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.     

ptr-MIR156a启动子克隆及特征分析

miR156a在火炬松、烟草、拟南芥中的表达与病原菌侵染密切相关。为了研究miR156a在杨树与病原菌互作过程中分子机制,以毛果杨全基因组DNA为材料,预测ptr-MIR156a启动子的大概区域,设计特异性PCR引物,克隆了ptr-MIR156a上游启动子区500、1 000和1 500 bp片段,并进行顺式作用元件分析,然后分别构建了绿色荧光蛋白(GFP)报告基因植物表达载体,最后通过原生质体的瞬时表达体系对其进行了活性检测。结果表明,1 500 bp片段活性最高,1 000 bp片段次之,500 bp片段活性最低。     

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．     

甘蓝型油菜Toc33基因启动子的克隆及序列分析

叶绿体是植物细胞中重要的细胞器，大部分叶绿体蛋白质都是由核基因组编码，在细胞质中合成分子量较大的前体蛋白，转运至叶绿体实施其功能．TOC33／TOC34是叶绿体上发现的一个外膜蛋白转运器构件蛋白，它与TOC159、TOC75和TOC64相互作用，构成了叶绿体外被膜上的一个蛋白转运器．目前已从豌豆（Pisumsa tizrurn）、拟南芥（Arabidopsis thaliana）、玉米（Zea mays）、小立碗藓（Physcomitrella patens）、诸葛菜（Orychophragmus violaceus）和油菜（Brassica napus）克隆到TOC33或TOC34的cDNA或DNA的编码区．与其功能研究相比，Toc33基因的表达调控研究较少，该基因5’端调控区域的克隆及序列分析均未见报道．为此，在本实验室已经克隆到甘蓝型油菜Toc33基因编码区的基础上，采用单引物PCR方法进行染色体步移，克隆出Toc33基因的启动子，为进一步研究Toc33基因的转录调控机制奠定基础．     

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.     

Structural characterization of the Sry 5’ regulatory region of bovine

By amplification of the polymerase chain reaction (PCR), the two fragments of 680 bp and 640 bp of 5’ regulatory region of Chinese bovine Sry gene were subcloned into pUC18. Sequence analysis shows that the fragment of total 1040 bp, which is highly conserved within bovine species, contains a start code ATG and a core promoter.     

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 ofArabidopsis profilin2 promoter on its vascular specific expression

Based on the published sequence of profilin2 promoter ofArabidopsis 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 withgus (uidA) gene respectively. Constructed plant expression vectors were individually transferred intoKalanchoe laciniata and transgenic plants regenerated. GUS histochemical 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 inhibitedgus gene expression. Fragment 3 (−597—−1 bp) is considered as a basic domain of profilin2.     

G to A substitution in the distal CCAAT box of the A gamma-globin gene in Greek hereditary persistence of fetal haemoglobin

The sequence 5'TTGGPyCAAT 3' (the 'CCAAT box') is a constituent of the promoter region of many eukaryotic and prokaryotic genes and is believed to play a part in promoter function. A characteristic of the two fetal human globin genes (A gamma and G gamma) is a duplication of a 12-base pair (bp) sequence containing the CCAAT box. Here we report a G----A substitution in the TTG sequence of the distal CCAAT box of the A gamma-globin gene in an individual with the A gamma (Greek) type of hereditary persistence of fetal haemoglobin (HPFH). This represents the first report of a natural mutation of the CCAAT box in a eukaryotic gene. The fact that this transition is associated with inappropriate expression of the A gamma gene in adult life suggests that the CCAAT box (or its surrounding sequences) may have a role in the developmental control of gamma-globin genes.     

人IDE基因启动子生物信息学分析

对人IDE基因启动子进行生物信息学分析以获得人IDE基因启动子、CpG岛及转录因子结合位点特征.从UCSC基因组数据库成功获得人IDE基因5’调控区2 000 bp序列.Promoter 2.0、FPROM、NNPP预测人IDE基因分别有3个、2个、6个启动子.Relative profile score threshold选择80%、85%、90%、95%、100%时,JASPAR预测该序列存在5170、1771、454、87和5个可能的转录因子结合位点.Relative profile score threshold选择80%,搜索到6个潜在的TCF7L2转录因子结合位点.采用进化足迹法,LAGAN预测方法获得位于人和小鼠同源IDE基因启动子保守区域相同位置的转录因子结合位点为14个,包含转录因子SPI-1、cap、c-FOS、FREAC-3、c-ETS、Cdxa、HSF2等.发现一个CpG岛,位于1 303～1 705 bp 之间,大小为403 bp.人IDE基因启动子、CpG岛及转录因子结合位点的生物学信息学分析,为下一步基因表达调控实验奠定了基础.     

双靶向溶瘤腺病毒对肿瘤细胞及正常细胞的杀伤差异性研究

为了降低溶瘤腺病毒对正常细胞的杀伤作用，提高临床应用上的安全性，构建了一种双靶向溶瘤增殖型腺病毒AdCN103，以人端粒酶逆转录酶（hTERT）启动子代替野生型腺病毒E1A自身的启动子，同时在E1A区缺失保守区域CR2的24 bp.并将AdCN103与两种相应的单靶向溶瘤增殖型腺病毒AdCN101和AdCN102，以及野生型WtAd5相比较，通过MTT,结晶紫以及病毒子代复制实验，观察它们对肿瘤细胞和正常细胞的杀伤性差异.结果表明，AdCN103只能严格地在肿瘤细胞中复制，对肿瘤细胞有较好的杀伤作用，对正常细胞的杀伤性较野生型腺病毒及单靶向腺病毒都弱.实验证明AdCN103能作为新一代的安全的双靶向溶瘤腺病毒载体应用于肿瘤治疗.     

中国明对虾卵黄蛋白原基因启动子克隆与分析

为了探明中国明对虾卵黄蛋白原基因启动子表达调控机制,利用DNA步移法克隆了中国明对虾卵黄蛋白原基因启动子及其上游调控序列,总长1 100bp.分析表明,在基因转录起始位点上游-30～-24bp处有1个TATA box,未发现有CAAT box和GC box.同时,在上游调控区还存在有多个可能影响启动子转录活性的顺式作用元件,如NF-κB,YY1和SP1等转录因子结合位点.这些结果为深入研究中国明对虾卵黄蛋白积累及卵子发生过程奠定了基础.     

油菜MAP激酶基因BnMPK6基因的克隆及表达分析

从陇油6号油菜中克隆得到了一种新的MAP激酶基因BnMPK6,全长1521 bp,其中包括1185 bp的开放阅读框,81 bp的5′非翻译区(5′UTR),255 bp的3′非翻译区(3′UTR).与拟南芥AtMPK6同源性达86.9%,因此命名为BnMPK6(GenBank登录号为HQ156228).该基因编码394个氨基酸的蛋白质,分子量44.8 kDa,等电点为5.13.实时荧光定量PCR结果表明该基因表达量随着低温胁迫时间的增长而增高,说明BnMPK6基因受低温胁迫诱导表达.