茶树咖啡碱合成酶基因cDNA在烟草中的表达

为了在烟草中合成咖啡碱,将咖啡碱合成酶(Tea caffeine synthase,TCS)基因cDNA全序列克隆到双元载体pBI121中,通过农杆菌叶盘转化法将TCS基因成功转入烟草,结果得到72株转基因烟草植株。对其中3株转基因植株进行PCR检测和PCR-Southern检测,证实TCS基因已整合到基因组中;RT-PCR和Northern分析显示,TCS在这些植株中均能正常转录。用从转基因植株中提取的粗酶液进行体外酶促反应,能催化7-甲基黄嘌呤和可可碱向咖啡碱的转变,由此表明,转基因烟草植株中能产生具有正常生物学活性的TCS,但从3株转基因烟草中均未检测到咖啡碱。     

凤冈锌硒绿茶中7种儿茶素及咖啡因含量特征

采用HPLC方法同时测定了26个凤冈锌硒绿茶样品中7种儿茶素儿茶素(+C)、没食子儿茶素(GC)、没食子儿茶素没食子酸酯(GCG)、表儿茶素(EC)、表没食子儿茶素(EGC)、表没食子儿茶素没食子酸酯(EGCG)、表儿茶素没食子酸酯(ECG))和咖啡因(CAF)的含量。结果表明,凤冈锌硒绿茶中儿茶素总含量平均值为20.08%,其中EGCG含量最高;儿茶素平均品质指数为556;咖啡因的含量平均值为3.9%。该研究可为凤冈锌硒绿茶品质的进一步研究提供依据。     

Genetic parameters of several biochemical compounds from green coffee, Coffea canephora

A factorial crossing scheme of Cofflea canephora (two parents from the Congolese group crossed to 14 parents of the Guinean group) was used to evaluate genetic parameters of several biochemical compounds, bean weight and crop outturn (ration of dry bean weight 10 fresh berry weight). For most characters studied, additive genetic effects were preponderant. Narrow-sense heritability was high for caffeine content (h²_ns= 0.80), fat matter content (h²_ns, = 0.74), bean weight (h²_ns= 0.73) and crop outturn (h²_ns= 1). It was intermediate for trigonelline (h²_ns= 0.38) and chlorogenic acid (h²_ns= 0.36) content. Only sucrose content had a low narrow-sense heritability (h²_ns=0.11). There were few genetic and enviromnental correlations, Consequences for breeding, in relation to coffee drinking quality, are discussed.     

Effect of Chelated Vanadium Spray on Concentration of Vanadium and Organic Compounds in Tea Shoots

The study aimed to develop vanadium tea products and evaluated the potential of chelated vanadium （V） absorption by tea shoots and effect of vanadium accumulation on tea quality during the first crop season.Chelated V application with three times foliar sprays at 5 days intervals starting from 15 days before plucking showed a significant increase in V content of new young shoots,which was much effective at higher concentration.However,one or two times sprays had no increase in V content regardless of spray concentration compared to non-treated.Despite V accumulation in the treated at 3 to 9 times higher that of than the non-treated plants,there were no significant changes in contents of total amino acids,theanine,caffeine,total polyphenols,crude fiber,and cathechins between the treated and the non-treated.     

用超临界CO2脱除绿茶浓缩液中咖啡碱的工艺研究

对绿茶浓缩液中咖啡碱的超临界CO₂萃取工艺进行了研究，通过4因素正交试验探讨了浓缩液的浓度、操作压强、操作温度、萃取时间对咖啡碱脱除率的影响。结果表明，超临界CO₂萃取技术可以有效地脱除绿茶浓缩液中大部分的咖啡碱，在此基础上完成了用超临界CO₂脱除绿茶浓缩液中咖啡碱的连续作业试验，从而获得了加工脱咖啡碱绿茶浓缩液或速溶绿茶的新工艺。     

Effect of adding different levels of caffeine in the extender on some biochemical constituents,enzymatic activities and physical characteristics of chilled and frozen ram semen

Two experiments were carried out to determine the efficiency of supplementation of ram semen extender with caffeine on chilled storage and frozen capacity of spermatozoa. In the first experiment, eighty ejaculates were collected by an artificial vagina from five adult Barki rams, aged 2–3 years and weighted 45.0 ± 2.0 kg throughout the experimental period (January to February 2017). The ejaculates were pooled and diluted (1:10) with tris‐citric egg yolk extender and were split into five groups. Group 1 served as control, whereas groups 2‐5 were supplemented with 0.1, 0.2, 0.3 and 0.4 mM caffeine. All diluted semen specimens were evaluated for physical characteristics immediately after dilution (T₀) and throughout preservation period of 48 hr at 4°C. Simultaneously, oxidative stress and indices such as total antioxidant capacity (TAC), malondialdehyde concentrations (MDA) and alkaline transaminase (AKP) concentrations and value of resazurin reduction test (RRT) were determined. In the second experiment, the raw pooled ejaculates were diluted (1:10) with glycerolated tris‐citric egg yolk extender, receiving the previously mentioned caffeine levels. The post‐thaw assessment of cryopreserved spermatozoa, in all groups, was conducted by a computer‐assisted sperm analysis (CASA) system. The results revealed that adding caffeine to ram semen extender at low (0.1 mM) or medium (0.2 mM) levels had positive impact on both physical characteristics of ram sperm and the enzymatic activities compared to the other semen groups. Caffeine supplementation also enhanced post‐thaw sperm dynamics, which implies its potential as an exogenous antioxidant supplement.     

Highly efficient induction of triploid Pacific abalone <Emphasis Type="Italic">Haliotis discus hannai</Emphasis> by caffeine treatment

ABSTRACT:   In order to develop a highly efficient method for mass production of triploid Pacific abalone Haliotis discus hannai , caffeine treatment that is safe and inexpensive was optimized. To suppress the first meiotic division, fertilized eggs were exposed to either a 10- or 15-mM caffeine solution for 24 min beginning at 12 min after fertilization. In most treated batches, the rates of cleaved eggs showed no significant difference from the control batches. However, in most treated batches, the rates of occurrence of normal larvae and the survival rates of the early juveniles were significantly lower than those of the controls. The triploid rates at 6 days to 11 months after settlement in all the treated batches were extremely high (91–100%). There was no significant difference in the mean triploid rates between 10- and 15-mM caffeine treatments. These results suggest that both treatment conditions were conducive to triploid abalone production. One live 2n/3n mosaic specimen was found in the treated batches. However, since the frequency of mosaic was extremely low, the mosaicism would probably not have an adverse effect on the stable production of triploid abalones.     

RNAi沉默CsHB1降低茶树叶片愈伤组织咖啡碱积累

以‘英红9号’茶树叶片为材料,克隆HD-Zip转录因子基因CsHB1,原核表达CsHB1重组蛋白,利用本氏烟草进行CsHB1表达蛋白的亚细胞定位;构建CsHB1的RNAi转基因载体,经农杆菌介导转化‘英红9号’茶树叶片愈伤组织,并对沉默CsHB1愈伤组织系进行基因表达分析和咖啡碱含量测定。结果表明,克隆基因与NCBI登记的‘龙井43’茶树Cs HB1(MF033534)有6个碱基差异,但编码相同的氨基酸序列;原核诱导表达获得CsHB1的49 kD不可溶蛋白;将CsHB1蛋白亚细胞定位于细胞核和细胞质。在沉默表达CsHB1愈伤组织中,CsHB1表达显著下降,催化茶叶咖啡碱合成的N–甲基转移酶基因yhNMT1表达下调,愈伤组织中咖啡碱含量显著降低。推测转录因子基因CsHB1的沉默抑制了靶基因yhNMT1的表达,进而降低‘英红9号’愈伤组织中咖啡碱的合成积累。     

Tea plant (Camellia sinensis L.) roots secrete oxalic acid and caffeine into medium containing aluminum

We examined the response of the tea plant (Camellia sinensis L.) to aluminum (Al) exposure under sterile conditions, focusing specifically on the secretion of low molecular mass organic compounds from roots. After germination in agar medium, tea seedlings together with medium were placed on agar containing 0.4?mM Al with 0.2% hematoxyline (hematoxylin-Al medium). The purple color of the hematoxylin-Al medium was observed to fade gradually, until none of the color remained 6 days later. The tea seedlings were then treated with simple calcium solution (0.2?mM, at pH 4.2) containing AlCl₃, which ranged in concentration from 0 to 0.8?mM, for 24?hrs. The amount of oxalate secreted into the medium increased as the external Al concentration increased, while the concentrations of malate and citrate in the medium remained unchanged. Oxalate secretion started within 30?min after Al exposure and increased linearly thereafter. The findings demonstrated that oxalate was a key compound in the Al-tolerance mechanism employed by the tea plant, which detoxifies Al³⁺ externally in the rhizosphere. In addition to oxalate, caffeine was also secreted by tea roots in response to Al exposure. It is possible that caffeine excretion from the roots of tea plants may stimulate root growth through the inhibition of callose deposition in root tips.     

从信阳毛尖中提取咖啡因的工艺优化研究

以信阳毛尖为材料,通过单因素及正交试验,进行从信阳毛尖中提取咖啡因的工艺优化研究。结果表明:最佳的提取工艺为乙醇浓度为80%,提取时间为3 h,沉淀剂为氧化钙,咖啡因提取得率为0.594%。