Isolation of a vernalization-related cDNA clone (VRC) using mRNA differential display in winter wheat

Vernalization is an essential factor which affects the flowering development in cold-requiring plants. There is a key stage of nucleic acid and protein metabolism in the process of vernalization in winter wheat. To probe into the molecular determinants of vernalization, we examined mRNA populations in differently-treated plumules of winter wheat (Triticum aestivum L. cv Yanda 1817) using mRNA differential display. One vernalizationrelated cDNA clone (VRC), VRC54, was identified and was only expressed at the key stage of 20 d vernalization, rather than at other stages of nonvernalization, 4 d vernalization and devernalization. Northern blot and sequence analysis indicated that VRC54 was a novel vernalization-related clone found in higher plant which not only might play an important role in the floral induction in vernalization-requiring plants but also was different from the cold-acclimatized genes.     

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

Based on the cDNA fragment sequence of vernalization-related geneverc203 cloned by differential screening in our lab, the 5′ primer has been designed. The cDNA 3′ end ofver203 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 withHordeum vulgare jesmonate-induced protein gene. It is suggested that this gene might be related to the signal transduction mediated by jamonate.     

Cloning and sequencing of cDNA fragment of gene of wheat (Triticum aestivum. L) induced by dehydration

cDNA fragment of the gene (dehydration induced,di1) of wheat (Triticum aestivum. L) induced by 30% PEG-6000 (−1.13 MPa) treatment was isolated with mRNA differential display technique. Northern blot analysis showed that the expression ofdi1 gene improved at 10 h reached the highest at 48 h under 30% PEG-6000 treatment. cDNA fragment ofdi1 gene has been cloned and sequenced (211 bp). DNA sequence analysis shows that there is no homologue in GenBank todi1 cDNA.     

中华绒螯蟹卵巢新基因EJO6的全长cDNA克隆和序列分析

利用RACE技术从中华绒螫蟹卵巢获得了新基因EJO6(Eriocheir japonica ovary gene 6,EJO6)的全长cDNA序列(GenBarnk 检索号：AYl85922)。该cDNA序列长度为1250bp,开放阅读框为690bp,编码229个氨基酸。根据氨基酸序列计算的相对分子质量和等电点分别为24270和11．9。同时用生物信息学的方法对该基因的结构和功能进行了初步分析。     

人类Neuritin cDNA的克隆和表达

从人胎脑cDNA文库筛选出一条1618bp的cDNA。此cDNA含有一个426bp的最大开放阅读框,编码一个142个氨基酸的蛋白质,预测分子质量为15.3ku。与目前数据库中序列比较,该cDNA与鼠neuritin基因同源性达98％。多组织Northern blot分析显示neuritin在脑组织高度表达。neuritin cDNA的读框片段正确插入到pQE40表达载体中,获得了预期的表达产物,并初步得到了其纯化蛋白。     

中华绒螯蟹卵巢新基因EJO5的全长cDNA克隆和序列分析

利用RACE技术从中华绒螯蟹卵巢获得了新基因EJO5 (Eriocheirjaponicaovarygene 5 ,EJO5 )的全长cD NA序列 (GenBank检索号 :AY185 92 1) .该cDNA序列长度为 14 2 5bp ,开放阅读框为 5 85bp ,编码 194个氨基酸 .根据氨基酸序列计算的相对分子质量和等电点分别为 2 2 3 44和 9 5 .用生物信息学的方法未能得到对该基因功能预测的信息 .     

人胎盘G-蛋白Rab3a cDNA的克隆

为了获取全长的小分子G-蛋白Rab3a,以用于研究Rab3a与其他蛋白相互作用关系,本实验以人胎盘总cDNA为模板,PCR扩增到人Rab3a cDNA全编码区。产物回收后克隆于质粒pYESTrp2的BamHI/XhoI位点,测序结果表明,本实验获得的Rab3a cDNA包含了起始和终止密码子。与PCR引物设计的参照Rab3a比较有5个核苷酸变异,与翻译的氨基酸序列完全一致。由此表明本实验获得的Rab3a cDNA可用于进一步研究。     

Cloning and expression analysis of MBLL cDNA

The mbl (muscleblind) gene of Drosophila encodes a nuclear protein which contains two Cys3His motifs. The mutation of mbl gene will disturb the differentiation of all the Drosophila's photoreceptors. Primers have been designed according to human EST086139, which is highly homologous to mbl gene. Human fetal brain cDNA library has been screened and a novel cDNA clone has been obtained. The 2595 bp cDNA, designated MBLL (muscleblind-like), contains an open reading frame which encodes 255 amino acids and has 4 Cys3His motifs (GenBank Acc. AF061261). The amino acids sequence shares high homology to Drosophila's mbl. The Northern blot and RNA dot blot hybridization of 43 human adult tissues and 7 fetal tissues show that MBLL is a widely expressed gene, but the expression amounts differ in these tissues.     

Cloning and characterization of rice RH3 gene induced by brown planthopper

Histones are basic low molecular weight proteins found in all eukaryotic genomes. The histones include five classes of basic proteins (H1, H2A, H2B, H3 and H4) that interact with each other and nuclear DNA to form the nucleosome. The H3 and H4 histone proteins are highly conserved and form the central tetrameric block of the core-nucleosome. Histone H3 has several post-transcrip- tional modifications such as methylation, acetylation, phosphonation, and ADP-ribosylation and it plays impor…     

应用3’RACE技术扩增仿刺参C型凝集素基因及实验条件的优化

从GenBank上调取刺参C型凝集素（C-type Lectin）基因序列EST,根据此序列设计RACE引物,采用PCR扩增技术得到了仿刺参C-type Lectin基因序列（EST）,根据这段EST序列设计1个基因特异引物（GSPF）,与通用引物（UPM）扩增,成功地克隆到了该基因的3’末端序列.同时,对仿刺参C型凝集素基因3’克隆的实验条件进行了优化.该扩增片段长度为670bp,与已知序列重叠部分为417bp.经测序和比对发现该段序列与预期的目标基因的序列一致.     

辣椒抗菌蛋白cDNA的分离与表达的初步分析

本研究从经辣椒疫霉侵染处理的辣椒叶片cDNA文库中分离获得了一个辣椒抗菌蛋白cDNA阳性克隆,其长度为255bp,含有长度为85个氨基酸的开放读码框架,与其它植物抗菌蛋白或几丁质酶有不同程度的同源性,推测为辣椒抗菌蛋白,命名为CansLTPS.cDNA-AFLP分析表明,CansLTPS的转录受UV-B照射和辣椒疫霉侵染的诱导,外源脱落酸(ABA)、乙烯(ETH)、水杨酸(SA)等可不同程度的诱导CansLTPS基因的转录,而MeJA处理对CansLTPS基因的转录表达影响不大,表明CansLTPS基因可应答生物和非生物逆境胁迫,其上游可能涉及ABA、ETH、SA等介入的信号传递途径.     

黄蜀葵查尔酮合成酶基因AmCHS克隆及序列分析

从黄蜀葵(Abelmoschus manihot) 花瓣中通过简并引物克隆得到查尔酮合成酶基因cDNA 核心序列,根据核心序列设计特异引物,再应用5′RACE 和3′RACE 技术分别扩增该序列的5′末端和3′末端序列,最后通过序列拼接获得黄蜀葵查尔酮合成酶基因（AmCHS）cDNA全序列.序列分析结果表明AmCHS cDNA编码区长1170 bp,编码389个氨基酸.其氨基酸序列与其它已知高等植物CHS基因具有很高的同源性,并且包含了CHS具有的活性位点和催化位点等保守性位点.     

人类RING型锌指蛋白DTX2的全长cDNA克隆与序列分析

鉴定了一个人类WWE与RING型锌指基因DTX2,位于人染色体7q11.23. 通过RACE获得的全长cDNA显示,DTX2编码包含两个WWE区域与一个RING锌指区域的蛋白.     

人EC-SOD的克隆及高效表达

将编码人胞外超氧化物歧化酶（EC-SOD）成熟肽的cDNA插入含T7启动子的质粒pET-28a( )中构建表达质粒pET-EC-SOD，表达菌株用1mmol/L异丙基硫代-β-D半乳糖苷（IPTG）诱导表达3h-5h后，产生较多的重组人EC-SOD，并形成包含体。SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析表明，表达的重组蛋白占菌体可溶性蛋白质的26%以上。经纯化和复性后的EC-SOD比活为每毫克纯化酶蛋白1200U。将EC-SOD转染粉纹夜蛾Tn-5Bl-4细胞，经扩增后在细胞内进行表达。SOS-PAGE分析结果表明，粉纹夜蛾细胞中表达一相对分子质量约为28ku的特异蛋白质带，Western blot分析表明，该特异条带即为EC-SOD蛋白，连苯三酚自氧化法测得表达产物比活为每毫克细胞裂解物260U。     

结核分枝杆菌H37Ra FadD3基因克隆与表达研究

为探索FadD3在结核分枝杆菌胆固醇分解代谢中的作用,从结核分枝杆菌H37Ra的全基因组中克隆了FadD3基因,并在大肠杆菌BL21中表达.根据NCBI公布的结核分枝杆菌全基因组序列设计一对引物,PCR扩增FadD3基因.PCR扩增产物与克隆载体pGEM3Zf(+)进行拼接,得到重组基因FadD3-pGEM3Zf(+),再转化到大肠杆菌DH5ɑ得到克隆.PCR检测阳性克隆,再与表达载体pYUB28b拼接,得到重组质粒FadD3-pYUB28b.阳性重组质粒亚克隆到大肠杆菌BL21宿主菌进行自体诱导表达.经过表型筛选及鉴定分析,已成功构建了重组表达质粒FadD3-pYUB28b.SDS-PAGE和Western blotting证实,重组基因FadD3-pYUB28b在大肠杆菌BL21中有表达产物.实验结果表明,以pYUB28b为表达载体,FadD3重组基因在大肠杆菌表达体系于温度分别为18,28 ℃有包涵体形式的表达蛋白,在37 ℃无表达产物.     

AtGT-3b基因克隆及原核表达与纯化

GT-3b转录因子是一个受NaCl和病原体诱导表达的GT-1-like转录因子,它能与GT-1 cis-element( GAAAAA)相互作用,促进下游基因的表达,在植物耐盐中起着重要的调节作用.通过分离了拟南芥(Arabidopsis thaliana)AtGT-3b基因,克隆到原核表达载体pCold TF中,并在大肠杆菌(Escherichia coli)BL21中进行融合表达;通过纯化得到AtGT-3b融合蛋白,以期用于研究其与GT-1顺式作用元件在体外的相互作用.