生命科学研究的若干重大进展

简要介绍了生命科学研究的若干重大进展,包括我国基因剔除技术获重大突破、小麦小染色体研究取得重大进展、利用基因工程技术制造新的微生物以及脊椎神经生长开发将使残疾人恢复肢体运动等.     

几丁质酶基因在转基因烟草中表达效率的检测

采用氨基葡萄糖法检测转基因烟草植株叶片几丁质酶活力，结果表明几丁质酶基因在转基因植株中酶活力增强，与β—1,3-葡聚糖酶基因的协同作用下酶活性最强，几丁质酶活力的测定对外源基因表达效率的检测是可行的．     

甘蓝磷酯酶D HKD2功能区基因片段的克隆与反义表达载体的构建

以甘蓝(Brassiaca oleracea)为材料，取幼叶分离mRNA，反转录合成cDNA，以cDNA第一链为模板，通过PCR扩增，获得甘蓝磷酯酶D(Phosphorate　Lipid　Dehydrolase，PLD)HKD2功能区的基因片段．对其进行Blast分析，结果表明，分离的目的片段核苷酸序列与Genbank中报道的甘蓝PLD基因相比同源率为99．7％，只有2个碱基发生改变．将得到的PLD基因片段插入植物表达载体pAT940，构建了PLD基因反义表达载体pATC—rPLD，为下一步进行抗逆转基因作物选育打下基础．     

极端嗜热厌氧纤维素分解菌基因文库的构建及其内切葡聚糖酶基因片段的克隆

以从云南邦拿掌温泉中分离、纯化的高温厌氧纤维素分解菌邦2菌(Cadicellulosiruptor)为材料,制备其总DNA,经限制性核酸内切酶EcoRⅠ部分酶切后,在T4DNA连接酶的作用下与经EcoRⅠ完全酶切、去磷酸化的质粒载体pUC18连接,然后转化E.coliJM109,建立了邦2的基因文库,经筛选鉴定得到6.3×103个重组子;重组子经刚果红平板验证:约有23.5%菌落呈现透明圈;重组子经EcoRⅠ酶切验证显示:重组质粒均含有外源DNA插入片段.结果表明已克隆到邦2菌纤维素酶系中的内切葡聚糖酶基因(ED基因)片段.     

Genetic Diversity in Populations of Sepiella maindroni Using 16S rRNA Gene Sequence Analysis

Part of the 16S rRNA gene is amplified with PCR and sequenced for 5 populations of common Chinese cuttlefish Sepiella maindroni:three from the South China Sea,one from East China Sea and one from Japan.The result shows that a total of 5 nucleotide positions are found to have gaps or insertions of base pairs among these individuals,and 13 positions are examined to be variable in all the sequences,which range from 494 to 509 base pairs.All of the individuals are grouped into 7 haplotypes (h1-h7).No marked genetic difference is osberved among those populations.All of the individuals from Nagasaki belong to h1 and the h3 haplotype is found only in the coastal waters of China.A→←G transition in Nucleotide 255 is suggested to be taken as a kind of genetic marker to identify the populations distributed in East-South China Sea and the Nagasaki waters of Japan.     

基因疫苗及影响其免疫效果的因素

基因疫苗被视为“第3代疫苗“,是目前疫苗研究领域的热点.本文回顾了基因疫苗的诞生,阐述了其优越性,重点讨论了增强其免疫效应的策略包括合理的载体设计、细胞因子及共刺激因子与抗原的共表达及适宜的接种途径等.     

Recent progress in polymer-based gene delivery vectors

The gene delivery system is one of the three components of a gene medicine, which is the bottle neck of current gene therapy. Nonviral vectors offer advantages over the viral system of safety, ease of manufacturing, etc. As important nonviral vectors, polymer gene delivery systems have gained increasing attention and have begun to show increasing promising. In this review, the fundamental and recent progress of polymer-based gene delivery vectors is reviewed.     

N-terminal of L protein of vesicular stomatitis virus contains a new signal sequence

The L protein (241kD) of vesicular stomatitis virus (VSV) is the most important snbnnit of the replication complex. The existence of specific localization signal in the L protein was investigated by making recombinant constructs expressing truncated mutants of the L protein fused to green fluorescent protein (GFP) in transient transfection assays. The chimeric genes encoding varied N-terminal of L and GFP gene were put under the control of T7 promoter or CMV promoter. The fusion proteins were transiently expressed in BHK-21, COS-7, CHO or Hep G2 cells. When more than 120 residues were deleted or only 96 residues were kept on the N-terminal, the fusion proteins were shown to be distributed throughout the cells, cytoplasm and nucleus under the confocal microscope. However, other chimeric proteins with 120 or more amino acids were dotted and distributed in the perinuclear regions. And the fusion protein with 96—120 aa has the similar distribution. A thirteen-residue peptide QGYSFLHEVDKEA (108—120) was identified as localization signal, whose function would be absolutely distributed with the deficiency of D or V. Our results show that there is an independent localizing signal in N-terminal domain of L protein of VSV and this functional signal is conserved in different cell lines.     

Building of hFⅨ transgenic mice by spermatogenic cells

Human FⅨ expression vector pCMVⅨ was packaged by effectene^TM reagent and injected into mice seminiferous tubules with glass pipettes.The expressional frame of pCMVⅨ was examined by PCR and Southern blotting among 41 progenies.There were 2(4%) mice being integrated with hFⅨ gene into chromosomes.4.6ng/mL of hFⅨ protein was expressed in plasma of one mouse,which was tested by ELISA.We demonstrated that building of transgenic animals by spermatogonial stem cells is an efficient method.Meanwhile,it has also been proved to be an alternative choice for mammary gland bioreactor.     

表达千穗谷Ah—AMP基因的转基因烟草抗病性研突

通过PCR从苋属植物千穗谷(Amarathus hypochondriacus)的总DNA中扩增出苋菜抗菌肽(Ah—AMP)的核基因片段，序列分析结果表明该基因长261bp，编码一个由86个氨基酸组成的Ah—AMP前体多肽。在构建Ah—AMP基因的植物表达载体pBinAH916后，通过根癌土壤杆菌介导方法转化了烟草。转化再生植株和T1代转基因烟草的PCR和Southern blot分析表明，AA—AMP基因已整合到烟草的染色体中，并为单拷贝整合。T1代转基因烟草的Nouthern blot分析结果表明，AA—AMP基因在转基因烟草中至少在mRNA水平上能正常表达。对T0代转基因烟草进行烟草青枯病的抗病性试验，筛选出两株抗性较强的植株。对T1代抗青枯病的统计分析发现，其抗病性比起始品种SRl分别提高了2．24和1．62个级别；其病情指数比起始品种降低49．6％和37．3％。对黑烃病也表现一定的抗性，主要表现在推迟发病时间，减缓发病速度上。这些结果表明AA—AMP基因在植物抗病基因工程研究中可能是一个有潜在应用价值的基因。