兔防御素NP-1基因转化百合的研究

本研究以百合鳞片为外植体,用叶盘法将兔防御素NP-1基因导入百合中,经培养获得再生植株。通过抗生素筛选和PCR分析,证实为转基因植株。同时,对农杆菌转化过程中的一些影响因素进行了探讨。     

抗菌肽的分类及作用机理研究进展

抗菌肽是广泛存在于自然界生物中的具有广谱抗菌、抗病毒、抑制杀伤肿瘤细胞等作用的多肽。简要概述了抗菌肽在分类进化及作用机理方面的研究进展。     

兔防御素基因酵母表达的研究

防御素是由动植物体内产生的一种多肽类抗菌、抗病毒活性物质,本研究根据巴斯德毕赤 酵母偏爱密码子的特性,设计兔防御素NP2基因,并构建巴斯德毕赤酵母的表达质粒pGAPZα-NP2,通 过LiCl法转入巴斯德毕赤酵母中,筛选和检测结果表明,兔防御素NP2基因已经成功地转入巴斯德毕 赤酵母,并得到表达。该实验可为兔防御素NP2抗菌抗病毒的研究和开发奠定理论和物质基础。     

Defensin抗菌肽的序列特性分析

对defensin抗菌肽进行了生物信息学分析.一级结构表明其具有典型的昆虫防御素特征.二级结构分析表明defensin抗菌肽中主要的结构元件为α-螺旋,但有例外.信号肽及导肽分析结果显示defensin抗菌肽均属于分泌型蛋白.通过对不同物种defensin抗菌肽的分析结果表明,非阳离子抗菌肽的作用机制可能是疏水作用力起主导作用或者是对细菌胞内酶结构与功能、对细菌胞内信号转导的影响有关.     

紫藤种子总RNA提取方法研究

分离提取高质量的RNA是基因表达、调控和基因工程等研究的基础。植物组织中含有大量RNA酶、多糖和酚类等杂质,影响了高质量总RNA的获得。应用改良的CTAB法提取紫藤种子中的总RNA,添加了PVP、β-巯基乙醇和水饱和酚氯仿后能有效地除去大部分杂质,获得高质量的紫藤种子总RNA。3'RACE实验结果表明,以此总RNA反转录获得的cDNA为模板,可成功扩增紫藤种子中的防御素基因。     

Antifungal proteins: targets,mechanisms and prospective applications

All organisms have evolved several defence systems in order to protect themselves against bacteria, fungi and viruses. Higher organisms have developed a complex network of humoral and cellular responses, called adaptive immunity. A second defence system, innate immunity, was discovered in the early 1980s, consisting of small cationic peptides with a broad antimicrobial spectrum. These proteins act immediately at sites of infection or inflammation. The production of proteins with antimicrobial activity was not limited to higher organisms but was also found in insects, plants and microorganisms. During the last 2decades a broad range of proteins with very different structural features have been isolated and characterised from differing organisms ranging from bacteria to human beings. Over 500cationic membrane-acting proteins with antimicrobial and antifungal activities have been identified to date. Apart from these proteins, a very large number of antifungal proteins active on the fungal cell wall, on enzymes of the cell wall synthesis machinery, the plasma membrane and on intracellular targets have been characterised.Received 17 June 2003; received after revision 4 August 2003; accepted 18 August 2003     

家蝇防御素defensin cDNA的克隆及在毕赤酵母中的表达

提取家蝇总RNA,RT-PCR扩增编码家蝇防御素defensin的cDNA,克隆并测定了其序列,将该cDNA序列与酵母表达载体pPICZαA重组,构建酵母分泌型表达载体pPICZα-de,电激法转化毕赤酵母(Pichia pasto-ris)受体菌GS115。经表型筛选和PCR鉴定证明目的片段已稳定整合到酵母染色体基因组中。在含不同浓度的Zeoc in平板上筛选到高拷贝整合的转化子,阳性克隆经甲醇诱导表达,用Tric ine-SDS-PAGE确定了表达产物的正确性,琼脂孔穴平板扩散法、比浊法测定了表达产物的活性,对金黄色葡萄球菌Staphylococcus aureus具有一定的杀菌活性。     

Expression of rabbit defensin NP-1 gene in transgenic tobacco plants and its activity against bacterial wilt

A cDNA-derived 200 bp fragment encoding a rabbit defensin NP-1 mature peptide was ligated into the GUS-less pBH21 to yield pBIC-35NP1. Tobacco leaf discs were genetically transformed with anAgrobacterium tumefaclens strain harbouring pBIC-3SNP1. Northern blot analysis showed that the defensin NP-1 expression cassette was normally transcribed in transgenic plants. Resistance tests demonstrated that expression of native defensin NP-1 can confer partial resistance to the bacterial wilt pathogen,Ralstonia solanacearum.     

Robust and regulatory expression of defensin A gene driven by vitellogenin promoter in transgenic Anopheles stephensi

The use of genetically modified mosquitoes to reduce or replace field populations is a new strategy to control mosquito-borne diseases. The precondition of the implementation of this strategy is the ability to manipulate the genome of mosquitoes and to induce specific expression of the effector molecules driven by a suitable promoter. The objective of this study is to evaluate the expression of defensin A gene of Anopheles sinensis under the control of a vitellogenin promoter in transgenic Anopheles ste- phensi. The regulatory region of Anopheles gambiae vitellogenin was cloned and subcloned into transfer vector pSLFa consisting of an expression cassette with defensin A coding sequence. Then, the expression cassette was transferred into transformation vector pBac[3xP3-DsRedafm] using Asc I di- gestion. The recombinant plasmid DNA of pBac[3xP3DsRed-AgVgT2-DefA] and helper plasmid DNA of phsp-pBac were micro-injected into embryos of An. stephensi. The positive transgenic mosquitoes were screened by observing specific red fluorescence in the eyes of G1 larvae. Southern blot analysis showed that a single-copy transgene integrated into the genome of An. stephensi. RT-PCR analysis showed that the defensin A gene expressed specifically in fat bodies of female mosquitoes after a blood meal. Interestingly, the mRNA of defensin A is more stable compared with that of the endogenous vitellogenin gene. After multiple blood meals, the expression of defensin A appeared as a reducible and non-cycling type, a crucial feature for its anti-pathogen effect. From data above, we concluded that the regulatory function of the Vg promoter and the expression of defensin A gene were relatively con- served in different species of anopheles mosquitoes. These molecules could be used as candidates in the development of genetically modified mosquitoes.     

绵羊防御素sBD-1 cDNA的克隆及序列分析

从成年绵羊舌表皮组织分离提取总RNA，经反转录PCR（RT-PCR)扩增出绵羊防御素sBD-lcDNA，将扩增片段回收，重组插入克隆载体T—vector，经限制性酶切鉴定和DNA序列测定，结果扩增出cDNA215bp，与发表的绵羊sBD-lcDNA序列完全相同。该cDNA编码以个氨基酸，其中信号肽与前片段相同，成熟肽位于前片段的羧基端，由38个氨基酸残基组成。