霍山石斛HMGR基因的克隆及其定量表达分析

获得甲羟戊酸生物合成途径中的关键酶基因——3-羟基-3-甲基戊二酰辅酶A还原酶基因的信息是确定该基因与倍半萜类石斛碱含量相关性的重要基础工作。本研究采用RT-PCR结合RACE技术,以霍山石斛原球茎为材料,克隆得到霍山石斛HMGR基因的全长c DNA序列,其在Gen Bank中的登录号为KF011508。HMGR基因全长2 240 bp,包含144 bp的5′-UTR,407 bp的3′-UTR(含poly A),开放阅读框为1 689 bp,共编码562个氨基酸。生物信息学分析表明:该蛋白可能是定位于细胞质的一种疏水性不稳定蛋白,无信号肽,不含卷曲螺旋结构,具有14个功能位点、27个磷酸化位点,其二级结构由α螺旋、β折叠和无规则卷曲组成。系统进化树分析表明,该基因与黄姜HMGR基因亲缘关系最近,聚为同一类,主要负责催化萜类化合物生物合成途径中的辅酶A和甲羟戊酸的生成。q PCR结果显示:霍山石斛HMGR基因在茎中的表达量最高,而根中的表达量最低。     

马银花愈伤组织RoWUS基因及其启动子的克隆与表达分析

WUSCHEL(WUS)基因是植物干细胞的标志基因。采用同源克隆法结合RACE技术从马银花愈伤组织中克隆得到Ro WUS c DNA全长序列,采用染色体步移法克隆得到该基因的启动子序列,登录号分别为KF365488、KF861578。马银花Ro WUS c DNA全长1 123 bp,编码302个氨基酸;DNA序列2 001 bp,包含2个内含子和3个外显子;启动子序列3 122 bp。生物信息学分析表明:Ro WUS亚细胞定位于细胞核,是不稳定的亲水蛋白,无信号肽,具有跨膜结构,包含homeodomain功能位点。系统进化树分析结果表明:Ro WUS单独形成一个分支,与葡萄、大豆和苜蓿的亲缘关系最近。对Ro WUS的启动子进行分析表明,该启动子除了含有丰富的TATA-box和CAAT-box等基本元件以外,还含有多个光响应元件、逆境胁迫响应元件、激素应答元件和其他功能元件。q PCR结果表明:Ro WUS基因在马银花愈伤组织不同生长时期中呈先上升后下降的趋势,在第5个继代周期时表达量最高。外源赤霉素和脱落酸浓度为15 mg/L时表达量最高,说明Ro WUS基因在该浓度时对赤霉素和脱落酸的响应最强。     

猪胚胎干细胞的分离克隆

本文从分化抑制物、培养基的选择内细胞团的分离,胚胎干细胞的分离及鉴定方法等方面系统地综述了猪胚胎干细胞分离克隆的研究进展;并对其未来的应用前景进行展望。     

伊氏锥虫HGPRT基因cDNA的克隆及其在E.coli中的表达

根据引物设计的一般原则，参照伊氏锥虫HGPRT基因的核苷酸序列设计、合成一对引物，PCR扩增伊氏锥虫HGPRT基因cDNA。低熔点琼脂糖回收：PCR产物，并将其克隆至pGEM—T Easy载体中，经酶切、PCR鉴定和序列分析，获得重组中间质粒pGEM／HGPRT。重组中间质粒pGEM／HGPRT经Nco I和Sal I双酶切，回收目的片段HGPRT，以非融合形式插入原核表达质粒pBV220构建表达质粒pBV／HGPRT，转化大肠杆菌DH5a，42℃诱导表达，聚丙烯酰胺凝胶电泳和薄层扫描分析，表达产物HGPRT的分子量约为23kD，与理论推算值相符，表达率占总菌体蛋白的19％，经间接ELISA检测，表达产物能被伊氏锥虫阳性血清所识别。     

Cloning and Characterization of a Family of Disease Resistance Gene Analogs from 6VS of Haynaldia villosa

In the present study, microdissection of 6VS and the cloning of the resistance gene analogs (RGA) from them were reported. The 6VS were microdissected with needle and 10 types of resistance gene analogs were obtained by PCR with degenerate oligonucleotide primer designed according to resistance genes. They were designated as Hvrgak1-Hvrgak10, GenBank accession numbers are AF387113-AF387121,AY040671- AY040672. Identity among RGAs was about 10-50%, and identity with cloned R gene from plants was 5-20%. Southern hybridization analysis results showed 3 RGAs, Hvrgak2, Hvrgak4, and Hvrgak5 were linked with wheat powdery mildew resistance. These RGAs may be used as direct entrance or probes for cloning the disease resistance genes.     

甘蓝型油菜oleosin基因片段的克隆及序列分析

以油菜(Brassica napus)基因组DNA为模板,通过设计一对特异性引物,利用多聚酶链式反应(PCR)扩增获得oleosin基因片段。将其克隆到E.coli质粒上进行序列分析。结果表明,该基因由878个碱基组成,含一个内含子,编码193个氨基酸。不计附加的18bp凝血酶切割序列,与已报道的序列相比较,核苷酸序列及推导出的氨基酸序列的同源性分别为79.20%和91.21%。推测的氨基酸序列构成的蛋白质具有oleosin的基本结构特点,包含3个结构区域:两性N末端区(anamphipathicN-terminalre鄄gion),中心疏水区(acentralhydrophobicdomain),两性C-末端区(anamphipathicC-terminaldomain)。     

黄伞菌株中三磷酸甘油醛脱氢酶基因片段的分离鉴定与保守性验证

本研究以黄伞菌丝cDNA为模板，设计简并引物，经PCR扩增出两条片段SLI和S12，经过回收、重扩增、克隆与鉴定后进行测序分析，结果发现SI．1的序列在Genebank中与三磷酸甘油醛脱氢酶（Glyceraldehyde-3-Dhosphate dehydrogease，GPD）基因的同源性达到85％，根据SLI序列设计引物，以黄伞菌丝体、原基、菇蕾、子实体菌柄、子实体菌盖的cDNA为模板进行PCR反应以验证其保守性，结果显示此片段在不同分化发育阶段表达量相同。     

The role of genetic engineering in livestock production

In this paper we discuss the use of genetic engineering in livestock production. We examine the main two different aspects of genetic engineering: cloning and transgenesis. After commenting what has been expected from both techniques in livestock production in the last 25 years, the practical difficulties for implementing cloning and transgenesis are examined. Apart from technical difficulties, problems derived from the detection of genetically superior animals and evaluation of the clones and the transgenic animals make these techniques less interesting than they appear to be. Most of the observed variability of the economically interesting traits is not genetic, genetic evaluation needs a large number of animals and cloning success will represent a serious loss of genetic variability and the loss of the flexibility needed for markets in constant evolution. There is a risk in transgenic animals of production of new intermediate biochemical products that may be toxic, allergenic or carcinogenic. The benefits produced by transgenic animals hitherto hardly justify this risk. The expectations that genetic engineering produced 25 years ago should be re-examined, considering the risks and the high investment required.     

家蚕G蛋白γ1亚基(BmGγ1)的克隆及其谷胱甘肽硫转移酶融合蛋白的表达与纯化

异三元G蛋白是真核细胞感知外界信号后将信号传递到胞内的重要分子,参与生物体广泛的信号转导。为了研究家蚕体内G蛋白的生理功能及其作用机制,运用生物信息学方法预测了家蚕G蛋白γ1亚基(Gγ1)的序列,设计引物验证预测序列后,克隆了家蚕Gγ1的序列,再通过酶切克隆至表达载体pET-41b(+)后,导入E.coliBL21宿主菌中,经异丙基β-D-硫代半乳糖苷(IPTG)诱导表达重组谷胱甘肽硫转移酶(glutathione s-transferase,GST)融合蛋白,并亲和层析纯化表达产物。家蚕Gγ1重组GST融合蛋白经SDS-PAGE电泳和Western blot分析,在分子质量约36 kD处出现特异性蛋白条带,重组蛋白经GST亲和层析柱纯化后,得到了高纯度的融合蛋白,说明已经成功克隆到家蚕Gγ1基因,并在E.coliBL21中高效表达。