玉米候选抗病相关基因片段的克隆

根据已经克隆的植物抗病基因和候选抗病基因的保守序列P-loop、Kinase-2及GLPLAL设计一系列简并引物，利用同源序列扩增法，对玉米的基因组DNA进行PCR扩增，并对5个扩增产物的克隆进行测序．测序结果在Gen—Bank内进行BLAST检索，发现A9克隆序列与玉米BAC库中的206C17克隆的部分序列有很高的相似性，并且距离GenBank内注册的玉米抗锈病基因rpl位点中的rpl-3基因、rpl-4基因分别约有66Kb、20Kb，且A9克隆序列在玉米基因组中是单拷贝的．这为玉米抗锈病性状的分子标记辅助选择和抗锈病基因的克隆奠定了良好的基础。     

基于中间件的企业应用集成

在企业的信息化发展过程中，形成的信息孤岛已成为现代企业信息共享的一个制约因素。笔者对当前企业应用集成的现状、方法及存在的问题进行了分析和讨论，通过结合中间件技术和Web技术特点，探讨了一个基于中间件技术的企业应用集成框架。该集成框架以中间件系统为核心构建分布式应用平台，克服了传统应用集成方法的不足，有效地解决了企业内部的跨平台应用间的集成问题，并为企业发展电子商务提供支持。     

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。     

Inhibitory effect of the adenovirus type 5 E1A protein expressed in the yeast system of the human tumor cell growth

Adenovirus 5 type E1A as a tumor suppressor gene can inhibit tumor growth and enhance the censitivity of chemotherapy and radiotherapy.E1A have the ability to integrate into the host genome,resulting in long-time expres-sion that induces Rb gene inactivation and animal cells im-mortalization.This prompted us to select the E1A protein for treatment of cancer in order to overcome the limitations of E1A gene therapy.Thus,we firstly comstructed E1A eu-caryotic expression vector (pPIC9/E1A),transformated the pichia pastoris yeast cells(GS115) and screened the high-expressing recombinant strains.The positive yeast strains were cultured in the shake flask,and induced for 3d.The crude E1A protein was purified using two steps of col-umu chromatography on HiTrap Q and HiTrap SP.The pu-rified E1A protein was identified by SDS-PAGE and Western blot.E1A protein was mostly located at cellular unclear when Cheriot delivered E1A protein into cells.The analysis in vitro indicated that the E1A protein arrested LN686 cell cycle at G2/M phase,and significantly inhibited the growth of LN686 tumor cells.The current studies firstly provided an experimental basis to further develop E1A protein for tumor treatment.     

DNA分子标记在果树上的应用

介绍了在果树上常用的DNA分子标记的原理和特点，并对其在果树的分子遗传图谱和指纹图谱的构建，品种鉴别、系谱分析和分类，基因定位与分子标记辅助选择等方面的应用进行了综述。