TAIL-PCR的改良及其在分离小麦基因启动子中的应用

在经典TAIL-PCR(Thermal asymmetric interlaced PCR)的基础上, 对其进行了如下四处改进: 用10个碱基的RAPD引物代替16个碱基的随机兼并引物作为PCR中的随机引物; 将较低特异性循环的复性温度由44°C降至29°C; 增加5个高特异性反应循环, 减少5个较低特异性反应循环; 用单引物对第三轮PCR产物进行初步鉴定。利用改进的TAIL-PCR方法分离了小麦X基因的5′未知的侧翼序列, 与GUS基因融合后转入拟南芥, 通过组织化学检测分析表明分离到的5′侧翼序列具有启动子功能, 同时说明改进的TAIL-PCR能更好地应用到较复杂基因启动子的分离。     

Cloning, characterization and promoter analysis of S-RNase gene promoter from Chinese pear (Pyrus pyrifolia)

The 5'-flanking region of the S(12)-, S(13)-, S(21)-RNase with a length of 854 bp, 1448 bp and 1137 bp were successfully isolated by TAIL-PCR from genomic DNA from 'Jinhua', 'Maogong' (Pyrus pyrifolia) and 'Yali' (Pyrus bretschneideri) genomic DNA. Sequence alignment and analysis of S(13)-, S(12)-, S(21)-RNase gene promoter sequences with S(2)-, S(3)-, S(4)-, S(5)-RNase 5'-flanking sequences indicated that a homology region of about 240 bp exists in the regions just upstream of the putative TATA boxes of the seven Chinese/Japanese pear S-RNase genes. Phylogenetic tree suggests that the homology region between the Chinese/Japanese pear and apple S-RNase gene promoter regions reflects the divergence of S-RNase gene was formed before the differentiation of subfamilies. Full length and a series of 5'-deletion fragments-GUS fusions were constructed and introduced into Arabidopsis thaliana plants. GUS activity were detected in S(12)-pro-(1 to 5)-GUS-pBll01.2 transgenic pistils and progressively decreased from S(12)-pro-1-GUS-pBI l01.2 to S(12)-pro-5-GUS-pBll01.2. No GUS activity was detected in S(12)-pro-6-GUS-pBll01.2 transgenic pistil and other tissues of non-transformants and all transgenic plants. The result suggested S(12)-RNase promoter is pistil specific expression promoter.     

大花美人蕉查尔酮异构酶基因的cDNA克隆和序列分析

以高等植物查尔酮异构酶（CHI）基因的保守区域CKFVKFT、KFTAIGV、AVKWKGK及GPFEKFT、IIGKI-IGV设计简并引物和采用逆转录．聚合酶链式反应（RT-PCR）以及温度非对称交互PCR（TAIL．PCR）方法,从大花美人蕉花瓣组织中扩增查尔酮异构酶基因（倒）的全长cDNA（678bp）,编码226个氨基酸,其氨基酸组成与其它已知的高等植物CHI基因具有很高的同源性,与葡萄、草莓、丁香、柑桔、矮牵牛、洋葱及玉米的同源性分别为82％、79％、80％、80％、79％、81％和76％。     

TAIL-PCR方法快速分离Xcc致病相关基因序列

以mini-Tn5 gfp-km转座子中nptⅡ片段作为探针,对已获得的五株野油菜黄单胞菌野油菜黑腐病致病型（Xcc)非致病突变体进行了Southern blot分析,结果表明,这五株突变体确由mini-Tn5 gfp-km转座子插入致病相关基因所致,且为单拷贝不同位点的插入。提取这五株突变体总DNA作为模板,采用改进的热不对称交错PCR（TAIL－PCR）方法从其中克隆到了各自转座子插入区侧翼序列,对这些侧翼序列进行了序列测定并将分析结果与GenBank database及Xcc全基因组序列做了比较,结果表明,五个侧翼序列所在的基因确与Xcc致病性有关。这种改进后的TAIL－PCR方法为突变体特别是转座子插入突变体中目的基因的克隆提供了一种简要高效的新方法。     

麻疯树MIPS基因启动子的分离及在烟草原生质体中瞬时表达活性分析

根据麻疯树MIPS基因序列,设计特异性的巢式引物,运用TAIL-PCR法两次步移得到MIPS基因5＇端侧翼序列,序列分析显示含有多个胁迫应答相关元件,如ABRE、HSE等。以该序列为基础,PCR扩增得到5个5＇端不同长度的缺失片段,分别插入pBI221载体置换CaMV35S启动子,构建的表达载体在PEG介导下转入烟草叶片原生质体进行瞬时表达,检测GUS报告基因的活性。经GUS活性荧光定量检测发现,分离到的MIPS基因侧翼序列5＇端不同缺失片段都能启动GUS报告基因表达,启动活性最高的是WQ1区（-565bp）,核心区位于-565～-449bp。在100μmol·L-1ABA诱导下启动活性增强,但不同区段的增长幅度不同。WQ1区增长幅度最大,比未处理时提高41.4%。     

Common carp metallothionein-1 gene: cDNA cloning, gene structure and expression studies

Metallothionein-1 (MT-1) cDNA clones were isolated from a common carp (Cyprinus carpio) uninduced hepatopancreas cDNA library. Northern blot assay using the common carp (cc) MT-1 cDNA as a probe showed high fold induction of ccMT mRNA levels in the intestine and kidney following exposure to Cd2+ and Zn2+. Using polymerase chain reaction (PCR), primers designed from the cDNA sequences allowed the isolation of ccMT-1 gene fragments including the 5'-flanking region. The 600 bp 5'-flanking region of ccMT-1 gene carries four putative metal regulatory regions, one AP1, two SP1, one c-Jun site, and a TATA box. The 5'-flanking region of the ccMT-1 gene obtained was a functional promoter responding to the administration of various metal ions as well as hydrogen peroxide (H2O2) and lipopolysaccharide (LPS). When tested in primary cultures of cc hepatocytes, Zn2+ had the highest fold (20 times) induction of the 600 bp cloned ccMT-1 gene promoter, followed by Cu2+, Hg2+, Ni2+ and Pb2+ (4-5-fold inductions); H2O2 and LPS had a 6-7-fold induction. In conclusion, the ccMT-1 is a constitutively expressed MT and its gene promoter is inducible by various metal ions and chemical agents.     

Molecular cloning and expression analysis of the replacement histone H3 gene of Italian ryegrass (Lolium multiflorum)

The replacement histone H3 gene and its 5'-flanking sequence were isolated from Italian ryegrass by polymerase chain reaction and inverse polymerase chain reaction, respectively. Expression analysis showed that this gene is constitutively expressed in the entire plant. The expression level in leaves was found to be significantly low when compared with that in other tissues. However, the gene expression level in leaves was increased by the treatment with abscisic acid and abiotic stresses such as cold, heat and high-salinity (NaCl). The motif search of the 5'-flanking sequence of the replacement histone H3 gene revealed the presence of several potential cis-acting elements that could respond to the above-mentioned abiotic stresses. In addition to defence-related elements, we also found type I and II-/III-like elements, which are highly conserved motifs in the 5'-regulatory sequence of plant histone genes that are expressed specifically during the S-phase. Experiments using transgenic Italian ryegrass plants proved that the isolated 5'-flanking sequence of the replacement histone H3 gene, which was fused to a beta-glucuronidase reporter gene, was fully functional for inducing gene expression under various abiotic stress conditions.     

The gene for the major cuticular wax-associated protein and three homologous genes from broccoli (Brassica oleracea) and their expression patterns

The gene for the major protein (WAX9) found in surface wax of broccoli, designated wax9D , and three homologous genes ( wax9A, B and C ) were isolated from a genomic library using the previously isolated cDNA encoding the WAX9 protein as the probe; all four genes were sequenced. Genomic Southern blot analysis using the WAX9 cDNA as a probe showed the presence of at least four homologous genes in broccoli genome. The sequence of the originally isolated WAX9 cDNA matched with that of gene D . All four genes have an intron two codons before the stop codon. The putative promoter regions of the four genes, beyond the first 200 bp immediately 5' to the translation start sites, are quite different. Essential elements such as TATA and CAAT boxes and several regions homologous to the promoter regions of other plant ltp genes were identified. The expression patterns of the genes were determined by RT-PCR with gene-specific primers and sequencing of the PCR products. All the genes were expressed in leaves and flower buds. While genes A, B and D also were expressed in stems and open flowers, expression of gene C was not detected in these organs. None of them were expressed in roots. The 972 bp 5'-flanking region of wax9D when fused to β-glucuronidase (GUS) gene, directed tissue-specific GUS expression in transgenic tobacco plants; GUS expression was found in the epidermis of leaves, stems and flower petals, sepals, trichomes, and ovules but not in roots.     

Effect of flanking regions on the expression of the human interleukin-2 chromosomal gene in a murine myeloma cell line]

A human interleukin-2 (IL-2) gene was isolated from genomic DNA library. The isolated gene with 5'- and 3'-flanking sequences of various lengths was inserted into plasmids derived from the retroviral vector pPSneo. The recombinant plasmids were transfected into myeloma X63Ag8-653 cells. The transfected cells, harbouring the IL-2 gene with the shortened (to position -165) or totally deleted 5'-flanking sequence, constitutively expressed biologically active IL-2. Deletion of 3'-flanking region on did not affect the IL-2 expression.     

从水稻Ac/Ds插入突变体扩增Ds侧翼序列的最适TAIL-PCR引物

温度非对称交互PCR(TAIL-PCR)技术已广泛应用于从多种生物体系克隆侧翼于已知序列的DNA片段的分子操作中,并极大地促进了反向遗传学研究。但是,可能由于不同物种间基因组大小和序列存在显差异,在采用该技术进行转座元件Ds水稻插入突变体鉴定过程中,常因TAIL-PCR反应的稳定性差而影响突变体筛选效率。有鉴于此,根据Ds核苷酸序列设计了分别对应或互补于Ds插入元件两端长度不同的12个特异引物组成32个组合,在大量预试验基础上与6个不同简并性(32～256)的随机简并引物分别组合进行TAIL-PCR反应,较系统地研究了引物特性对以水稻基因组DNA为模板的TAIL-PCR反应效率的影响。结果发现,第一反应采用长序列特异引物(36～40mer)可显提高扩增特异性,随机简并引物的简并度对反应的影响显。还选择出两个适于从水稻Ds插入突变体基因组高效扩增出Ds插入侧翼片段的最优特异引物组合和最适简并引物。应用本研究结果可显地提高TAIL-PCR技术筛选水稻插入突变体的效率。     

Recombination of two homologous MHC class III genes of the mouse (C4 and Slp) that accounts for the loss of testosterone dependence of sex-limited protein expression

In most mouse strains, expression of a gene encoding sex-limited protein (Slp), an isotype of the fourth component of complement (C4), is induced by testosterone, or the gene is not expressed at all; however, in some wild-derived strains carrying H-2w7, H-2w16, or H-2w19 haplotype, Slp is expressed constitutively in the same way as C4. To examine the structural basis for the testosterone-independent expression of Slp, 41 overlapping clones together encoding the S region were isolated from C3H.W7 mouse (H-2w7) cosmid library. Five C4-related genes each spanning approximately 16 kb were identified among the cluster of cosmid clones and were isolated for structural study. One of the genes (C4w7) hybridized with the C4-specific oligonucleotide probe but not with the Slp-specific oligonucleotide probe, whereas the other genes (Slpw7a, Slpw7b, Slpw7c, and Slpw7d) hybridized only with the Slp-specific probe. Restriction mapping of these genes and sequencing of the selected regions of 5'-flanking regions of the genes were performed, and the results were compared with the data obtained with the C4 and Slp genes of FM (H-2d) and B10.BR (H-2k). These studies showed that three of the C4-related genes of C3H.W7 (Slpw7b, Slpw7c, and Slpw7d) are C4-Slp recombinant genes comprising a 5'-region derived from C4 gene and a 3'-region derived from Slp gene. It is suggested that 5'-flanking region derived from C4 in these C4-Slp recombinant genes accounts for testosterone-independent expression of Slp in C3H.W7 mouse.