Identification of candidate SNPs for drug induced toxicity from differentially expressed genes in associated tissues

The full-length cDNA sequence (1158 bp) encoding a ribosomal L5 protein, designated as TaL5, was firstly isolated from common wheat (Triticum aestivum L.) using the rapid amplification of cDNA ends method (RACE). The open reading frame (ORF) of TaL5 gene was 906 bp, and its deduced amino acid sequence (301 residues) shared high similarity to those of other higher plant L5 proteins. TaL5 protein contained a putative 5S binding region (74 amino acids). TaL5 DNA sequence was further cloned, and sequence analysis showed that it contained 7 introns and 8 exons. Predicated using TargetP software, TaL5 protein was putatively located in mitochondria and contains a transit peptide of 12 amino acids. During grain filling period, temporal expression pattern of TaL5 gene was approximately consistent with the rates of starch accumulation in grains. Additionally, TaL5 gene was dramatically induced by salt, drought and freezing stresses, exogenous abscisic acid (ABA) and salicylic acid (SA) in wheat seedlings. These implied that TaL5 gene could function in growth, development and abiotic stresses in wheat plants.     

Cloning and sequencing of cDNAs encoding a pathogen-induced putative peroxidase of wheat (Triticum aestivum L.)

We report here the complete amino acid sequence of a pathogen-induced putative peroxidase from wheat (Triticum aestivum L.) as deduced from cDNA clones representing mRNA from leaves infected with the powdery mildew fungus Erysiphe graminis. The protein consists of 312 amino acids, of which the first 22 form a putative signal sequence, and has a calculated pI of 5.7. Sequence comparison revealed that the putative wheat peroxidase is most similar to the turnip (Brassica rapa) peroxidase, with which it shares 57% identical and 13% conserved amino acids.     

Molecular cloning,sequence and expression analysis of <Emphasis Type="Italic">ZmArf2</Emphasis>, a maize ADP-ribosylation factor

A full-length cDNA encoding a maize GTP-binding protein of the ADP-ribosylation factor family was cloned by suppression subtractive hybridization and an in silico cloning approach. The cDNA was 938 bp in length and contained a complete ORF of 612 bp, which encodes a protein of 203 amino acid residues. Its deduced amino acids sequence had an 83% identity with that of a GTP-binding protein in rice. The gene was designated ZmArf2. The ZmArf2 gene consists of G1, G2, G3, G4 and G5 boxes, and Switch I and Switch II regions. Eight nucleotides differed and five amino acids changed between the popcorn inbred N04 and the dent corn inbred Dan232. One changed amino acid was in the G1 box. RT-PCR analysis showed that ZmArf2 expression increased in the early stages of endosperm development and was not tissue-specific.     

Cloning of a cytosolic ascorbate peroxidase gene from Lycium chinense Mill. and enhanced salt tolerance by overexpressing in tobacco

To evaluate the physiological importance of cytosolic ascorbate peroxidase (APX) in the reactive oxygen species (ROS)-scavenging system, a full-length cDNA clone, named LmAPX, encoding a cytosolic ascorbate peroxidase was isolated from Lycium chinense Mill. using homologous cloning, then the expression of LmAPX under salt stress was investigated. After sequencing and related analysis, the LmAPX cDNA sequence was 965 bp in length and had an open reading frame (ORF) of 750 bp coding for 250 amino acids. Furthermore, the LmAPX sequence was sub-cloned into prokaryotic expression vector pET28a and the recombinant proteins had a high expression level in Escherichia coli. Results from a southern blot analysis indicated that three inserts of this gene existed in the tobacco genome encoding LmAPX. Compared with the control plants (wild-type and empty vector control), the transgenic plants expressing the LmAPX gene exhibited lower amount of hydrogen peroxide (H₂O₂) and relatively higher values of ascorbate peroxidase activity, proline content, and net photosynthetic rate (P_n) under the same salt stress. These results suggested that overexpression of the LmAPX gene could decrease ROS production caused by salt stress and protect plants from oxidative stress.     

大蕉苯丙氨酸解氨酶基因的克隆、鉴定和表达分析

为了研究苯丙氨酸解氨酶基因与大蕉(Musa ABB cv. Dongguandajiao)抗枯萎病的关系,利用 RT-PCR 和 RACE技术克隆了大蕉苯丙氨酸解氨酶基因全长 cDNA。此 cDNA 长 1 300 bp,包含一个长为 1 191 bp,编码 397 个氨基酸的完整开放阅读框(ORF),推导的氨基酸序列与水稻 PAL 基因氨基酸序列同源性达 89%,将此基因命名为 M-PAL。Southern杂交结果表明大蕉中存在一个包含 4-5 个 PAL基因的基因家族,将此基因克隆到大肠杆菌表达载体 pET32(a )中,表达的蛋白质分子量大小与推导的相一致,并且表达的蛋白质表现出 PAL 酶活性。对接种香蕉枯萎病菌 4 号生理小种(Fusarium oxysporumf. sp. cubense (FOC) race 4 )后大蕉叶片中 M-PAL基因的转录谱进行研究表明,在接种枯萎病菌后,M-PAL基因在叶片中的转录水平提高,因此推测 M-PAL基因的表达可能与香蕉枯萎病抗性相关。     

Molecular characterization of salt-stress-associated protein in citrus: protein and cDNA sequence homology to mammalian glutathione peroxidases

A gene encoding for a citrus salt-stress-associated protein (Cit-SAP) was cloned from Citrus sinensis salt-treated cell suspension. The gene, designated csa, was isolated from a cDNA expression library. The partial amino acid sequence of the protein, as well as that deduced from the nucleotide sequence of csa, revealed a considerable homology to mammalian glutathione peroxidase (GP), and to clone 6P229 from tobacco protoplasts. The increased expression of Cit-SAP in NaCl-treated cultured citrus cells and in citrus plants irrigated with saline water, and its similarity to GP, raise the possibility that one of the effects of salt stress in plants may be the increase of the level of free radicals.     

Molecular Cloning of cDNA for Trehalase from the European Honeybee,Apis mellifera L., and Its Heterologous Expression in Pichia pastoris

cDNA encoding the bound type trehalase of the European honeybee was cloned. The cDNA (3,001 bp) contained the long 5′ untranslated region (UTR) of 869 bp, and the 3′ UTR of 251 bp including a poly(A) tail, and the open reading frame of 1,881 bp consisting of 626 amino acid residues. The M _r of the mature enzyme comprised of 591 amino acids, excluded a signal sequence of 35 amino acid residues, was 69,177. Six peptide sequences analyzed were all found in the deduced amino acid sequence. The amino acid sequence exhibited high identity with trehalases belonging to glycoside hydrolase family 37. A putative transmembrane region similar to trehalase-2 of the silkworm was found in the C-terminal amino acid sequence. Recombinant enzyme of the trehalase was expressed in the methylotrophic yeast Pichia pastoris as host, and displayed properties identical to those of the native enzyme except for higher sugar chain contents. This is the first report of heterologous expression of insect trehalase.     

葡萄风信子MaANS基因及启动子的克隆与分析

该研究根据转录组测序结果,在葡萄风信子(Muscari armeniacum) ‘亚美尼亚’中克隆到花青素合酶(ANS)基因的cDNA与DNA序列,该基因命名为MaANS。采用荧光实时定量分析MaANS时空表达模型,同时利用染色体步移法克隆到MaANS上游1 044 bp的一段序列。信息学分析表明：MaANS开放阅读框为1 065 bp,编码355个氨基酸;DNA与cDNA的一致性为89.62%,DNA序列在ATG下游515~594 bp之间插入1个79 bp内含子;启动子序列在-70 bp位置有1个TATA-box,有多个光响应元件及MYB结合位点等。荧光实时定量分析表明,MaANS基因在花中优势表达,并且在完全着色期表达量最高。该研究结果为深入研究MaANS基因功能、分析葡萄风信子着色机理奠定了基础。     

Isolation and characterisation of a cDNA encoding a novel cytosolic ascorbate peroxidase from potato plants (Solanum tuberosum L.)

Ascorbate peroxidase (APX) is one of the key enzymes of the plant antioxidant system playing, along with catalase, a central role in hydrogen peroxide scavenging. An approach to further increase the knowledge about cytosolic APX gene organization can be achieved by isolating and characterisating new cDNAs, thus providing new insights about the physiological roles and regulation of these enzymes. A partial cDNA clone (corresponding to the 3’ untranslated region), cytosolic ascorbate peroxidase-related, was isolated from potato sprouts by RT-PCR. Database analysis retrieved several expressed sequence tags (ESTs) coding potato cytosolic ascorbate peroxidase, that were used to infer the complete cDNA sequence. The deduced amino acid sequence revealed high homologies with other plant cytosolic ascorbate peroxidases, confirming the reliability of the virtual cDNA. Northern blot analysis revealed the existence of a single band related to the isolated cDNA and the southern blotting results allowed the elaboration of a possible gene organization.     

Cloning and sequencing of a cDNA encoding ascorbate peroxidase fromArabidopsis thaliana

A cDNA clone encoding ascorbate peroxidase (AP, EC 1.11.1.11) was isolated from a phage gt11 library of cDNA fromArabidopsis thaliana by immunoscreening with monoclonal antibodies against the enzyme, and then sequenced. The cDNA insert hybridized to a 1.1 kb poly(A)⁺ RNA from leaves ofA thaliana. Genomic hybridization suggests that the cDNA obtained here corresponds to a single-copy gene. The N-terminal amino acid sequence ofArabidopsis AP was determined by protein sequencing of the immunochemically purified enzyme, and proved to be homologous to the N-terminal amino acid sequence of the chloroplastic AP of spinach. The predicted amino acid sequence of the mature AP ofA. thaliana, deduced from the nucleotide sequence, consists of 249 amino acid residues, which is 34% homologous with cytochromec peroxidase of yeast, but less homologous with other plant peroxidases. Amino acid residues at the active site of yeast cytochromec peroxidase are conserved in the amino acid sequence ofArabidopsis AP. The poly(dG-dT) sequence, which is a potential Z-DNA-forming sequence, was found in the 3 untranslated region of the cDNA.     

Molecular cloning and characterization of a mannose-binding lectin gene from <Emphasis Type="Italic">Pinellia cordata</Emphasis>

Using RNA extracted from Pinellia cordata young leaves and primers designed according to the conserved regions of Araceae lectins, the full-length cDNA of Pinellia cordata agglutinin (PCL) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of pcl was 1,182 bp and contained a 768 bp open reading frame (ORF) encoding a lectin precursor of 256 amino acids. Through comparative analysis of pcl gene and its deduced amino acid sequence with those of other Araceae species, it was found that pcl encoded a precursor lectin with signal peptide. PCL is a mannose-binding lectin with three mannose-binding sites. Semi-quantitative RT-PCR analysis revealed that pcl is expressed in all tested tissues including leaf, stem and bulbil, but with the highest expression in bulbil. PCL protein was successfully expressed in Escherichia coli with the molecular weight expected.     

Identification and molecular characterization of a novel y-type Glu-Dt 1 glutenin gene of Aegilops tauschii

A novel y-type high-molecular-weight glutenin subunit possessing a slightly faster mobility than that of subunit 1Dy12 in SDS-PAGE, designated 1Dy12.1^t in Aegilops tauschi, was identified by one- and two-dimensional gel and capillary electrophoresis. Its coding gene at the Glu-D ^t 1 locus was amplified with allele-specific-PCR primers, and the amplified products were cloned and sequenced. The complete nucleotide sequence of 2,807 bp containing an open reading frame of 1,950 bp and 857 bp of upstream sequence was obtained. A perfectly conserved enhancer sequence and the –300 element were present at positions of 209–246 bp and 424–447 bp upstream of the ATG start codon, respectively. The deduced mature protein of 1 Dy12.1^t subunit comprised 648 amino acid residues and had a Mr of 67,518 Da, which is slightly smaller than the 1Dy12 (68,695 Da) but larger than the 1Dy10 (67,495 Da) subunits of bread wheat, respectively, and corresponds well with their relative mobilities when separated by acid-PAGE. The deduced amino acid sequence indicated that the 1Dy12.1^t subunit displayed a greater similarity to the 1Dy10 subunit, with only seven amino acid substitutions, suggesting that this novel gene could have positive effect on bread-making quality. A phenetic tree produced by nucleotide sequences showed that the x- and y-type subunit genes were respectively clustered together and that the Glu-D ^t 1y12.1 gene of Ae. tauschii is closely related to other y-type subunit genes from the B and D genomes of hexaploid bread wheat.Communicated by H.F. Linskens     

Cloning and Molecular Analysis of cDNA Encoding a Carboxymethylcellulase of the Yeast Cryptococcus flavus

A cDNA copy for carboxymethylcellulase (CMCase 1) of the yeast Cryptococcus flavus was cloned by screening an expression cDNA library with anti-CMCase 1 antibody. The sequence of the cDNA had an open reading frame of 1023 bp that encoded a preprotein of 341 amino acids with a molecular weight of 35,698. The putative precursor begins with a hydrophobic segment that possibly acts as a signal sequence for secretion, which is followed by a presumed prosequence and a sequence consistent with the N-terminal amino acid sequence of secreted CMCase 1. No potential N-glycosylation site was found in the sequence of putative pro-CMCase 1. Comparison of the deduced protein sequence shows that the C. flavus CMCase 1 is partially homologous to the Trichoderma reesei endoglucanase EGIII. Alignment of the cDNA copy and the chromosomal DNA showed seven putative introns of 45 to 134 bp. When introduced into E. coli, the cDNA directed the synthesis of CMCase 1 as seen by CMCase activity and Western blotting using anti-CMCase 1 antibody.     

独一味苯丙氨酸解氨酶基因的克隆与表达分析

以独一味叶片为材料,采用RT-PCR和RACE方法克隆了独一味苯丙氨酸解氨酶基因（PAL）的全长cDNA,命名为LrPAL基因。测序结果表明,LrPA L基因全长2 298 bp,含有1个2 145 bp的完整开放阅读框（ORF）,编码714个氨基酸。蛋白序列分析表明,其包含典型的PAL活性中心序列（GTITASGDLVPLSYIA）,与其他植物的PAL蛋白有很高的同源性。系统进化树分析表明,独一味LrPAL与唇形科植物的PAL蛋白聚为一类,说明两者的亲缘关系较近。用 Real-Time PCR方法检测发现,LrPAL基因在独一味的叶中表达量最高,茎中表达量最少。研究结果推测,从独一味中克隆获得的苯丙氨酸解氨酶基因（LrPAL）是典型的PAL家族成员,在独一味各组织发育过程中具有重要功能。