激光微束切割小冰麦异附加系染色体的研究

利用激光微束,并选用适当的功率密度可将小冰麦异附加系花粉细胞染色体切割成2～3个片段,这个技术的建立为激光微束应用于染色体片段DNA微克隆及基因定位提供可能。     

麻雀(Passer montanus saturatus Stejneger)冬季集群与性腺发育的关系

本文用改变麻雀实验种群光照周期的方法,探讨了集群状况与其腺性发育的关系。实验结果表明:增加光照时间能使处于性休止期的性腺发育,但其发育程度不仅与延长光照时间而且与集群的状况有关;在相同的光照条件下,群鸟的性腺发育比单饲鸟显著。由此可见,麻雀冬季集群具有激发性腺发育的生物学意义。     

Purification and characterization of a novel thermostable extracellular protein tyrosine phosphatase from Metarhizium anisopliae strain CQMa102

An extracellular phosphatase was purified to homogeneity from the entomopathogenic fungus Metarhizium anisopliae with a 41.0% yield. The molecular mass and isoelectric point of the purified enzyme were about 82.5 kDa and 9.5 respectively. The optimum pH and temperature were about 5.5 and 75 degrees C when using O-phospho-L-tyrosine as substrate. The protein displayed high stability in a pH range 3.0-9.5 at 30 degrees C and was remarkably thermostable at 70 degrees C. The purified enzyme showed high activity on O-phospho-L-tyrosine and protein tyrosine phosphatase substrate monophosphate (a specific substrate of protein tyrosine phosphatase). Although one peptide of the phosphatase shared identity with one alkaline phosphatase of Neurospora crassa, its substrate specificity and inhibitor sensitivity indicate that the enzyme is a protein tyrosine phosphatase.     

Identification of an extracellular acid trehalase and its gene involved in fungal pathogenesis of Metarizium anisopliae

Trehalose is the main sugar in the haemolymph of insects and is a key nutrient source for an insect pathogenic fungus. Secretion of trehalose-hydrolysing enzymes may be a prerequisite for successful exploitation of this resource by the pathogen. An acid trehalase [EC 3.2.1.28] was purified to homogeneity from a culture of a locust-specific pathogen, Metarhizium anisopliae, and its properties were characterized. The gene (ATM1) of this acid trehalase was also isolated. The pure enzyme can efficiently hydrolyze haemolymph trehalose into glucose in vitro. The new acid trehalase appearing in the haemolymph of Locusta migratoria infected with M. anisopliae had the same pI and substrate specificity as the purified fungal acid trehalase, and the concentration of trehalose in the haemolymph decreased sharply after infection. RT-PCR also revealed the ATM1 gene's expression in the haemolymph of the infected insects. Our results indicated that the acid trehalase may serve as an "energy scavenger" and deplete blood trehalose during fungal pathogenesis.     

优化重叠PCR法进行单链抗体基因扩增和点突变

利用重叠PCR技术扩增单链抗体基因或位点突变是抗体文库构建或稳定表达的关键和难点,国内外文献未见其方法学的系统报道.以不同VH、VL和Linker基因为拼接模板进行重叠PCR,针对影响重叠PCR扩增的拼接类型,引物设计,反应条件等进行优化.结果表明两段重叠连接比三段更容易实现,且扩增效果好;引物的互补序列长度一般应大于15 bp,且在18～24 bp 时扩增效果最好;退火温度在52～60℃,Mg2+浓度在1.5～2.5 mM时对拼接的效果影响较小;直接或间接使用拼接模板均可以实现重叠PCR的扩增.利用优化策略,首次构建了抗除虫菊酯的scFv基因文库并引入抗XAC糖蛋白scFv基因的点突变,为除虫菊酯抗体文库构建和抗XAC重组抗体的稳定表达奠定了基础.     

1994年国家自然科学基金生态学科资助项目

１９９４年国家自然科学基金生态学科资助项目陈领,陆仲康（国家自然科学基金委员会北京,１０００８３）ＥＣＯＬＯＧＩＣＡＬＰＲＯＪＥＣＴＳＳＵＰＰＯＲＴＥＤＢＹＴＨＥＮＡＴＩＯＮＡＬＳＣＩＥＮＣＥＦＯＵＮＤＡＴＩＯＮ（１９９４）遵循：“控制规模、提高强度...     

中国生态学的研究现状及发展趋势──近年来国家自然科学基金委员会生态学科资助项目分析

本文系统地分析了近年来国家自然科学基金委生态学科受理基金项目的批准率及资助强度,分析了项目负责人及主要参加者的年龄结构和职称结构及其动态变化,以资助项目的题目及关键词出现的频率为依据,探讨了近年来我国生态学的发展趋势。重点研究领域和生态学的研究热点。     

A method to construct cDNA library of the entomopathogenic fungus, <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>, in the hemolymph of the infected locust

A method was developed to construct cDNA library of pathogenic fungus in the blood of the infected insect for cloning the fungal genes expressed in the host. This method is designed to take advantage of the obvious difference between the cell structures and components of the pathogen cells and that of the host cells. The host blood cells only have cell membrane, which can be disrupted by using SDS/proteinase K (PK). The fungal cells grown in the animal blood have cell wall, which can protect the fungal cell from the disruption of SDS/proteinase K (PK). By this method, the blood cells were disrupted by SDS/proteinase K (PK) and then the released animal RNA and DNA were digested completely with RNase and DNase. Therefore, the fungi grown in the blood were harvested without any contamination of host RNA and DNA. The pure fungi harvested from the infected blood can be used for mRNA extraction and cDNA library construction. The purity of the fungal mRNA was confirmed by PCR and RT-PCR with specific primer pairs for the host and specific primer pairs for the fungus, respectively, and the clones of cDNA library constructed by using the fungal mRNA was also analyzed. The results showed that there was no detectable contaminated insect DNA or RNA existing in the fungal mRNA. Randomly selected cDNA clones from cDNA library were sequenced and analyzed against GenBank using Blastx; no selected sequences had significant similarity with insects’ genes in comparison with the data of GenBank. The results further confirmed that the method to purify the pathogenic fungus from the host animal is reliable and the mRNA extracted from the fungus is eligible for cDNA library construction, and other molecular analysis including RT-PCR. This method may be applied to other pathogenic fungi and their host animals.     

Regulation of extracellular acid phosphatase biosynthesis by culture conditions in entomopathogenic fungusMetarhizium anisopliae strain CQMa102

Metarhizium anisopliae is an imperfect entomopathogenic fungus. Once invading into its host,M. anisopliae needs to absorb basic nutrients such as phosphorus from the host haemolymph. A large number of phosphorylated compounds in haemolymph cannot be directly utilised by the fungal cell and must be hydrolysed into available form by phosphatase before ingested. Aims of this paper were to investigate optimum fermentation conditions for production of acid phosphatase and phosphatase isoenzymes byMetarhizium anisopliae. The optimum fermentation conditions were: glucose, 20 g/l; (NH₄)₂SO₄, 2 g/l; casein, 4 g/l; MgSO₄, 0.5 g; KCl, 0.5 g; microelement salt solution, 10 ml; inoculum size, 1×10⁷ spores per 100 ml medium; initial medium pH, 6.0. Under these conditions, the highest total acid phosphatase activity was 3.05 U/ml in 4 days at 27 °C and 160 rpm. Synthesis of the acid phosphatase was repressed by 0.01% inorganic phosphate in culture medium. The spectrum of isoenzymes produced byM. anisopliae varied depending on the phosphorus source employed in the culture. A specific isoform with pI 9.45 was induced by casein, and another isoform of pI 8.21 was induced by phytic acid and disodium phenyl phosphate.