建立以TK基因为标记将外源基因导入臭曲霉（Asper—gillus...

A new system for selection of transformed Aspergillus foetidus was reported. In this system, TK- A. foetidus which were constructed by homologous recombination of mutated TK gene of vaccinia virus with TK gene of A. foetidus were screened by adding BUdR in agar plates. Conditions for screen of TK+ A. foetidus strain, transformation of A. foetidus and selection of transformed TK- A. foetidus have been studied. By using this system, several transformed A. foetidus which contained HBsAg gene derived bf a promoter H8 cloned from genomic DNA of A. foetidus were isolated. It was demonstrated that HBsAg gene was integrated into the chromosome DNA of A. foetidus by Southern blot after many passages of spores. ELISA showed that HBsAg was positive in the growth medium (p/n = 20). The 22 nm particles which were very similar to the HBsAg particles in human serum were found in the growth medium by immunoelectromicroscope. Western blot also gave the specific bands. All these data showed that HBsAg gene was expressed in A. foetidus and the products were secreted into the growth medium. The selection system using TK gene as marker could generally be used to study the expression of foreign gene in A. foetidus.     

Cloning of α-amylase gene from Schwanniomyces occidentalis and expression in Saccharomyces cerevisiae

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臭曲霉(Aspergillus foetidus)启动子H8片段克隆及其序列

使用原核及真核菌通用的挑选启动子克隆载体pJGI,从臭曲霉菌体总DNA中分离到具有较好活性的启动子H8片段。根据Southern blot证明该启动子来自臭曲霉菌;H8全片段DNA序列分析结果表明其含有7 50bp,内含典型真核启动子功能序列(TA TA box)及增强子GTGG：TTTAAAG的相似序列,分析证实是一个新的臭曲霉启动子。初步实验表明该启动子不但在臭曲霉菌内有启动功能;并第一次证明来自臭曲霉菌的启动子在原核细胞中也具有启动子活性,能够表达完整的Lacz基因。     

极端嗜盐菌16SrDNA的PCR扩增

四株嗜盐菌Haloarcula vallismortis(EM201)、Haloferax denitrificans(EM303)、A_5和B_2已通过一对特定引物用PCR技术从总DNA中扩增出各自的16SrDNA片段,分子大小在1.47kd左右.DNA杂交也表明这些PCR产物具有嗜盐菌的同源性.     

美味侧耳病毒及其生化特性

自美味侧耳(Pleurotus sapidus)子实体中分离到两类病毒颗粒,一是球形,直径为25nm(少数为19、32am);一是杆状,具较深锯齿状亚基,大小为12～14×40～600nm,病毒具核蛋白吸收峰,最大吸收为E257nm,最小为242nm,ISCO蔗糖密度梯度离心中出现3个峰。 病毒具有分子量为44000和22000两条主要衣壳多肽,自子实体中提取到分子量约1.2×10~(?)道尔顿的dsRNA,可能是球形病毒的基因组,病毒与同属真菌糙皮侧耳(P,ostreatus)和德平菇(P,floride)球形病毒之间有血清学关系。     

关于TMV外壳蛋白质信使RNA的分离及其体外翻译产物分析的初步研究

从感染TMV普通株的烟叶中提取总RNA,并经Sepharose 6B层析分部得到7个分部。各分部中各种RNA的分布情况用凝胶电泳检查,当在含[14C]-蛋白质水解液小麦胚无细胞保温液中加入病叶总RNA或层析后得到的IV分部的RNA时,在凝胶电泳放射自显影图谱上出现放射性相当强的带,该带之泳动率与[14C]-标记的天然TMV外壳蛋白质相同。加入病毒颗粒的RNA或用甲醛处理的病毒颗粒RNA就不出现。在同样条件下,改用[9H]-组氨酸代替[14C]-蛋白质水解液,在荧光放射目显影图谱上此带也不出现。因此认为此带即是新合成的TMV外壳蛋白。凝胶电泳图谱表明,IV分部是低分子量RNA富集的一个分部,其中RNA,和RNA．可能相当前人报道的LMC。值得注意的是,用不连续的凝胶电泳IV分部,RNA,和RNA,给出7个带,似乎LMC区是个复杂的多分散的区域,究竟耶个带才真正是TMV外壳蛋白质的mRNA,有待进一步研究。     

葡萄卷叶病毒的纯化,血清学研究及其在脱毒组培苗检测中的应用

将具有典型葡萄卷叶病(Grapevine leafroll diseas,GLRD)症状的葡萄组织,经差速和硫酸铯—蔗糖密度梯度离心,提纯了GLRV,并制备了兔抗血清。电镜下可观察到长度从600～2000nm的线形病毒颗粒,其中以1400nm左右为主。免疫电镜结果表明线形病毒颗粒能被美国的NY-1分离株抗血清(Ⅲ型)所修饰。在间接ELISA中提纯制品与GLRV的Ⅲ、Ⅳ、Ⅱ型抗血清均能产生免疫反应。与Ⅲ型抗血清产生较强的免疫反应,Ⅳ型次之,Ⅱ型最弱。在SDS-免疫双扩散实验中病组织韧皮部粗提液与GLRV的Ⅲ,Ⅳ、Ⅱ型抗血清均产生免疫沉淀线。从而推测我国葡萄园内的葡萄卷叶病很可能由2种或3种卷叶病毒感染所致.采用A蛋白夹心酶联免疫吸附试验(PAS-ELISA)检测葡萄试管苗,Ⅲ型抗血清和自制抗血清的平行测试结果基本相符,共获得11个生食葡萄和10个山葡萄品种的脱葡萄卷叶病毒和扇叶病毒的组培苗,扩繁后田间试种表现出良好的农艺性状。