一种用少量标本快速构建老年性白内障消减cDNA文库的方法

为从少量标本中获得含较多大片段的、高质量的老年性白内障消减cDNA文库,利用磁珠分离、生物素标记的改良消减杂交法获得差异cDNA,利用选择性PCR法扩增其中大片段差异cDNA,从而成功构建老年性白内障消减cDNA文库.在文库中随机挑取的22个克隆中,1 000 bp以上的片段有7个,占31.8%,750 bp以上有15个,占68.2%.所得cDNA片段较大,可以满足下一步研究需要.改良消减杂交法结合选择性PCR法可以从少量标本中快速有效地获得大片段高质量的消减cDNA文库.     

Identification and expression analysis of cold-regulated genes from the cold-hardy Citrus relative Poncirus trifoliata (L.) Raf.

Citrus is a cold-sensitive genus and most commercially important varieties of citrus are susceptible to freezes. On the other hand, Poncirus trifoliata (L.) Raf. is an interfertile Citrus relative that can tolerate temperatures as low as −26°C when fully cold acclimated. Therefore, it has been used for improving cold tolerance in cold-sensitive commercial citrus rootstock varieties and in attempts to improve scion varieties. In this study, cDNA libraries were constructed from both 2-day cold-acclimated and from non-acclimated Poncirus seedlings using a subtractive hybridization method with the objective of identifying cold-regulated genes. A total of 192 randomly picked clones, 136 from the cold-induced library and 56 from the cold-repressed library, were sequenced. The majority of these clones showed sequence homology to previously identified cold-induced and/or environmental stress-regulated genes in Arabidopsis. In addition, some of them shared homology with cold and/or environmental stress-induced genes previously identified in other herbaceous and woody perennial plants and some showed no homology with sequences in GenBank. When these 192 cDNAs were analyzed by reverse northern blot with cold-acclimated and non-acclimated probes, 92 of the cDNAs displayed significantly increased expression, ranging from 2 to 49-fold, during cold acclimation; all 92 were from the cold-induced library. Surprisingly no clones displayed significantly repressed expression in response to cold. Analysis of a number of selected genes individually in northern blots of mRNA from cold-acclimated and non-acclimated plants largely confirmed the reverse northern analysis, verifying induction of expression of selected cDNAs in response to cold. The results showed that subtractive hybridization is an efficient method for identification of cold-induced genes in plants with limited sequence information available. This study also revealed that genes induced during cold acclimation of the cold-hardy citrus relative P. trifoliata are similar to those in Arabidopsis, indicating that similar pathways may be present and activated during cold acclimation in woody perennial plants.     

A system for the directed evolution of the insecticidal protein from Bacillus thuringiensis

Theoretically, the activity of AB-type toxin molecules such as the insecticidal toxin (Cry toxin) from B. thuringiensis, which have one active site and two binding site, is improved in parallel with the binding affinity to its receptor. In this experiment, we tried to devise a method for the directed evolution of Cry toxins to increase the binding affinity to the insect receptor. Using a commercial T7 phage-display system, we expressed Cry1Aa toxin on the phage surface as fusions with the capsid protein 10B. These recombinant phages bound to a cadherin-like protein that is one of the Cry1Aa toxin receptors in the model target insect Bombyx mori. The apparent affinity of Cry1Aa-expressing phage for the receptor was higher than that of Cry1Ab-expressing phage. Phages expressing Cry1Aa were isolated from a mixed suspension of phages expressing Cry1Ab and concentrated by up to 130,000-fold. Finally, random mutations were made in amino acid residues 369–375 in domain 2 of Cry1Aa toxin, the mutant toxins were expressed on phages, and the resulting phage library was screened with cadherin-like protein-coated beads. As a result, phages expressing abnormal or low-affinity mutant toxins were excluded, and phages with high-affinity mutant toxins were selected. These results indicate that a method combining T7 phage display with selection using cadherin-like protein-coated magnetic beads can be used to increase the activity of easily obtained, low-activity Cry toxins from bacteria.     

西藏八角莲与桃儿七根及根茎SSH文库的建立

以西藏八角莲(Dysosma tsayuensis Ying)和桃儿七[Sinopodophyllum hexandrum(Royle)Ying]为材料,构建其根及根茎的SSH文库,从中筛选鬼臼类植物属种间与鬼臼毒素生物合成相关的差异表达基因。从文库中随机挑取201个阳性克隆测序后得到183条ESTs。去除载体序列和冗余序列,聚类拼接得到17个西藏八角莲的unique ESTs。经BLAST同源比较和功能查寻,有功能注释的unique ESTs共12个,占70.6%,所编码的蛋白涉及光合作用、合成代谢、转录调控等功能;无功能注释和匹配结果的共5个,占29.4%。该研究成功构建了西藏八角莲和桃儿七SSH文库,为进一步揭示鬼臼毒素生物合成途径及其调控机制奠定了基础。     

Antibody-free peptide substrate screening of serine/threonine kinase (protein kinase A) with a biotinylated detection probe

Being different from anti-phosphotyrosine antibodies, anti-phosphoserine- or anti-phosphothreonine-specific antibodies with high affinity for the detection of serine/threonine kinase substrates are not readily available. Therefore, chemical modification methods were developed for the detection of phosphoserine or threonine in the screening of protein kinase substrates based on β-elimination and Michael addition. We have developed a biotin-based detection probe for identification of the phosphorylated serine or threonine residue. A biotin derivative induced a color reaction using alkaline phosphate-conjugated streptavidin that amplified the signal. It was effective for the detection and separation of the target peptide on the resin. The detection probe was successfully used in identifying PKA substrates from peptide libraries on resin beads. The peptide library was prepared as a ladder-type, such that the active peptides on the colored resin beads were readily sequenced with the truncated peptide fragments by MALDI-TOF/MS analysis after releasing the peptides from the resin bead through photolysis.     

Identification of fungal (Magnaporthe grisea) stress-induced genes in wild rice (Oryza minuta)

To identify fungal stress-related genes in wild rice, Oryza minuta, we constructed a subtracted library using suppression subtractive hybridization in combination with mirror orientation selection. DNA chips containing 960 randomly selected cDNA clones were applied by reverse Northern analysis to eliminate false positive clones from the library and to prescreen differentially expressed genes. In total, 377 cDNA clones were selected on the basis of their signal intensities and expression ratios. Sequence analyses of these 377 cDNA fragments revealed that 180 of them (47.7%) represented unique genes. Of these180 cDNAs, 89 clones (49.6%) showed significant homologies to previously known genes, while the remaining 91 did not match any known sequences. The putative functions of the 180 unique ESTs were categorized by aligning them with MIPS data. They were classified into seven different groups using microarray data-derived expression patterns and verified by Northern blotting.Abbreviations ER: Endoplasmic reticulum - EST: Expressed sequence tag - MIPS: Munich Information Center for Protein Sequences - MOS: Mirror orientation selection - NCBI: National Center for Biotechnology Information - omfi: Oryza minuta fungal-stress induced - PCD: Programmed cell death - PDI: Proteins disulfide isomerase - SSH: Suppression subtractive hybridization Communicated by I.S. ChungK.S. Shim and S.K. Cho contributed equally to this work.     

Suppression subtractive hybridization cloning of cDNAs of differentially expressed genes in dovetree (<Emphasis Type="Italic">Davidia involucrata</Emphasis>) bracts

A subtracted cDNA library forDavidia involucrata was constructed using suppression subtractive hybridization (SSH). mRNA isolated from young leaves was used as a “driver,” and mRNAs isolated from young bracts were used as “testers.” The differentially expressed cDNA fragments in bracts were identified by differential screening. Of the 16 clones selected randomly from the screened library, 8 were known genes found in GenBank, and 2 had no similar sequences. Northern blot analysis revealed that the expression level of P1A5 cDNAs selected randomly was dominantly expressed in bracts. This indicates that SSH can be used to clone differentially expressed cDNAs inD. involucrata bracts.     

Monoclonal antibodies for the detection of Puccinia striiformis urediniospores

The fungal pathogen Pst causes yellow rust disease in wheat plants leading to crop losses. The organism spreads by releasing wind-dispersed urediniospores from infected plants. In this study a library of novel monoclonal antibodies (mAbs) was developed against Pst urediniospores. Nine mAb-producing cell lines were cloned and their cross-reactivities characterised against a panel of airborne fungal spores representing genera commonly found in the same environment as Pst. Two specific mAbs were used to develop a competitive ELISA (Pst mAb4) and a subtractive inhibition ELISA (Pst mAb8). Standard curves for both assays had good intra- and interday reproducibility. The subtractive inhibition ELISA had greater sensitivity with a detection limit of 1.5 × 10⁵ spores ml⁻¹. Cross-reactivity studies of Pst mAb8 in the subtractive inhibition ELISA, showed reaction with other Puccinia spores only, suggesting that common epitopes exist within this genus. The biosensor-compatible Pst mAb8 assay principle developed in this study has the potential to be implemented in future ‘label-free’ in-the-field systems for Pst detection.     

Late Activation of Interferon-Induced Genes IFI-54K and IFI-56K in Human RH Cells Infected with Tick-borne Encephalitis Virus

The genes that were induced and suppressed in human embryonic kidney cell line RH upon the infection with tick-borne encephalitis virus were studied by the method of subtractive hybridization. The expression of interferon-induced genes IFI-54K and IFI-56K in the infected cells was found to increase 50–100-fold.     

Early developmentally regulated genes in the arbuscular mycorrhizal fungus Glomus mosseae: identification of GmGIN1, a novel gene with homology to the C-terminus of metazoan hedgehog proteins

The life cycle of the obligate biotrophic arbuscular mycorrhizal fungi comprises several well-defined developmental stages whose genetic determinants are still unknown. With the aim of understanding the molecular processes governing the early developmental phase of the AM fungal life cycle, a subtractive cDNA library was constructed using a suppressive subtractive hybridization technique. The library contains more than 600 clones with an average size of 500 bp. The isolated cDNAs correspond to genes up-regulated during the early development of the AM fungus Glomus mosseaeversus genes expressed in extraradical hyphae. The expression of several of the isolated genes was further confirmed by RT-PCR analysis. Among the isolated clones, a novel gene named GmGIN1 only expressed during early development in G. mosseae was found. The full-length GmGIN1 cDNA codes for a protein of 429 amino acids. The most interesting feature of the deduced protein is its two-domain structure with a putative self-splicing activity. The N-terminal domain shares sequence similarity with a novel family of GTP binding proteins while the C-terminus has a striking homology to the C-terminal part of the hedgehog protein family from metazoa. The C-terminal part of hedgehog proteins is known to participate in the covalent modification of the N-terminus by cholesterol, and in the self-splicing activity which renders the active form of the protein with signalling function. We speculate that the N-terminal part of GmGIN1, activated through a similar mechanism to the hedgehog proteins, has GTP-binding activity and participates in the signalling events prior to symbiosis formation.     

镰刀菌诱导的泸定百合SSH文库构建及抗病相关基因筛选

该研究以泸定百合(Lilium sargentiae Wilson)为材料,构建其组培苗经百合尖孢镰刀菌侵染后的叶片SSH文库,从中筛选镰刀菌枯萎病抗病相关基因。从正向SSH文库中随机挑取300个单克隆测序后得到280条ESTs,进行功能比对分析后,除去未知功能、沉冗蛋白以及无同源序列,得到有功能的ESTs共168条,其功能涉及信号传导、蛋白质合成与代谢、抗病与防御、物质与能量代谢、转录相关等多种途径,其中有31条ESTs与抗病防御相关。从抗病防御相关基因中选取8条ESTs:过氧化氢酶、ATP结合盒转运蛋白(ABC transporter)、Kunitz型胰蛋白酶抑制剂4(Kunitz trypsin inhibitor 4)、丝氨酸乙醛酸氨基转移酶、多聚泛素、脂氧合酶I(Lipoxygenase I)、丝氨酸/苏安酸蛋白激酶(Serine/Threonine-protein kinase)、抗坏血酸过氧化物酶(Arabidopsis thaliana),通过RTPCR对其表达情况进行分析,发现经镰刀菌诱导后均为上调表达,推测它们可能参与了泸定百合镰刀菌枯萎病的抗病反应途径。     

ISOLATION OF PHASE-SPECIFIC COMPLE-MENTARY DNAS FROM CARPOSPOROPHYTES OF GRACILARIOPSIS LEMA-NEIFORMIS USING MAGNETIC BEADS AND PCR

Kamiya, M.¹, Moon, D. A.², Kawai, H.¹ & Goff, L. J.^{2 1}Kobe University Research Center for Inland Seas, 2746 Iwaya, Awaji-cho 656-2401 Japan; ²Department of Biology, University of California, Santa Cruz, CA 95064 USA Although the morphology and developmental patterns of carposporophyte stage have been investigated well, there are few molecular, genetic, or biochemical data about this stage. The greatest obstacle to this research has been that the conventional methods to isolate tissue-specific genes require a lot of tissues, but the carposporophyte is very tiny and mostly embedded in female gametophyte tissues. Recent advanced techniques have allowed the subtractive cloning of differentially expressed genes from small amounts of tissue or cells. We applied the subtractive hybridization method using magnetic beads and PCR to the analysis of phase-specific cDNAs from carpo-sporophytes of Gracilariopsis lemaneiformis (Gracilariales, Rhodophyta). A hundred cystocarps were dissected to isolate gonimoblast tissues, and total RNAs were extracted from the gonimoblast tissues and the female gametophyte branches, respectively. Messenger RNAs were captured on paramagnetic oligo-dT beads, followed by first-strand cDNA synthesis on the beads. Three rounds of subtractive hybridization between the amplified second-strand carposporophyte cDNA in solution and the first-strand gametophyte cDNA attached to magnetic beads were sufficient to remove common genes present in both gametophyte and carposporophyte stages. A specific PCR product from the nuclear GAPDH gene was readily amplified from gametophyte and carposporophyte cDNA, but no amplification was observed using the subtracted carposporophyte cDNA as template. This control PCR product demonstrates that the hybridization steps successfully removed the common GAPDH cDNA, which is found in both stages, giving confidence that the remaining genes cloned from the subtracted carposporophyte cDNA library are stage-specific.     

Immobilization of Pseudomonas delafieldii with magnetic polyvinyl alcohol beads and its application in biodesulfurization

Pseudomonas delafieldii was immobilized in magnetic polyvinyl alcohol (PVA) beads using a hydrophilic magnetic fluid, which was prepared by a co-precipitation method. The beads had distinct super-paramagnetic properties and were compared with immobilized cells in non-magnetic PVA beads. Their desulfurizing activity was increased slightly from 8.7 to 9 mmol sulfur kg(-1) (dry cell) h(-1). The main advantages was that the magnetic immobilized cells maintain a high desulfurization activity and remain in good shape after 7 times of repeated use, while the non-magnetic immobilized cells could only be used for 5 times. Furthermore, the magnetic immobilized cells could be easily collected or separated magnetically from the biodesulfurization reactor.     

Identification of protein-binding peptides by direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of peptide beads selected from the screening of one bead-one peptide combinatorial libraries

A fast and inexpensive strategy for the identification of peptide ligands by direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of peptide beads screened from one bead-one peptide combinatorial libraries is herein described. Streptavidin was used as the model protein. A combinatorial library of 6561 peptides was synthesized on ChemMatrix resin by the divide-couple-recombine method. 4-Hydroxymethylbenzoic acid was used as the linker and five residues of Gly were incorporated at the C termini to increase the final peptide molecular weight. Positive control peptides with the HPQ motif and negative control peptides without the HPQ motif evidenced that the linker and the five residues of Gly have neither impaired the specific binding nor facilitated unspecific binding. After screening the library, positive beads were isolated and washed with 8M guanidine hydrochloride. The beads were sliced into two or four pieces, deposited onto the stainless steel MALDI sample plate, and treated with ammonia vapor to release the peptides. In addition, 26 beads picked at random from the library were subjected to the same treatment. All samples were analyzed by MALDI-TOF-MS and the peptides were unambiguously identified with very good reproducibility between the bead pieces, thus evidencing the good homogeneity of the bead. All sequences obtained from the screening contained HPQ.     

Identification and characterization of differentially expressed ESTs of <Emphasis Type="Italic">Gossypium barbadense</Emphasis> infected by <Emphasis Type="Italic">Verticillium dahliae</Emphasis> with suppression subtractive hybridization

The wilt defense reaction of cotton is a complicated continuous process and involves a battery of genes. In this study, we adopted the suppression subtractive hybridization (SSH) technique to isolate differentially expressed ESTs from Gossypium barbadense variety 7124 during the Verticillium wilt defense process. An array of 1165 clones from the subtractive library has been screened with reverse northern blotting, of which 131 ESTs were considered as overexpressed and 16 ESTs were downregulated. Sequence analysis and blast search showed that 83 ESTs were homologous to 45 unique sequences in the databases. Among all these differentially expressed ESTs, at least three kinds of genes were characterized. The majority of ESTs with a deduced identity as aerobic metabolism enzymes were strongly expressed in the infection process. Likewise, ESTs similar to those reported for pathogen-related protein genes were also picked out in this study. These ESTs, in combination with other kinase-like genes and a defensin-like EST, constituted an assembly of genes which responded during pathogenic infection. These results imply that sea-island cotton undergoes strong oxidative stress and results in a series of defense responses when attacked by V. dahliae. To our knowledge, this is the first report on the isolation of global ESTs during the sea-island cotton defense reaction.__________From Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 214–223.Original English Text Copyright © 2005 by Zuo, Wang, Wu, Chai, Sun, Tang.This article was submitted by the authors in English.