence on homologous recombination rates; removal of potassium ion from the MS medium results in a decrease in HRF.

14.     干燥利于侵染和愈伤分化;热击处理可降低或抑制农杆菌诱导的侵染细胞的死亡;饥饿处理利于侵染;钙离子利于适于转化的胚细胞的产生,银离子可以阻止乙烯诱导的组织坏死,硝酸铵、氯化钾和一些稀有元素(镧系)利于植物的生长和遗传转化。

15.     抗坏死物质(抗氧化剂):antioxidants: 聚乙烯吡咯烷酮 PVP (0.5 and 1 g/L), 半胱氨酸 L-cysteine (150 mg/L), 没石子酸gallic acid (1.5 mg /L), 谷胱甘肽glutathione 400 mg /L; 二巯基苏糖醇DTT (70 mg/L), 生物蝶呤biopterin (15 mg/L), 抗坏血酸ascorbic acid (150 mg/L50μM,分子量176.12), and 柠檬酸citric acid (150 mg/L), Sodium thiosulfate (Na2S2O3),158 mg/L, 其中DTT的效果很好。

16.     抗褐化:苯酚甲醛离子交换树脂Amberlite XAD-4 is mainly used for the removal of 芳香族aromatic hydrocarbons such as phenols and pesticides from wastes. However, they have also been utilized for both extraction of specific phytochemicals produced in vitro and removal of toxic compounds negatively affecting explant viability in plant cell cultures

17.     0.5  g/L  2-(4-morpholino)-ethanesulfonic acid (MES, 2-吗啉乙磺酸), 调节氢离子浓度,促进植物生长。

 

18.     在侵染液和选择培养基中加入100 μM  L -cysteine半胱氨酸(分子量 121.16)即12.1 mg/L,也可以100-400 mg/L,可以提高转化效率和利于抗性愈伤的恢复生长。 in the selection medium enhanced the frequency of transformation and transgenic plant recovery

大豆侵染:子叶节作为外植体,dip-wounding 手术刀沾菌后切掉主芽,去除顶端优势,促进AM的生长;再用菌液浸泡。

19.     恢复培养基resting和选择培养基 selection I II 也需要加MES 0.5g/L +Vc+硝酸银

 

20.     侵染液:1/2 NB基本培养基+2mg/L 2,4-D+proline 0.7g/L+CH 2000mg/L+myo-inositol 2g/L+谷氨酰胺2 g/L +68.5g/L sucrose+36g/L glucose  pH 5.2  高温灭菌后4度保存,使用前加抽滤除菌的L -cysteine半胱氨酸150 mg/L + silver nitrate 4.4 mg/L + Vc 150mg/L +100-200  μM AS+10 mL/L  10% Pluronic F68

低盐利于侵染,1/10盐浓度可以大大提高侵染率(高粱可达17.5%),但愈伤容易死亡,1/2盐浓度再加抗氧化剂3.3mM(400 mg/L)cystein1mM(154 mg/L)DDT高粱可达10%,操作人performer和载体、基因、启动子等影响转化率,拷贝数和表达情况。

伤口和快速生长的时候分泌小分子酚类信号物质利于侵染;低温利于4SS型分泌通道的形成,所以集菌时要15-20,悬浮菌液也要10-20放置20分钟;

1M蔗糖342 g/L处理愈伤12小时后,再侵染,会增加转基因的拷贝数。高渗透压预处理提高转化率,侵染前用25%的蔗糖处理3-5个小时。

培养愈伤时,培养基表面放上滤纸再放愈伤,可以防止褐化,可能是利于褐化物质扩散和干燥。

Effect of L-glutamine on Agrobacterium Growth

The effect of L-glutamine on bacterial growth in presence of patchouli extract was examined. Among the concentrations of L-glutamine tested, 2.0 g/l was found effective in promoting bacterial growth in presence of inhibitory concentration (15 mg/ml) of patchouli callus extract (Fig. 5c, d). In the absence of callus extract, media supplemented with L-glutamine at all tested concentrations showed Agrobacterium growth similar to the growth observed in AB minimal media devoid of L-glutamine.

Addition of 0.1% Tween 20 and 2 g/l L-glutamine to Agrobacterium infection medium counteracted the bactericidal effect and significantly increased the T-DNA delivery to explants.

Vir proteins suppress the host innate immune system;

农杆菌与植物互作的主要信号:phenolics (acetosyringone), aldose monosaccharides (glucose), acidic pH(5.5) and low phosphate, 其中酚类是必需的,其它信号能增加农杆菌对酚的敏感性,扩大信号作用。

农杆菌受到植物受伤信号分子(酚类)的诱导后,通过VirA-VirG信号系统激活毒性蛋白(Vir)的表达,诱导表达产生的VirD1VirD2VirC1 VirC2 形成松弛体,具有松弛酶和核酸内切酶活性的VirD2 能在特定的位点上(LB34个碱基之间)T-DNA中的一条链从Ti 质粒上切割下来,并且以共价键的形式结合在单链T-DNA(RB)5’-端,形成VirD2-单链T-DNAnVirE2 复合物,该复合物在VirD5, VirE2, VirE3VirF的作用下通过由VirB VirD4组成的type IV secretion system (T4SS)从细菌进入到植物细胞内,T链复合体在VirE2结合蛋白VIP1VirE3VirE2作用下形成超T链复合体并向细胞核靠近,到达植物细胞核后,在VIP1结合蛋白VBFVirF的作用下,VIP1 VirE2从复合体上解离下来,并通过蛋白酶体SCF (Skp1-Cul1-F-box protein)ubiquitin E3 ligase complex降解。

T-DNA整合到植物基因组主要是通过宿主的DNA修复机制完成的。染色体修饰在DNA修复中起着重要的作用,构成核小体的八聚体的组蛋白H2A, H2B, H3H4N-端尾巴容易被进行翻译后的修饰,包括磷酸化、甲基化、乙酰化和泛素化等,这些修饰与T-DNA插入有关系。

染色质的状态感受高温(热击):H2A-2脱离染色质

DSB诱导因子如X射线,能提高T-DNA的插入;

瞬时表达rare-cutting restriction enzyme能提高T-DNA的插入;

Gram-negative bacterium is a broad-host range plant pathogen, tumour-inducing (Ti) plasmid transfer DNA (T-DNA), The expression of T-DNA-encoded bacterial genes in the host cell results in the production of enzymes that catalyse the synthesis of plant hormones, which are responsible for tumour growth and the formation of novel aminoacid–sugar conjugates, termed as opines. As opines can serve as carbon and sometimes nitrogen sources for Agrobacterium to the exclusion of most other microorganisms, they provide a selective advantage for this species.

Disarmed plasmids, the genes responsible for tumourous growth have been removed, ensuring that the transformed cells can be regenerated into fertile plants that transmit the engineered DNA to their progeny

 

VirA is a membrane-bound sensor and VirG is the intracellular response regulator

On signal sensing, the histidine kinase VirA activates VirG through transferring its phosphate to a particular aspartate of VirG, thereby activating VirG to function as a transcription factor. Phosphorylated VirG then binds at specific 12 bp DNA sequences of the vir gene promoters (vir boxes), thereby activating transcription.

cells in the root elongation zone were found to be the most highly transformable (Yi et al, 2002). Cells of this non-meristematic zone are not undergoing a normal cell cycle, but endoreduplication.

Cell division activity 对转化是很重要的。

chvA, chvB, and pscA (exoC), which are involved in the synthesis and/or localisation of periplasmic b-1,2 glucan,利于农杆菌和植物附着;

soluble pectic plant cell wall fractions decreases both the specific binding of Agrobacterium to plant cells and tumour-induction frequencies

The VirB complex belongs to the class of type IV secretion systems (T4SS), which are found across a broad range of Gram-negative bacteria and are involved in the conjugative transfer of plasmids between bacteria as well as the translocation of Vir factors from pathogens to host cells during infection

VirB1–11 and VirD4 and is required for virulence.

 

 

 

 

 

 

 

分化培养基

MS+烟酸5 mg/L +VB6 10 mg/L+VB1 10 mg/L+肌醇100 mg/L+CH 2 g/L+NAA 0.2 mg/L+kinetin 2 mg/L+sorbitol 30 g/L+sucrose 30g/L+gelrite 3 g/L

 

 

 



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