根癌农杆菌介导脂肪酶在无孢黑曲霉中的高效表达

黑曲霉(Aspergillus niger)具有高效的蛋白表达和分泌能力.为实现异源蛋白在无孢黑曲霉中的高效重组表达,在优化遗传筛选标记的基础上,建立了根癌农杆菌介导的、以潮霉素抗性基因为筛选标记的黑曲霉转化体系.利用该体系介导米黑根毛霉脂肪酶(RML)转化黑曲霉SH-1,通过PCR鉴定、酶活检测、镍柱亲和层析及SDS-PAGE电泳检测等确定成功获得了RML的黑曲霉转化子,并通过发酵条件优化实现了RML的高效表达,对硝基苯酚比色法测得转化子酶活最高可达15 U/mL,是其在酵母中表达水平的10倍以上.     

Ti质粒对甘薯叶片和愈伤组织的转化

用根癌农杆菌菌株T_(37)和B_6S_3感染甘薯叶片和愈伤组织,在无激素的MS培养基上诱发产生了转化愈伤组织。两菌株对愈伤组织的转化率明显高于对叶片的转化率。电泳结果表明,转化组织中均具有LpDH或NpDH活性,证明Ti质粒上的T-DNA已转移到甘薯细胞的基因组中。同时还进行了转化组织的过氧化物同工酶分析。     

根癌农杆菌法转化烟草的条件探索

 利用含有四环素阻遏蛋白,具有卡娜霉素抗性的烟草种子为实验材料,采用根癌农杆菌介导转化中的叶圆盘法,将外源基因导入烟草愈伤组织,建成可诱导表达细胞质和细胞核CaM/CaM结合多肽的转基因烟草体系.在获得转基因植物的过程中,通过比较影响根癌农杆菌转化频率的各种因素,表明植物材料的选择和培养,农杆菌的培养和稀释,植物材料与农杆菌共培养的程度及转基因植株的筛选条件是影响农杆菌转化效率的重要因素.     

禾木科作物基因转化研究进展

本文从四个方面对禾本科作物特别是水稻的基因转化的新近研究成果进行了评述。作者认为，研究工作取得了不少成果，但尚未建立起一整套具体的操作模式．     

树状多节孢HQD_(33)遗传转化体系建立的影响因素研究

利用根癌农杆菌(Agrobacterium tum efaciens)介导的转化方法,以紫杉醇产生菌———树状多节孢HQD33的分生孢子为受体材料,分析影响转化效率的主要因素.获得高效的遗传转化体系,转化率最高可达1.3×10-4.PCR检测表明,T-DNA已整合进树状多节孢HQD33的基因组中,而且大多数都是以单拷贝的形式整合,转化子都能够稳定遗传.结果表明,根癌农杆菌菌株类型、菌株生长状态、分生孢子浓度、共培养时间以及乙酰丁香酮的诱导等因素对转化效率都具有重要影响.通过对以上因素的分析,为建立紫杉醇产生菌———树状多节孢HQD33稳定的遗传转化体系奠定了基础.     

Introduction of pokeweed antiviral protein cDNA into Brassica napus and acquisition of transsenic plants resistant to viruses

Using cotyledonary petioles as explants, the PAP cDNA controlled by wound-in- ducible promoter has been introduced into Brassica napus by coculture with Agrobacterium tumefaciens and laser microbeam puncture respectively. Regenerated plants resistant to gen-tamycin have been selected out. PCR amplification and Southern blotting analysis indicated that PAP cDNA together with wound-inducible promoter had been integrated into Brassica genome with transformation frequencies of 2.0% and 1.7% for two transformation methods respectively. The test of virus challenge showed that these transgenic Brassica plants were resistant in different degrees to mechanically inoculated TuMV.     

Mutant acetolactate synthase （ALS） gene as a selectable marker for Agrobacterium-mediated transformation of soybean

Soybean is one of the crops most difficult to be manipulated in vitro. Although several soybean marker genes, all the selectable markers used were from bacteria origin. To find suitable selectable marker gene from plant origin for soybean transformation, a mutant acetolactate synthase （ALS） gene from Arabidopsis thaliana was tested for Agrobacterium-mediated soybean embryo axis transformation with the herbicide Arsenal as the selective agent. Transgenic soybean plants were obtained after the herbicide se- lection and the To transgenic lines showed resistance to the herbicide at a concentration of 100 g/ha. ALS enzyme assay of To transgenic line also showed higher activity compared to the wild type control plant. PCR analysis of the T1 transgenic lines confirmed the integration and segregation of the transgene. Taken together, our results showed that the mutant ALS gene is a suitable selectable marker for soybean transformation.     

发根农杆菌Ri质粒诱导的黄芪发状根根冠细胞亚显微结构研究

对膜荚黄芪发状根根冠细胞的亚显微结构进行研究发现：发状根根尖没有分化出典型的根冠结构；根冠平衡囊细胞中淀粉分布无极性，散地细胞质中；细胞中未发现粗面内质网而仅有滑面内质网的存在；高尔基体分泌功能不旺盛，无肥大的潴泡；细胞壁较薄，纵向壁略加厚，其间无粘液积累；根冠外周细胞逐渐老化，但细胞壁不溶解，老化的细胞不脱落，且根冠细胞层数不增加。