小菊锌指蛋白基因cDNA全长克隆及表达分析

目的：克隆菊花耐盐碱相关锌指蛋白基因,并进行盐胁迫和品种间差异的表达分析。方法：从大量菊花资源中筛选出抗盐碱品系小菊‘阳光’,利用RT—PCR从经150mmol／L碳酸钠处理的小菊‘阳光’叶片中分离得到一个锌指蛋白cDNA全长克隆,用Northern杂交检测其在不同盐处理和不同品种小菊中的表达。结果：获得了全长794bp的基因CmSTZF（GenBank接受号为DQ864730）,编码区为170个氨基酸残基。Blast分析表明,CmSTZF含有AN1型锌指结构,序列模式为C-X2-C—X（9—12）-C-X（1-2）-C-X4-C-X2-H-X5-H-X-C,由Cys^110-Cys^113-Cys^131-His^134及Cys^124-Cys^126-Cys^142-His^140分别围绕锌离子与其他氨基酸共同组成2个锌指四面体结构;在第67～76及第94～104氨基酸残基序列间存在核定位信号。同源性比较发现,CmSTZF与水稻OsISAPI具有54％的同源性,而二者的锌指保守区相似性达100％。Cluster分析表明,小菊CmSTZF锌指蛋白与水稻的2种逆境反应蛋白亲缘关系最近,归属同一类逆境功能蛋白。在150mmol／L碳酸钠胁迫下,耐盐小菊‘阳光’锌指蛋白表达量明显高于非耐盐小菊‘神韵’,表明CmSTZF锌指蛋白基因在盐碱胁迫下起重要的调控作用。结论：克隆了小菊耐盐碱相关的锌指蛋白基因CmSTZF,其在耐盐小菊‘阳光’中的表达量高于非耐盐小菊‘神韵’,这为小菊锌指蛋白基因CmSTZF耐盐碱功能分析奠定了基础。     

拟南芥根特异表达基因启动子在烟草中的表达

以拟南芥为材料,利用PCR技术分离pyk10启动子序列,构建了该启动子GUS植物表达载体,农杆菌介导转化烟草,分析该基因在烟草中的表达,以明确拟南芥根特异表达基因pyk10启动子在烟草中的表达特性.结果表明:克隆的pyk10启动子与已报道的pyk10启动子一致性为100%,GUS基因在烟草的根部特异表达,表明该启动子为根部特异表达启动子,为揭示植物根的发生、分化和发育机制,以及培育抗根部病虫害和营养高效利用型转基因烟草奠定了基础.     

Alternative splicing of flowering regulatory gene LFY in Arabidopsis thaliana

为研究拟南芥成花调控基因LFY,我们采用RT-PCR方法分离克隆了三种选择性剪接的片段,分别命名为LFY1239,LFY1263和LFY1275.序列分析表明LFY1263包含一个大小为1 263 bp的开放阅读框,与之前报道的LFY基因片段大小相同,而LFY1239在第一外显子的3'端缺失了36 bp,LFY1275在第一内含子的3'末端插入了12 bp.对几种片段表达部位的分析显示,LFY1239只能在营养生长期的莲座叶中表达,而LFY1263和LFY1275在营养生长期和花期的花器官和莲座叶中都可以检测到,并且,LFY1263呈现出主导地位,LFY1275与LFY1263表达的比例表现为花器官高于莲座叶,该比例的变化可能预示着与成花调控有关.     

Cloning and characterization of a cDNA encoding serine palmitoyltransferase in Arabidopsis thaliana

The first and committed step in de novo sphingolipid synthesis is catalysed by serine palmitoyltransferase (EC 2.3.1.50), which condenses serine and palmitoyl-CoA to form 3-ketosphinganine in a pyridoxal-5'-phosphate-dependent reaction. We have isolated and characterized a cDNA clone from Arabidopsis thaliana that is homologous to yeast and mammalian LCB2. For a functional identification, the A. thaliana homologous cDNA was expressed in Escherichia coli, which resulted in significant production of new sphinganine in E. coli cells.     

Cloning and characterization of rac-like cDNAs from Arabidopsis thaliana

The Rho family of GTPases are in higher eukaryotes divided into 3 major subfamilies; the Rho, Rac and Cdc42 proteins. In plants, however, the Rho family is restricted to one large family of Rac-like proteins. From work with mammalian phagocytes the Rac proteins are known to activate a multicomponent NADPH-dependent oxidase which results in accumulation of H₂O₂, a process termed oxidative burst. In plants a similar oxidative burst is observed and plays an important role in its defence against pathogen infections, suggesting a similar role for the plant Rac-like proteins. The Rho family of GTPases proteins are also involved in control of cell morphology, and are also thought to mediate signals from cell membrane receptors.In a broad search for members of the Ras superfamily in plants, several new small GTP-binding proteins were found. We report here the identification and molecular cloning of 5 rac-like cDNAs from Arabidopsis thaliana, Arac1–5. The Rac-like proteins deduced from the cDNA sequences all share 80–95% homology, but show considerably more diversity on the nucleotide level, indicating that this is an ancient gene family. Four of the rac genes were found to be expressed in all tissues examined, but one gene, Arac2, was expressed exclusively in the root, hypocotyl and stem. Our results show that the rac gene family in A. thaliana consists of at least 10 different genes.     

Construction of an equalized cDNA library from Arabidopsis thaliana

Using a kinetic approach, a cDNA library composed of almost equal representations of all genes expressed in the aerial parts of 2-week-old Arabidopsis was constructed. A cDNA was synthesized with an oligo dT primer containing a Not l site. A linker containing the nucleotide sequence of Sse 8387I which recognizes octanucleotides was added at the ends of the synthesized cDNA. The cDNA was amplified by the polymerase chain reaction (PCR), denatured, and reassociated under modified conditions. Thereafter, the remaining single-stranded DNA was converted to double-stranded DNA and amplified by PCR. These equalization steps were repeated three times and the products were cloned unidirectionally into a plasmid vector. Equalization was evaluated by colony hybridization and DNA sequencing. This approach will be applicable to construct a cDNA library suitable for subtraction, differential screening, and expression screening, especially for mRNA species present at very low concentrations in a few cells of a specific tissue.     

拟南芥psy基因cDNA的克隆及其植物表达载体的构建

为了获得胚乳组织特异性表达八氢番茄红素的转基因小麦,以拟南芥幼叶RNA为模板,由特异型引物通过RT-PCR一步法得到大小约为1.3kb的基因片段,将此片段连接在克隆载体pMD18-T进行测序,结果表明,该基因片段为八氢番茄红素合成酶基因(psy)cDNA片段。将psy基因片段正向插入植物表达载体pLRPT中高分子量麦谷蛋白亚基基因1Dx5启动子与nos终止子之间,pLRPT载体无1Dx5基因开放阅读框,运用菌落PCR对重组子进行筛选与鉴定,说明拟南芥psy基因已正确插入pL-RPT,成功构建了植物表达载体pLRPTPSY。     

Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana.

We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein. The plant U1 snRNP 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the amino-terminal region as compared to human U1 snRNP 70K protein. Comparison of the deduced amino acid sequence of the plant U1 snRNP 70K protein with the amino acid sequence of animal and yeast U1 snRNP 70K protein showed a high degree of homology. The plant U1 snRNP 70K protein is more closely related to the human counter part than to the yeast 70K protein. The carboxy-terminal half is less well conserved but, like the vertebrate 70K proteins, is rich in charged amino acids. Northern analysis with the RNA isolated from different parts of the plant indicates that the snRNP 70K gene is expressed in all of the parts tested. Southern blotting of genomic DNA using the cDNA indicates that the U1 snRNP 70K protein is coded by a single gene.     

Cloning of a cDNA encoding ATP sulfurylase from Arabidopsis thaliana by functional expression in Saccharomyces cerevisiae.

ATP sulfurylase, the first enzyme in the sulfate assimilation pathway of plants, catalyzes the formation of adenosine phosphosulfate from ATP and sulfate. Here we report the cloning of a cDNA encoding ATP sulfurylase (APS1) from Arabidopsis thaliana. APS1 was isolated by its ability to alleviate the methionine requirement of an ATP sulfurylase mutant strain of Saccharomyces cerevisiae (yeast). Expression of APS1 correlated with the presence of ATP sulfurylase enzyme activity in cell extracts. APS1 is a 1748-bp cDNA with an open reading frame predicted to encode a 463-amino acid, 51,372-D protein. The predicted amino acid sequence of APS1 is similar to ATP sulfurylase of S. cerevisiae, with which it is 25% identical. Two lines of evidence indicate that APS1 encodes a chloroplast form of ATP sulfurylase. Its predicted amino-terminal sequence resembles a chloroplast transit peptide; and the APS1 polypeptide, synthesized in vitro, is capable of entering isolated intact chloroplasts. Several genomic DNA fragments that hybridize with the APS1 probe were identified. The APS1 cDNA hybridizes to three species of mRNA in leaves (1.85, 1.60, and 1.20 kb) and to a single species of mRNA in roots (1.85 kb).     

Molecular cloning of cDNA encoding N-acetylglucosaminyltransferase II from Arabidopsis thaliana

N-acetylglucosaminyltransferase II (GnTII, EC 2.4.1.143) is a Golgi enzyme involved in the biosynthesis of glycoprotein-bound N-linked oligosaccharides, catalysing an essential step in the conversion of oligomannose-type to complex N-glycans. GnTII activity has been detected in both animals and plants. However, while cDNAs encoding the enzyme have already been cloned from several mammalian sources no GnTII homologue has been cloned from plants so far. Here we report the molecular cloning of an Arabidopsis thalianaGnTII cDNA with striking homology to its animal counterparts. The predicted domain structure of A. thalianaGnTII indicates a type II transmembrane protein topology as it has been established for the mammalian variants of the enzyme. Upon expression of A. thalianaGnTII cDNA in the baculovirus/insect cell system, a recombinant protein was produced that exhibited GnTII activity.     

Antimutagenic activity of flavonoids from Chrysanthemum morifolium

A methanol extract from the flower heads of Chrysanthemum morifolium showed a suppressive effect on umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide). The methanol extract was re-extracted with hexane, chloroform, ethyl acetate, butanol, and water. The ethyl acetate fraction showed a suppressive effect. Suppressive compounds in the ethyl acetate fraction were isolated by silica gel column chromatography and identified as the flavonoids acacetin (1), apigenin (2), luteolin (3), and quercetin (4) by EI-MS, IR, and (1)H and 13C NMR spectroscopy. Compounds 1-4 suppressed the furylfuramide-induced SOS response in the umu test. Compounds 1-4 suppressed 60.2, 75.7, 90.0, and 66.6% of the SOS-inducing activity at a concentration of 0.70 micromol/ml. The ID50 (50% inhibitory dose) values of 1-4 were 0.62, 0.55, 0.44, and 0.59 micromol/ml. These compounds had the suppressive effects on umu gene expression of the SOS response against other mutagens, 4-nitroquinolin 1-oxide (4NQO) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which do not require liver-metabolizing enzymes. These compounds also showed the suppression of SOS-inducing activity against the other mutagens aflatoxin B1 (AfB1) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), which require liver-metabolizing enzymes, and UV irradiation. In addition to the antimutagenic activities of these compounds against furylfuramide, Trp-P-1 and activated Trp-P-1 were also assayed by the Ames test using S. typhimurium TA100.     

拟南芥IQM2 cDNA 的克隆与生物信息学分析

拟南芥IQM2由At3g13600编码,是IQM家族的第二个成员,但在各种分子生物学相关的文献和数据库中都找不到其cDNA序列。本研究采用RACE和RT-PCR技术,克隆得到拟南芥IQM2基因的全长cDNA序列。该cDNA长2245 bp,其开放阅读框长1818 bp,编码1个由605个氨基酸残基组成的多肽链。生物信息学分析表明,IQM2蛋白含有一个IQ基序,属于钙不依赖性钙调素结合蛋白;其N端与豌豆重金属诱导蛋白6（HMIP6）有较高的同源性,而IQ基序则分布在HMIP6结构域内部;其C端与栝楼天花粉蛋白（trichosanthin）N端具有较高的同源性。