一种新型双向启动子--棉花曲叶病毒启动子的分离、序列分析与功能初探

以棉花曲叶病毒(CLCuV)侵染的烟草叶片组织总DNA为模板,通过聚合酶链反应扩增CLCuV双向启动子片段并插入克隆载体.序列分析和同源性比较表明,克隆的启动子长436bp,与目前发现的9种CLCuV株系的启动子序列均不相同,同源性最高达99.32%.将启动子片段分别以不同方向与GUS报告基因和nos终止子融合,构建了瞬时表达载体.通过基因枪法将质粒载体导入烟草和棉花叶片细胞中进行瞬时表达,结果表明,互补链基因方向启动子属强启动子,在叶肉及维管组织均有较高的活性;病毒链基因方向启动子表达活性较低.本文初步证实分离的互补链基因启动子可作为新型强启动子应用于双子叶植物尤其棉花的遗传转化.     

部分缺失的棉花曲叶病毒互补链基因启动子具有超强活性

棉花曲叶病毒（CLCuV)互补链基因启动子是一种新近鉴定的启动子,它能驱动外源基因在植物体内高效表达,为了研究其最佳启动子区域,对启动子5端进行了一系列缺失,得到5种不同长度的启动子片段与GUS基因融合的植物表达载体,继而导入根癌农杆菌,采用叶盘法转化烟草,并检测转基因植株的GUS活性,实验结果表明,自启动子5端缺失至翻译起始位点上游－287,－271时启动子活性分别是全长启动子的5倍,3倍。首次对棉花曲叶病毒互补链基因启动子的功能区进行了分析比较,发现缺失负调控元件的启动子比全长启动子具有更强的活性,平均活性是CaMV35S启动子的12倍,暗示该启动子具有应用潜力。     

一种新型双向启动子——棉花曲叶病毒启动子的分离,序列分析与功？…

以棉花曲叶病毒（ＣＬＣｕＶ）侵染的烟草叶片组织ＤＮＡ为模板,通过聚合酶链反应扩增ＣＬＣｕＶ双向启动子片段并子片段并插入克隆载体。序列分析和同源性比较表明,克隆的启动子长４３６ｂｐ,与目前发现的９种ＣＬＣｕＶ株系的启动子序列均不相同,同源性最高达９９．３２％。将启动子片段分别以不同方向与ＧＵＳ报告基因和ｎｏｓ终止子融合,构建了瞬时表达载体。通过基因枪法将质粒载体导入烟草和棉花叶片细胞中进行瞬时表达。     

心肌组织特异性表达质粒的构建及表达

应用ＰＣＲ方法从大鼠基因组ＤＮＡ中扩增出心肌组织特异性顺式作用元件－－心肌肌钙蛋白Ｃ（Ｃａｒｄｉａｃ Ｔｒｏｐｏｎｉｎ Ｃ，ｃＴｎＣ）增强子／启动子序列，构建成ｃＴｎＣ启动子驱动ＬａｃＺ基因的真核表达质粒ｐｃＴｎＣβ。Ｉｎ Ｖｉｔｒｏ和Ｉｎ Ｖｉｖｏ试验证明该调控元件能驱动外源性基因在心肌细胞中特异性表达。     

乙肝病毒融合表面抗原SA—28基因在酵母中的表达

利用基因工程技术，先将乙肝病毒表面抗原Ｓ－ｐｒｅＳ１融合基因ＳＡ－２８置于酵母杂合启动子ＡＤＨ２－ＳＵＣ２的控制下，然后将ＳＡ－２８基因的表达单元插入高稳定质粒ｐＨＣｌ１的ＢａｍＨＩ位点，构建成表达质粒ＹＦＤ１５０和ＹＦＤ１５０－ｏ并将其转化酿酒酵母Ｙ１９。对转化子表达ＳＡ－２８基因的研究表明：表达受葡萄糖浓度调控；表达产物具有Ｓ和ｐｒｅＳ１双重抗原性，并形成密度为１．２０－１．２２ｇ／ｍｌ的颗粒     

抗CD20嵌合抗体Fab'片段在大肠杆菌中高效表达

利用ＰＣＲ方法从抗ＣＤ２０ＳｃＦｖ表达载体上扩增重链可变区、轻链可变区基因,然后将ＶＨ、ＶＬ基因重组到Ｆａｂ’表达载体中,构建成抗ＣＤ２０嵌合抗体Ｆａｂ’片段表达载体ｐＹＺＦｃｄ２０,用ｐＹＺＦｃｄ２０转化大肠杆菌１６Ｃ９,在１６Ｃ９菌中分泌表达可溶性抗ＣＤ２０Ｆａｂ’片段,经分离纯化获得具有ＣＤ２０抗原特异结合活性的Ｆａｂ’片段,竞争性竞争荧光抑制实验表明,抗ＣＤ２０Ｆａｂ’片段竞争性抑亲本属源     

牛αsl乳酪蛋白基因上游调控序列的克隆及序列分析

应用ＰＣＲ技术从国内小牛胸腺基因组ＤＮＡ中克隆了１．０ｋｂ牛αｓｌ酪蛋白基因的上游调控序列，并进行了序列分析，发现有少量的缺失或突变，其ＴＡＴＡ框和ＣＡＡＴ框均未发生变异，表明已成功克隆了其启动子序列，这为乳腺定位表达的研究奠定了基础。     

牛as1乳酪蛋白基因上游调控序列的克隆及序列分析

应用ＰＣＲ技术从国内小牛胸腺基因组ＤＮＡ中克隆了１．０ｋｂ牛ａｓ１酪蛋白基因的上游调控序列，并进行了序列分析，发现有少量的缺失或突变，其ＴＡＴＡ框和ＣＡＡＴ框均未发生变异，表明已成功克隆了其启动子序列，这为乳腺定位表达的研究奠定了基础。     

酵母人工染色体载体的改造和修饰

将能在植物中表达的烟草花叶病毒ＣａＭＶ３５Ｓ启动子、ＧＵＳ基因、潮霉素（Ｈｙｇｒｏｍｙｃｉｎ）基因及ＭＯＳ终止子组建到酵母人工染色体（ＹＡＣ）载体，构建了具有能在植物细胞中表达和选择ｐＹＡＶ、ＧＵＳ、ｐＪＳ９７／ｐＪＳ９８－ＨＹ ＹＡＣ载体系体系统和一个标记ＹＡＣ克隆的ｐＵＲ４３－ＨＹ质粒。     

Certain Biominerals in Leaves Function as Light Scatterers

Cystoliths are amorphous calcium carbonate bodies that form in the leaves of some plant families. Cystoliths are regularly distributed in the epidermis and protrude into the photosynthetic tissue, the mesophyll. The photosynthetic pigments generate a steep light gradient in the leaf. Under most illumination regimes the outer mesophyll is light saturated, thus the photosynthetic apparatus is kinetically unable to use the excess light for photochemistry. Here we use micro‐scale modulated fluorometry to demonstrate that light scattered by the cystoliths is distributed from the photosynthetically inefficient upper tissue to the efficient, but light deprived, lower tissue. The results prove that the presence of light scatterers reduces the steep light gradient, thus enabling the leaf to use the incoming light flux more efficiently. MicroCT and electron microscopy confirm that the spatial distribution of the minerals is compatible with their optical function. During the study we encountered large calcium oxalate druses in the same anatomical location as the cystoliths. These druses proved to have similar light scattering functions as the cystoliths. This study shows that certain minerals in the leaves of different plants distribute the light flux more evenly inside the leaf.     

Isolation of a pollen specific promoter in tritordeum

The promoter is an important cis-acting element in regulating gene expression. Tissue specific promoters play a key role in genetic engineering. A promoterless plasmid containing UidA gene was transformed into tritordeum by barmbadment. Histochemical analysis of various tissues in transgenic tritordeum was carried to examine tissue-specific expression of GUS activity. The pollen-specific promoter was trapped and identified successfully in a transformant line. PCR method was used to isolate this pollen-specific promoter with DNA extracted from leaves as templates, using primer P1 of rice pollen specific promoter as forward primer, a fragment of UidA gene as the reverse primer P2. By sequencing and analyzing the amplified 1052bp DNA fragment from PCR reaction, a part of UidA gene and a flanking sequence were obtained. Some essential elements of plant promoters were found in the sequence. To determine the function of this promoter, the cloned promoter fragment was fused with uidA gene, then cloned into a plasmid and transformed into Triticum durum. The transgenic plant transformed by this vector showed GUS expression only in pollen. Therefore a pollen-specific promoter was trapped and isolated successfully, and it could be used in gene regulation study and genetic engineering.     

一种新的钙依赖性的蛋白激酶基因CP4的克隆及其表达研究

在利用人ACA(Anti-Centromere/Kinetochore Autoantibody)血清研究拟南芥ACA相关蛋白的过程中克隆了钙依赖性的蛋白激酶基因家族的一个不典型基因，称为CP4，GenBank注册号为AF130252。Southern杂交结果表明，拟南芥基因组中至少存在两种以上等位形式的CP4。原位杂交结果表明，CP4 mRNA在拟南芥的花和根中高表达，在果实和叶片中没有表达。此外还构建了CP4的高效表达载体，为进一步研究CP4的功能打下了基础。     

Release of diltiazem hydrochloride from hydrophilic matrices of polyethylene oxide and carbopol

The mucoadhesion, swelling, and drug release behavior of polyethylene oxide (PEO) and carbopol (CP) matrices were studied using a water soluble model drug diltiazem hydrochloride. The mucoadhesive strength of the matrices increased with increase in polymer content. The results showed that PEO was more mucoadhesive than CP. Mucoadhesion of the tablets was dependent upon the swelling. Swelling was ascertained by measuring the axial and radial expansion of matrix tablets following exposure to media of physiological ionic strength. There was a marked increase in the swelling index of matrices containing high polymer content of PEO as compared to CP. Drug release kinetics were found to be closely related to dissolution and swelling properties of the matrices. The release was found to be non-fickian with n (release exponent) values ranging from 0.45-0.58. At a constant polymer content (15.84% w/w), the main contributing factor for the mucoadhesion, swelling, and release was the amount of PEO.     

炭纸上原位生长CNF/CP复合体的氧气电催化反应性能

利用催化气相化学沉积(Catalytic chemical vapor deposition,CCVD)法在炭纸上原位生长得到CNF/CP复合体,并对这种复合体的物理化学性能和氧气电催化还原反应(Oxygen reduction reaction,ORR)性能进行了研究.结果表明:纳米炭纤维较为均匀地分散在炭纸上,其中纳米炭纤维具有窄的直径分布.所制CNF/CP复合体具有较大的比表面积和独特的中孔结构;相对于炭纸,CNF/CP复合体的端面碳原子和基面碳原子比例较高.另外,CNF/CP还具有较高的ORR反应活性,其ORR为2电子反应过程,原因可以归结于纳米炭纤维独特的微结构.同时,CNF/CP也具有较高的交换电流密度和较正的平衡电压.     

转抗菌肽B基因水稻植株的获得与鉴定

构建了一个适合在水稻中表达含有抗菌肽Ｂ基因的转化载体，应用基因枪转化法将其导入水稻未成熟胚，获得了一些转基因水稻植株。根据对选择标记基因和目的基因的分子检测和抗病性测定，证明抗菌肽Ｂ基因已整合入转化水稻基因组并在转基因水稻中表达了抗病性。     

人凝血因子IX在脆壁克鲁维酵母中的表达与分泌

通过改造人凝血因子IX基因(h-FIX)使其与乳酸克鲁维酵母(Kluyveromyces lactis)的分泌信号序列(Kss)融合，并置于脆壁克鲁维酵母(K．fragilis)LAC4基因调控型启动子的控制之下，构建成h-FIX基因表达盒。将此表达盒插入克鲁维酵母的整合质粒，得到了一个高稳定的h-FIX基因整合表达载体pHKB202-FP。经摇瓶培养并以半乳糖诱导此表达载体转化的酵母菌株，ELISA检测显示酵母上清液中的人凝血因子IX表达量可达到约200ng/ml。此结果表明人凝血因子IX蛋白首次成功地在脆壁克鲁维酵母中得到了表达和分泌。这对于促进人凝血因子IX的基因工程产业化具有实际意义。