人NLK上游启动子区的克隆与鉴定

初步鉴定并分析NLK上游启动子,为研究其转录调控打下基础。对NLK基因翻译起始位点上游约1 958 bp的序列分别进行生物信息学分析,以PCR技术扩增以上序列并测序。将PCR所得到的NLK上游片段进一步克隆到PGL-3basic载体中,构建荧光素酶报告基因质粒PGL-3basic-luc。通过荧光素酶报告基因实验检测上述启动子的活性。成功构建了包含NLK基因上游启动子序列的荧光报告系统,经荧光素酶报告基因实验证明该重组质粒体具有转录活性。构建的NLK基因上游启动子报告基因载体为进一步研究NLK基因的转录调控机制奠定了基础。     

人MTERF3基因启动子的克隆及其荧光素酶报告基因载体的构建

人MTERF3基因编码线粒体基因转录和能量代谢的负调控因子。采用PCR技术对人MTERF3基因5'侧翼上游1251 bp启动子序列进行扩增,并将其克隆至荧光素酶表达载体p GL6-TA,构建人MTERF3基因启动子荧光素酶报告基因质粒。经酶切、测序鉴定后,将其用脂质体转染体外培养的HEK293细胞株,利用双荧光素酶测定系统检测其表达活性。研究结果表明,克隆获得的1251 bp DNA序列与Gen Bank报道的一致,且插入方向正确。含人MTERF3基因启动子的报告基因荧光素酶的表达活性显著提高(P0.05),约为对照组(空载体p GL6-TA)的9.8倍。本研究通过对人MTERF3基因启动子的克隆及其荧光素酶表达载体构建与表达活性的测定,为进一步阐明人MTERF3基因表达的调控机制奠定实验基础。     

异源四倍体鲫鲤Sox4基因部分序列及其5′端调控区启动子片段的克隆与分析

通过PCR技术从异源四倍体鲫鲤基因组DNA中分离到Sox4基因部分基序.序列分析表明,其5′端调控区具有多种启动子所特有的序列元件,如TATA框,CAAT框等,据此推测,它具有启动子活性.采用高保真PCR方法克隆了异源四倍体鱼Sox4基因5′端调控区600bp启动子片段,将其插入启动子缺失的增强绿色荧光蛋白(EGFP)表达载体pEGFP-1中,构建重组载体pAtsox4EGFP-1.通过瞬时转染HeLa细胞,在倒置荧光显微镜下可检测到绿色荧光.结果表明,克隆的Atsox4基因5′端调控区具有启动子活性,并成功构建了含四倍体鱼Sox4基因启动子片段的报告基因载体,为以后研究Sox4基因表达调控的分子机制奠定了基础.     

斜纹夜蛾核型多角体病毒日本株(C3)p10基因的克隆及序列分析

从斜纹夜蛾核多角体病毒(Spodoptera litura mukieapsid nucleopolyhedrovirus，Splt MNPV)日本株(C3)基因组中克隆了p10基因．核苷酸序列分析表明，该克隆片断涵盖了318bp的读码框及5’端启动子区191bp和3’端终止区62bp的序列．在起始密码子ATG上游-63～-59bp处有一杆状病毒晚期和晚晚期启动子基序TAAG．联配分析表明，Splt MNPV日本株(C3)的核苷酸序列和氨基酸序列与其它9种核多角体病毒的同源性有较大差异．以p10基因为基础绘制的杆状病毒分子进化树将家蚕核多角体病毒(Bombyx mori nuchopolyhedrovirus，BmNPV)与苜蓿丫纹夜蛾核多角体病毒(Autographa californica multicapcid nucleopolyhedrovirus，AcMNPV)归到同一分枝，这与以gp37基因和egt基因为基础绘制的杆状病毒分子进化树结果一致．     

人MiTERF3基因启动子的克隆及其荧光素酶报告基因载体的构建

人MiTERF3基因编码线粒体基因转录和能量代谢的负调控因子。采用PCR技术对人MiTERF3基因5''侧翼上游1251 bp启动子序列进行扩增,并将其克隆至荧光素酶表达载体pGL6-TA,构建人MiTERF3基因启动子荧光素酶报告基因质粒。经酶切、测序鉴定后,将其用脂质体转染体外培养的HEK293细胞株,利用双荧光素酶测定系统检测其表达活性。研究结果表明,克隆获得的1251 bp DNA序列与GenBank报道的一致,且插入方向正确。含人MiTERF3基因启动子的报告基因荧光素酶的表达活性显著提高（P<0.05）,约为对照组（空载体pGL6-TA）的9.8倍。本研究通过对人MiTERF3基因启动子的克隆及其荧光素酶表达载体构建与表达活性的测定,为进一步阐明人MiTERF3基因表达的调控机制奠定实验基础。     

麻疯树异戊烯基焦磷酸异构酶基因(IPI)启动子的克隆及瞬时表达分析

本文以麻疯树基因组DNA为模板,通过PCR扩增得到麻疯树异戊烯基焦磷酸异构酶(IPI)基因(JcIPI)起始密码子上游1 536bp的启动子序列.利用在线软件PLACE和PlantCARE分析表明该序列除具备TATA-Box、CAAT-Box等启动子基本元件外,还含有AuxRR-core、TATC-box、MBS、HSE等特异性元件.为确定启动子核心启动区域,构建JcIPI启动子283、550、970、1 276和1 536bp的5′端缺失片段,并将其分别驱动pBI121载体的葡萄糖苷酸酶(GUS)基因,构建植物表达载体.采用农杆菌介导法转化烟草,在烟草叶片中进行瞬时表达分析.GUS酶活测定结果显示,五个启动子缺失片段都具有启动子活性,并随长度增加而增强.     

含PGK启动子慢病毒表达质粒的构建与鉴定

为了构建一个含有人源PGK1(human phosphoglycerate kinase 1)启动子的慢病毒表达载体 pL-PGK-GFP.采用PCR从人组织中扩增PGK1基因的启动子部分,再用酶切-连接的方法将扩增的启动子区片段亚克隆入慢病毒表达质粒pL-EGFP中,再用测序、酶切和瞬时表达的方法进行鉴定.结果是下游的eGFP基因在PGK1启动子驱动下,在293FT细胞中表达绿色荧光蛋白报告基因,这表明成功构建了慢病毒表达质粒pL-PGK-GFP.扩增的537bp PGK1启动子片段具有一定的启动效率,能在HIV来源的慢病毒载体中驱动下游目的基因的表达.     

Pinb基因启动子生物信息学分析及载体构建

运用生物信息学预测并分析小麦硬度基因Pinb的启动子,利用无缝克隆构建胚乳特异表达载体.采用生物信息学软件预测Pinb基因5’端上游调控区3056bp序列进行启动子预测与分析,克隆启动子,利用无缝克隆技术构建Pinb基因的胚乳特异表达的重组质粒,重组质粒经测序鉴定构建正确.Pinb基因5’上游-3060bp至-4bp区域内存在启动子活性,在-2419bp至-500bp间启动子活性分值均在0.8以上,其中-550bp至-500bp区启动子片段分值最高,达到0.99.在-2862bp处和-362bp处各有一个转录起始位点.在-544bp处有一个CAAT盒信号,在-541bp处有一个TATA盒信号.在-2362bp至-2256bp、-1704bp至-1559bp和-537bp至-361bp处各有一个CpG岛.取最有可能的757bp(-803bp至-47bp)启动子片段构建重组质粒(LBLV232),质粒经酶切和测序验证正确.最终,成功构建Pinb基因启动子报告基因表达载体,为系统研究该基因启动子活性提供前期基础.     

拟南芥VHA-c3基因的特异性表达和调节

利用RT-PCR技术检测VHA-c基因在拟南芥中的表达，结果表明VHA-c3基因在拟南芥的果荚、花、叶、茎和根中都有表达，但是，在叶中的表达量远远高于其它的组织.以GUS基因作为报告基因构建了不同长度的VHA-c3基因启动子缺失突变体，利用农杆菌介导的瞬时表达系统检测GUS基因的表达，研究发现在VHA-c3基因起始密码子上游2812-2 234 bp之间的区域內存在着控制VHA-c3基因高表达的转录调控元件.     

Pax5启动子的克隆及其在人淋巴瘤细胞系HL-60中转录因子的确定

克隆Pax5基因的启动子,插入荧光素酶报告基因载体中,并检测其活性;采用PCR技术从人淋巴瘤细胞系HL-60基因组中扩增出Pax5启动子,插入荧光素酶报告基因载体pGL3-basic中,确定所扩增的DNA序列,在293T细胞中检测其活性;测序结果表明扩增的Pax5启动子序列正确,活性实验表明构建的报告基因具有启动子活性,转录因子SP1和Runx1能以剂量依赖的方式提高Pax5报告基因的转录活性;克隆了Pax5启动子,并发现转录因子SP1和Runx1能够调控Pax5的转录。     

AcMNPV p95基因一段339 bp序列在病毒复制中的作用

杆状病毒苜蓿银纹夜蛾核型多角体病毒(AcMNPV)的p95基因编码的P95蛋白是病毒核衣壳的组成部分,也是虫体经口感染相关因子PIF(Per os Infectivity Factor)复合体的组成部分.前期研究表明,完整的P95蛋白对病毒的复制是非必需的,其中一段450bp的序列可以部分弥补p95基因的作用.本研究将p95基因一段339bp的片段(Core339,AcMNPV基因组位置:69 576~69 914bp)以正反两个方向分别插入到p95基因敲除的AcMNPV基因组中多角体位点的polyA之后,构建了两株重组病毒vP95K/EGFP/339+和vP95K/EGFP/339-.该两株病毒能在Sf9细胞中正常复制,复制水平与p95基因正常的AcEGFP相当,但复制速度较慢.重组病毒感染细胞的Western blot分析未检测到P95蛋白的表达.这一结果表明P95蛋白的缺失对病毒在细胞中的复制没有显著影响,但核心片段Core339对于病毒复制具有关键作用.     

Kas基因启动子区的克隆及功能初步分析

利用TAIL-PCR技术，克隆到了与辣椒素合成有关的胎座特异表达基因——3-酮酯酰．ACP合成酶基因（Kas）上游400bp的调控区域．将其全长片段与GUS基因连接构建植物表达载体并转化烟草．GUS组织化学染色表明，克隆到的440bp片段具有启动子活性．对该片段进行序列分析发现，在起始密码子ATG上游存在2个TATA-box，分别为-316～-311位的TATAAA和-224～-219位的TATAAA；在TATA-box上游还存在1个位于-378～-374处的CAAT-box，序列为CCAAT．该研究旨在为利用基因调控辣椒素的生物合成，提高辣椒果实中的辣椒素含量奠定基础．     

Promoter activities of genes cpcT2 and cpcS2 in Nostoc PCC 7120

To study the promoter activities of genes cpcT 2 and cpcS 2,several upstream DNA fragments of these two genes with different lengths were selected.These fragments were fused with promoterless gfp(reporter gene) for constructing five recombinant plasmids.Then,these recombinant plasmids were transferred into Nostoc PCC 7120 by conjugation.The promoter activities of genes cpcT 2 and cpcS 2 were determined by observing the fluorescence of green fluorescent protein(GFP) with a fluorescence microscope.The result showed that the 1 300 bp(-1 300 to 0 bp) and 2 600 bp(-2 600 to 0 bp) upstream fragments of cpcT 2 had strong promoter activity and the promoter activity of the 680 bp(-680 to 0 bp) fragment was weaker than that of two above fragments of cpcT 2.The 2 000 bp(-2 000 to 0 bp) upstream fragment of cpcS 2 revealed weak promoter activity.This showed that a strong promoter of cpcT 2 was located in the upstream fragment between-680 and-1 300 bp.     

GCC box in<Emphasis Type="Italic">Arabidopsis PDF1.2</Emphasis> promoter is an essential and sufficient cis-acting element in response to MeJA treatment

The expression of Arabidopsis PDF1.2 gene isregulated by jasmonic acid (JA) and ethylene (ET). It also has been well documented that GCC box is an element responsive to ET, however, the responsive mechanism of JA in such plant defense gene expression is unclear. In this paper, the authors define the essential cis-acting element in PDF1.2 promoter responsive to methyl jasmonate (MeJA) through fragment deletions and site-directed mutageneses combiningAgrobacterium-mediated transient reporter gene expression in tobacco leaves. Firstly, the MeJA inducible expression o fPDF1.2 was confirmed by using the upstream -1.86 kb fragment of PDFI.2 gene. Secondly, the upstream -300— -243 bp fragment of the promoter was evidenced to respond to MeJA. To further characterize this promoter region, three point mutations were introduced into the -300— -243 bp fragment of the promoter. This result showed that the mutation of GCC box abolished MeJA induction, whereas the mutations of the G box-like and the imperfect palindrome sequence did not significantly decrease MeJA inducible effect, indicating that GCC box in PDFI.2 is essential for MeJA induction. The sufficient responsiveness to MeJA of this GCC box was further investigated by 4xGCC fused upstream to the CaMV 35S minimal promoter. This result suggested that the fused promoter was able to activate reporter gene expression in response to MeJA. Thus these results indicate that the GCC box in PDFI.2 is an essential and sufficient element to confer MeJA induction.     

杨树木质部特异性定位表达4CL1启动子的结构与表达特性

4-香豆素COA连接酶(4CL1)是木质素代谢途径中的一个关键酶,对该基因启动子的表达特性与调控元件进行了研究:首先,对毛白杨4CL1启动子进行了生物信息学分析,结果表明该启动子包括3个顺式作用元件,box P(CCTTCACCAACCCCC),box A(CCGTTC),box L(TCTCACCAACC),这3个顺式作用元件在已知的木质素代谢途径相关酶系如苯丙氨合成酶(PAI)和4CL中普遍存在;其次,运用PCR方法对该启动子进行了剪切,获得一个长393 bp的启动子片断,该启动子片断包括以上3个顺式作用元件;最后,将该启动子片段与GUS报告基因构建了植物表达载体并转化烟草,成功获得转基因再生苗,结果发现转基因烟草的茎木质部呈现GUS染色阳性.研究结果表明,一个393 bp长度的4CL1启动子片断足以介导外源基因在木质部特异性定位表达.     

短柄五加(Acanthopanax brachypus)rbcL基因的结构分析

克隆了含完整短柄五加rbcL基因的3.2kb EcoRI片段,测定了该基因的核苷酸序列.所测核苷酸序列总长度为1924bp,其中编码区1428bp,编码475个氨基酸的蛋白质.测定的基因5’上游区共278bp,包含原核性质-35区(TTGCGC),-10区(TACAAT)及类似真核的TATA box元件(TATATA).5’前导区长194bp,其中SD序列为GGAGG,紧邻起始密码子上游.测定的3’下游区共218bp,含2个相邻的转录后可形成茎环结构的反向重复序列.短柄五加rbcL基因编码区推导的氨基酸序列与烟草、菠菜、豌豆、苜蓿、玉米、水稻、松树、地钱、衣藻和Anacystis的同源性分别为93.5％、94.11％、94.53％、94.74％、89.68％、92.21％、92.21％、92.63％、87.58％和80.84％.本文还对不同植物rbcL基因的启动区及部分5’和3’非编码区进行了比较分析.     

Effect of 5'-deletion of Arabi-dopsis profilin2 promoter on its vascular specific expression

Based on the published sequence of profilin2 promoter of Arabidopsis thaliana, a full-length promoter (1667 bp) was amplified by PCR. The 5' -end deletion fragments with length of 1380, 1153, 969 and 597 bp were then fused with gus (uidA.) gene respectively. Constructed plant expression vectors were individually transferred into Kalan-choe laciniata and transgenic plants regenerated. GUS his-tochemical assay confirmed that the full-length promoter Pfn1.7 was vascular-specific. Deletion assays showed that profilin2 promoter could be divided into three parts. Deletion of fragment 1 ( -1667--1380 bp) resulted in constitutive expression, suggesting that element(s) responsible for vascular-specific expression might exist in this region. Fragment 2 located at -1153 - -597 bp strongly inhibited gus gene expression. Fragment 3 ( -597 - -1 bp) is considered as a basic domain of profilin2.     

Nucleotide sequence of polyhedrin gene of LsNPV and analysis of baculovirus polyhedron proteins

The intact 741 bp polyhedrin gene of LsNPV was sequenced by Silver, Sequencing System, and shares 90.6% and 97.0% nucleotide identity, 97.2% and 97.6% amino acid identity with PfNPV and MdNPV polh genes respectively. The 14 bp conservative sequence with the core element GTAAG, is located in the 5′untranslated region of the gene. The polh gene was predicted to encodes a 246 amino acid residures with molecular weight of 29.0 kd, in which the number of acidic amino acids and alkaline amino acids was roughly equal resulting in almost no charges in polyhedrin protein molecule and hence occlusion body. It gives a valuable implication that ionic bonds as well as hydrophobic bonds and hydrogen bond may play an important role in the crystallization of polyhedrin, by comparing amino acid variation of twenty—one polyhedrin. The comparsion of promoter regions of polyhedrin gene and class II gene shown that they are very similar, but also have differences in GC content. This could explain that both categories of gene are highly expressed, and polyhedrin genes are expressed more higher than class II gene. Wang Jiawang: born in 1962, Doctor of science. Present address: Cancer Institute, CAMS, Beijing 100005