用复制酶基因cDNA探针检测表达病毒外壳蛋白基因马铃薯中的PLRV

利用马铃薯卷叶病毒（ＰＬＲＶ）复制酶基因３′端长约６００ｂｐ的ｃＤＮＡ，用缺口平移方法进行３２Ｐ同位素标记，制备成ｃＤＮＡ探针，通过核酸斑点杂交，对提纯的马铃薯卷叶病毒（ＰＬＲＶ）、病毒ＲＮＡ、ＰＬＲＶ感染的马铃薯汁液，表达ＰＬＲＶＣＰ基因的马铃薯叶片及块茎芽进行了检测．结果表明，３２Ｐ标记的复制酶基因ｃＤＮＡ探针特异性强、灵敏度高．可测得提纯病毒的最低含量为４０８ｎｇ／ｍｌ，病毒ＲＮＡ最低量为２７．８ｐｇ／ｍｌ，未转ＣＰ基因接种ＰＬＲＶ的马铃薯叶片提取液的最高稀释度为１３７５．接种ＰＬＲＶ的转ＣＰ基因的抗性马铃薯块茎芽和叶片提取液的最高稀释度为０～１／１５．此探针和只转入病毒外壳蛋白基因，不接种ＰＬＲＶ的转基因马铃薯汁液不发生反应．表明，探针只与ＰＬＲＶ基因组ＲＮＡ特异反应，而不与外壳蛋白基因及其ｍＲＮＡ反应，从而为表达ＣＰ基因的转基因马铃薯中ＰＬＲＶ的检测和抗病性鉴定建立了一种可靠的灵敏的分子生物学技术．此项技术已用于转ＣＰ基因马铃薯Ｄｅｓｉｒｅ，Ｆａｖｏｒｉｔａ，乌盟６０１和虎头的抗病性鉴定     

Monitoring gene expression by cDNA array

A new method designated cDNA array was developed by hybridization of quantitatively arrayed DNA samples isolated randomly from a cDNA library with probes reverse-transcribed from mRNAs of different sources or treatments. The gene expression patterns of 1 000 randomly chosen clones from an Arabidopsis library were analyzed with green seedlings versus suspension cells and seedlings irradiated under UV light. Northern blot and sequence analysis of some differentially expressed clones confirmed the results revealed by cDNA array, indicating that this method is efficient and reliable to monitor gene expression.     

Cloning and characterization of vernalization-related gene (ver203F) cDNA 3' end

Based on the cDNA fragment sequence of vernalization-related gene verc203 cloned by differential screening in our lab, the 5' primer has been designed. The cDNA 3' end of ver203 gene (1 197 bp) has been cloned by the RACE method. And it is identified by Northern blotting that its expression is special in vernalization treatment. After comparing the sequence in the nucleotide sequence databases of Genbank, EMBL and DDBJ, the gene has homology with Hordeum vulgare jesmonate-induced protein gene. It is suggested that this gene might be related to the signal transduction mediated by jamonate.     

人锰超氧化物歧化酶cDNA基因的克隆

利用人胚胎肝组织提取总ＲＮＡ,从总ＲＮＡ 中分离纯化出ｍ ＲＮＡ,经逆转录得到ｃＤＮＡ 第一条链,并以之为模板和相应寡聚脱氧核苷酸为引物,进行ＰＣＲ 合成人锰超氧化物歧化酶ｃＤＮＡ 基因,将所得ｃＤＮＡ 克隆到质粒ｐＢｌｕｅｓｃｒｉｐｔＳＫ 上,转化大肠杆菌ＤＨ５α细胞,进行诱导表达筛选,将筛选的克隆进行ｃＤＮＡ 全序列测定     

与大豆叶片衰老相关的cDNA的克隆

植物叶片衰老是一个受遗传控制的细胞降解过程,最终导致叶片的死亡。一些研究结果已表明,蛋白酶基因的表达与叶片衰老过程相关,其中一些基因的表达具有衰老特异性。以半胱氨酸蛋白基因特异性引物和寡聚ｄＴ为引物,用ＲＴ－ＰＣＲ方法分别扩增来源于绿叶和诱导衰老叶片ｍＲＮＡ的互补ＤＮＡ（ｃＤＮＡ）,然后进行琼脂糖凝胶电泳,差异显示出一条衰老叶片样品所特有的ｃＤＮＡ带,将此ｃＤＮＡ进行了克隆和序列分析。分析结果证明     

溴氰菊酯胁迫下小菜蛾差异表达基因的筛选与分析

通过双向cDNA RDA,即分别以敏感品系为检测子(tester)、抗溴氰菊酯品系为驱赶子(driver)和以抗溴氰菊酯品系为检测子(tester)、敏感品系为驱赶子(driver)筛选2种品系中的差异表达基因,初步分析小菜蛾敏感品系和抗性品系之间的遗传差异.经过3轮消减杂交后,将所得的差异片段克隆于PMD 18-T载体,氨苄筛选阳性克隆,经菌落PCR鉴定后送样测序.得到2条序列与GenBank数据库中的部分已知序列有一定同源性,其中1条序列与编码S3a蛋白基因有较高的同源性.     

云南疣粒野生稻部分cDNA片段的分离和注释

从疣粒野生稻cDNA扩增文库中随机挑取500个噬菌斑,通过载体环化,选择120个菌样进行测序,获得的95条序列,分别采用Blast、ORFfinder、UniGene和EntrezGene系统等软件进行序列分析,结果为:与栽培稻日本晴序列比较匹配碱基数>400 bp的占27.37%;确定可阅读框(>100 bp,具有起始和终止密码子)的占90%;与拟南芥的功能基因比较,相似性大于60%的有46个;确定功能、代谢过程和编码蛋白部位的cDNA片段分别有34个、31个和31个.     

Cloning, Characterization, and Expression Analysis of Calreticulin from Pearl Oyster Pinctada fucata

Calreticulin is a unique calcium-binding protein with multiple functions mostly located in the sar-coplasmic/endoplasmic reticulum.A large amount of calcium is absorbed from the medium and transported to mineralization sites during biomineralization in pead oyster.This paper describes the cloning of the full-length cDNA of calreticulin from Pinctada fucata,namely PCRT.PCRT encodes a deduced 414-amino acid protein,which includes a predicted 17- amino acid signal peptide and an endoplasmic reticulum retrieval sequence HDEL.The protein shows 63%-76% sequence identity and shares some common characteristics with calreticulins from other species.Semi-quantitative RT-PCR indicates that PCRT is ubiquitously ex-pressed in all tissues tested with the highest expression in the hemolymph and the mantle.In situ hybridiza-tion analysis of PCRT in the mantle showed strong signals in the inner fold,the inner side of middle fold,and the inner side of outer fold of the mantle epithelium.All these results suggest PCRT might be involved in Ca2+ transport and storage during oyster biomineralization.