酵母PH081在酸性磷酸酯酶基因表达调控中的作用

利用酵母ＰＨＯ８１与大肠杆菌产β－半乳糖苷酶的融合基因,系统地研究了ＰＨＯ８１基因在酵母酸性磷酸醋酶的不同调控因子的单缺失株和双缺失株中的表达规律,它与酸性磷酸酯酶基因ＰＨＯ５和ＰＨＯ１１的控制机制相似。构建了ＡＤＨ１启动子控制下ＰＨＯ８１表达质粒。在ＰＨＯ８１在细胞内组成型表达时,酸性磷酸酯酶基因的表达仍受无机磷的控制,揭示ＰＨＯ８１蛋白可能在不同浓度无机磷条件发生变构。ＰＨＯ８１在酸性磷酸酯酶基因表达系统中是一个中介因子。在此基础提出了一个酸性磷酸酯酶基因调控的分子模型。     

大肠杆菌亮氨酰－tRNA合成酶LeuRS67R的纯化及其动力学研究

本实验室已得到的亮氨酰－ｔＲＮＡ合成酶（ＬｅｕＲＳ）基因,与文献相比,６７位氨基酸残基由Ｈｉｓ变为Ａｒｇ,此酶被定名为ＬｅｕＲＳ６７Ｒ。我们从该基因与ｐＵＣ１９重组质粒的大肠杆菌ＴＧ１转化子ＴＧ１－９１中得到ＬｅｕＲＳ的高表达,粗抽液中ＬｅｕＲＳ的表达量在转化子中比在宿主菌ＴＧ１中高２０倍以上。用三步拉层析得到电泳一条带的酶,其比活为１７８９单位／毫克。测定其动力学常数,氨酰化活力对Ｌｅｕ、ＡＴＰ的Ｋｍ值分别为０．０２７ｍｍｏｌ／Ｌ、０．４７ｍｍｏｌ／Ｌ,Ｋｃａｔ值分别为３．５～５．１ｓ－１。ＡＴＰ－ＰＰｉ交换活力对Ｌｅｕ、ＡＴＰ的Ｋｍ值分别为０．０３ｍｍｏｌ／Ｌ、１．０ｍｍｏｌ／Ｌ,Ｌｃａｔ值分别为１４０～１５５ｓ－１。此结果与从野生型大肠杆菌Ｋ－１２中提纯的ＬｅｕＲＳ的动力学常数差别很小,６７位氨基酸残基在与活性中心无直接关系的域可能是大肠杆菌的种间差异。     

具有中等毒性的马氏钳蝎神经毒素的纯化和初步晶体学研究

运用柱层析技术对产自淅川和常德的马氏钳蝎毒素进行分离纯化,得到８种哺乳动物神经毒素。运用制备型等电聚焦电泳技术,对常德样品中具有中等毒性的蝎神经毒素（ＢｍＫ５）进一步纯化,获得了高纯度样品。两个产地的蝎毒素ＢｍＫ５均已经成功地获得了大晶体。空间群均为Ｐ２１２１２１,晶胞参数分别为：ａ＝３８．４６埃,ｂ＝３７．２８埃,ｃ＝３６．９７埃（淅川）;ａ＝３８．４４埃,ｂ＝３７．５５埃,ｃ＝３６．８３埃（常德）。对两个产地的晶体分别收集了２．１埃（淅川）和１．６２埃（常德）分辨率的衍射数据。     

用蛋白质工程方法改变葡萄糖异构酶最适pH和最适温度

用寡核苷酸诱导的定点突变方法构建了葡萄糖异构酶基因的突变体（Ｎ１８４Ｄ和Ａ１９８Ｃ）。含突变体的重组质粒ｐＴＫＤ－ＧＩ１（Ｎ１８４Ｄ）和ｐＴＫＤ－ＧＩ２（Ａ１９８ｃ）在Ｅ．ｃｏｌｉＫ３８菌株中表达,用ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ ＦＦ和Ｓｅｐｈａｃｒｙｌ Ｓ－３００ＨＲ柱层析分离纯化突变酶。与野生型葡萄糖异构酶比较实验表明：（１）突变酶Ｎ１８４Ｄ的最适ｐＨ值下降了１个单位;等电点下降了０．６个单位     

薄层扫描法测定绞股蓝中人参皂甙Rb_1

用高效薄层扫描法测定了绞股蓝中人参皂甙Ｒｂ_１的含量,并进行分离、纯化,再用酸解法水解,测得其结合糖为葡萄糖。     

几种细胞分裂素诱导石竹外植体直接再分化成花芽的机理

用ＭＳ＋２,４－Ｄ０．１ｍｇ／Ｌ为基本培养基,分别附加不同浓度的ＺＴ、ＢＡ和ＫＴ,对石竹的叶片、茎和花等外植体直接再分化成花芽的试验表明：叶片的外植体在有ＢＡ时可再分化出只有红色花瓣的不正常花芽;在含ＺＴ的培养基中再分化出具有１～２片叶或无叶的花芽,其花有两性花,雌花和仅单个雌蕊的不正常花,两性花可发育成结籽的果实;ＫＴ未能诱导花芽分化。茎外植体,３种ＣＴＫ都不能诱导出花芽。花等外植体仅在ＫＴ３ｍｇ／Ｌ的培养中再分化出无叶的不正常花芽,ＺＴ和ＢＡ的处理均不能诱导出花芽。     

A Selective Medium for the Isolation and Quantification of Bradyrhizobium japonicum and Bradyrhizobium elkanii Strains from Soils and Inoculants

The ecological examination of members of the family Rhizobiaceae has been hampered by the lack of a selective medium for isolation of root nodule bacteria from soil. A novel non-antibiotic-containing medium has been developed which allows selective isolation of Bradyrhizobium japonicum and B. elkanii strains from soil and inoculants. The medium, BJSM, is based on the resistance of B.japonicum and B. elkanii strains to more than 40 μg of the metals ions Zn²⁺ and Co²⁺ per ml. BJSM does not allow growth of Rhizobium sp. strains. We used BJSM to isolate bacteria from a Hubbard soil and from several commercially prepared soybean inoculants. Ninety-eight percent of the isolates obtained from Hubbard soil nodulated Glycine max cv. Kasota, and between 55 and 95% of the isolates from the commercial inoculants had the ability to nodulate soybeans. Numbers of bradyrhizobia obtained by using BJSM, strain-specific fluorescent antibodies, and the most-probable-number plant infection assay indicated that the three techniques were comparable in quantifying B. japonicum strains in soils and inoculants, although most-probable-number counts were generally 0.5 order of magnitude greater than those obtained by using BJSM. Results of our studies indicate that BJSM is useful for direct isolation and quantification of B. japonicum and B. elkanii from natural soils and inoculants. This medium may prove to be an important tool for autecological and enumeration studies of diverse populations of bradyrhizobia and as a quality control method for soybean inoculants.     

Hypomethylated sequences: Characterization of the duplicate soybean genome

Soybean is believed to be a diploidized tetraploid generated from an allotetraploid ancestor. In this study, we used hypomethylated genomic DNA as a source of probes to investigate the genomic structure and methylation patterns of duplicated sequences. Forty-five genomic clones from Phaseolus vulgaris and 664 genomic clones from Glycine max were used to examine the duplicated regions in the soybean genome. Southern analysis of genomic DNA using probes from both sources revealed that greater than 15% of the hypomethylated genomic regions were only present once in the soybean genome. The remaining ca. 85% of the hypomethylated regions comprise duplicated or middle repetitive DNA sequences. If only the ratio of single to duplicate probe patterns is considered, it appears that 25% of the single-copy sequences have been lost. By using a subset of probes that only detected duplicated sequences, we examined the methylation status of the homeologous genomes with the restriction enzymes MspI and HpaII. We found that in all cases both copies of these regions were hypomethylated, although there were examples of low-level methylation. It appears that duplicate sequences are being eliminated in the diploidization process. Our data reveal no evidence that duplicated sequences are being silenced by inactivation correlated with methylation patterns.