经大肠杆菌感染的中国家蚕cDNA文库的构建

用建立非表达文库的方法,从大肠杆菌感染后９ｈ的中国家蚕的ｍＲＮＡ合成ｃＤＮＡ片段,重组入噬菌体λｇｔ１０的ＥｃｏＲＩ的位点之间,所构成的基因非表达文库库容量为２×１０６重组子,经大肠杆菌Ｃ６００与Ｃ６００ｈｌｆ菌株平皿测定,重组率为７６％．     

人HCUTA全长cDNA的克隆与原核表达

人ＨＣＵＴＡ是最近克隆的一个新的全长cＤＮＡ,编码蛋白可能参与人铜离子容受系统。 ＪＣＩＴＡ的全区用〗ＣＲ拉示后。克隆到ｐＥＴ２８ａ＋表达载体中,在Ｅ．ｃｏｌｉ中获得了高效表达,表达的ＨＣＵＴＡ重组蛋白细菌总蛋白的２８．３％。ＨＣＵＴＡｃＤＮＡ的ＧｅｎＢａｎｋ接收号为ＡＦ１０６９４３。     

RBM13 cDNA的克隆及其表达谱分析

从人胎脑cDNA文库中克隆到一条长3479bp的cDNA,它含一个长903bp的开放阅读框架,拟编码一个300个氨基酸的蛋白质,其分子质量为35397u,等电点为5．35,与酵母MAK16蛋白的同源性为41％,该编码蛋白有一个双侧核定位信号motif和一个RNA结合motif,将这一新cDNA序列推导的蛋白命名为RNA结合基序蛋白13（RBM13）,基因定名为RBM13,用RBM13基因的cDNA探针进行Northern杂交,检测到3．6kb和2．2kb二种长度的转录本。在心脏,骨骼肌,肾脏和肝脏中有较高表达。     

大鼠4．5S snRNA的cDNA序列对荧光素酶基因表达的影响

利用脂质体转染法将大鼠4．5S snRNA cDNA重组载体转染小鼠肾离体培养细胞,连续培养48h,96h和144h检测荧光酶活性发现,4．5S snRNA cDNA插入表达载体的方向不同对荧光素酶基因表达的影响无差异,重组体转化的细胞中荧光素酶基因表达的活性与对照组相比提高2－2．2倍,4．5S snRNA cDNA对荧光素酶基因表达的影响,随转染细胞培养时间的延长而表达活性迅速下降,其下降速度实验组比对照组要快得多。     

Molecular evolution of scorpion a-toxins----Accelerated substitutions and functional divergence

Scorpion a-toxins are a family of toxic proteins with similar scaffold, but possess divergent pharmacological properties. Analysis of cDNA sequences reveals that the numbers of nucleotide substitutions per site ( K ) for 5' and 3' UTRs are smaller than those per synonymous site ( Ks) for the mature peptide-coding sequences, whereas the numbers of nucleotide substitutions per nonsynonymous site (-Ka) are close to or larger than Ks values for relevant pairs of cDNAs. These results, together with phylogenetic analysis, indicate that scorpion a-toxins have evolved by accelerated substitutions in the mature toxin regions. In addition, the 15 amino acids, absolutely conserved in all the scorpion a-toxins described so far. are mostly located in molecular interior, which may be involved in structural constraints for stabilizing the CSa(3 fold in evolution of these molecules. Four hot spot mutation sites in the molecular surface are found to distribute in the putative functional regions of a-toxins. suggesting that positive Darwinian selection drives the accelerated evolution of scorpion a-toxins. These findings reasonably explain the relationship between three-dimensional structure conservation and functional divergence of scorpion a-toxins and are of important value in guiding us in our engineering experiments to obtain higher affinity ligands to Na+ channels.     

人RNA解旋酶基因家族新成员DVH的克隆与初步功能研究

一条长3446bp 的人类新基因cDNA已从胎脑cDNA文库中被克隆,该cDNA克隆包长2583bp的开放读框,推测编码一条长860个氨基酸残基的蛋白质,预测分子质量为96．8ku,通过同源性比较及profile搜索,预测氨基酸序列显示出DExH-BOX RNA解旋酶基因家族特有的7保守基序及已知RNA解旋酶氨基酸序列的较高同源性,进一步通过蛋白质结构模型同已知RNA解旋酶结构的比较,可以认定其为一条人类RNA解旋酶基因家族新成员,通过基因编码的氨基酸序列中基序II D－E－V－H残基将其命名为DVH（DEVHbox Helicase),生物信息学手段结合DVH基因表达谱分析结果可推定DVH可能在胎儿发育过程中起作用,由于解旋基因家族同人类遗传疾病关系密切,以上结果可指导DVH基因的进一步功能研究并提示该,基因作为疾病侯选基因的可能性。     

Identification and functional analysis of 23 novel box C／D snoRNAs from Oryza sativa

In a cDNA library generated from rice small nuclear RNAs,30box C/D small nucleolar RNAs (snoRNAs) were identiffied through preliminary screen.Except 7 known snoRNAs such as U14,all snoRNAs were identified in rice for the first time experimentally.Among the 23 novel snoRNAs,11 snoRNAs appear rice-specific,6 snoRNAs are unique to plants,the remaining 6 snoRNAs have their counterparts in both Arabidopsis and yeast or mammals according to the conserved antisense sequencs that guide 2‘-O-ribose methylation of rRNA,17 of the 23 novel snoRNAs were predicted to guide 24 2‘‘-O-ribose methylations at the specificsites of rice 5.8S,18S,25S rRNAs,among which 19 methylated sites were determined by primer extension at low dNTP concentrations.The remaining 6 snoRNAs devoid of rRNA antisense elements may represent novel snoRNA species in rice.The results show that constructing a cDNA library from small nuclear RNAs is an effective experimental approach for novel snoRNA is identification.The novel snoRNAs are important in elucidating the genomic organization and expression of plant snoRNA genes and the mechanism through which 2‘‘-O-ribose methylations took place in rRNAs.     

蕨晚期配子体发育相关cDNA的克隆和初步研究

利用差异显示PCR技术比较蕨早、晚期配子体(颈卵器期)在mRNA水平上基因表达的差异. 经Reverse-Northern杂交证实获得了6个和颈卵器发育相关的cDNA,对其克隆并测序. 用BLAST和DNAStar分析了各片段间的序列同源性,发现这些序列与水稻、拟南芥、大肠杆菌和人的一些序列具有同源性.     

五指山小型猪PMSA6基因cDNA克隆及生物信息学分析

为了对海南五指山小型猪蛋白酶体亚基α型6（proteasome subunit alpha type 6,PMSA6）cDNA基因进行克隆和生物信息学分析,笔者以构建的五指山小型猪外周血白细胞cDNA文库为材料,采用菌落PCR方法,克隆得到PMSA6全长cDNA,并向GenBank递交了该序列（登录号：FJ358606）．同时运用生物信息学软件对该基因核苷酸序列进行了分析,并预测了其编码蛋白的理化性质及二级结构等．生物信息学分析表明：该cDNA全长1029bp,5’非翻译区长96bp,3’非翻译区长192bp,含有一个741bp完整的开放阅读框,编码246个氨基酸．该蛋白的分子量为37．680kD,等电点为8．76．进一步比对分析发现,五指山小型猪PMSA6基因的核酸序列及其氨基酸序列与人、牛等哺乳动物具有很高的相似性．本研究成功克隆了海南五指山小型猪P肛SA6基因cDNA,并进行了相关生物信息学分析,为进一步研究PMSA6在动物体内的作用机理奠定了基础．     

植酸酶基因表达与调控机制研究(Ⅱ)──番茄幼苗cDNA文库构建与植酸酶基因筛选

采用异硫氰酸胍法从发芽３～４ｄ的番茄幼苗中提取出总ＲＮＡ,用Ｏｌｉｇｏ（ｄＴ）纤维素亲和层析分离纯化ｍＲＮＡ．以ｍＲＮＡ为模板,Ｏｌｉｇｏ（ｄＴ）为引物用逆转录法合成双链ｃＤＮＡ．将ｃＤＮＡ与ＥｃｏＲＩ接头连接后克隆到表达载体λｇｔ１１的单一ＥｃｏＲＩ位点,经体外包装后感染宿主菌Ｙ１０９０,成功地构建了完整的番茄幼苗ｃＤＮＡ文库．用颜色筛选法筛选重组子,然后用植酸酶抗体做探针进行免疫筛选,得到两个阳性克隆．挑取阳性克隆噬菌斑扩增后纯化ＤＮＡ,经琼脂糖凝胶电泳鉴定,显示确有外源ＤＮＡ存在．该插入片断序列测定正在进行．