热休克转录因子（σ^32）基因的克隆及其在大肠杆菌中的表达

用ＰＣＲ方法获得大肠杆菌热休克蛋白转录因子σ＾３２的编码基因ｒｐｏＨ，并将基克隆至含有Ｐｔａｃ启动子的表达载体ＰＵＨＥ中。     

碱基关联与基因表达水平的关系

研究了Ｅ．ｃｏｌｉ和Ｙｅａｓｔ基因编码区内碱基关联与基因表达水平的关系，结果表明：１）单碱基构成与基因表达水平有一定的关系；２）紧邻、次紧邻碱基间的关联（ｔ≤２）与基因表达水平有较好的线性关系；３）密码子第１位碱基的构成与基因表达水平有关系；４）密码子内１、３位和２、３位碱基之间的关联熵与基因表达水平有很强的关系；５）密码子内各位碱基与相邻密码子第１位碱基的关联熵与基因表达水平有很好的线性关系．     

在大肠杆菌中利用反义RNA抑制乙肝病毒（HBV）X基因的表达

将乙肝病毒（ＨＢＶ）ａｙｗ株完整的Ｘ基因正向重组到原核表达质粒ｐＢＶ－２２１的ＰＬ启动子下游，得到能表达Ｘ蛋白的重组质粒ｐＢＶ－ＨＢＶ（＋）；同时将Ｘ基因反向重组到原核表达质粒ｐＢＶ－２２０的ＰＬ启动子下游，得到能转录Ｘ基因反义ＲＮＡ的重组质粒ｐＢＶ－ＨＢＸ（－）。利用这两个质粒，构建出能同时转录Ｘ基因ｍＲＮＡ和反义ＲＮＡ的重组质粒ｐＥＸ。ＡＮ－ＨＢＸ，并在原核水平上，证实了反义ＲＮＡ对Ｘ基因的表     

反义RNA阻抑基因表达的精确模型——萤火虫荧光素酶基因-爪蟾卵母细胞系统

80年代建立的“反义基因操作技术”不仅打破了通过基因突变认识基因的局限,成为了解基因功能新的有效工具.而且,还拓宽了借助基因操作治疗疾病和改良植物品质的应用途径。虽然,至今仍未取得真核细胞能利用自身存在的反义RNA调节基因表达的证据,但是,人工构建的反义RNA或反义基因能有效地抑制细胞内源或外源基因的暂时性或稳定性表达已有越来越多的报道.只是对抑制机理,最适抑制条件等仍了解很少.本文报道含荧光素     

吗啡耐受时大鼠脑内胆囊收缩素基因表达加速

大鼠胆囊收缩素(cholecystokinin,CCK)基因编码长度为115个氨基酸的前胆囊收缩素原(preproCCK),近于羧基端的8个氨基酸残基肽就是八肽胆囊收缩素(CCK-8)。多次注射吗啡引起其镇痛作用逐渐减弱而出现耐受。资料表明,应用CCK抗血清或受体拮抗剂去除中枢CCK-8的作用可翻转或延缓吗啡耐受,表明脑内CCK-8作为一种抗阿片物质参与吗啡耐受的形成。本文欲了解在吗啡耐受形成过程中,CCK基因表达是否加速,从而进一步论证脑内CCK在吗啡耐受形成中所起的作用。     

Expression of lysine-rich protein gene and analysis of lysine content in transgenic wheat

Expression vector pBPC102, which carries winged bean lysine-rich protein (wblrp) gene and dihydropicolinate synthase (DHDPS) gene, was transferred into hexaploid winter wheat cv. Jinghua No.l, Jing411, You899 and Yangnongl5 explants of immature inflorescence and immature embryos by particle bombardment. More than 100 transgenic plants were obtained under the selection of s-(2-aminoethyl)-L-cysteine (AEC). Confirmed transgenic plants of To and TI generation by PCR and PCR-Southern blotting analyses showed successful integration of wblrp gene into wheat genome. Analysis of transgenic plant lines of T2 by Northern dot-blotting showed good expression of wblrp gene in offspring seed. The content of free lysine in leaves, contents of bound lysine and total proteins in seeds of T2 transgenie wheat lines were determined and analyzed. Among 34 tested transgenic lines, levels of free lysine content in leaves of 9 transgenic lines are 2~3times higher than un-trans-formed wild-type cultivars. Among 17 analyzed transgenic lines, bound lysine content of 4 transgenic lines is more than 10% higher than that of wild-type cultivars. Our research suggests that introducing wblrp gene into wheat is an effective way to improve its nutrition quality.     

β葡聚糖酶杂合基因bglHAM的克隆及在P.pastoris中的表达

采用PCR的方法,以Bacillus macerans总DNA为模板,构建出β-1,3-1,4葡聚糖酶杂合基因bglHAM,与巴斯德毕赤酵母表达载体pPIC9K重组,得到重组质粒pPIC9K-HAM,电脉冲转化法转化酵母菌GS115,KM71,在MM,MD平板上筛选表型,YPD-G418平板上筛选多拷贝重组子,重组菌株经甲醇诱导后,刚果红平板染色法检测到β-葡聚糖酶活性,SDS-PAGE证明表达产物的分子量约为24kD,摇瓶诱导培养筛选到的重组菌株,胞外每mL发酵液最高酶活达240U。     

Cloning and expressional analyses of a cinnamoyl CoA reductase cDNA from rice seedlings

Cinnamoyl CoA reductase (CCR: EC 1.2.1.44),the entry-point enzyme of the llgnin specific biosynthetic pathway, catalyzes the conversion of cinnamoyl CoA esters to their corresponding dnnamaldehydes. Multiple sequence alignment showed that the deduced polypeptide shared 70% similarity and 30% sequence identity at the amino acid level with defined CCR genes from other plant species and they all contain the common signature sequences thought to be the catalytic site as well as the putative NADP binding domain.Using a conserved OsCCR cDNA fragment as the probe for library screening, we isolated the genomic DNA that covered the whole coding region of OsCCR with total length of 3045bp including 4 introns and 5 exons. The open reading frame for our OsCCR gene coBtAin~ 337 amino adds. Northern blot indicated that OsCCR was expressed in different organs with the highest level found in stems. In situ hybridization results showed that OsCCR mRNA was localized mainly along the vascular bundles in stems and leaves, and also in lateral roots that was differentiating from the tiilering node. We conclude that the vascular-localized expression of OsCCR gene may suggest its possible involvement in llgnin biosynthesis. Cloning and characterization of OsCCR will help to clarify how llgniflcations in plants are regulated and will provide a physical basis for creating genetically engineered rice plants with optimal lignin contents.