Cells of Dunaliella salina from the log phase of growth were broken by sonication on ice-water-bath and the chloroplasts were isolated by centrifugation through sucrose gradient. Osmolarity of the growth medium, the suspending medium and the sucrose gradient was kept identical to minimize changes in chloroplast volume and mitochondrial entrapment. The isolated intact chloroplasts were stable to washing with buffered medium. Isolated chloroplast yield and purity was dependent on cell culture condition; a cycle of 16 hours light and 8 hours dark with continuous high CO2 was optimum. Isolated chloroplasts were about 70% intact by microscopic examination. The enzyme activity of glycerol-3-phosphate dehydrogenase was well retained in isolated chloroplasts.
A copper-inducible, Cre-loxP recombination-mediated DNA excision system has been developed in transgenic tobacco plants. The copper inducible system derived from yeast was used for the control of the expression of the Cre recombinase. Upon copper induction, the GVS reporter gene expression unit flanked by two direct lox sites was excised from the transgenic tobacco genome. Quantitative fluorometric GUS assays, Northern blot and PCR analyses showed a high-efficient, copper-dependent and Cre-loxP mediated DNA recombination in all the tested transgenic lines. The copper inducible foreign gene excision might be of great potential in genetic control of transgenic crops.
PENG Xiang-lei, LIANG Bin, CHEN Ming, HU Yuan-lei and LIN Zhong-ping(National Key Laboratory of Protein Engineering and Plant Genetic Engineering , Peking University , Beijing 100871 , P. R. China)
A full-length cDNA of dehydrin BcDh2 from Boea crassifolia and its antisense nucleotidesequence have been transferred into tobacco (Nicotiana tabacum) NC89 under the controlof a caulifower mosaic virus 35S promoter. Under a progressive water stress, photosyntheticrate, transpiration rate and stomatal conductance of the sense and antisense plantsreduced, and those of the control reduced much more. Photosynthetic rate, transpirationrate and stomatal conductance of all plants tested increased significantly 24 hourslater after recoveried water supply, and those of the sense and antisense plants werehigher than control. These indicated that overexpression of a dehydrin gene in tobaccomay improve tolerance to water stress for plants, however, antisense BcDh2 gene intransgenic plant did not influence physiological conditions. The results of germinationexperiment of the transgenic seeds showed that on MS medium with different concentrationPEG (8000), sense seed could more endure drought than control, while antisense seed wassensitive to drought. The results suggested that the overexpression of a dehydrin genein tobacco might improve the tolerance to water stress for plants.
以橄榄球2号草地早熟禾种子为外植体,研究了不同培养基、不同类型的生长调节剂（2,4-D,BA等）对愈伤组织诱导、增殖继代、幼苗分化及植株再生的影响。结果表明,在愈伤组织诱导培养基中附加10 mg/L Ag NO3抑制了橄榄球2号叶芽生长,促进了愈伤组织的诱导效果;HNP2培养基诱导频率明显高于HNP3培养基,是橄榄球2号的最适诱导培养基;愈伤组织的增殖继代方面激素配比宜采用附加适当浓度的6-BA,TDZ的N19,N21,N22这3个培养基;继代1~2次的愈伤在分化培养基R1上培养分化率可达78.4%,再生频率可达30%~50%,为后期相关抗性基因的转化提供了良好的受体系统。
